Hippeastrum hybridum assisted bioreduction of Hydrogen tetrachloroaurate (III) trihydrate: Multifaced application

Hippeastrum hybridum assisted bioreduction of Hydrogen tetrachloroaurate (III) trihydrate: Multifaced application | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hippeastrum hybridum assisted bioreduction of Hydrogen tetrachloroaurate (III) trihydrate: Multifaced application Naila Sher, Mushtaq Ahmed, Nadia Mushtaq, Rahmat Ali Khan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1639345/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nanotechnology is concerned with the production of nanoparticles (NPs) with restricted sizes and shapes through facile, straightforward, and medicinally active phytochemical routes. This study aims to develop an easy and justifiable method for the synthesis of Hippeastrum hybridum (HH) induced gold NPs ( HH-AuNPs) and then to investigate the effects of these NPs as a free radical scavenger, an inhibitor of the two enzymes i-e Alpha-amylase (α-amylase) and acetylcholinesterase (AChE). UV-Vis spectrum at 576 nm with maximum absorbance at 1.96 confirmed the HH-AuNPs formation. Fourier transform infrared spectroscopy (FT-IR) conforms to the peaks for the functional groups of HH extract and on the surface of HH-AuNPs that are involved in the synthesis and stability of the HH-AuNPs. The average size of 10.72 nm was calculated using four major peaks 38.02°, 44.29°, 64.37°, and 77.58° of X-Rays Diffraction (XRD) analysis. The scanning electron microscope (SEM) analysis confirmed the presence of spherical shaped, monodispersed, and huge density HH-AuNPs with an average size of 30 nm. Energy dispersive X-ray (EDX) confirmed the intense sharp peak at 3.1 keV showing that Au was the main element (48.08%). The HH-AuNPs showed an excellent inhibitory efficacy against free radicals, α-amylase, and AChE as compared to HH extract and HAuCl 4 .3H 2 O salt. Our results suggest that HH-AuNPs exhibited significant antioxidant, Antidiabetic, and antialzheime activities in a concentration-dependent manner as compared to HAuCl 4 .3H 2 O and plant extract. However, further investigations are recommended to be able to minimize potential risks of application. Hippeastrum hybridum nanotechnology AuNPs biological activities Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction Nanotechnology is one of the modern techniques of material science which have received much importance in the last many years. It is concerned with the production of nanoparticles (NPs) with restricted sizes and shapes of materials at the nanometer range and is used for the welfare of mankind [ 1 ]. Nanoscience is the science in which we have studied phenomena and management of nanomaterials having different properties than those having a larger scale [ 2 , 3 ]. Application of nanoscience and technology used in applied sciences and as well as material sciences [ 4 ]. The importance of NPs science directly depends on the nano size of NPs; because of the sizes, these particles have made their importance in many fields such as medicine, food industries, agricultural wound dressing, chronic ulcers, and oxide fuel batteries for energy storage, cosmetics, and garments [ 5 , 6 ]. Nowadays, metallic NPs of gold (Au), silver (Ag), copper (Cu), zinc (Zn), titanium (Ti), magnesium (Mg), etc. have been synthesized [ 7 – 9 ]. All Au and Ag have attracted considerable attention in imaging catalysis, sensing, optics, and biomedical devices [ 10 ]. NPs can be synthesized in many chemical and physical ways, but these processes generate hazardous byproducts [ 11 ]. Green nanotechnology, which uses biological organisms, plant biomass or extracts considered an alternative to the conventional chemical and physical methods in a clean, non-toxic, ecologically sound, and environment-friendly manner [ 12 , 13 ]. The use of plant materials for the synthesis of NPs could be more advantageous because it does not require elaborate processes as various biomolecules like Ascorbic acids, Citric acid, flavonoids, dehydrogenases, phenols, saponins, and tannins in the plants play a vital role in Ag and Au reduction [ 7 , 14 , 15 ]. Other compounds that have been used in the formation of AuNPs with antidiabetic properties include chitosan, chondroitin sulfate, tyrosine, and tryptophan [ 16 , 17 ]. Alpha-amylase, (α-amylase) is an important enzyme in the human body responsible for the metabolism of starch i.e., it converts polysaccharides such as starch and glycogen into disaccharides and oligosaccharides. The inhibition of α-amylase by NPs slows down the carbohydrate digestion and can control the reduction in glucose absorption rate [ 18 ]. Like the metal-reducing ability of plant extracts, the antioxidants activity has been associated with the plants’ phenolic [ 19 ]. [ 20 ] argued that antioxidants possess free radical scavenging properties, hence, they play role in promoting health and preventing diseases. Several antioxidant activities [ 21 – 24 ] have been carried out on AuNPs through the green route. Antioxidants extracted from plants play an important role in the prevention of Alzheimer's disease (AD). AD is the most common form of dementia among elderly people that causes problems with memory, thinking, and behavior. Maintaining the levels of acetylcholine (ACh) by inhibition of the acetylcholinesterase enzyme (AChE) is an important strategy to treat AD [ 25 ]. AChE inhibitors like tacrine, donepezil, and galantamine are well-known drugs in the treatment of AD which have separated from herbal sources. Although several reports on the screening of AChE inhibitors from herbal sources have been made [ 26 – 28 ], no attention has been given to inhibitor activity of plant-mediated AuNPs so far. Hippeastrum hybridum (HH) is an ornamental bulbous flowering plant belonging to the family Amaryllidaceae, it has large and showy flowers with many bright colors and is commonly known as Royal Dutch Amaryllis [ 29 ]. Normally it produces 2–3 bulblets in a year of growth [ 30 ]. HH plant is commonly used in physiological and ecological research, but the extent of their genomic and genetic resources remains limited [ 31 ]. Today, a vast majority of plants used in traditional medicine in Pakistan have not been evaluated for their synthesis of AuNPs. The present investigation was, therefore, undertaken to evaluate its AuNPs synthesis, and then evaluated for antioxidant, anti-cholinesterase, and antidiabetic potential. 2. Materials And Methods 2.1. Materials Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 .3H 2 O) obtained from Central Chemical Lahore, was used as the source of AuIII ions. HH plant was collected in September from district Bannu Khyber Pakhtunkhwa and was identified by Dr. Tahir Iqbal, at the Department of Botany, University of Science and Technology Bannu. Potassium ferricyanide, ferric chloride, Trichloroacetic acid, Sulphuric acid, Sodium phosphate, Ammonium molybdenum, ascorbic acid, DPPH (1,1-Diphenyl-2-picrylhydrazyl), H2O2 (Hydrogen peroxide), ABTS (2, 2 azobis, 3-ethyl benzothiazoline-6-sulphonic acid, potassium persulfate (K 2 S 2 O 8 ), Potato starch, sodium acetate buffer, sodium potassium tartrate, 3, 5 dinitro salicylic acid (DNSA), standard (Glucophage), α-amylase, AChE, ACh, DTNB [5,5´-dithiobis(2-nitro-benzoic acid)], bovine serum albumin, and Coomassie Brilliant blue R-250 were purchased from Sigma (USA). Sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Neon Comercial LTDA (Brazil); and Tris (hydroxymethyl aminomethane) from Vetec (Brazil). All other reagents used were of analytical grade. 2.2. Plant’s extraction After identification the plant was washed using water, shade dried, and ground to a fine powder. About 250 g of the fine plant powder was mixed with 70% methanol in 1:3 and kept on an orbital shaker at 120 rpm for 12 h and then placed at room temperature for 7 days, thus after the 7 days the plant is extracted, and filtered by using Whatman filter paper No 1 and concentrated with the help of the rotary evaporator, after the concentration the extra methanol was evaporated at 37°C to obtain a pure crude methanolic extract of sample and was then reserved in the refrigerator at 4°C for more studies [ 32 ]. 2.3. Synthesis of HH induced gold nanoparticles (HH-AuNPs) HH-AuNPs were synthesized from HH plant extract by following the standard protocol [ 33 ]. About 10 mM (0.01 M) solution of HAuCl 4 .3H 2 O was prepared in 50 mL to deionize water. The 10 mM HAuCl 4 .3H 2 O was further diluted 10 times to obtain a 1 mM HAuCl 4 .3H 2 O solution. 0.1 M NaOH, ≥ 98%, and 0.1M HCl were used to adjust the pH. An aqueous solution of HH extracts was prepared by dissolving 1 gm of plant extract in 100 mL of deionized water. For dissolution, it is gently stirred on a magnetic stirrer for about 1 hr. After the complete dissolution, it was centrifuged at 6000 rpm for 30 min. The supernatant was collected for activity and the pallets were discarded. The plant supernatant (50 mL) was mixed with the 500 mL of 1 mM HAuCl 4 .3H 2 O solution of pH 4. The resulting solution changed from golden yellow to crimson red and then finally to ruby red at an optimized ratio after a few hrs at pH 4 and 40ºC temperature. This change in coloration indicated the formation of AuNPs. The solution was then stored for 24 hrs for the complete settlement of NPs and was then monitored using UV–Visible spectrophotometer. The colloidal suspension thus obtained was centrifuged by cold centrifuge at -4°C at 10,000 rpm for 10 min and the pellet was obtained after discarding supermen’s. The synthesized NPs were lyophilized to obtain the powder form. The powdered is further characterized and tested for different biological activities. 2.4. Factors affecting synthesis rate, size, and shape of HH-AuNPs AuNPs synthesis was determined by using different intrinsic factors such as pH, HAuCl 4 .3H 2 O concentration, HH extract concentration, Temperature, Time, and stability time. To study the effect of basic and acidic conditions pH of the reaction mixture was maintained from 4–12 by using 0.1 M NaOH and 0.1 M HCl solution. To study the effect of HH extract concentration on AuNPs synthesis its concentration varied from 0.5, 1, 1.5, and 2 mL. To study the effect of HAuCl 4 .3H 2 O salt concentration; its concentration varied to 0.25, 0.5, 1, and 1.5 mM. To study the temperature effect AuNPs synthesis was carried out under different temperature ranges (20, 40, 60, 80, and 100ºC). To study the time of completion of the reaction AuNPs were synthesized at different time intervals (1 hr, 2 hrs, 3hrs, and 24 hrs). The synthesized AuNPs stability was studied after 1 day, 3 months, and 6 months. 2.5. Characterization of HH-AuNPs HH-AuNPs concentration in the aqueous solution was definite by using SHIMADZU UV SPECTROPHOTOMETER (UV-1800). The purified HH-AuNPs and HH plants extract was examined for the presence of different phytochemicals by using Fourier Transform-Infrared (FT-IR) Shimadzu (IR Prestige-21) spectrometer (Japan). The crystalline nature of the HH-AuNPs was determined by using the JDX-3532 (JEOL JAPAN) X-ray diffractometer (XRD) with λ-1.54 Aº wavelength. The size and shape of HH-AuNPs were determined by using JEOL Scanning Electron Microscope (SEM) Model JSM-5910 (Japan). The presence of elemental Au in synthesized HH-AuNPs was determined by using electron diffraction X-ray spectroscopy (EDX). 2.6. Biological activities 2.6.1. Antioxidant assays 2.6.1.1. Ferric-Reducing Antioxidant Power screening The reducing power potential of HAuCl4.3H2O, HH extract, and HH-AuNPs was done by following [ 34 ] method with a slight modification. About 2 mL samples (HAuCl4.3H2O, HH extract, and HH-AuNPs ), 2 mL of 10 mg/mL potassium ferricyanide, and 2 mL of 0.2 phosphate buffer (pH 6.6) were mixed and followed by incubation for 20 min at 50°C. After incubation 2 mL of 100 mg/mL Trichloroacetic acid was mixed with the solution. About 2 mL of the above solution was mixed with 0.4 mL of 0.1% ferric chloride and 2 mL of deionized H2O followed by incubation for 10 min. Absorbance was observed at 700 nm by spectrophotometer. All samples were run in triplicate. The %age was determined by using the formula (i); Ac is the control absorbance and As is the sample absorbance 2.6.1.2. Ammonium molybdenum assay The Ammonium molybdenum antioxidant potential of HAuCl4.3H2O salt, HH, and HH-AuNPs was carried by following [ 35 ] procedure. About 1 mL of different concentrations of HAuCl4.3H2O, HH extract, and HH-AuNPs (40–160 µg/mL) and 9 mL of (28 mM sodium phosphate, 600 mM Sulphuric acid, and 4 mM Ammonium molybdenum) were mixed in test tubes. The test tubes were capped with aluminum foil and followed by incubation for 90 min at 95ºC in a water bath. After 90 min of incubation, the mixture was then cool to room temperature and absorbance was noted at 695 nm by spectrophotometer. All samples were run in triplicate. The %age scavenging of Ammonium molybdenum was deliberated by using the formula (i). 2.6.1.3. DPPH activity 1,1-Diphenyl-2-picrylhydrazyl (DPPH) antioxidant potential was carried by following [ 36 ] method. Stock solutions (1 mg/mL) of HAuCl 4 .3H 2 O, HH, and HH-AuNPs were prepared in deionized water which was further diluted into (40, 80,100, and 160 µg/mL). Standard ascorbic acid is also prepared in a similar concentration. About 200 µL from different concentrations of HAuCl 4 .3H 2 O, HH, HH-AgNPs, and the standard was mixed with 800 µL of DPPH (1.5 mg/50mL methanol) and then incubated for 30 min in dark a room temperature. Absorbance spectra were recorded at 517 nm by using a UV spectrophotometer against water as a reference. All samples were run in triplicate. The %age scavenging was deliberated by using the equation (i). 2.6.1.4. Hydrogen peroxide scavenging (H 2 O 2 ) The H 2 O 2 scavenging activity was analyzed by [ 37 ] method with certain modifications. About 200 µL from various concentrations (40 to160 µg/mL) of HAuCl 4 .3H 2 O, HH, and HH-AuNPs in deionized water, 400 µL of 2 mM H 2 O 2 , and 400 µL of 50 mM phosphate buffer (pH 7.4) were mixed and followed by the incubation for 20 minutes at 35ºC. The absorbance was recorded by using a spectrophotometer at 610 nm against phosphate buffer as blank. All samples were run in triplicate. The %age was determined by using the equation (i). 2.6.1.5. ABTS screening assay The ABTS free radical scavenging activity of HAuCl 4 .3H 2 O, HH extract, and HH-AuNPs was accomplished by [ 38 ] procedure with slight modification. About 7 mM of ABTS solution and 2.45 mM of potassium persulfate (K 2 S 2 O 8 ) solution was prepared in deionized water. These two solutions were mixed and allowed for overnight incubation, dark coloration indicated the existence of ABTS•+ free radicals in the solution. The optical density of the mixture was determined using a spectrophotometer and was brought to 0.700 (± 0.02) by the addition of more solvent. About 300 µL of HAuCl 4 .3H 2 O, HH extract, and HH-AuNPs (40 to 160 µg/mL) and standard mixed with 300 µL of (K 2 S 2 O 8 + ABTS) mixture. The absorbance was recorded immediately after mixing the solution at 734 nm by using a spectrophotometer. All samples were run in triplicate. The %age scavenging was deliberated by using the equation (i). 2.6.2. Anti-α-amylase activity Inhibition of α-amylase activity was determined using 3,5 dinitro salicylic acid (DNSA) [ 39 ]. To an obtained starch solution (1% w/v) 1 gram of potato starch was dissolved in 100 mL of 16 mM C 2 H 3 NaO 2 (Sodium acetate) buffer. To obtained enzyme solution 0.5 mg/mL α-amylase from stock (250 units/mL) was dissolved in 1 mL dH 2 O. Sodium potassium tartrate and DNSA (96 mM) mixtures were used as a calorimetric reagent. The stock solution of HAuCl4.3H2O, HH extract, HH-AuNPs, and standard (Glucophage) was prepared at 1 mg/mL and was further diluted into different sub-solutions i.e. 25, 50, 75, and 100 µg/mL. Samples were added to 250 µL of α-amylase. The mixture was pre-incubated at 25°C for 10 min and 250 µL of 1% starch prepared in 20 mM sodium phosphate buffer (pH 6.9) was added. The reaction mixtures were incubated at 25°C for 10 min. The reactions were stopped by incubating the mixture in a boiling water bath for 5 min after adding 250 µL from the combined mixture of DNSA and sodium potassium tartrate. The reaction mixtures were cooled to room temperature, diluted to 1:5 ratios with deionized water, and absorbance was measured in a spectrophotometer (double beam UV-1602, BMS-spectrophotometer) at 450 nm. The Glucophage served as a positive control. All samples were run in triplicate. The percentage of inhibition of enzyme activity was calculated by using the formula (i). 2.6.2.1. Mode of α-amylase inhibition assay The mode of inhibition of α-amylase is determined as described before [ 40 ]. For α-amylase, the enzyme solution (250 units/mL) was pre-incubated with samples (25, 50, 75 and 100 µg/mL). The reactions were started by adding 100, 200, and 300 mg of potato starch and continued at 25°C for 10 min. The reactions were stopped by adding 0.25 mL of DNSA followed by boiling for 5 min. The reaction mixtures were cooled to room temperature, diluted to a 1:5 ratio with dH 2 O, and absorbance was measured in a spectrophotometer (double beam UV-1602, BMS-spectrophotometer) at a 450 nm Double reciprocal plot (1/V versus 1/[S]) where V is reaction velocity and [S] is substrate concentration was plotted. The mode of inhibition was determined by analyzing the Lineweaver-Burk plot using Michaelis-Menten kinetics [ 41 ]. Michaelis constants (K m ) were determined by two different plots of 1/V vs. 1/S [ 41 ] and V vs. V/S [ 42 , 43 ]. The K i and KI values were obtained using the Cornish-Bowden plot of S/V vs. [I] and Dixon plot 1/V vs. [I] [ 44 , 45 ] respectively. IC 50 was determined by percentage residual activity and percentage inhibition versus concentration of HH extract and HH-AuNPs. 2.6.3. Anti-cholinesterase activity 2.6.3.1. Venom Venom from live Bungarus sindanus snakes was squeezed out manually, lyophilized immediately, and stored at − 20°C for further use. The study was approved by the Departmental Ethical Approval Committee, ref. n. Biotech/Ethic/110. 2.6.3.2. Anti-cholinesterase assay AChE activity was determined by the method of [ 46 ] modified by [ 47 ] using a double beam spectrophotometer UV-1602, BMS biotechnology medical service. Hydrolysis rates (V) were measured at various acetylthiocholine (S) concentrations (0.05–1 mM) in a 1 mL assay mixture with 50 mM phosphate buffer, pH 7.4, and 10 mM DTNB at 25oC. About 20 µL of diluted snake venom was also added and the reaction mixture was incubated for 5 minutes at 37 o C. The enzyme-substrate reaction immediately started upon the addition of different concentrations of substrate. The hydrolysis was scrutinized by the formation of thiolate di-anion of DTNB every 15 seconds for 90 seconds using a spectrophotometer. The amount of the yellow color develops is a measure of the activity of AChE. All samples were run in triplicate. 2.6.3.3. Protein determination The protein content of the enzyme preparation was assayed by the method of Bradford [ 48 ] using bovine serum albumin as a standard. 2.6.3.4. Kinetic determinations The interaction of HH extract/HH-AuNPs, and AChE was determined using the [ 41 ] double reciprocal plot, by plotting 1/V against 1/[S] analyzed over a range of ACh concentrations (0.05–1 mM) in the absence and presence of extract (10, 20, and 30 µg/mL). A double reciprocal plot (1/V versus 1/[S]) where V is reaction velocity and [S] is substrate concentration was plotted. The mode of inhibition was determined by analyzing the Lineweaver-Burk plot using Michaelis-Menten kinetics [ 41 ]. Michaelis constants (Km ) were determined by two different plots of 1/V vs. 1/S [ 41 ] and V vs. V/S [ 42 , 43 ]. The Ki and KI values were obtained using the Cornish-Bowden plot of S/V vs. [I] and Dixon plot 1/V vs. [I] [ 44 , 45 ] respectively. IC50 was determined by percentage residual activity and percentage inhibition versus concentration of HH extract and HH-AuNPs. 2.7. Statistical Analysis Data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA, which was followed by post-hoc analysis (Duncan multiple range test) [ 49 ]. The coefficient of correlation was determined by using Statistics (version 8.1 USA). The difference was considered to be significant for P < 0.05 3. Results 3.1. Synthesis of HH-AuNPs 3.1.1. UV-visible spectrophotometric analysis of HH-AuNPs The aqueous AuNPs exhibited ruby red color due to the SPR. After mixing the HH aqueous extract with HAuCl 4 .3H 2 O the color of the solution start to change from golden yellow to crimson red and then finally to ruby red indicating the bioreduction of the Au + by HH extract. The scale of the length of the spectrophotometer ranged from 200 to 800 nm. Figure 1 indicated 576 nm, absorption bands of the HH-AuNPs, at higher absorption of 1.96 after 24 hrs of incubation at 40ºC. 3.1.2. Factors affecting the synthesis of AuNPs In the present study; the synthesis of HH-AuNPs was studied under different factors. According to a pH study, HH-AuNPs were synthesized at different basic and acidic conditions (pH 4 to 12). No sharp band was observed in the range of 500–600 nm at higher pH of 8 to 12. The bands become sharp and sharp with decreasing the pH and a final sharp peak of 576 nm with a maximum absorbance of 1.96 was reported at pH 4. See Fig. 2a. According to the HH extract concentration studies different peaks i-e 556, 576, 548, and 550 were observed with a maximum absorbance of 0.6893, 1.96, 1.9135, and 1.7776 at the volume of 0.5, 1, 1.5, and 2 mL respectively. See Fig. 2b. The UV-vis spectra of HH-AuNPs aqueous medium at different HAuCl4.3H2O concentrations (0.25–1.5 mM) were noted in the range of 200 to 800 nm wavelength which indicated broader bands at 0.25, 0.5, and 1.5 mM with low absorbance but a sharp peak 576 nm with maximum absorbance 1.96 was obtained at 1 mM (See Fig. 2c). HH-AuNPs synthesis was studied by incubating the reaction mixture at different temperature ranges (20 to 100ºC). A sharp band at 576 nm was obtained at 40ºC with a maximum absorbance of 1.96, but with an increase in temperature beyond 40ºC the band becomes broad and broader i-e 558, 546, and 567 nm with a maximum absorbance of 1.0505 0.9098, and 0.7089 were reported at 60, 80, and 100ºC respectively. See Fig. 2d. Figure 2e indicated the effect of reaction time on the synthesis of HH reduced AuNPs. Broad peaks with lower absorption appeared after 1hr, 2hrs, and 3hrs of the stirring (685, 654, and 500 nm with a maximum absorbance of 0.251, 0.182, and 0.54 respectively). Due to the continuous reduction of Au ions by HH extract the absorption peak increases over time. A final clear sharp peak of 576 nm at high absorbance 1.96 was observed after 24 hrs of the HH and Au ions reaction. The stability of the HH reduced AuNPs was studied at different periods (1 day, 3 months, and 6 months). A sharp peak of 576 nm with a maximum absorbance of 1.96 appeared after 1 day of AuNPs formation; but after 3 and 6 months this peak become broad i-e 558 nm after 3 months and 567 nm after 6 months, with low absorbance of 1.0505 and 0.7089 respectively. (See Fig. 2f). 