Biofabrication of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves with their anticancer, antibacterial, and antioxidant activities

Biofabrication of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves with their anticancer, antibacterial, and antioxidant activities | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Biofabrication of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves with their anticancer, antibacterial, and antioxidant activities View ORCID Profile Ashish Gupta , Brajesh Chandra Pandey , Mohd Yaseen , Renu Kushwaha , Jaya Verma , Pratima Chaudhary , Partha Pratim Manna , Rajesh Kumari Manhas , Ida Tiwari , View ORCID Profile Nishi Kumari doi: https://doi.org/10.1101/2025.06.05.653664 Ashish Gupta a Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University , Varanasi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ashish Gupta Brajesh Chandra Pandey b Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University , Varanasi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mohd Yaseen c Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University , Varanasi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Renu Kushwaha d Department of Botany, Mahila Mahavidyalaya, Banaras Hindu University , Varanasi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jaya Verma e Department of Microbiology, Guru Nanak Dev University , Amritsar, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pratima Chaudhary f Department of Zoology, Institute of Sciences, Banaras Hindu University , Varanasi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Partha Pratim Manna g Department of Zoology, Institute of Sciences, Banaras Hindu University , Varanasi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rajesh Kumari Manhas h Department of Microbiology, Guru Nanak Dev University , Amritsar, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ida Tiwari i Department of Chemistry, Institute of Sciences, Banaras Hindu University , Varanasi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nishi Kumari Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nishi Kumari For correspondence: kumaridrnishi{at}yahoo.co.in nishi.kumari5{at}bhu.ac.in Abstract Full Text Info/History Metrics Preview PDF Abstract In this study, we describe the cytotoxic and antibacterial capabilities of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves. Here, we used an environmentally friendly method to synthesize GNPs and then characterized them using techniques such as transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectroscopy, XRD, and Fourier transform infrared spectroscopy. The Surface Plasmon Resonance of GNPs was identified by an absorption peak at 533 nm in the UV-visible spectroscopic studies. The biosynthesized GNPs had an average size of 83 nm and were spherical in shape, according to TEM examination. It’s interesting to note that the biosynthesized GNPs exhibited antibacterial action against a variety of bacterial isolates, both Gram-positive and negative. By using an MTT assay, growth inhibition and the cytotoxic effect of the synthesized GNPs against Dalton’s lymphoma cell lines were also evaluated. 1 Introduction Nanotechnology is a developing science that has potential applications in many areas and is typically utilized to enhance people’s quality of life. A quick, environmentally benign, and readily scaled-up approach is being widely recognized as the biological production of nanoparticles. In recent years, the production of metal nanoparticles using plants has received substantial study and has come to be recognized as an environmental friendly and effective method for utilizing microorganisms as practical nanofactories ( Singh, 2016 ). Metal nanoparticles made from plant extracts are monodispersible and stable when synthetic factors like pH, temperature, incubation time, and mixing ratio are controlled. For the synthesis of NPs, a variety of techniques have been reported, including sol-gel, microwave ( Hayes, 2004 ), hydrothermal ( Lee, 2004 ), flame spray ( Yin, 2002 ), sonochemical, and coprecipitation techniques ( Terribile, 1998 ). Elephant apple, also known as Dillenia indica L., is a perennial medium-sized tree that grows in temperate, tropical, and subtropical regions. From Madagascar to Fiji Island, the genus “Dillenia” disperses throughout the northern and southern Himalayan slopes as well as southwestern China ( Hoogland, 1952 ). The sub-Himalayan regions, West Bengal, Madhya Pradesh, Assam, North-Eastern India, and South Indian States are where you can find this tree in India. Dillenia indica (Elephant apple), a fruit that is underutilized, is abundantly produced in northeastern India ( Prakash, 2016 ). It is traditionally used in Ayurveda to relieve anxiety, stomach discomfort, and exhaustion ( Krishnan, 2020 ). The leaves are also play important role including insecticidal ( Reddy, 2010 ), antimicrobial ( Gupta, 2024 ), antioxidant, anticancer, antidiabetic, anti-inflammatory ( Barua, 2018 ) etc. activity. The present study tried to explore