3.2. FT-IR analysis of HE extract and HH-AuNPs The FTIR spectrum of HH extracts and HH-AuNPs (prepared in water) is given in Fig. 3 . The data on the peak values and the probable functional groups (obtained by FTIR analysis) present in the HH extract and HH-AuNPs are presented in Table 1 . The characteristic absorption band were exhibited in the range 3400-3200cm − 1 (for O-H stretch), 2935–2915cm − 1 (for –CH (CH 2 ) vibration), 2865-2845cm − 1 (for –CH (CH 2 ), 2260-2100cm − 1 (for C ≡ C stretch), 2100-1800cm − 1 (for C = O frequency), 1740-1725cm − 1 (for C = O stretch), 1650-1600cm − 1 (for C = O stretch), 1410-1310cm − 1 (for O-H bend), 1340-1250cm − 1 (for CN stretch), 1100-1000cm − 1 (for Phosphate ion), 995-850cm − 1 (for P-O-C stretch), 800-700cm − 1 (for C-Cl stretch), 700-600cm − 1 (for C-Br stretch), and 690-550cm − 1 (for C-Br stretch) were exhibited by HH extract and HH-AuNPs. Table 1 FTIR Interpretation of compounds in HH whole plant extract and HH-AuNPs S. No Wave number cm -1 [Reference article] Wave number cm -1 [HH-plant] Wave number cm -1 [HH-AuNPs Functional group assignment Phyto compounds Identified 1 3400 − 3200 3266.48 3324.90 O-H stretch Poly Hydroxy compound 2 2935–2915 2917.21 2922.72 Asymmetric stretching of –CH (CH 2 ) vibration Saturated aliphatic compound-Lipids 3 2865 − 2845 2855.98 2852.04 Symmetric stretching of –CH (CH 2 ) vibration, Lipids, protein 4 2260 − 2100 2259.95 2245.12 Carbon-Carbon triple bond Terminal alkynes 5 2100 − 1800 1985.34 1963.9 Carbonyl compound frequency Transition metal carbonyls 6 1740 − 1725 1740.10 1729 C = O stretch Aldehyde compound 7 1650 − 1600 1608.02 1638.20 C = O stretching vibration, Ketone group Ketone compound 8 1410 − 1310 1410 1410 O-H bend, Alcoholic group Phenol or tertiary alcohol 9 1340 − 1250 1290.95 1250 CN stretch Aromatic primary amine 10 1100 − 1000 1035.52 1010.16 Phosphate ion Phosphate compound 11 995 − 850 852.68 852.1 P-O-C stretch Aromatic phosphates 12 800 − 700 743.11 714.42 C-Cl stretch Aliphatic Chloro compound 13 700 − 600 676.003 622.61 C-Br stretch Aliphatic bromo compounds 14 690 − 550 571.91 555.2 Halogen compounds (Bromo-compounds) Aliphatic Bromo compounds 3.3. XRD analysis of HH-AuNPs XRD is a technique that is used for determining the size and crystalline nature of the sample. In the present study, the HH-AuNPs were analyzed by XRD. Figure 4 indicated 4 Bragg reflections at angles of 38.02°, 44.29°, 64.37°, and 77.58° which are corresponded to the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1) respectively. These reflections can be indexed conferring to the face of the face-centered cubic crystal structure of Au ion. The “d’ (interplanar spacing) and “a” (Miller constants) values were calculated by using the Debye-Sherrer’s equation (i) and (ii) respectively; Results are tabulated in Table 2 . Table 2 Determination of Interplaner spacing and lattice constant of HH-AuNPs S. no 2θ Value Element plane Interplaner spacing (d) Lattice constants (a 0 ) 1 2 3 4 38.02 44.29 64.37 77.58 Au Au Au Au 1 1 1 2 0 0 2 2 0 3 1 1 2.36 Å 2.06 Å 1.44 Å 1.22 Å 4.08 Å 4.08 Å 4.07 Å 4.04 Å Table 3 The HH and HH-AuNPs concentrations providing 50% inhibition (IC 50 ) values of the different antioxidants activities. IC 50 (µg/mL) Assays Ascorbic Acid HH extract HH AuNPs Ferric reducing Molybdenum scavenging DPPH scavenging H 2 O 2 scavenging ABTS scavenging 79 ± 0.06 48 ± 0.11 52.38 ± 0.21 51.18 ± 0.3 40.059 ± 0.0.05 151 ± 0.13 91.48 ± 0.13 156 ± 0.31 136 ± 0.21 154.12 ± 0.03 95.17 ± 0.025 58.5 ± 0.051 136.17 ± 0.071 105.66 ± 0.062 144.82 ± 0.072 Table 4 Effect of Glucophage, HH extract and HH-AuNPs on K m and V max of α-amylase. HH AuNPs Concentrations (µg) K m (mg) V max (µmol α-amylase/min/mg protein) %Decrease 0 25 50 75 100 66.171 66.63 67.03 66.77 66.85 0.0265 0.023 0.0225 0.0215 0.0184 0 13.20 15.094 18.96 30.56 A HH extract Concentrations (µg) K m (mg) %Increase V max (µmol α-amylase/min/mg protein) 0 25 50 75 100 68.50 75.8 91.4 111.64 126.33 0 10.65 33.43 62.9 84.37 0.0269 0.0264 0.0264 0.0271 0.0267 B Glucophage Concentrations (µg) K m (mg) %Decrease V max (µmol α-amylase/min/mg protein) %Decrease 0 25 50 75 100 194.56 115.78 80.49 67.78 60.012 0 40.49 58.74 56.6 69.15 0.0566 0.0346 0.0252 0.0216 0.0172 0 38.86 55.47 61.83 69.61 C Table 5 Effect of HH extract, HH AuNPs, and Glucophage on K Iapp and V maxiapp of α-amylase. The V maxiapp and K Iapp were determined from Dixon plot of 7A and 7B for α-amylase. The V maxiapp is equal to the reciprocal of y-axis intersection of each line for each potato starch concentration while K Iapp is equal to the x-axis intersection in Dixon plot. HH-AuNPs [Potato starch] (mg) K Iapp (µg) V maxiapp (µg / min / mg) % Decrease 100 200 300 261.74 260.22 263.78 0.016 0.018 0.022 0 12.5 37.5 A HH Extract [Potato starch] (mg) K Iapp (µg) % Increase V maxiapp (µg / min / mg) 100 200 300 139.53 317.64 634 0 127.64 354.33 0.20 0.20 0.22 B Glucophage K Iapp (µg) % Decrease V maxiapp (µg / min / mg) % Increase 128.30 106.95 69.44 0 16.64 46 0.0198 0.025 0.037 0 26.26 89 C Table 6 Comparative study of kinetic parameters of α-amylase inhibition by HH extract, HH-AuNPs, and Glucophage; K i , inhibition constant; K I , dissociation constant of the α-amylase–Pottao starch–HH complex into the α-amylase–Pottao starch complex and free HH; K m , Michaelis–Menten constant and IC 50, 50% inhibitory concentration. Parameters HH AuNPs HH Extract Glucophage K i (µg) K I (µg) K m (mg) IC 50 (µg) 25 261.91 66 44.33 ± 0.042 26 364 68.06 56 ± 0.003 12 101.56 195 37 ± 0.13 Table 7 Influence of HH and HH-AuNPs on K m and V max of Bungarus Sindanus (Krait) venom AChE. S.no HH-AuNPs HH extract Concentrations (µg) K m (mM) % Decrease V max (µmol / min per mg protein) % Decrease K m (mM) V max (µmol / min per mg protein) % Decrease 0 0.197 0 56.94 0 0.049 157.33 0 10 0.1025 47.96 29.98 47.34 0.049 62.85 60.05 20 0.059 70.05 19.79 65.24 0.053 48.47 69.19 30 0.050 74.61 15.93 72.02 0.049 40.89 74.01 Table 8 Effect of HH extract and HH-AuNPs on K Iapp and V maxiapp of Bungarus Sindanus (Krait) venom AChE. The V maxiapp and K Iapp were determined from Dixon plot of Fig. 12 for snake venom, AChE. The V maxiapp is equal to the reciprocal of y-axis intersection of each line for each AcSCh concentration while K Iapp is equal to the x-axis intersection in Dixon plot. S.no HH-AuNPs HH extract [ASCh] (mM) K Iapp (mM) %Decrease V maxiapp (µmol / min per mg protein) %Decrease K Iapp (mM) V maxiapp (µmol / min per mg protein) % Decrease 0.05 0.1 0.25 0.5 1 80.75 55.02 39.123 28.46 23.81 0 32 51.55 64.75 70.51 11.62 16.08 22.34 33.78 41.73 0 38.38 92.25 190.70 259.122 36.49 36 36.13 36.24 36.47 38.29 57.38 60 68.46 77.23 0 49.85 56.6 78.79 104.42 Table 9 Study of kinetic parameters of AChE inhibition by HH-AuNPs and HH extract. K i , inhibition constant; K I , dissociation constant of the AChE–ASCh–HH complex into the AChE–ASCh/BSCh complex and free HH; K m , Michaelis–Menten constant, and IC 50, 50% inhibitory concentration. Parameters HH-AuNPs HH extract K i ( µg) K I ( µg) K m (mM) IC 50 ( µg) 23 45.43 0.195 16.66 ± 0.011 32 36.25 0.052 19.1 ± 0.021 The average crystalline size of HH induced AuNPs is calculated by using the Debye-Sherrer’s formula (iii); D is the average crystalline size, k is a geometric factor (0.9), λ is the wavelength of the X-ray radiation source and β is the angular FWHM (full-width at half maximum) of the XRD peak at the diffraction angle θ. For the four major peaks i-e 38.02°, 44.29°, 64.37°, and 77.58° the average calculated crystalline size was found to be 10.72 nm. 3.4. SEM analysis of HH-AuNPs In the present study; HH-AuNPs were screened by SEM to determine their surface morphology. SEM results showed that the HH-AuNPs are monodispersed, spherical-shaped, and are in high density (Fig. 5 ). The average particle size was calculated using Nano measurer software by marking 15 particles and was found to be 30 nm (0.03 µm). 3.5. EDX analysis of HH-AuNPs The elemental constituents and relative abundance of the biosynthesized AuNPs were obtained from EDX analysis as shown in Fig. 6 . EDX spectrometers confirmed the presence of elemental Au signal of the AuNPs with 48.08%. The vertical axis displays the number of X-ray counts while the horizontal axis displays energy in KeV. Identification lines for the major emission energies for Au are displayed and these correspond with peaks in the spectrum, thus giving confidence that Au has been correctly identified in HH-AuNPs. Thus the EDX spectrum discloses the purity and the complete chemical composition of HH-AuNPs. The other elements served as capping organic agents bound to the surface of the HH-AuNPs. 3.6. Antioxidant assays 3.6.1. Ferric reducing power assay (FRPA) In the present study; different concentrations (40, 80, 120, and 160µg/mL) of HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid were tested for ferric reducing activity. The obtained results indicated that all the tested samples showed ferric reducing potential in a dose-dependent manner and showed maximum scavenging of 16.92 ± 0.014 (HAuCl 4 .3H 2 O), 55.2 ± 0.035% (HH extract), and 77.2 ± 0.014 (HH-AuNPs); while the positive control ascorbic acid showed 84.4 ± 0.016% scavenging at the highest 160 µg/mL concentration (Fig. 7a). All the samples i-e HH extract, HH-AuNPs, and Ascorbic acid cause 50% scavenging (IC 50 ) at 151 ± 0.13, 95.17 ± 0.025, and 79 ± 0.06 µg/mL respectively; while the HAuCl 4 .3H 2 O does not cause 50% inhibition. (See the Table. 3). 3.6.2. Ammonium molybdenum assay Different concentrations (40–160 µg/mL) of HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid were tested for their Ammonium molybdenum scavenging assay (See Fig. 7b). HAuCl 4 .3H 2 O salt, HH extracts, HH-AuNPs, and ascorbic acid showed maximum scavenging potential of 22 ± 0.0014, 62.1 ± 0.0021, 74.3 ± 0.021, and 88.8 ± 0.001% at 160 µg/mL. The calculated IC 50 values were found to be 91.48 ± 0.13 for HH extract, 58.5 ± 0.051 for HH-AuNPs, and 48 ± 0.11 µg/mL for ascorbic acid respectively; while the HAuCl 4 .3H 2 O does not cause 50% scavenging of Ammonium molybdenum (See the Table. 3). 3.6.3. DDPH scavenging assay Different concentrations of HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid (40, 80, 120, and 160µg/mL) were tested for their DPPH scavenging activity. The obtained results indicated that HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid showed DPPH scavenging potential in a dose-dependent manner and showed maximum scavenging of 21 ± 0.007, 55 ± 0.059, 58.6 ± 0.51, and 90 ± 0.0014% % at 160µg/mL respectively (Fig. 7c). All the samples except HAuCl 4 .3H 2 O salt showed 50% DPPH scavenging activity. The calculated IC 50 (50% inhibition) values were found to be 156 ± 0.31for HH extract, 136.17 ± 0.071 for HH-AuNPs, and 52.38 ± 0.03 µg/mL for Ascorbic acid respectively. Results are tabulated in Table. 3. 3.6.4. Hydrogen peroxide scavenging (H 2 O 2 ) In the current research work; different concentrations (40–160 µg/mL) of HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid were tested for their H 2 O 2 scavenging activity (Fig. 7d). HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid showed maximum scavenging potential of 21 ± 0.021, 56.6 ± 0.0021, 62 ± 0.041, and 76.6 ± 0.0014% at 160 µg/mL with IC 50 values of 136 ± 0.21 for HH extract, 105.66 ± 0.062 for HH-AuNPs, and 51.18 ± 0.3 µg/mL for ascorbic acid respectively; while the HAuCl 4 .3H 2 O does not cause 50% inhibition of H 2 O 2 . (See the Table. 3). 3.6.5. ABTS screening assay The present study indicated the ABTS scavenging potential of HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid at various concentrations (40–160 µg/mL). Figure 7e indicated that HAuCl 4 .3H 2 O salt, HH extract, HH-AuNPs, and ascorbic acid scavenge ABTS free radicals in dose-dependent manner and indicated 21 ± 0.0014, 55.1 ± 0.004, 59 ± 0.05, and 90 ± 0.002% inhibition at the highest concentration (160 µg/mL) with an IC 50 of 154.12 ± 0.03 for HH extract, 144.82 ± 0.072 for HH-AuNPs, and 40.059 ± 0.0.05µg/mL for ascorbic acid respectively; while HAuCl 4 .3H 2 O salt does not show 50% scavenging of ABTS free radicals. (See the Table. 3). 3.7. Antidiabetic activity 3.7.1. Anti-α-amylase activity The HAuCl 4 .3H 2 O salt, extracts of HH, HH-AuNPs, and standard drug Glucophage were tested to evaluate theirs in vitro inhibition abilities against α-amylase. All the samples inhibit α-amylase in dose-dependent manner (25, 50, 75, and 100 µg/mL) and cause maximum inhibition of 7 ± 0.0014% (HAuCl 4 .3H 2 O salt), 66 ± 0.003% (HH extract), 71 ± 0.032% (HH-AuNPs), and 85 ± 0.0014% (Glucophage) respectively at fixed (1%) substrate concentration (Fig. 8 ). 3.7.2. Effects of HH, HH-AuNPs, and Glucophage on K m and V max The effect of HH, HH-AuNPs and Glucophage on K m and V max of α-amylase were calculated. Statistical analysis indicated that HH-AuNPs cause a non-competitive type of inhibition K m to remain constant and V max decrease from 13.20 to 30.56% (Fig. 9a), similarly HH causes a competitive type of inhibition of α-amylase i-e. K m increases from 10.65 to 84.37%, while V max remained constant (Fig. 9b), while in the case of Glucophage uncompetitive type of inhibition was observed i-e. Both K m and V max decreased from 40.49 to 69.15 and 38.86 to 69.61% respectively (Fig. 9c). Results are presented in Table. 4A, 4B, and 4C. 3.7.3. Effects of HH, HH-AuNPs, and Glucophage on K Iapp and V maxiapp In α-amylase, K Iapp remained constant while V maxiap was decreased from 12.5 to 37.5% for HH-AuNPs, while for HH extract the K Iapp was found to increase from 127.64 to 354.33% while V maxiapp remained constant with the increase of substrate concentration (100–300 mg). In the case of Glucophage, the K Iapp was found to decrease from 16.64 to 46% while V maxiapp decreased from 26.26-to 89% (Table. Table. 5a, 5b, and 5c). The values were calculated from Fig. 10a, 10b, and 10c respectively. 3.7.4. Determination of K m K m values for the hydrolysis of the substrate (potato starch) by α-amylase, were calculated by using the Lineweaver-Burk plot and were found to be 66, 68.06, and 195 mg for HH-AuNPs, HH, and Glucophage respectively. The values are presented in Table. 6. 3.7.5. Determination of K I and K i K I (constant of α-amylase–Potato starch–HH-AuNPs, HH extract/Glucophage complex into α-amylase–Potato starch complex and HH-AuNPs, HH extract/Glucophage) was estimated to be 261.91 (Fig. 10a), 364 µg (Fig. 10b), and 101.56 µg (Fig. 10c) for α-amylase respectively, while Ki (inhibitory constant) was estimated to be 25, (Fig. 11a), 26 (Fig. 11b) and 12 µg (Fig. 11c) for HH-AuNPs, HH extract, and Glucophage respectively. The values are tabulated in Table. 6. 3.7.6. Determination of IC 50 HH-AuNPs, HH extract, and Glucophage cause 50% inhibition (IC 50 ) against α-amylase at a concentration of 44.33 ± 0.042, 56 ± 0.003, and 37 ± 0.13 µg/mL, respectively. The values are tabulated in Table. 6. 3.8. Anti-acetylcholinesterase activity At fixed substrate acetylthiocholine (ACh) concentration (0.5 mM) HAuCl 4 .3H 2 O salt, HH plant, and HH-AuNPs exerted 23 ± 0.057, 59 ± 0.003, and 61 ± 0.314% inhibition against the snake krait venom AChE at maximum 30 µg/mL concentration in 1 mL assay mixture (Fig. 12 a). 3.8.1. Determination of IC 50 The concentration of HH extract and HH-AuNPs that cause 50% inhibition (IC 50 ) of AChE enzyme activity were found to be 19.1 ± 0.021 and 16.66 ± 0.011 µg/mL respectively; while no any concentration of HAuCl 4 .3H 2 O salt cause 50% inhibition of AChE (Fig. 12 b). 3.8.2. Effects of HH on K m and V max The effect of HH and HH-AuNPs on K m and V max of AChE were calculated. HH caused a non-competitive type of inhibition the K m values remained constant and V max decreased from 60.05–74.01%, while HH-AuNPs caused an uncompetitive type of inhibition in both K m and V max decreased from 47.96–74.61 and 47.34–72.02% respectively (Fig. 13 a and 13 b). Values are presented in (Table. 7). 3.8.3. Effects of HH on K Iapp and V maxiapp In snake venom AChE, K Iapp was found to remain constant, while V maxiapp decreased from 49.85 to 104.42% HH extract, while in the case of HH-AuNPs K Iapp decreased from 32-70.51% and V maxiapp decreased from 38.38-259.122% with an increase of substrate (0.05–1 mM) (Table. 8). The values were calculated from Fig. 14a and 14b. 3.8.4. Determination of K m K m values for the hydrolysis of substrate by AChE were calculated by using a Lineweaver-Burk plot and were found to be 0.195 and 0.052 mM for HH-AuNPs and HH extract, respectively. The values are presented in Table. 9. 3.8.5. Determination of K I and K i K I (constant of AChE–AcSC–HH-AuNPs/HH extract complex into AChE–AcSC complex and HH-AuNPs/HH) was estimated to be 44.43 and 36.25 µg (Fig. 14a and 14b) for AChE, while K i (inhibitory constant) was estimated to be 23 and 32 µg for HH-AuNPs and HH extract respectively, (Fig. 15a and 15b). The values are presented in Table. 9. 4. Discussion The synthesis and characterization of NPs and their applications represent a rapidly growing concept and an emerging trend in science and technology [ 50 ]. The use of plant materials for the synthesis of NPs could be more advantageous because it does not require elaborate processes [ 51 ]. The UV–vis spectroscopy technique can be used to determine the synthesis and stability of AuNPs in an aqueous solution due to their characteristic absorption in the range of 500–600 nm [ 52 , 53 ]. AuNPs exhibited ruby red color in the aqueous solution due to the excitation of surface plasmon vibrations in the metal NPs which give rise to the surface plasmon resonance (SPR) band [ 54 , 55 ]. The plasmon resonance (PR) can be pictured as a ‘‘wave’’ of electrons sloshing over the surface of a metal NPs. As a result, an enhanced electromagnetic field at and near the metal NPs surface is set up. The position of the plasmon band (extinction spectrum) is best measured on a conventional UV–vis spectrophotometer and appears as a band with extremely high extinction coefficients [ 56 ]. In the present study, when 1 mL of HH aqueous extract was added to 1 mM HAuCl 4 .3H 2 O solution at pH 4 the color of the solution start to change from golden yellow to crimson red and then finally to ruby red at an optimized ratio after 24 hrs of incubation at 40ºC. The appearance of the ruddiness color in an aqueous medium is considered the first indication of colloidal AuNPs formation [ 53 , 54 ]. This color change is due to the reduction of the Au + into HH-AuNPs by the active molecules of the HH aqueous extract such as phenolic, alkaloids, saponins, amino acids, proteins, etc. The absorption spectrum of the aqueous solution revealed a peak at 576 nm with a maximum absorbance of 1.96 after 24 hrs. Synthesis of the AuNPs is affected by different factors such as plant extract volume, HAuCl 4 .3H 2 O, pH, temperature, time, etc. [ 57 ]. An ideal pH is required for synthesis of controlling the shape and size of NPs [ 58 , 59 ]. The role of pH is significant in changing the size and shape of NPs. Numbers of studies have shown the stability of AuNPs at acidic pH while many achieved stable suspension in the basic region. Furthermore, the synthesis reaction, size, shape, and stability of AuNPs could be controlled by adjusting the initial pH value of the reaction mixture. In the present study, the reaction was performed at different pH ranges from 4 to 12 to identify the effect of pH on the formation of HH-reduced AuNPs. It was detected that the absorbance of the solution increased while changing the initial pH of the solution. With the increase in pH, the SPR band was also blue-shifted to 576 nm at pH 4. The AuNPs were quite stable in an acidic medium; however, a gradual decrease in UV–vis peak intensities showed less stability with an increase in pH of the colloidal solution from 4 to 12. Peak broadening and redshift were noted at pH 10 and 11. UV-vis results suggested that no reaction occurred in the basic region which is in agreement with the previous studies [ 54 ]. It was might be due to the deprotonation of hydroxyl and carboxyl groups present in extracts [ 60 ]. AuNPs synthesized using banana peel extract were stable at a pH value of 2–5 supporting the results of the present study [ 61 ]. A broader range of pH i.e. 2–11 were taken to synthesize AuNPs using oil palm mill effluent and pH 3 was observed optimum to achieve definite shapes particles [ 62 ]. Medicinal plants are a rich source of secondary metabolites that act as reducing, capping, and stabilizing agents for NPs. However, the composition of these active secondary metabolites varies from plant to plant depending on the nature, part, type of plant, and method followed for the extraction of these metabolites [ 63 ]. In the present study, the SPR peak is blue-shifted from 553 to 576 nm as the added volume of HH extract increased from 0.5 to 1mL; the blue shift of SPR peaks is a sign of the production of small size NPs. The shift towards shorter wavelengths with decreasing NPs size is associated with frequencies of oscillation of different free electrons in the conduction band [ 64 ]. The SPR band absorbance increased with an increasing volume of HH extract, which reveal the higher production of AuNPs. This is due to the availability of more reducing agents for the Au ions bioreduction [ 65 ]. But the further increase in the volume of HH extract from 1.5 to 2 mL the redshift of SPR was recorded, this redshift is a sign of large size NPs. HH contains active molecules such as carbohydrates, flavones, terpenoids, alkaloids, and proteins that were testified to be responsible for the bio-reduction of Au + to Au o . Proteins and terpenoids are believed to play an important role in AuNPs biosynthesis through the reduction of Au ions, and carbohydrates provide a coating of AuNPs [ 66 , 67 ]. The HH extract produces more AuNPs than other plant extracts credited to the availability of the larger amount of reducing agents in the extract, such as flavonoid and its antioxidant activity [ 67 ]. Varying concentrations of HAuCl 4 .3H 2 O (0.25 to 1.5 mM) were prepared while the other factors were kept constant i-e HH extract (1 mL), pH (4), temperature (40ºC), and time (24 hrs). At 0.25 and 0.5 mM concentrations of HAuCl 4 .3H 2 O broad peaks were revealed; while at increased HAuCl 4 .3H 2 O concentration (1 mM) a sharp peak of 576 nm with maximum absorbance 1.96 was reported but beyond this concentration the peak becomes broad. Thus it can be reported from the present results that the absorption peak intensity increase with an increase in HAuCl 4 .3H 2 O salt concentration. All the results of the present study are in good covenant with the results reported in the literature [ 68 ]. Temperature is another factor that plays a crucial role in tuning the size and shape of AuNPs. The effect of temperature on the SPR feature of metal NPs is a critical factor in the pure and applied science of the NPs [ 69 , 70 ]. In the present work, the effect of temperature on AuNPs synthesis was studied at five different temperatures, that is, 20ᵒC, 40ᵒC, 60ᵒC, 80ᵒC, and 100ᵒC, and the UV-vis spectra were recorded after 24 hrs. From the results, it is clear that the rate of AuNPs increase with increasing temperature. Earlier, similar findings related to the increase in the reaction rate of AuNPs synthesis with an increase in reaction temperature have also been reported by [ 71 , 72 ]. According to the UV-vis spectra of AuNPs synthesized at different temperatures, it is reported that there is a blue shift to 576 nm with a maximum absorbance of 1.96 at 40ᵒC in comparison to the AuNPs synthesized at 20ᵒC. The results suggest that the higher temperature leads to an increase in the activation energy of the molecules and a faster rate of reaction. As a result, there is a decrease in the size of synthesized AuNPs and, hence, monodispersed small NPs are formed without undergoing the phase of particle size growth [ 73 ]. However, the present study shows that a further increase in reaction temperature (60ᵒC to 100ᵒC) and an increase in the size of AuNPs were reported as is evident from the sharp and narrow SPR peaks with increased sphericity. These results are consistent with the previous findings discussing the same in context to the increased reaction rate of AuNPs synthesis upon increasing the reaction temperature. The high reaction temperature leads to a rapid nucleation process of metallic NPs involving the enhanced consumption of most of the metal ions with the least secondary reduction of the preformed nuclei [ 73 , 74 ]. It has been shown that an optimum temperature could help control the rate of AuNPs synthesis and the uniform NPs can be synthesized under optimum pH at different reaction temperatures [ 75 ]. The possible role of Phyto-components present in aqueous plant extract responsible for mediating and stabilizing the nanoparticles was depicted using FTIR analysis. A perusal of scientific studies reports FTIR as one of the ideal tools to predict functional moieties. In the present investigation, vibrational stretch occurring at different peaks which corresponds to polyhydroxy, phenol, carboxyl, proteins, lipids, amide, alkynes, alkene etc. Scientific studies on FTIR analysis of plant-mediated NPs report that different functional moieties like hydroxyl, carboxyl, and amide are responsible for the reduction of metal ions to produce NPs [ 76 , 77 ]. The obtained result of FTIR analysis is by previous findings [ 78 ]. Interestingly, in the plant-mediated synthesis of NPs, the Phyto-components also play important role in the stabilization of NPs which is very crucial for rendering its applicative properties. These results also coincide with reports of earlier findings [ 79 , 80 ]. XRD is used for the phase identification and characterization of the crystal structure of the AuNPs. XRD analysis of HH-AuNPs shows four distinct peaks at 38.02°, 44.29°, 64.37°, and 77.58° which are corresponded to the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1) respectively. The mean size for HH-AuNPs was calculated using Debye-Sherrer’s equation is 10.72 nm. The “d” and “a” values were calculated by using Debye-Sherrer’s equation. To fulfill Bragg’s Law the incidence theta (θ) must vary with the change in “d” values which showed that as the value of θ increases the “d” values of the atomic layers decrease. Similar results were also reported by [ 7 , 81 ]. SEM was used to confirm the production and examine the morphology characterization at the nm to µm scale of the obtained AuNPs [ 82 ]. In the present study, the SEM data revealed that the AuNPs were spherical with an average particle size of 30 nm. SEM analysis shows uniformly distributed HH-AuNPs that indicate the stabilization of AuNPs by HH extract capping agents. Green synthesis of spherical shape AuNPs with particle size range from 21 to 45 nm was carried out by using Stevia rebadiauna leaf extracts are in good accordance with the results of the present research work [ 81 ]. The elemental composition was determined using EDX [ 83 , 84 ]. The percentage of Ag metal in the present study was found to be appreciable. The EDX analysis showed the percentage relative composition of Au signal of the AuNPs with 48.08%. The other elements served as capping organic agents bound to the surface of the AuNPs [ 85 ]. This recognition was made because of the registered energy, which is characteristic of AuNPs. [ 86 ] reported the biosynthesis and characterization of AuNPs using extracts of Tamarindus indica L leaves are in good accordance with the results of the present study. Oxidative stress has been linked to the cause of many deadly diseases such as Neurological disorder, Parkinson disease, mild cognitive impairment, and aging, etc the managing of which is costly [ 87 ]. Thus, plant extracts and NPs with antioxidant, antidiabetic, and anticholinesterase properties will be greatly beneficial [ 88 ]. Antioxidants are substances that can inhibit or delay the oxidation of a substrate when present in low concentrations. Due to the relationship of oxidative stress to other diseases, we, therefore, investigated the antioxidant capacities of HH extract, and the corresponding HH-AuNPs. Five essays; Ferric reducing antioxidant power, AmmoniuM Molybdate, DPPH, H 2 O 2 , and ABTS were carried out. The mechanism with which ferric reducing operates is known as single electron transfer (SET), whereby an antioxidant transfers an electron to the corresponding cation, which would neutralize it [ 89 ]. Thus the reducing capacity of AuNPs might be due to their quick electron transferring ability, which makes AuNPs suitable for biosensors [ 90 ]. In the present study, strong ferric reducing activities were exhibited by HH-AuNPs (77.2 ± 0.014% with IC 50 95.17 ± 0.025µg/mL) as compared to HH extract (55.2 ± 0.035% with 151 ± 0.13 µg/mL IC 50 ) and HAuCl 4 .3H 2 O solution (16.92 ± 0.014 with IC 50 = 0), while lower scavenging then ascorbic acid (84.4 ± 0.016% with IC 50 = 79 ± 0.06 µg/mL). This inhibition is due to the HH extract hydroxyl groups attached to aromatic rings, which will perfectly participate in oxidation during the process. It is well known that phenolics have strong antioxidant activities [ 91 ]. However, since the ferric reducing mechanism is by electron transfer, the hydroxyl groups of the HH extract might be interacting with the NPs, thereby limiting the site for the oxidation process. High activities in close ranges were demonstrated for AuNPs in ferric reducing assay which might be due to the size, shape, and the surrounding environment of NPs. Therefore, it is expected that AuNPs behave somewhat differently since the smaller sized particles have a higher surface area. Previously, NPs with smaller-sizes were reported to show enhanced activity in comparison to relatively plant extract [ 59 ]. Ammonium molybdate is an ammonium salt composed of ammonium and molybdate ions. It has a role as a poison as it contains a Molybdate. It can be used as a free radical in the antioxidant assay. In the present study, all the four samples of HAuCl 4 .3H 2 O solution, HH extract, HH-AuNPs, and ascorbic acid were tested for their Ammonium Molybdate scavenging; HAuCl 4 .3H 2 O solution, HH extract, HH-AuNPs, and ascorbic acid showed maximum scavenging potential of 22 ± 0.0014, 62.1 ± 0.0021, 74.3 ± 0.021, and 88.8 ± 0.001% at 160 µg/mL. The calculated IC 50 values were found to be 91.48 ± 0.13 for HH extract, 58.5 ± 0.051 for HH-AuNPs, and 48 ± 0.11 µg/mL for ascorbic acid respectively; while the HAuCl 4 .3H 2 O solution does not cause 50% scavenging. The scavenging potential of HH-AuNPs is higher as compared to HAuCl 4 .3H 2 O solution and HH extract [ 92 ]. DPPH is a free radical which changes its color from violet to yellow on reduction by a hydrogen or electron [ 93 ]. In the DPPH scavenging assay, the compounds which can reduce DPPH are considered antioxidants. By DPPH radical scavenging assay, it was found that AuNPs were able to react with free oxygen radicals and hence, possessed strong antioxidant activity as compared to HH extract and HAuCl 4 .3H 2 O solution (HAuCl 4 .3H 2 O solution of 21 ± 0.007, HH extract 55 ± 0.059, HH AuNPs 58.6 ± 0.51, and Ascorbic acid 90 ± 0.0014% % at 160µg/mL). The biological activity of NPs is depending upon the aspect ratio of particles. NPs with a high aspect ratio have been demonstrated to exhibit good antioxidant properties [ 94 ]. The result was also confirmed by the finding of [ 92 , 95 ] who showed that a higher concentration of AuNPs significantly showed high scavenging capacity as compared to Acinetobacter sp. Similar results were also reported by [ 96 , 97 ]. H 2 O 2 is an unstable inorganic compound that damages the cell membrane in the living organism. It is also known as oxidant or dioxide and can be used as free radicals in antioxidant activity. An anti-oxidant compound donates an electron to H 2 O 2 ions and thus neutralizes it to water [ 93 ]. In the present study synthesized HH-AuNPs were able to scavenge 62 ± 0.041% using 160 µg/mL concentrations in comparison to HAuCl 4 .3H 2 O solution and HH extract which scavenge 21 ± 0.021 and 56.6 ± 0.0021% respectively. The calculated IC 50 values were found to be 136 ± 0.21 for HH extract, 105.66 ± 0.062 for HH-AuNPs, and 51.18 ± 0.3 µg/mL for ascorbic acid. [ 97 ] reported that AuNPs can catalyze the rapid decomposition of H 2 O 2 . H 2 O 2 scavenging activity shown by AuNPs is almost equal to other embedded 3,6-dihydroxyflavone AuNPs, used to enhance the antioxidant activity (Medhe et al., 2014). On the other hand, a clear trend can be observed for the ABTS assay. This is probably because of the difference in the mechanism of operation between the assays. ABTS is largely operating on hydrogen atom transfer. The trend in the ABTS results is such that individual AuNPs demonstrated better antioxidant capacity relative to their respective precursors. Recent research reports [ 19 , 91 , 98 ] supported the above submission. AuNPs biosynthesized from Halymenia dilatata also demonstrated higher antioxidant activity than the starting plant extract [ 99 ]. Thus biosynthesis of NPs, has been reported for its antioxidant capacity [ 100 ]. These antioxidant compounds might get adsorbed onto the active surface of NPs. The surface reaction phenomenon of these biosynthesized NPs (due to adsorbed antioxidant moiety onto the surface) and the high surface area to volume ratio of NPs generate a tendency to interact and scavenge the free radical [ 101 ]. NPs donate electrons to free radicals due to which the free radical becomes stable. The enhanced potential of the HH-AuNPs is due to their control size, shape, and type due to which they became more reactive as compared to the plant extract [ 92 ]. Dietary antioxidants have been hypothesized to have a protective effect against α-amylase. The human salivary α-amylase digests the starch into small fragments with two or three pieces. Hence, the inhibition of the α-amylase enzyme could control the carbohydrate metabolism which also decreases the amount of glucose absorption. It seems plausible that a sufficient intake of antioxidants plays an important role in protection against type 2 diabetes [ 102 ]. In the present study the observed inhibitory percentage of α-amylase by HAuCl 4 .3H 2 O solution, HH extract, HH-AuNPs, and standard drug Glucophage are shown. The α-amylase inhibitory activity of HH-AuNPs exhibited the highest inhibitory activity with 56 ± 0.003 µg/mL IC 50 when compared to HAuCl 4 .3H 2 O solution and HH extract. The positive control Glucophage has revealed the potent α-amylase inhibitory activity with the IC 50 value of 37 ± 0.13 µg/mL. This behavior is similar to that reported by [ 21 ], therefore, closely related behaviors in the same experimental conditions are obvious. Accordingly, the improved antidiabetic performance of AuNPs over their precursor extracts has been reported by [ 103 , 104 ]. The IC 50 values of α-amylase of HH-AuNPs form also showed improved activity in agreement with previous studies [ 103 ]. The inhibiting powers of the nanomaterials may be a function of size and shape. In our results, HH-AuNPs had a size of 10.72 nm. These values are similar to the size of AuNPs in previous investigations [ 21 ]. Similarly, [ 105 ] affirmed that spherical AuNPs of sizes 20 and 40 nm in diameter induced the west Nile virus better than those of other sizes and shapes. This may be the reason for the improved enzymatic activity of AuNPs. The enzyme kinetics competitively revealed inhibition for HH on α-amylase ( K m is increased, whereas V max remains the same). These findings indicate that some of the α-amylase inhibitory components in HH extract may be structural analogs of the substrate that compete for binding at the active site of α-amylase, while in the case of HH-AuNPs effect on α-amylase a non-competitive type of inhibition was reported ( Km remained constant and V max is decreased), this suggests that some of the α-amylase inhibitory components in this HH-AuNPs resulting from HH extract bind only to the enzyme-substrate complex and may alter the active site of the enzyme. α-amylase inhibitors delay the rate of carbohydrate digestion, thereby providing an alternative therapeutic option for modulation of postprandial hyperglycemia [ 106 ]. In diabetic patients with a sustained reduction of hyperglycemia is shown to decrease the risk of developing microvascular and macrovascular diseases and their associated complications [ 107 ]. The HH extract exhibited higher inhibitory activity towards the α-amylase as opposed to many other hypoglycemic plants reported in previous studies [ 108 ]. In comparison, the first time in vitro hypoglycemic assessment of HH-AuNPs indicates higher bioactive properties. The enhanced activity of AuNPs obtained in the α-amylase assessment may be due to their high surface area to volume ratio, thus increasing the surface area phenomenon (promoting the electron transfer reaction) and may increase the pharmacokinetics from a biological view. The effects of oral hypoglycemic drugs depend on several pharmacokinetic factors such as absorption, metabolism, and excretion, and the actions of drugs begin inside the cells, it is believed that AuNPs' small size is easily carried across the cell membrane by transport proteins and may exhibit prolonged effects in bio-systems [ 109 ]. Given the α-amylase inhibitory effects of HH-AuNPs, the results obtained in this study are coherent with previous studies [ 110 ]. Antioxidants such as vitamin C and vitamin E have been associated with AChE inhibition and play an important role in AD treatment [ 102 ] and patients with AD on take high doses of antioxidants will have a slow cognitive deterioration rate [ 111 ]. Inhibition of the AChE is considered promising in AD disease treatment and various researches are focused on new inhibitors from the herbal resources [ 27 , 112 – 114 ]. To our knowledge, no attention has been given to AuNPs synthesized using the plant for the treatment of AD. The HAuCl 4 .3H 2 O solution, HH extract, and HH-AuNPs synthesized using the HH extract were screened for their in vitro AChE inhibitory activity. Their IC 50 values are found to be 19.1 ± 0.021 for HH extract and 16.66 ± 0.011 µg/mL for HH-AuNPs while no concentration of HAuCl 4 .3H 2 O solution cause 50% inhibition of AChE. Under the same conditions, the IC 50 values of HH-AuNPs showed a remarkable increase in activity toward AChE than extract. This finding is noteworthy because AD is associated with AChE deficiency and AuNPs could be potential new AChE inhibitors [ 85 ]. The good anti-AChE potential of AuNPs as compared to HH extract is due to the size and shape that may find useful in the AD treatment [ 115 ]. According to Line Weaver Burk Plot HH-AuNPs causes an uncompetitive type of inhibition of AChE (both K m and V max decrease), while HH extract caused a non-competitive type of inhibition. From the kinetic study, it is reported that AuNPs primarily cause the inhibition by interaction or adsorption with the AChE enzyme. The exact mechanism by which AuNPs inhibit AChE remains unknown. The binding affinity of NPs to AChE might be due to the lipophilicity of the NPs and the hydrophobicity of the enzyme environment in ChE molecules. Another possible mechanism of AChE inhibition might be due to the adsorption of AChE on the surface of NPs resulting in conformational changes, and surface coverage leading to the inactivation of the enzyme as reported by [ 116 ]. Inactivation of the enzyme by NPs depends on physicochemical properties like shape, size, curvature, and surface functional groups. [ 12 ] proposed that inhibition by NPs is primarily caused by adsorption or interaction with AChE protein and partly by dissolved metal ions. 5. Conclusion In this study, an environmentally facile, compassionate, straightforward, and medicinally active phytochemical route synthesized colloidal HH-AuNPs from the HH extract an indigenous plant found in abundance in Pakistan. These HH-AuNPs were characterized by the following techniques, UV-Vis spectroscopy, FTIR, SEM, EDX, and XRD. HH-AuNPs showed excellent antioxidant and inhibitory enzymatic properties than their respective HH crude extract and HAuCl 4 .3H 2 O salt. These observations, plus evidence of their potent antioxidant and enzymatic activity from active molecules rich plants such as HH indicate the value of further studies. Thus, the synthesis of AuNPs based drugs with greater targeted activity combined with medicinal phytochemicals derived from the HH extract may result in unprecedented opportunities directed at the discovery of a cheaper and more beneficial therapy for oxidants, type 2 diabetes, and Alzheimer’s diseases Declarations Acknowledgments The author wishes to thank the Higher Education Commission of Pakistan (HEC-PAK) for financial support of project No: 20-5082/NRPU/R&D/HEC. Conflict of Interest The authors do not have any conflict of interest regarding this article and its publication. References C.C.I. Kavitha, G. Indira, Green synthesis, characterization and antimicrobial activity of silver nanoparticles using Morindapubscens J. E. Smith root extract. J. Sci. Innov. Res. 5 (3), 83–86 (2016) A. Chanda, D. 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Ercetin, A. Kahraman, F. Celep, G. Akaydin, B. Sener, M. Dogan, 2013. s.., , Assessment of anticholinesterase and antioxidant properties of selected sage (Salvia) species with their total phenol and flavonoid content, Ind. Crop. Prod. 41 21-30 N. Dorosti, F. Jamshidi, Plant-mediated gold nanoparticles by Dracocephalum kotschyi as anticholinesterase agent: Synthesis, characterization, and evaluation of anticancer and antibacterial activity. J. Appl. Biomed. 14 (3), 235–245 (2016) A.A. Vertegel, R.W. Siegel, J.S. Dordick, Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme Langmuir. 20 6800-6807 (2004) Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1639345","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":106327446,"identity":"7f329429-58d9-47c7-9a9d-b2a17eb7f162","order_by":0,"name":"Naila Sher","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Naila","middleName":"","lastName":"Sher","suffix":""},{"id":106327447,"identity":"75d0108d-ea6e-450c-a93b-e311c75fc0af","order_by":1,"name":"Mushtaq Ahmed","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYHACNhAhx8bffICBsYFYLQcYGIz5JI4lkKYlcR5DjgFxWnRnpD97/KHGmrGN4cw3iZ87bOQY2A8f3YBPi9mNHHODA8fSmdmYe7dJ9p5JM2bgSUu7QUALm8QBtsNsbAxnt0nwth1ObJDgMSOgJf2ZxIF/h3nYGHKeSf4lTkuCmcTBtsMSQC1s0sTZcuaNmcTZvnQDNoljxtaybWnGbAT9chzosIpv1vXz+5sf3nzbZiPHz374GF4tUMAMIlgkQCQbEcrhWpg/EKl6FIyCUTAKRhgAAAizTGAeSNN+AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0286-326X","institution":"University of Science and Technology Bannu","correspondingAuthor":true,"prefix":"","firstName":"Mushtaq","middleName":"","lastName":"Ahmed","suffix":""},{"id":106327448,"identity":"2ca38bb3-5420-4364-86b5-857c9c1f4d28","order_by":2,"name":"Nadia Mushtaq","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Mushtaq","suffix":""},{"id":106327449,"identity":"55fdb13f-a240-4e7b-9749-461e9431f88b","order_by":3,"name":"Rahmat Ali Khan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rahmat","middleName":"Ali","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2022-05-09 18:30:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1639345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1639345/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22313015,"identity":"c02afc61-7d22-4aed-83c7-3bb4287c3775","added_by":"auto","created_at":"2022-06-06 16:55:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45450,"visible":true,"origin":"","legend":"\u003cp\u003eUV–vis absorption spectrum of HH-AuNPs.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/ad47a8f6d831f50b12acce55.jpg"},{"id":22313019,"identity":"5b29d139-e9ee-41f4-befe-77467aa4ac23","added_by":"auto","created_at":"2022-06-06 16:55:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":154569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(2a, 2b, 2c, 2d, 2e, and 2f)\u003c/strong\u003e UV–spectra of HH-AuNPs with different pH, HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, the volume of extract, temperature, time, and stability.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/2724b32814fbfb4282d1778d.jpg"},{"id":22313908,"identity":"f48efedf-0b0e-4dae-b9a4-f964a961dbc7","added_by":"auto","created_at":"2022-06-06 17:00:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(3a and 3b) \u003c/strong\u003e\u0026nbsp;FT-IR analysis of HH extract and HH-AuNPs.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/a4deadc8f2af9b376f5a5863.jpg"},{"id":22313910,"identity":"14a9a588-02b4-4368-81a7-291f5b6318f5","added_by":"auto","created_at":"2022-06-06 17:00:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41952,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern analysis of HH-induced AgNPs.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/b471a4cbc9b88eedc861e0e6.jpg"},{"id":22314245,"identity":"1fe4e4d3-f0ed-41a4-b942-e08d154026d0","added_by":"auto","created_at":"2022-06-06 17:05:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":67640,"visible":true,"origin":"","legend":"\u003cp\u003eSEM analysis and SEM calculated the size of HH-AuNPs.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/d4fe8105e0c6fe6718efd703.jpg"},{"id":22314247,"identity":"45cb9486-27ec-4136-8a98-a9b980fa1d75","added_by":"auto","created_at":"2022-06-06 17:05:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99224,"visible":true,"origin":"","legend":"\u003cp\u003eEDX analysis of HH-AuNPs.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/b3cc4d5ebcc0c6e286349660.jpg"},{"id":22313017,"identity":"b5b9d33e-f679-4be3-97d8-3bf9fe6b091a","added_by":"auto","created_at":"2022-06-06 16:55:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":181850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(7a, 7b, 7c, 7d, and 7e)\u003c/strong\u003e Ferric reducing power assay (7A), Ammonium molybdenum (7B), DPPH scavenging (7C), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e inhibition activity (7D), and ABTS scavenging potential (7E) of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, and HH-AuNPs.\u003cem\u003e \u003c/em\u003eData are expressed as mean ± standard deviation (SD; n = 3).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/795ac3a8ab2994e13c196793.jpg"},{"id":22314246,"identity":"7d74ae7f-a579-44f1-9147-cc0127de95ba","added_by":"auto","created_at":"2022-06-06 17:05:56","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":90294,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro α-amylase Inhibitory activity of HH-AuNPs, HH extract, and Glucophage. Data are expressed as mean ± standard deviation (SD; n = 2).\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/d3c067eb999b46bdddfdfd47.jpg"},{"id":22313021,"identity":"7b12022b-4506-4c3e-a93d-aea4ab914780","added_by":"auto","created_at":"2022-06-06 16:55:56","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":100259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(9a, 9b, and 9c) \u003c/strong\u003eHH-AuNPs caused non-competitive (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e constant and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax \u003c/em\u003e\u003c/sub\u003edecrease), HH\u003cem\u003e \u003c/em\u003eextract caused competitive type of inhibition (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e increase and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e remain constant), while Glucophage cause an uncompetitive type of inhibition (both \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e \u003c/sub\u003edecrease). Data are expressed in the form of Lineweaver–Burk (reciprocal of enzyme velocity versus reciprocal of potato starch) plot. The results represent the mean of three different experiments done in triplicate by using different concentrations of extract as shown in the legend boxes.\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/17c47aabc9ea586dda8d9732.jpg"},{"id":22313025,"identity":"7a89bc84-c84e-4a11-b905-0e08660febfa","added_by":"auto","created_at":"2022-06-06 16:55:56","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":102154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(10a, 10b, and 10c)\u003c/strong\u003e The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e were determined for HH-AuNPs, HH extract, and Glucophage from Dixon plots for α-amylase. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e is equal to the reciprocal of the y-axis intersection of each line for each potato starch concentration while \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e is equal to the x-axis intersection in the Dixon plot.\u003c/p\u003e","description":"","filename":"Fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/578295037a1ff143193f1d6c.jpg"},{"id":22313914,"identity":"79c87bb1-cdf7-4ab8-a05d-2c51eb2423ab","added_by":"auto","created_at":"2022-06-06 17:00:56","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":96831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(11a, 11b, and 11c) \u003c/strong\u003eThe \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (inhibition constant) was obtained for α-amylase using the Cornish-Bowden plot of S/V vs. [I].\u003c/p\u003e","description":"","filename":"Fig11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/b8f6cf2473915b7b7ddfea02.jpg"},{"id":22313912,"identity":"accc79e2-46f9-4a37-a162-d888e223ac9e","added_by":"auto","created_at":"2022-06-06 17:00:56","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":93394,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of AChE in the presence and absence of HH-AuNPs and HH extract in the dose-dependent mode was measured at 421 nm by using fixed 0.5 mM ACh concentration with 50 mM phosphate buffer (pH 7.4) and 10 mM DTNB in 1 mL assay which were preincubated for 10 min before ACh addition. The experiments were repeated three times; the obtained results were similar in all three cases and significantly different from the control. * P \u0026lt; 0.05.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/f4094d7bb74d29c1141e3517.jpg"},{"id":22314248,"identity":"4ad74c66-4753-4c99-afce-6bc20b74fa9a","added_by":"auto","created_at":"2022-06-06 17:05:56","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":74789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(13a and 13b)\u003c/strong\u003e Both HH-AuNPs and HH extract caused a non-competitive type of inhibition (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e remain constant and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e decrease) of krait snake venom AChE. Data are expressed in the form of Lineweaver–Burk (reciprocal of enzyme velocity versus reciprocal of AcSCh) plot. The results represent the mean of three different experiments done in triplicate by using different concentrations of HH extract.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/3f6dd8789b42cf7267c8c157.jpg"},{"id":22313029,"identity":"c9cad3fb-6f4f-411e-8a2e-505ccfaeb693","added_by":"auto","created_at":"2022-06-06 16:55:56","extension":"jpg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":76481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(14a and 14b)\u003c/strong\u003e The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp \u003c/em\u003e\u003c/sub\u003efor\u003csub\u003e\u003cem\u003e \u003c/em\u003e\u003c/sub\u003eboth HH-AuNPs and HH extract were determined from Dixon plots for AChE. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e is equal to the reciprocal of the y-axis intersection of each line for each AcSCh concentration while \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e is equal to the x-axis intersection in the Dixon plot.\u003c/p\u003e","description":"","filename":"Fig14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/7e07c62181a259e1b8df0543.jpg"},{"id":22313028,"identity":"245b340b-5465-48c8-8115-65f97b377e4e","added_by":"auto","created_at":"2022-06-06 16:55:56","extension":"jpg","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":74666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(15a and 15b)\u003c/strong\u003e The \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e \u003c/em\u003e(inhibition constant) was obtained for AChE using the Cornish-Bowden plot of S/V vs. [I] for both HH-AuNPs and HH extract.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/694a7211a7ffba7ae87b1cfb.jpg"},{"id":22314257,"identity":"4badf1b3-486e-42aa-8eb7-9dad46e3d5ec","added_by":"auto","created_at":"2022-06-06 17:06:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2049520,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1639345/v1/7527b53a-a0ef-401b-a7f7-1737808da415.pdf"}],"financialInterests":"","formattedTitle":"Hippeastrum hybridum assisted bioreduction of Hydrogen tetrachloroaurate (III) trihydrate: Multifaced application","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanotechnology is one of the modern techniques of material science which have received much importance in the last many years. It is concerned with the production of nanoparticles (NPs) with restricted sizes and shapes of materials at the nanometer range and is used for the welfare of mankind [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nanoscience is the science in which we have studied phenomena and management of nanomaterials having different properties than those having a larger scale [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Application of nanoscience and technology used in applied sciences and as well as material sciences [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The importance of NPs science directly depends on the nano size of NPs; because of the sizes, these particles have made their importance in many fields such as medicine, food industries, agricultural wound dressing, chronic ulcers, and oxide fuel batteries for energy storage, cosmetics, and garments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNowadays, metallic NPs of gold (Au), silver (Ag), copper (Cu), zinc (Zn), titanium (Ti), magnesium (Mg), etc. have been synthesized [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. All Au and Ag have attracted considerable attention in imaging catalysis, sensing, optics, and biomedical devices [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. NPs can be synthesized in many chemical and physical ways, but these processes generate hazardous byproducts [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Green nanotechnology, which uses biological organisms, plant biomass or extracts considered an alternative to the conventional chemical and physical methods in a clean, non-toxic, ecologically sound, and environment-friendly manner [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The use of plant materials for the synthesis of NPs could be more advantageous because it does not require elaborate processes as various biomolecules like Ascorbic acids, Citric acid, flavonoids, dehydrogenases, phenols, saponins, and tannins in the plants play a vital role in Ag and Au reduction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Other compounds that have been used in the formation of AuNPs with antidiabetic properties include chitosan, chondroitin sulfate, tyrosine, and tryptophan [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Alpha-amylase, (α-amylase) is an important enzyme in the human body responsible for the metabolism of starch i.e., it converts polysaccharides such as starch and glycogen into disaccharides and oligosaccharides. The inhibition of α-amylase by NPs slows down the carbohydrate digestion and can control the reduction in glucose absorption rate [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLike the metal-reducing ability of plant extracts, the antioxidants activity has been associated with the plants\u0026rsquo; phenolic [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] argued that antioxidants possess free radical scavenging properties, hence, they play role in promoting health and preventing diseases. Several antioxidant activities [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] have been carried out on AuNPs through the green route. Antioxidants extracted from plants play an important role in the prevention of Alzheimer's disease (AD). AD is the most common form of dementia among elderly people that causes problems with memory, thinking, and behavior. Maintaining the levels of acetylcholine (ACh) by inhibition of the acetylcholinesterase enzyme (AChE) is an important strategy to treat AD [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. AChE inhibitors like tacrine, donepezil, and galantamine are well-known drugs in the treatment of AD which have separated from herbal sources. Although several reports on the screening of AChE inhibitors from herbal sources have been made [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], no attention has been given to inhibitor activity of plant-mediated AuNPs so far.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHippeastrum hybridum\u003c/em\u003e (HH) is an ornamental bulbous flowering plant belonging to the family Amaryllidaceae, it has large and showy flowers with many bright colors and is commonly known as Royal Dutch Amaryllis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Normally it produces 2\u0026ndash;3 bulblets in a year of growth [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. HH plant is commonly used in physiological and ecological research, but the extent of their genomic and genetic resources remains limited [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Today, a vast majority of plants used in traditional medicine in Pakistan have not been evaluated for their synthesis of AuNPs. The present investigation was, therefore, undertaken to evaluate its AuNPs synthesis, and then evaluated for antioxidant, anti-cholinesterase, and antidiabetic potential.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eHydrogen tetrachloroaurate (III) trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO) obtained from Central Chemical Lahore, was used as the source of AuIII ions. HH plant was collected in September from district Bannu Khyber Pakhtunkhwa and was identified by Dr. Tahir Iqbal, at the Department of Botany, University of Science and Technology Bannu. Potassium ferricyanide, ferric chloride, Trichloroacetic acid, Sulphuric acid, Sodium phosphate, Ammonium molybdenum, ascorbic acid, DPPH (1,1-Diphenyl-2-picrylhydrazyl), H2O2 (Hydrogen peroxide), ABTS (2, 2 azobis, 3-ethyl benzothiazoline-6-sulphonic acid, potassium persulfate (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e), Potato starch, sodium acetate buffer, sodium potassium tartrate, 3, 5 dinitro salicylic acid (DNSA), standard (Glucophage), α-amylase, AChE, ACh, DTNB [5,5\u0026acute;-dithiobis(2-nitro-benzoic acid)], bovine serum albumin, and Coomassie Brilliant blue R-250 were purchased from Sigma (USA). Sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Neon Comercial LTDA (Brazil); and Tris (hydroxymethyl aminomethane) from Vetec (Brazil). All other reagents used were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant\u0026rsquo;s extraction\u003c/h2\u003e \u003cp\u003eAfter identification the plant was washed using water, shade dried, and ground to a fine powder. About 250 g of the fine plant powder was mixed with 70% methanol in 1:3 and kept on an orbital shaker at 120 rpm for 12 h and then placed at room temperature for 7 days, thus after the 7 days the plant is extracted, and filtered by using Whatman filter paper No 1 and concentrated with the help of the rotary evaporator, after the concentration the extra methanol was evaporated at 37\u0026deg;C to obtain a pure crude methanolic extract of sample and was then reserved in the refrigerator at 4\u0026deg;C for more studies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3. Synthesis of HH induced gold nanoparticles (HH-AuNPs)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eHH-AuNPs were synthesized from HH plant extract by following the standard protocol [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. About 10 mM (0.01 M) solution of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO was prepared in 50 mL to deionize water. The 10 mM HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO was further diluted 10 times to obtain a 1 mM HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution. 0.1 M NaOH, \u0026ge;\u0026thinsp;98%, and 0.1M HCl were used to adjust the pH. An aqueous solution of HH extracts was prepared by dissolving 1 gm of plant extract in 100 mL of deionized water. For dissolution, it is gently stirred on a magnetic stirrer for about 1 hr. After the complete dissolution, it was centrifuged at 6000 rpm for 30 min. The supernatant was collected for activity and the pallets were discarded. The plant supernatant (50 mL) was mixed with the 500 mL of 1 mM HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution of pH 4. The resulting solution changed from golden yellow to crimson red and then finally to ruby red at an optimized ratio after a few hrs at pH 4 and 40\u0026ordm;C temperature. This change in coloration indicated the formation of AuNPs. The solution was then stored for 24 hrs for the complete settlement of NPs and was then monitored using UV\u0026ndash;Visible spectrophotometer. The colloidal suspension thus obtained was centrifuged by cold centrifuge at -4\u0026deg;C at 10,000 rpm for 10 min and the pellet was obtained after discarding supermen\u0026rsquo;s. The synthesized NPs were lyophilized to obtain the powder form. The powdered is further characterized and tested for different biological activities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Factors affecting synthesis rate, size, and shape of HH-AuNPs\u003c/h2\u003e \u003cp\u003eAuNPs synthesis was determined by using different intrinsic factors such as pH, HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO concentration, HH extract concentration, Temperature, Time, and stability time. To study the effect of basic and acidic conditions pH of the reaction mixture was maintained from 4\u0026ndash;12 by using 0.1 M NaOH and 0.1 M HCl solution. To study the effect of HH extract concentration on AuNPs synthesis its concentration varied from 0.5, 1, 1.5, and 2 mL. To study the effect of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt concentration; its concentration varied to 0.25, 0.5, 1, and 1.5 mM. To study the temperature effect AuNPs synthesis was carried out under different temperature ranges (20, 40, 60, 80, and 100\u0026ordm;C). To study the time of completion of the reaction AuNPs were synthesized at different time intervals (1 hr, 2 hrs, 3hrs, and 24 hrs). The synthesized AuNPs stability was studied after 1 day, 3 months, and 6 months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Characterization of HH-AuNPs\u003c/h2\u003e \u003cp\u003eHH-AuNPs concentration in the aqueous solution was definite by using SHIMADZU UV SPECTROPHOTOMETER (UV-1800). The purified HH-AuNPs and HH plants extract was examined for the presence of different phytochemicals by using Fourier Transform-Infrared (FT-IR) Shimadzu (IR Prestige-21) spectrometer (Japan). The crystalline nature of the HH-AuNPs was determined by using the JDX-3532 (JEOL JAPAN) X-ray diffractometer (XRD) with λ-1.54 A\u0026ordm; wavelength. The size and shape of HH-AuNPs were determined by using JEOL Scanning Electron Microscope (SEM) Model JSM-5910 (Japan). The presence of elemental Au in synthesized HH-AuNPs was determined by using electron diffraction X-ray spectroscopy (EDX).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Biological activities\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Antioxidant assays\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e2.6.1.1. Ferric-Reducing Antioxidant Power screening\u003c/h2\u003e \u003cp\u003eThe reducing power potential of HAuCl4.3H2O, HH extract, and HH-AuNPs was done by following [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] method with a slight modification. About 2 mL samples (HAuCl4.3H2O, HH extract, and HH-AuNPs ), 2 mL of 10 mg/mL potassium ferricyanide, and 2 mL of 0.2 phosphate buffer (pH 6.6) were mixed and followed by incubation for 20 min at 50\u0026deg;C. After incubation 2 mL of 100 mg/mL Trichloroacetic acid was mixed with the solution. About 2 mL of the above solution was mixed with 0.4 mL of 0.1% ferric chloride and 2 mL of deionized H2O followed by incubation for 10 min. Absorbance was observed at 700 nm by spectrophotometer. All samples were run in triplicate. The %age was determined by using the formula (i);\u003c/p\u003e \n\u003cp\u003e\u003cimg 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\u003cp\u003eAc is the control absorbance and As is the sample absorbance\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003ch2\u003e2.6.1.2. Ammonium molybdenum assay\u003c/h2\u003e \u003cp\u003eThe Ammonium molybdenum antioxidant potential of HAuCl4.3H2O salt, HH, and HH-AuNPs was carried by following [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] procedure. About 1 mL of different concentrations of HAuCl4.3H2O, HH extract, and HH-AuNPs (40\u0026ndash;160 \u0026micro;g/mL) and 9 mL of (28 mM sodium phosphate, 600 mM Sulphuric acid, and 4 mM Ammonium molybdenum) were mixed in test tubes. The test tubes were capped with aluminum foil and followed by incubation for 90 min at 95\u0026ordm;C in a water bath. After 90 min of incubation, the mixture was then cool to room temperature and absorbance was noted at 695 nm by spectrophotometer. All samples were run in triplicate. The %age scavenging of Ammonium molybdenum was deliberated by using the formula (i).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.6.1.3. DPPH activity\u003c/h2\u003e \u003cp\u003e1,1-Diphenyl-2-picrylhydrazyl (DPPH) antioxidant potential was carried by following [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] method. Stock solutions (1 mg/mL) of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO, HH, and HH-AuNPs were prepared in deionized water which was further diluted into (40, 80,100, and 160 \u0026micro;g/mL). Standard ascorbic acid is also prepared in a similar concentration. About 200 \u0026micro;L from different concentrations of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO, HH, HH-AgNPs, and the standard was mixed with 800 \u0026micro;L of DPPH (1.5 mg/50mL methanol) and then incubated for 30 min in dark a room temperature. Absorbance spectra were recorded at 517 nm by using a UV spectrophotometer against water as a reference. All samples were run in triplicate. The %age scavenging was deliberated by using the equation (i).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e \u003ch2\u003e2.6.1.4. Hydrogen peroxide scavenging (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eThe H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity was analyzed by [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] method with certain modifications. About 200 \u0026micro;L from various concentrations (40 to160 \u0026micro;g/mL) of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO, HH, and HH-AuNPs in deionized water, 400 \u0026micro;L of 2 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 400 \u0026micro;L of 50 mM phosphate buffer (pH 7.4) were mixed and followed by the incubation for 20 minutes at 35\u0026ordm;C. The absorbance was recorded by using a spectrophotometer at 610 nm against phosphate buffer as blank. All samples were run in triplicate. The %age was determined by using the equation (i).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003ch2\u003e2.6.1.5. ABTS screening assay\u003c/h2\u003e \u003cp\u003eThe ABTS free radical scavenging activity of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO, HH extract, and HH-AuNPs was accomplished by [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] procedure with slight modification. About 7 mM of ABTS solution and 2.45 mM of potassium persulfate (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e) solution was prepared in deionized water. These two solutions were mixed and allowed for overnight incubation, dark coloration indicated the existence of ABTS\u0026bull;+ free radicals in the solution. The optical density of the mixture was determined using a spectrophotometer and was brought to 0.700 (\u0026plusmn;\u0026thinsp;0.02) by the addition of more solvent. About 300 \u0026micro;L of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO, HH extract, and HH-AuNPs (40 to 160 \u0026micro;g/mL) and standard mixed with 300 \u0026micro;L of (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ABTS) mixture. The absorbance was recorded immediately after mixing the solution at 734 nm by using a spectrophotometer. All samples were run in triplicate. The %age scavenging was deliberated by using the equation (i).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Anti-α-amylase activity\u003c/h2\u003e \u003cp\u003eInhibition of α-amylase activity was determined using 3,5 dinitro salicylic acid (DNSA) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To an obtained starch solution (1% w/v) 1 gram of potato starch was dissolved in 100 mL of 16 mM C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e3\u003c/sub\u003eNaO\u003csub\u003e2\u003c/sub\u003e (Sodium acetate) buffer. To obtained enzyme solution 0.5 mg/mL α-amylase from stock (250 units/mL) was dissolved in 1 mL dH\u003csub\u003e2\u003c/sub\u003eO. Sodium potassium tartrate and DNSA (96 mM) mixtures were used as a calorimetric reagent. The stock solution of HAuCl4.3H2O, HH extract, HH-AuNPs, and standard (Glucophage) was prepared at 1 mg/mL and was further diluted into different sub-solutions i.e. 25, 50, 75, and 100 \u0026micro;g/mL. Samples were added to 250 \u0026micro;L of α-amylase. The mixture was pre-incubated at 25\u0026deg;C for 10 min and 250 \u0026micro;L of 1% starch prepared in 20 mM sodium phosphate buffer (pH 6.9) was added. The reaction mixtures were incubated at 25\u0026deg;C for 10 min. The reactions were stopped by incubating the mixture in a boiling water bath for 5 min after adding 250 \u0026micro;L from the combined mixture of DNSA and sodium potassium tartrate. The reaction mixtures were cooled to room temperature, diluted to 1:5 ratios with deionized water, and absorbance was measured in a spectrophotometer (double beam UV-1602, BMS-spectrophotometer) at 450 nm. The Glucophage served as a positive control. All samples were run in triplicate. The percentage of inhibition of enzyme activity was calculated by using the formula (i).\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section4\"\u003e \u003ch2\u003e2.6.2.1. Mode of α-amylase inhibition assay\u003c/h2\u003e \u003cp\u003eThe mode of inhibition of α-amylase is determined as described before [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. For α-amylase, the enzyme solution (250 units/mL) was pre-incubated with samples (25, 50, 75 and 100 \u0026micro;g/mL). The reactions were started by adding 100, 200, and 300 mg of potato starch and continued at 25\u0026deg;C for 10 min. The reactions were stopped by adding 0.25 mL of DNSA followed by boiling for 5 min. The reaction mixtures were cooled to room temperature, diluted to a 1:5 ratio with dH\u003csub\u003e2\u003c/sub\u003eO, and absorbance was measured in a spectrophotometer (double beam UV-1602, BMS-spectrophotometer) at a 450 nm Double reciprocal plot (1/V versus 1/[S]) where V is reaction velocity and [S] is substrate concentration was plotted. The mode of inhibition was determined by analyzing the Lineweaver-Burk plot using Michaelis-Menten kinetics [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Michaelis constants \u003cem\u003e(K\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) were determined by two different plots of 1/V vs. 1/S [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and V vs. V/S [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eKI\u003c/em\u003e values were obtained using the Cornish-Bowden plot of S/V vs. [I] and Dixon plot 1/V vs. [I] [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] respectively. IC\u003csub\u003e50\u003c/sub\u003e was determined by percentage residual activity and percentage inhibition versus concentration of HH extract and HH-AuNPs.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Anti-cholinesterase activity\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section4\"\u003e \u003ch2\u003e2.6.3.1. Venom\u003c/h2\u003e \u003cp\u003eVenom from live \u003cem\u003eBungarus sindanus\u003c/em\u003e snakes was squeezed out manually, lyophilized immediately, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for further use. The study was approved by the Departmental Ethical Approval Committee, ref. n. Biotech/Ethic/110.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section4\"\u003e \u003ch2\u003e2.6.3.2. Anti-cholinesterase assay\u003c/h2\u003e \u003cp\u003eAChE activity was determined by the method of [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] modified by [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] using a double beam spectrophotometer UV-1602, BMS biotechnology medical service. Hydrolysis rates (V) were measured at various acetylthiocholine (S) concentrations (0.05\u0026ndash;1 mM) in a 1 mL assay mixture with 50 mM phosphate buffer, pH 7.4, and 10 mM DTNB at 25oC. About 20 \u0026micro;L of diluted snake venom was also added and the reaction mixture was incubated for 5 minutes at 37\u003csup\u003eo\u003c/sup\u003eC. The enzyme-substrate reaction immediately started upon the addition of different concentrations of substrate. The hydrolysis was scrutinized by the formation of thiolate di-anion of DTNB every 15 seconds for 90 seconds using a spectrophotometer. The amount of the yellow color develops is a measure of the activity of AChE. All samples were run in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section4\"\u003e \u003ch2\u003e2.6.3.3. Protein determination\u003c/h2\u003e \u003cp\u003eThe protein content of the enzyme preparation was assayed by the method of Bradford [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] using bovine serum albumin as a standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section4\"\u003e \u003ch2\u003e2.6.3.4. Kinetic determinations\u003c/h2\u003e \u003cp\u003eThe interaction of HH extract/HH-AuNPs, and AChE was determined using the [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] double reciprocal plot, by plotting 1/V against 1/[S] analyzed over a range of ACh concentrations (0.05\u0026ndash;1 mM) in the absence and presence of extract (10, 20, and 30 \u0026micro;g/mL). A double reciprocal plot (1/V versus 1/[S]) where V is reaction velocity and [S] is substrate concentration was plotted. The mode of inhibition was determined by analyzing the Lineweaver-Burk plot using Michaelis-Menten kinetics [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Michaelis constants \u003cem\u003e(Km\u003c/em\u003e) were determined by two different plots of 1/V vs. 1/S [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and V vs. V/S [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The \u003cem\u003eKi\u003c/em\u003e and \u003cem\u003eKI\u003c/em\u003e values were obtained using the Cornish-Bowden plot of S/V vs. [I] and Dixon plot 1/V vs. [I] [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] respectively. IC50 was determined by percentage residual activity and percentage inhibition versus concentration of HH extract and HH-AuNPs.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA, which was followed by post-hoc analysis (Duncan multiple range test) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The coefficient of correlation was determined by using Statistics (version 8.1 USA). The difference was considered to be significant for P\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Synthesis of HH-AuNPs\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. UV-visible spectrophotometric analysis of HH-AuNPs\u003c/h2\u003e \u003cp\u003eThe aqueous AuNPs exhibited ruby red color due to the SPR. After mixing the HH aqueous extract with HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO the color of the solution start to change from golden yellow to crimson red and then finally to ruby red indicating the bioreduction of the Au\u0026thinsp;+\u0026thinsp;by HH extract. The scale of the length of the spectrophotometer ranged from 200 to 800 nm. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicated 576 nm, absorption bands of the HH-AuNPs, at higher absorption of 1.96 after 24 hrs of incubation at 40\u0026ordm;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. Factors affecting the synthesis of AuNPs\u003c/h2\u003e \u003cp\u003eIn the present study; the synthesis of HH-AuNPs was studied under different factors. According to a pH study, HH-AuNPs were synthesized at different basic and acidic conditions (pH 4 to 12). No sharp band was observed in the range of 500\u0026ndash;600 nm at higher pH of 8 to 12. The bands become sharp and sharp with decreasing the pH and a final sharp peak of 576 nm with a maximum absorbance of 1.96 was reported at pH 4. See Fig.\u0026nbsp;2a. According to the HH extract concentration studies different peaks i-e 556, 576, 548, and 550 were observed with a maximum absorbance of 0.6893, 1.96, 1.9135, and 1.7776 at the volume of 0.5, 1, 1.5, and 2 mL respectively. See Fig.\u0026nbsp;2b. The UV-vis spectra of HH-AuNPs aqueous medium at different HAuCl4.3H2O concentrations (0.25\u0026ndash;1.5 mM) were noted in the range of 200 to 800 nm wavelength which indicated broader bands at 0.25, 0.5, and 1.5 mM with low absorbance but a sharp peak 576 nm with maximum absorbance 1.96 was obtained at 1 mM (See Fig.\u0026nbsp;2c). HH-AuNPs synthesis was studied by incubating the reaction mixture at different temperature ranges (20 to 100\u0026ordm;C). A sharp band at 576 nm was obtained at 40\u0026ordm;C with a maximum absorbance of 1.96, but with an increase in temperature beyond 40\u0026ordm;C the band becomes broad and broader i-e 558, 546, and 567 nm with a maximum absorbance of 1.0505 0.9098, and 0.7089 were reported at 60, 80, and 100\u0026ordm;C respectively. See Fig.\u0026nbsp;2d. Figure\u0026nbsp;2e indicated the effect of reaction time on the synthesis of HH reduced AuNPs. Broad peaks with lower absorption appeared after 1hr, 2hrs, and 3hrs of the stirring (685, 654, and 500 nm with a maximum absorbance of 0.251, 0.182, and 0.54 respectively). Due to the continuous reduction of Au ions by HH extract the absorption peak increases over time. A final clear sharp peak of 576 nm at high absorbance 1.96 was observed after 24 hrs of the HH and Au ions reaction. The stability of the HH reduced AuNPs was studied at different periods (1 day, 3 months, and 6 months). A sharp peak of 576 nm with a maximum absorbance of 1.96 appeared after 1 day of AuNPs formation; but after 3 and 6 months this peak become broad i-e 558 nm after 3 months and 567 nm after 6 months, with low absorbance of 1.0505 and 0.7089 respectively. (See Fig.\u0026nbsp;2f).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.2. FT-IR analysis of HE extract and HH-AuNPs\u003c/h2\u003e \u003cp\u003eThe FTIR spectrum of HH extracts and HH-AuNPs (prepared in water) is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The data on the peak values and the probable functional groups (obtained by FTIR analysis) present in the HH extract and HH-AuNPs are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The characteristic absorption band were exhibited in the range 3400-3200cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for O-H stretch), 2935\u0026ndash;2915cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for \u0026ndash;CH (CH\u003csub\u003e2\u003c/sub\u003e) vibration), 2865-2845cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for \u0026ndash;CH (CH\u003csub\u003e2\u003c/sub\u003e), 2260-2100cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for C\u0026thinsp;\u0026equiv;\u0026thinsp;C stretch), 2100-1800cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for C\u0026thinsp;=\u0026thinsp;O frequency), 1740-1725cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for C\u0026thinsp;=\u0026thinsp;O stretch), 1650-1600cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for C\u0026thinsp;=\u0026thinsp;O stretch), 1410-1310cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for O-H bend), 1340-1250cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for CN stretch), 1100-1000cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for Phosphate ion), 995-850cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for P-O-C stretch), 800-700cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for C-Cl stretch), 700-600cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(for C-Br stretch), and 690-550cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (for C-Br stretch) were exhibited by HH extract and HH-AuNPs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFTIR Interpretation of compounds in HH whole plant extract and HH-AuNPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWave number cm\u003csup\u003e-1\u003c/sup\u003e [Reference article]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWave number cm\u003csup\u003e-1\u003c/sup\u003e [HH-plant]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWave number cm\u003csup\u003e-1\u003c/sup\u003e [HH-AuNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFunctional group assignment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePhyto compounds Identified\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3400\u0026thinsp;\u0026minus;\u0026thinsp;3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3266.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3324.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO-H stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePoly Hydroxy compound\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2935\u0026ndash;2915\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2917.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2922.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAsymmetric stretching of \u0026ndash;CH (CH\u003csub\u003e2\u003c/sub\u003e) vibration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSaturated aliphatic compound-Lipids\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2865\u0026thinsp;\u0026minus;\u0026thinsp;2845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2855.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2852.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSymmetric stretching of \u0026ndash;CH (CH\u003csub\u003e2\u003c/sub\u003e) vibration,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLipids, protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2260\u0026thinsp;\u0026minus;\u0026thinsp;2100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2259.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2245.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCarbon-Carbon triple bond\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTerminal alkynes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2100\u0026thinsp;\u0026minus;\u0026thinsp;1800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1985.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1963.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCarbonyl compound frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransition metal carbonyls\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1740\u0026thinsp;\u0026minus;\u0026thinsp;1725\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1740.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAldehyde compound\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1650\u0026thinsp;\u0026minus;\u0026thinsp;1600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1608.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1638.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O stretching vibration, Ketone group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKetone compound\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1410\u0026thinsp;\u0026minus;\u0026thinsp;1310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO-H bend, Alcoholic group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePhenol or tertiary alcohol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1340\u0026thinsp;\u0026minus;\u0026thinsp;1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1290.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCN stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAromatic primary amine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1100\u0026thinsp;\u0026minus;\u0026thinsp;1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1035.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1010.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhosphate ion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePhosphate compound\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e995\u0026thinsp;\u0026minus;\u0026thinsp;850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e852.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e852.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP-O-C stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAromatic phosphates\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e800\u0026thinsp;\u0026minus;\u0026thinsp;700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e743.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e714.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC-Cl stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAliphatic Chloro compound\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e700\u0026thinsp;\u0026minus;\u0026thinsp;600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e676.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e622.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC-Br stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAliphatic bromo compounds\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e690\u0026thinsp;\u0026minus;\u0026thinsp;550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e571.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e555.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHalogen compounds (Bromo-compounds)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAliphatic Bromo compounds\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.3. XRD analysis of HH-AuNPs\u003c/h2\u003e \u003cp\u003eXRD is a technique that is used for determining the size and crystalline nature of the sample. In the present study, the HH-AuNPs were analyzed by XRD. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e indicated 4 Bragg reflections at angles of 38.02\u0026deg;, 44.29\u0026deg;, 64.37\u0026deg;, and 77.58\u0026deg; which are corresponded to the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1) respectively. These reflections can be indexed conferring to the face of the face-centered cubic crystal structure of Au ion. The \u0026ldquo;d\u0026rsquo; (interplanar spacing) and \u0026ldquo;a\u0026rdquo; (Miller constants) values were calculated by using the Debye-Sherrer\u0026rsquo;s equation (i) and (ii) respectively; \n\u003cp\u003e\u003cimg 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\u003cp\u003eResults are tabulated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetermination of Interplaner spacing and lattice constant of HH-AuNPs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. no\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2θ Value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElement\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eplane\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInterplaner spacing (d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLattice constants (a\u003csub\u003e0\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.02\u003c/p\u003e \u003cp\u003e44.29\u003c/p\u003e \u003cp\u003e64.37\u003c/p\u003e \u003cp\u003e77.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAu\u003c/p\u003e \u003cp\u003eAu\u003c/p\u003e \u003cp\u003eAu\u003c/p\u003e \u003cp\u003eAu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 1 1\u003c/p\u003e \u003cp\u003e2 0 0\u003c/p\u003e \u003cp\u003e2 2 0\u003c/p\u003e \u003cp\u003e3 1 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.36 \u0026Aring;\u003c/p\u003e \u003cp\u003e2.06 \u0026Aring;\u003c/p\u003e \u003cp\u003e1.44 \u0026Aring;\u003c/p\u003e \u003cp\u003e1.22 \u0026Aring;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.08 \u0026Aring;\u003c/p\u003e \u003cp\u003e4.08 \u0026Aring;\u003c/p\u003e \u003cp\u003e4.07 \u0026Aring;\u003c/p\u003e \u003cp\u003e4.04 \u0026Aring;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe HH and HH-AuNPs concentrations providing 50% inhibition (IC\u003csub\u003e50\u003c/sub\u003e) values of the different antioxidants activities.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAssays\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAscorbic Acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eHH extract\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eHH AuNPs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFerric reducing\u003c/p\u003e \u003cp\u003eMolybdenum scavenging\u003c/p\u003e \u003cp\u003eDPPH scavenging\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging\u003c/p\u003e \u003cp\u003eABTS scavenging\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003cp\u003e48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003cp\u003e52.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003cp\u003e51.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003cp\u003e40.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003cp\u003e91.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003cp\u003e156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003cp\u003e136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003cp\u003e154.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e \u003cp\u003e58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003cp\u003e136.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.071\u003c/p\u003e \u003cp\u003e105.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062\u003c/p\u003e \u003cp\u003e144.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.072\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Glucophage, HH extract and HH-AuNPs on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of α-amylase.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eHH AuNPs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentrations\u003c/p\u003e \u003cp\u003e(\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e(\u0026micro;mol\u003c/p\u003e \u003cp\u003eα-amylase/min/mg protein)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%Decrease\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e50\u003c/p\u003e \u003cp\u003e75\u003c/p\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.171\u003c/p\u003e \u003cp\u003e66.63\u003c/p\u003e \u003cp\u003e67.03\u003c/p\u003e \u003cp\u003e66.77\u003c/p\u003e \u003cp\u003e66.85\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0265\u003c/p\u003e \u003cp\u003e0.023\u003c/p\u003e \u003cp\u003e0.0225\u003c/p\u003e \u003cp\u003e0.0215\u003c/p\u003e \u003cp\u003e0.0184\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e13.20\u003c/p\u003e \u003cp\u003e15.094\u003c/p\u003e \u003cp\u003e18.96\u003c/p\u003e \u003cp\u003e30.56\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHH extract\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentrations\u003c/p\u003e \u003cp\u003e(\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%Increase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e(\u0026micro;mol\u003c/p\u003e \u003cp\u003eα-amylase/min/mg protein)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e50\u003c/p\u003e \u003cp\u003e75\u003c/p\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.50\u003c/p\u003e \u003cp\u003e75.8\u003c/p\u003e \u003cp\u003e91.4\u003c/p\u003e \u003cp\u003e111.64\u003c/p\u003e \u003cp\u003e126.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e10.65\u003c/p\u003e \u003cp\u003e33.43\u003c/p\u003e \u003cp\u003e62.9\u003c/p\u003e \u003cp\u003e84.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0269\u003c/p\u003e \u003cp\u003e0.0264\u003c/p\u003e \u003cp\u003e0.0264\u003c/p\u003e \u003cp\u003e0.0271\u003c/p\u003e \u003cp\u003e0.0267\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGlucophage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentrations\u003c/p\u003e \u003cp\u003e(\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%Decrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e(\u0026micro;mol\u003c/p\u003e \u003cp\u003eα-amylase/min/mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%Decrease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e50\u003c/p\u003e \u003cp\u003e75\u003c/p\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e194.56\u003c/p\u003e \u003cp\u003e115.78\u003c/p\u003e \u003cp\u003e80.49\u003c/p\u003e \u003cp\u003e67.78\u003c/p\u003e \u003cp\u003e60.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e40.49\u003c/p\u003e \u003cp\u003e58.74\u003c/p\u003e \u003cp\u003e56.6\u003c/p\u003e \u003cp\u003e69.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0566\u003c/p\u003e \u003cp\u003e0.0346\u003c/p\u003e \u003cp\u003e0.0252\u003c/p\u003e \u003cp\u003e0.0216\u003c/p\u003e \u003cp\u003e0.0172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e38.86\u003c/p\u003e \u003cp\u003e55.47\u003c/p\u003e \u003cp\u003e61.83\u003c/p\u003e \u003cp\u003e69.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of HH extract, HH AuNPs, and Glucophage on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e of α-amylase. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e were determined from Dixon plot of 7A and 7B for α-amylase. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e is equal to the reciprocal of y-axis intersection of each line for each potato starch concentration while \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e is equal to the x-axis intersection in Dixon plot.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabd\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eHH-AuNPs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e[Potato starch] (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g / min / mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Decrease\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003cp\u003e200\u003c/p\u003e \u003cp\u003e300\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e261.74\u003c/p\u003e \u003cp\u003e260.22\u003c/p\u003e \u003cp\u003e263.78\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003cp\u003e0.018\u003c/p\u003e \u003cp\u003e0.022\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e12.5\u003c/p\u003e \u003cp\u003e37.5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabe\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHH Extract\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e[Potato starch] (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% Increase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g / min / mg)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003cp\u003e200\u003c/p\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e139.53\u003c/p\u003e \u003cp\u003e317.64\u003c/p\u003e \u003cp\u003e634\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e127.64\u003c/p\u003e \u003cp\u003e354.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003cp\u003e0.20\u003c/p\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabf\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGlucophage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% Decrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g / min / mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Increase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e128.30\u003c/p\u003e \u003cp\u003e106.95\u003c/p\u003e \u003cp\u003e69.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e16.64\u003c/p\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0198\u003c/p\u003e \u003cp\u003e0.025\u003c/p\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e26.26\u003c/p\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative study of kinetic parameters of α-amylase inhibition by HH extract, HH-AuNPs, and Glucophage; \u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e, inhibition constant; \u003cem\u003eK\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e, dissociation constant of the α-amylase\u0026ndash;Pottao starch\u0026ndash;HH complex into the α-amylase\u0026ndash;Pottao starch complex and free HH; \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, Michaelis\u0026ndash;Menten constant and IC\u003csub\u003e50,\u003c/sub\u003e 50% inhibitory concentration.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHH AuNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHH Extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlucophage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g)\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eI\u003c/em\u003e\u003c/sub\u003e (\u0026micro;g)\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (mg)\u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e261.91\u003c/p\u003e \u003cp\u003e66\u003c/p\u003e \u003cp\u003e44.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003cp\u003e364\u003c/p\u003e \u003cp\u003e68.06\u003c/p\u003e \u003cp\u003e56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003cp\u003e101.56\u003c/p\u003e \u003cp\u003e195\u003c/p\u003e \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInfluence of HH and HH-AuNPs on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of \u003cem\u003eBungarus Sindanus\u003c/em\u003e (Krait) venom AChE.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.no\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eHH-AuNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eHH extract\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentrations\u003c/p\u003e \u003cp\u003e(\u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% Decrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e (\u0026micro;mol / min\u003c/p\u003e \u003cp\u003eper mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e% Decrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e (\u0026micro;mol / min\u003c/p\u003e \u003cp\u003eper mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e% Decrease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e157.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e62.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e48.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e69.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e72.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e74.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of HH extract and HH-AuNPs on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e of \u003cem\u003eBungarus Sindanus\u003c/em\u003e (Krait) venom AChE. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e were determined from Dixon plot of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e12\u003c/span\u003e for snake venom, AChE. The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e is equal to the reciprocal of y-axis intersection of each line for each AcSCh concentration while \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e is equal to the x-axis intersection in Dixon plot.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.no\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eHH-AuNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eHH extract\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e[ASCh] (mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e (mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%Decrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emaxiapp\u003c/sub\u003e (\u0026micro;mol / min per mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%Decrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e (mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emaxiapp\u003c/sub\u003e (\u0026micro;mol / min per mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e% Decrease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003cp\u003e0.1\u003c/p\u003e \u003cp\u003e0.25\u003c/p\u003e \u003cp\u003e0.5\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.75\u003c/p\u003e \u003cp\u003e55.02\u003c/p\u003e \u003cp\u003e39.123\u003c/p\u003e \u003cp\u003e28.46\u003c/p\u003e \u003cp\u003e23.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e32\u003c/p\u003e \u003cp\u003e51.55\u003c/p\u003e \u003cp\u003e64.75\u003c/p\u003e \u003cp\u003e70.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.62\u003c/p\u003e \u003cp\u003e16.08\u003c/p\u003e \u003cp\u003e22.34\u003c/p\u003e \u003cp\u003e33.78\u003c/p\u003e \u003cp\u003e41.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e38.38\u003c/p\u003e \u003cp\u003e92.25\u003c/p\u003e \u003cp\u003e190.70\u003c/p\u003e \u003cp\u003e259.122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36.49\u003c/p\u003e \u003cp\u003e36\u003c/p\u003e \u003cp\u003e36.13\u003c/p\u003e \u003cp\u003e36.24\u003c/p\u003e \u003cp\u003e36.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e38.29\u003c/p\u003e \u003cp\u003e57.38\u003c/p\u003e \u003cp\u003e60\u003c/p\u003e \u003cp\u003e68.46\u003c/p\u003e \u003cp\u003e77.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e49.85\u003c/p\u003e \u003cp\u003e56.6\u003c/p\u003e \u003cp\u003e78.79\u003c/p\u003e \u003cp\u003e104.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy of kinetic parameters of AChE inhibition by HH-AuNPs and HH extract. \u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e, inhibition constant; \u003cem\u003eK\u003c/em\u003e\u003csub\u003eI\u003c/sub\u003e, dissociation constant of the AChE\u0026ndash;ASCh\u0026ndash;HH complex into the AChE\u0026ndash;ASCh/BSCh complex and free HH; \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, Michaelis\u0026ndash;Menten constant, and IC\u003csub\u003e50,\u003c/sub\u003e 50% inhibitory concentration.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHH-AuNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHH extract\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e ( \u0026micro;g)\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eI\u003c/em\u003e\u003c/sub\u003e ( \u0026micro;g)\u003c/p\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e (mM)\u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e ( \u0026micro;g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003cp\u003e45.43\u003c/p\u003e \u003cp\u003e0.195\u003c/p\u003e \u003cp\u003e16.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003cp\u003e36.25\u003c/p\u003e \u003cp\u003e0.052\u003c/p\u003e \u003cp\u003e19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe average crystalline size of HH induced AuNPs is calculated by using the Debye-Sherrer\u0026rsquo;s formula (iii);\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/jpeg;base64,/9j/4AAQSkZJRgABAQEAYABgAAD/4RD6RXhpZgAATU0AKgAAAAgABAE7AAIAAAAQAAAISodpAAQAAAABAAAIWpydAAEAAAAgAAAQ0uocAAcAAAgMAAAAPgAAAAAc6gAAAAgAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAFNhY2hpbiBNYWhhcm51cgAABZADAAIAAAAUAAAQqJAEAAIAAAAUAAAQvJKRAAIAAAADNzEAAJKSAAIAAAADNzEAAOocAAcAAAgMAAAInAAAAAAc6gAAAAgAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA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\"\u003e\u003c/p\u003e\n\u003cp\u003eD is the average crystalline size, k is a geometric factor (0.9), λ is the wavelength of the X-ray radiation source and β is the angular FWHM (full-width at half maximum) of the XRD peak at the diffraction angle θ. For the four major peaks i-e 38.02\u0026deg;, 44.29\u0026deg;, 64.37\u0026deg;, and 77.58\u0026deg; the average calculated crystalline size was found to be 10.72 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.4. SEM analysis of HH-AuNPs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn the present study; HH-AuNPs were screened by SEM to determine their surface morphology. SEM results showed that the HH-AuNPs are monodispersed, spherical-shaped, and are in high density (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The average particle size was calculated using Nano measurer software by marking 15 particles and was found to be 30 nm (0.03 \u0026micro;m).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.5. EDX analysis of HH-AuNPs\u003c/h2\u003e \u003cp\u003eThe elemental constituents and relative abundance of the biosynthesized AuNPs were obtained from EDX analysis as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e. EDX spectrometers confirmed the presence of elemental Au signal of the AuNPs with 48.08%. The vertical axis displays the number of X-ray counts while the horizontal axis displays energy in KeV. Identification lines for the major emission energies for Au are displayed and these correspond with peaks in the spectrum, thus giving confidence that Au has been correctly identified in HH-AuNPs. Thus the EDX spectrum discloses the purity and the complete chemical composition of HH-AuNPs. The other elements served as capping organic agents bound to the surface of the HH-AuNPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Antioxidant assays\u003c/h2\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1. Ferric reducing power assay (FRPA)\u003c/h2\u003e \u003cp\u003eIn the present study; different concentrations (40, 80, 120, and 160\u0026micro;g/mL) of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid were tested for ferric reducing activity. The obtained results indicated that all the tested samples showed ferric reducing potential in a dose-dependent manner and showed maximum scavenging of 16.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 (HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO), 55.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035% (HH extract), and 77.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 (HH-AuNPs); while the positive control ascorbic acid showed 84.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016% scavenging at the highest 160 \u0026micro;g/mL concentration (Fig.\u0026nbsp;7a). All the samples i-e HH extract, HH-AuNPs, and Ascorbic acid cause 50% scavenging (IC\u003csub\u003e50\u003c/sub\u003e) at 151\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, 95.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025, and 79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u0026micro;g/mL respectively; while the HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO does not cause 50% inhibition. (See the Table. 3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2. Ammonium molybdenum assay\u003c/h2\u003e \u003cp\u003eDifferent concentrations (40\u0026ndash;160 \u0026micro;g/mL) of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid were tested for their Ammonium molybdenum scavenging assay (See Fig.\u0026nbsp;7b). HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extracts, HH-AuNPs, and ascorbic acid showed maximum scavenging potential of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014, 62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0021, 74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021, and 88.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001% at 160 \u0026micro;g/mL. The calculated IC\u003csub\u003e50\u003c/sub\u003e values were found to be 91.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 for HH extract, 58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051 for HH-AuNPs, and 48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u0026micro;g/mL for ascorbic acid respectively; while the HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO does not cause 50% scavenging of Ammonium molybdenum (See the Table. 3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e3.6.3. DDPH scavenging assay\u003c/h2\u003e \u003cp\u003eDifferent concentrations of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid (40, 80, 120, and 160\u0026micro;g/mL) were tested for their DPPH scavenging activity. The obtained results indicated that HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid showed DPPH scavenging potential in a dose-dependent manner and showed maximum scavenging of 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007, 55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.059, 58.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51, and 90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014% % at 160\u0026micro;g/mL respectively (Fig.\u0026nbsp;7c). All the samples except HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt showed 50% DPPH scavenging activity. The calculated IC\u003csub\u003e50\u003c/sub\u003e (50% inhibition) values were found to be 156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31for HH extract, 136.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.071 for HH-AuNPs, and 52.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;g/mL for Ascorbic acid respectively. Results are tabulated in Table. 3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e \u003ch2\u003e3.6.4. Hydrogen peroxide scavenging (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eIn the current research work; different concentrations (40\u0026ndash;160 \u0026micro;g/mL) of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid were tested for their H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity (Fig.\u0026nbsp;7d). HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid showed maximum scavenging potential of 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021, 56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0021, 62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041, and 76.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014% at 160 \u0026micro;g/mL with IC\u003csub\u003e50\u003c/sub\u003e values of 136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 for HH extract, 105.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062 for HH-AuNPs, and 51.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;g/mL for ascorbic acid respectively; while the HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO does not cause 50% inhibition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (See the Table. 3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section3\"\u003e \u003ch2\u003e3.6.5. ABTS screening assay\u003c/h2\u003e \u003cp\u003eThe present study indicated the ABTS scavenging potential of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid at various concentrations (40\u0026ndash;160 \u0026micro;g/mL). Figure\u0026nbsp;7e indicated that HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH extract, HH-AuNPs, and ascorbic acid scavenge ABTS free radicals in dose-dependent manner and indicated 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014, 55.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004, 59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, and 90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002% inhibition at the highest concentration (160 \u0026micro;g/mL) with an IC\u003csub\u003e50\u003c/sub\u003e of 154.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 for HH extract, 144.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.072 for HH-AuNPs, and 40.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0.05\u0026micro;g/mL for ascorbic acid respectively; while HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt does not show 50% scavenging of ABTS free radicals. (See the Table. 3).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Antidiabetic activity\u003c/h2\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003e3.7.1. Anti-α-amylase activity\u003c/h2\u003e \u003cp\u003eThe HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, extracts of HH, HH-AuNPs, and standard drug Glucophage were tested to evaluate theirs in vitro inhibition abilities against α-amylase. All the samples inhibit α-amylase in dose-dependent manner (25, 50, 75, and 100 \u0026micro;g/mL) and cause maximum inhibition of 7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014% (HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt), 66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003% (HH extract), 71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032% (HH-AuNPs), and 85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014% (Glucophage) respectively at fixed (1%) substrate concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section3\"\u003e \u003ch2\u003e3.7.2. Effects of HH, HH-AuNPs, and Glucophage on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe effect of HH, HH-AuNPs and Glucophage on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of α-amylase were calculated. Statistical analysis indicated that HH-AuNPs cause a non-competitive type of inhibition \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e to remain constant and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e decrease from 13.20 to 30.56% (Fig.\u0026nbsp;9a), similarly HH causes a competitive type of inhibition of α-amylase i-e. \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e increases from 10.65 to 84.37%, while \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e remained constant (Fig.\u0026nbsp;9b), while in the case of Glucophage uncompetitive type of inhibition was observed i-e. Both \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e decreased from 40.49 to 69.15 and 38.86 to 69.61% respectively (Fig.\u0026nbsp;9c). Results are presented in Table. 4A, 4B, and 4C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003e3.7.3. Effects of HH, HH-AuNPs, and Glucophage on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eIn α-amylase, \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e remained constant while \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiap\u003c/em\u003e\u003c/sub\u003e was decreased from 12.5 to 37.5% for HH-AuNPs, while for HH extract the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e was found to increase from 127.64 to 354.33% while \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e remained constant with the increase of substrate concentration (100\u0026ndash;300 mg). In the case of Glucophage, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e was found to decrease from 16.64 to 46% while \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e decreased from 26.26-to 89% (Table. Table. 5a, 5b, and 5c). The values were calculated from Fig.\u0026nbsp;10a, 10b, and 10c respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec41\" class=\"Section3\"\u003e \u003ch2\u003e3.7.4. Determination of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eK\u003c/em\u003e \u003csub\u003e \u003cem\u003em\u003c/em\u003e \u003c/sub\u003e values for the hydrolysis of the substrate (potato starch) by α-amylase, were calculated by using the Lineweaver-Burk plot and were found to be 66, 68.06, and 195 mg for HH-AuNPs, HH, and Glucophage respectively. The values are presented in Table. 6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec42\" class=\"Section3\"\u003e \u003ch2\u003e3.7.5. Determination of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eI\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eK\u003c/em\u003e \u003csub\u003e \u003cem\u003eI\u003c/em\u003e \u003c/sub\u003e (constant of α-amylase\u0026ndash;Potato starch\u0026ndash;HH-AuNPs, HH extract/Glucophage complex into α-amylase\u0026ndash;Potato starch complex and HH-AuNPs, HH extract/Glucophage) was estimated to be 261.91 (Fig.\u0026nbsp;10a), 364 \u0026micro;g (Fig.\u0026nbsp;10b), and 101.56 \u0026micro;g (Fig.\u0026nbsp;10c) for α-amylase respectively, while \u003cem\u003eKi\u003c/em\u003e (inhibitory constant) was estimated to be 25, (Fig.\u0026nbsp;11a), 26 (Fig.\u0026nbsp;11b) and 12 \u0026micro;g (Fig.\u0026nbsp;11c) for HH-AuNPs, HH extract, and Glucophage respectively. The values are tabulated in Table. 6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec43\" class=\"Section3\"\u003e \u003ch2\u003e3.7.6. Determination of IC\u003csub\u003e50\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eHH-AuNPs, HH extract, and Glucophage cause 50% inhibition (IC\u003csub\u003e50\u003c/sub\u003e) against α-amylase at a concentration of 44.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042, 56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003, and 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u0026micro;g/mL, respectively. The values are tabulated in Table. 6.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec44\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Anti-acetylcholinesterase activity\u003c/h2\u003e \u003cp\u003eAt fixed substrate acetylthiocholine (ACh) concentration (0.5 mM) HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt, HH plant, and HH-AuNPs exerted 23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057, 59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003, and 61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.314% inhibition against the snake krait venom AChE at maximum 30 \u0026micro;g/mL concentration in 1 mL assay mixture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e12\u003c/span\u003ea).\u003c/p\u003e \u003cdiv id=\"Sec45\" class=\"Section3\"\u003e \u003ch2\u003e3.8.1. Determination of IC\u003csub\u003e50\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe concentration of HH extract and HH-AuNPs that cause 50% inhibition (IC\u003csub\u003e50\u003c/sub\u003e) of AChE enzyme activity were found to be 19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021 and 16.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011 \u0026micro;g/mL respectively; while no any concentration of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt cause 50% inhibition of AChE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e12\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec46\" class=\"Section3\"\u003e \u003ch2\u003e3.8.2. Effects of HH on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe effect of HH and HH-AuNPs on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of AChE were calculated. HH caused a non-competitive type of inhibition the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values remained constant and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e decreased from 60.05\u0026ndash;74.01%, while HH-AuNPs caused an uncompetitive type of inhibition in both \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e decreased from 47.96\u0026ndash;74.61 and 47.34\u0026ndash;72.02% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e13\u003c/span\u003ea and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e13\u003c/span\u003eb). Values are presented in (Table. 7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec47\" class=\"Section3\"\u003e \u003ch2\u003e3.8.3. Effects of HH on \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eIn snake venom AChE, \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e was found to remain constant, while \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e decreased from 49.85 to 104.42% HH extract, while in the case of HH-AuNPs \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eIapp\u003c/em\u003e\u003c/sub\u003e decreased from 32-70.51% and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emaxiapp\u003c/em\u003e\u003c/sub\u003e decreased from 38.38-259.122% with an increase of substrate (0.05\u0026ndash;1 mM) (Table. 8). The values were calculated from Fig.\u0026nbsp;14a and 14b.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec48\" class=\"Section3\"\u003e \u003ch2\u003e3.8.4. Determination of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eK\u003c/em\u003e \u003csub\u003e \u003cem\u003em\u003c/em\u003e \u003c/sub\u003e values for the hydrolysis of substrate by AChE were calculated by using a Lineweaver-Burk plot and were found to be 0.195 and 0.052 mM for HH-AuNPs and HH extract, respectively. The values are presented in Table. 9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec49\" class=\"Section3\"\u003e \u003ch2\u003e3.8.5. Determination of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eI\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eK\u003c/em\u003e \u003csub\u003e \u003cem\u003eI\u003c/em\u003e \u003c/sub\u003e (constant of AChE\u0026ndash;AcSC\u0026ndash;HH-AuNPs/HH extract complex into AChE\u0026ndash;AcSC complex and HH-AuNPs/HH) was estimated to be 44.43 and 36.25 \u0026micro;g (Fig.\u0026nbsp;14a and 14b) for AChE, while \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (inhibitory constant) was estimated to be 23 and 32 \u0026micro;g for HH-AuNPs and HH extract respectively, (Fig.\u0026nbsp;15a and 15b). The values are presented in Table. 9.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe synthesis and characterization of NPs and their applications represent a rapidly growing concept and an emerging trend in science and technology [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The use of plant materials for the synthesis of NPs could be more advantageous because it does not require elaborate processes [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The UV\u0026ndash;vis spectroscopy technique can be used to determine the synthesis and stability of AuNPs in an aqueous solution due to their characteristic absorption in the range of 500\u0026ndash;600 nm [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. AuNPs exhibited ruby red color in the aqueous solution due to the excitation of surface plasmon vibrations in the metal NPs which give rise to the surface plasmon resonance (SPR) band [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The plasmon resonance (PR) can be pictured as a \u0026lsquo;\u0026lsquo;wave\u0026rsquo;\u0026rsquo; of electrons sloshing over the surface of a metal NPs. As a result, an enhanced electromagnetic field at and near the metal NPs surface is set up. The position of the plasmon band (extinction spectrum) is best measured on a conventional UV\u0026ndash;vis spectrophotometer and appears as a band with extremely high extinction coefficients [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In the present study, when 1 mL of HH aqueous extract was added to 1 mM HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution at pH 4 the color of the solution start to change from golden yellow to crimson red and then finally to ruby red at an optimized ratio after 24 hrs of incubation at 40\u0026ordm;C. The appearance of the ruddiness color in an aqueous medium is considered the first indication of colloidal AuNPs formation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. This color change is due to the reduction of the Au\u003csup\u003e+\u003c/sup\u003e into HH-AuNPs by the active molecules of the HH aqueous extract such as phenolic, alkaloids, saponins, amino acids, proteins, etc. The absorption spectrum of the aqueous solution revealed a peak at 576 nm with a maximum absorbance of 1.96 after 24 hrs.\u003c/p\u003e \u003cp\u003eSynthesis of the AuNPs is affected by different factors such as plant extract volume, HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO, pH, temperature, time, etc. [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. An ideal pH is required for synthesis of controlling the shape and size of NPs [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The role of pH is significant in changing the size and shape of NPs. Numbers of studies have shown the stability of AuNPs at acidic pH while many achieved stable suspension in the basic region. Furthermore, the synthesis reaction, size, shape, and stability of AuNPs could be controlled by adjusting the initial pH value of the reaction mixture. In the present study, the reaction was performed at different pH ranges from 4 to 12 to identify the effect of pH on the formation of HH-reduced AuNPs. It was detected that the absorbance of the solution increased while changing the initial pH of the solution. With the increase in pH, the SPR band was also blue-shifted to 576 nm at pH 4. The AuNPs were quite stable in an acidic medium; however, a gradual decrease in UV\u0026ndash;vis peak intensities showed less stability with an increase in pH of the colloidal solution from 4 to 12. Peak broadening and redshift were noted at pH 10 and 11. UV-vis results suggested that no reaction occurred in the basic region which is in agreement with the previous studies [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. It was might be due to the deprotonation of hydroxyl and carboxyl groups present in extracts [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. AuNPs synthesized using banana peel extract were stable at a pH value of 2\u0026ndash;5 supporting the results of the present study [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. A broader range of pH i.e. 2\u0026ndash;11 were taken to synthesize AuNPs using oil palm mill effluent and pH 3 was observed optimum to achieve definite shapes particles [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Medicinal plants are a rich source of secondary metabolites that act as reducing, capping, and stabilizing agents for NPs. However, the composition of these active secondary metabolites varies from plant to plant depending on the nature, part, type of plant, and method followed for the extraction of these metabolites [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In the present study, the SPR peak is blue-shifted from 553 to 576 nm as the added volume of HH extract increased from 0.5 to 1mL; the blue shift of SPR peaks is a sign of the production of small size NPs. The shift towards shorter wavelengths with decreasing NPs size is associated with frequencies of oscillation of different free electrons in the conduction band [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The SPR band absorbance increased with an increasing volume of HH extract, which reveal the higher production of AuNPs. This is due to the availability of more reducing agents for the Au ions bioreduction [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. But the further increase in the volume of HH extract from 1.5 to 2 mL the redshift of SPR was recorded, this redshift is a sign of large size NPs. HH contains active molecules such as carbohydrates, flavones, terpenoids, alkaloids, and proteins that were testified to be responsible for the bio-reduction of Au\u0026thinsp;+\u0026thinsp;to Au\u003csup\u003eo\u003c/sup\u003e. Proteins and terpenoids are believed to play an important role in AuNPs biosynthesis through the reduction of Au ions, and carbohydrates provide a coating of AuNPs [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The HH extract produces more AuNPs than other plant extracts credited to the availability of the larger amount of reducing agents in the extract, such as flavonoid and its antioxidant activity [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Varying concentrations of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO (0.25 to 1.5 mM) were prepared while the other factors were kept constant i-e HH extract (1 mL), pH (4), temperature (40\u0026ordm;C), and time (24 hrs). At 0.25 and 0.5 mM concentrations of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO broad peaks were revealed; while at increased HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO concentration (1 mM) a sharp peak of 576 nm with maximum absorbance 1.96 was reported but beyond this concentration the peak becomes broad. Thus it can be reported from the present results that the absorption peak intensity increase with an increase in HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt concentration. All the results of the present study are in good covenant with the results reported in the literature [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Temperature is another factor that plays a crucial role in tuning the size and shape of AuNPs. The effect of temperature on the SPR feature of metal NPs is a critical factor in the pure and applied science of the NPs [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. In the present work, the effect of temperature on AuNPs synthesis was studied at five different temperatures, that is, 20ᵒC, 40ᵒC, 60ᵒC, 80ᵒC, and 100ᵒC, and the UV-vis spectra were recorded after 24 hrs. From the results, it is clear that the rate of AuNPs increase with increasing temperature. Earlier, similar findings related to the increase in the reaction rate of AuNPs synthesis with an increase in reaction temperature have also been reported by [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. According to the UV-vis spectra of AuNPs synthesized at different temperatures, it is reported that there is a blue shift to 576 nm with a maximum absorbance of 1.96 at 40ᵒC in comparison to the AuNPs synthesized at 20ᵒC. The results suggest that the higher temperature leads to an increase in the activation energy of the molecules and a faster rate of reaction. As a result, there is a decrease in the size of synthesized AuNPs and, hence, monodispersed small NPs are formed without undergoing the phase of particle size growth [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. However, the present study shows that a further increase in reaction temperature (60ᵒC to 100ᵒC) and an increase in the size of AuNPs were reported as is evident from the sharp and narrow SPR peaks with increased sphericity. These results are consistent with the previous findings discussing the same in context to the increased reaction rate of AuNPs synthesis upon increasing the reaction temperature. The high reaction temperature leads to a rapid nucleation process of metallic NPs involving the enhanced consumption of most of the metal ions with the least secondary reduction of the preformed nuclei [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. It has been shown that an optimum temperature could help control the rate of AuNPs synthesis and the uniform NPs can be synthesized under optimum pH at different reaction temperatures [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe possible role of Phyto-components present in aqueous plant extract responsible for mediating and stabilizing the nanoparticles was depicted using FTIR analysis. A perusal of scientific studies reports FTIR as one of the ideal tools to predict functional moieties. In the present investigation, vibrational stretch occurring at different peaks which corresponds to polyhydroxy, phenol, carboxyl, proteins, lipids, amide, alkynes, alkene etc. Scientific studies on FTIR analysis of plant-mediated NPs report that different functional moieties like hydroxyl, carboxyl, and amide are responsible for the reduction of metal ions to produce NPs [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The obtained result of FTIR analysis is by previous findings [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Interestingly, in the plant-mediated synthesis of NPs, the Phyto-components also play important role in the stabilization of NPs which is very crucial for rendering its applicative properties. These results also coincide with reports of earlier findings [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eXRD is used for the phase identification and characterization of the crystal structure of the AuNPs. XRD analysis of HH-AuNPs shows four distinct peaks at 38.02\u0026deg;, 44.29\u0026deg;, 64.37\u0026deg;, and 77.58\u0026deg; which are corresponded to the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1) respectively. The mean size for HH-AuNPs was calculated using Debye-Sherrer\u0026rsquo;s equation is 10.72 nm. The \u0026ldquo;d\u0026rdquo; and \u0026ldquo;a\u0026rdquo; values were calculated by using Debye-Sherrer\u0026rsquo;s equation. To fulfill Bragg\u0026rsquo;s Law the incidence theta (θ) must vary with the change in \u0026ldquo;d\u0026rdquo; values which showed that as the value of θ increases the \u0026ldquo;d\u0026rdquo; values of the atomic layers decrease. Similar results were also reported by [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSEM was used to confirm the production and examine the morphology characterization at the nm to \u0026micro;m scale of the obtained AuNPs [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. In the present study, the SEM data revealed that the AuNPs were spherical with an average particle size of 30 nm. SEM analysis shows uniformly distributed HH-AuNPs that indicate the stabilization of AuNPs by HH extract capping agents. Green synthesis of spherical shape AuNPs with particle size range from 21 to 45 nm was carried out by using \u003cem\u003eStevia rebadiauna\u003c/em\u003e leaf extracts are in good accordance with the results of the present research work [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe elemental composition was determined using EDX [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. The percentage of Ag metal in the present study was found to be appreciable. The EDX analysis showed the percentage relative composition of Au signal of the AuNPs with 48.08%. The other elements served as capping organic agents bound to the surface of the AuNPs [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. This recognition was made because of the registered energy, which is characteristic of AuNPs. [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e] reported the biosynthesis and characterization of AuNPs using extracts of \u003cem\u003eTamarindus indica\u003c/em\u003e L leaves are in good accordance with the results of the present study.\u003c/p\u003e \u003cp\u003eOxidative stress has been linked to the cause of many deadly diseases such as Neurological disorder, Parkinson disease, mild cognitive impairment, and aging, etc the managing of which is costly [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Thus, plant extracts and NPs with antioxidant, antidiabetic, and anticholinesterase properties will be greatly beneficial [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Antioxidants are substances that can inhibit or delay the oxidation of a substrate when present in low concentrations. Due to the relationship of oxidative stress to other diseases, we, therefore, investigated the antioxidant capacities of HH extract, and the corresponding HH-AuNPs. Five essays; Ferric reducing antioxidant power, AmmoniuM Molybdate, DPPH, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and ABTS were carried out. The mechanism with which ferric reducing operates is known as single electron transfer (SET), whereby an antioxidant transfers an electron to the corresponding cation, which would neutralize it [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Thus the reducing capacity of AuNPs might be due to their quick electron transferring ability, which makes AuNPs suitable for biosensors [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, strong ferric reducing activities were exhibited by HH-AuNPs (77.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014% with IC\u003csub\u003e50\u003c/sub\u003e 95.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u0026micro;g/mL) as compared to HH extract (55.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035% with 151\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u0026micro;g/mL IC\u003csub\u003e50\u003c/sub\u003e) and HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution (16.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 with IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0), while lower scavenging then ascorbic acid (84.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016% with IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u0026micro;g/mL). This inhibition is due to the HH extract hydroxyl groups attached to aromatic rings, which will perfectly participate in oxidation during the process. It is well known that phenolics have strong antioxidant activities [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. However, since the ferric reducing mechanism is by electron transfer, the hydroxyl groups of the HH extract might be interacting with the NPs, thereby limiting the site for the oxidation process. High activities in close ranges were demonstrated for AuNPs in ferric reducing assay which might be due to the size, shape, and the surrounding environment of NPs. Therefore, it is expected that AuNPs behave somewhat differently since the smaller sized particles have a higher surface area. Previously, NPs with smaller-sizes were reported to show enhanced activity in comparison to relatively plant extract [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmmonium molybdate is an ammonium salt composed of ammonium and molybdate ions. It has a role as a poison as it contains a Molybdate. It can be used as a free radical in the antioxidant assay. In the present study, all the four samples of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution, HH extract, HH-AuNPs, and ascorbic acid were tested for their Ammonium Molybdate scavenging; HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution, HH extract, HH-AuNPs, and ascorbic acid showed maximum scavenging potential of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014, 62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0021, 74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021, and 88.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001% at 160 \u0026micro;g/mL. The calculated IC\u003csub\u003e50\u003c/sub\u003e values were found to be 91.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 for HH extract, 58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051 for HH-AuNPs, and 48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u0026micro;g/mL for ascorbic acid respectively; while the HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution does not cause 50% scavenging. The scavenging potential of HH-AuNPs is higher as compared to HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution and HH extract [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDPPH is a free radical which changes its color from violet to yellow on reduction by a hydrogen or electron [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. In the DPPH scavenging assay, the compounds which can reduce DPPH are considered antioxidants. By DPPH radical scavenging assay, it was found that AuNPs were able to react with free oxygen radicals and hence, possessed strong antioxidant activity as compared to HH extract and HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution (HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution of 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007, HH extract 55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.059, HH AuNPs 58.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51, and Ascorbic acid 90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014% % at 160\u0026micro;g/mL). The biological activity of NPs is depending upon the aspect ratio of particles. NPs with a high aspect ratio have been demonstrated to exhibit good antioxidant properties [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. The result was also confirmed by the finding of [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e] who showed that a higher concentration of AuNPs significantly showed high scavenging capacity as compared to \u003cem\u003eAcinetobacter\u003c/em\u003e sp. Similar results were also reported by [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is an unstable inorganic compound that damages the cell membrane in the living organism. It is also known as oxidant or dioxide and can be used as free radicals in antioxidant activity. An anti-oxidant compound donates an electron to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e ions and thus neutralizes it to water [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. In the present study synthesized HH-AuNPs were able to scavenge 62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041% using 160 \u0026micro;g/mL concentrations in comparison to HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution and HH extract which scavenge 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021 and 56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0021% respectively. The calculated IC\u003csub\u003e50\u003c/sub\u003e values were found to be 136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 for HH extract, 105.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062 for HH-AuNPs, and 51.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;g/mL for ascorbic acid. [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e] reported that AuNPs can catalyze the rapid decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity shown by AuNPs is almost equal to other embedded 3,6-dihydroxyflavone AuNPs, used to enhance the antioxidant activity (Medhe et al., 2014).\u003c/p\u003e \u003cp\u003eOn the other hand, a clear trend can be observed for the ABTS assay. This is probably because of the difference in the mechanism of operation between the assays. ABTS is largely operating on hydrogen atom transfer. The trend in the ABTS results is such that individual AuNPs demonstrated better antioxidant capacity relative to their respective precursors. Recent research reports [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e] supported the above submission. AuNPs biosynthesized from \u003cem\u003eHalymenia dilatata\u003c/em\u003e also demonstrated higher antioxidant activity than the starting plant extract [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. Thus biosynthesis of NPs, has been reported for its antioxidant capacity [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. These antioxidant compounds might get adsorbed onto the active surface of NPs. The surface reaction phenomenon of these biosynthesized NPs (due to adsorbed antioxidant moiety onto the surface) and the high surface area to volume ratio of NPs generate a tendency to interact and scavenge the free radical [\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. NPs donate electrons to free radicals due to which the free radical becomes stable. The enhanced potential of the HH-AuNPs is due to their control size, shape, and type due to which they became more reactive as compared to the plant extract [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDietary antioxidants have been hypothesized to have a protective effect against α-amylase. The human salivary α-amylase digests the starch into small fragments with two or three pieces. Hence, the inhibition of the α-amylase enzyme could control the carbohydrate metabolism which also decreases the amount of glucose absorption. It seems plausible that a sufficient intake of antioxidants plays an important role in protection against type 2 diabetes [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. In the present study the observed inhibitory percentage of α-amylase by HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution, HH extract, HH-AuNPs, and standard drug Glucophage are shown. The α-amylase inhibitory activity of HH-AuNPs exhibited the highest inhibitory activity with 56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u0026micro;g/mL IC\u003csub\u003e50\u003c/sub\u003e when compared to HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution and HH extract. The positive control Glucophage has revealed the potent α-amylase inhibitory activity with the IC\u003csub\u003e50\u003c/sub\u003e value of 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u0026micro;g/mL. This behavior is similar to that reported by [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], therefore, closely related behaviors in the same experimental conditions are obvious. Accordingly, the improved antidiabetic performance of AuNPs over their precursor extracts has been reported by [\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e, \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. The IC\u003csub\u003e50\u003c/sub\u003e values of α-amylase of HH-AuNPs form also showed improved activity in agreement with previous studies [\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. The inhibiting powers of the nanomaterials may be a function of size and shape. In our results, HH-AuNPs had a size of 10.72 nm. These values are similar to the size of AuNPs in previous investigations [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e] affirmed that spherical AuNPs of sizes 20 and 40 nm in diameter induced the west Nile virus better than those of other sizes and shapes. This may be the reason for the improved enzymatic activity of AuNPs. The enzyme kinetics competitively revealed inhibition for HH on α-amylase (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e is increased, whereas \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e remains the same). These findings indicate that some of the α-amylase inhibitory components in HH extract may be structural analogs of the substrate that compete for binding at the active site of α-amylase, while in the case of HH-AuNPs effect on α-amylase a non-competitive type of inhibition was reported (\u003cem\u003eKm\u003c/em\u003e remained constant and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e is decreased), this suggests that some of the α-amylase inhibitory components in this HH-AuNPs resulting from HH extract bind only to the enzyme-substrate complex and may alter the active site of the enzyme. α-amylase inhibitors delay the rate of carbohydrate digestion, thereby providing an alternative therapeutic option for modulation of postprandial hyperglycemia [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. In diabetic patients with a sustained reduction of hyperglycemia is shown to decrease the risk of developing microvascular and macrovascular diseases and their associated complications [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. The HH extract exhibited higher inhibitory activity towards the α-amylase as opposed to many other hypoglycemic plants reported in previous studies [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. In comparison, the first time \u003cem\u003ein vitro\u003c/em\u003e hypoglycemic assessment of HH-AuNPs indicates higher bioactive properties. The enhanced activity of AuNPs obtained in the α-amylase assessment may be due to their high surface area to volume ratio, thus increasing the surface area phenomenon (promoting the electron transfer reaction) and may increase the pharmacokinetics from a biological view. The effects of oral hypoglycemic drugs depend on several pharmacokinetic factors such as absorption, metabolism, and excretion, and the actions of drugs begin inside the cells, it is believed that AuNPs' small size is easily carried across the cell membrane by transport proteins and may exhibit prolonged effects in bio-systems [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. Given the α-amylase inhibitory effects of HH-AuNPs, the results obtained in this study are coherent with previous studies [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntioxidants such as vitamin C and vitamin E have been associated with AChE inhibition and play an important role in AD treatment [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e] and patients with AD on take high doses of antioxidants will have a slow cognitive deterioration rate [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e]. Inhibition of the AChE is considered promising in AD disease treatment and various researches are focused on new inhibitors from the herbal resources [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR113\" citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e]. To our knowledge, no attention has been given to AuNPs synthesized using the plant for the treatment of AD. The HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution, HH extract, and HH-AuNPs synthesized using the HH extract were screened for their in vitro AChE inhibitory activity. Their IC\u003csub\u003e50\u003c/sub\u003e values are found to be 19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021 for HH extract and 16.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011 \u0026micro;g/mL for HH-AuNPs while no concentration of HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO solution cause 50% inhibition of AChE. Under the same conditions, the IC\u003csub\u003e50\u003c/sub\u003e values of HH-AuNPs showed a remarkable increase in activity toward AChE than extract. This finding is noteworthy because AD is associated with AChE deficiency and AuNPs could be potential new AChE inhibitors [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. The good anti-AChE potential of AuNPs as compared to HH extract is due to the size and shape that may find useful in the AD treatment [\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e]. According to Line Weaver Burk Plot HH-AuNPs causes an uncompetitive type of inhibition of AChE (both \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e decrease), while HH extract caused a non-competitive type of inhibition. From the kinetic study, it is reported that AuNPs primarily cause the inhibition by interaction or adsorption with the AChE enzyme. The exact mechanism by which AuNPs inhibit AChE remains unknown. The binding affinity of NPs to AChE might be due to the lipophilicity of the NPs and the hydrophobicity of the enzyme environment in ChE molecules. Another possible mechanism of AChE inhibition might be due to the adsorption of AChE on the surface of NPs resulting in conformational changes, and surface coverage leading to the inactivation of the enzyme as reported by [\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e]. Inactivation of the enzyme by NPs depends on physicochemical properties like shape, size, curvature, and surface functional groups. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] proposed that inhibition by NPs is primarily caused by adsorption or interaction with AChE protein and partly by dissolved metal ions.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, an environmentally facile, compassionate, straightforward, and medicinally active phytochemical route synthesized colloidal HH-AuNPs from the HH extract an indigenous plant found in abundance in Pakistan. These HH-AuNPs were characterized by the following techniques, UV-Vis spectroscopy, FTIR, SEM, EDX, and XRD. HH-AuNPs showed excellent antioxidant and inhibitory enzymatic properties than their respective HH crude extract and HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt. These observations, plus evidence of their potent antioxidant and enzymatic activity from active molecules rich plants such as HH indicate the value of further studies. Thus, the synthesis of AuNPs based drugs with greater targeted activity combined with medicinal phytochemicals derived from the HH extract may result in unprecedented opportunities directed at the discovery of a cheaper and more beneficial therapy for oxidants, type 2 diabetes, and Alzheimer\u0026rsquo;s diseases\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author wishes to thank the Higher Education Commission of Pakistan (HEC-PAK) for financial support of project No: 20-5082/NRPU/R\u0026amp;D/HEC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors do not have any conflict of interest regarding this article and its publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eC.C.I. Kavitha, G. 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Dordick, Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme Langmuir. \u003cb\u003e20\u003c/b\u003e6800-6807 (2004)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hippeastrum hybridum, nanotechnology, AuNPs, biological activities","lastPublishedDoi":"10.21203/rs.3.rs-1639345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1639345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanotechnology is concerned with the production of nanoparticles (NPs) with restricted sizes and shapes through facile, straightforward, and medicinally active phytochemical routes. This study aims to develop an easy and justifiable method for the synthesis of \u003cem\u003eHippeastrum hybridum\u003c/em\u003e (HH) induced gold NPs \u003cb\u003e(\u003c/b\u003eHH-AuNPs) and then to investigate the effects of these NPs as a free radical scavenger, an inhibitor of the two enzymes i-e Alpha-amylase (α-amylase) and acetylcholinesterase (AChE). UV-Vis spectrum at 576 nm with maximum absorbance at 1.96 confirmed the HH-AuNPs formation. Fourier transform infrared spectroscopy (FT-IR) conforms to the peaks for the functional groups of HH extract and on the surface of HH-AuNPs that are involved in the synthesis and stability of the HH-AuNPs. The average size of 10.72 nm was calculated using four major peaks 38.02\u0026deg;, 44.29\u0026deg;, 64.37\u0026deg;, and 77.58\u0026deg; of X-Rays Diffraction (XRD) analysis. The scanning electron microscope (SEM) analysis confirmed the presence of spherical shaped, monodispersed, and huge density HH-AuNPs with an average size of 30 nm. Energy dispersive X-ray (EDX) confirmed the intense sharp peak at 3.1 keV showing that Au was the main element (48.08%). The HH-AuNPs showed an excellent inhibitory efficacy against free radicals, α-amylase, and AChE as compared to HH extract and HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO salt. Our results suggest that HH-AuNPs exhibited significant antioxidant, Antidiabetic, and antialzheime activities in a concentration-dependent manner as compared to HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO and plant extract. However, further investigations are recommended to be able to minimize potential risks of application.\u003c/p\u003e","manuscriptTitle":"Hippeastrum hybridum assisted bioreduction of Hydrogen tetrachloroaurate (III) trihydrate: Multifaced application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-06 16:55:53","doi":"10.21203/rs.3.rs-1639345/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"06b4e0f5-6cb8-4a84-b245-4f6e2722475e","owner":[],"postedDate":"June 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-06-16T14:12:17+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-06 16:55:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1639345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1639345","identity":"rs-1639345","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}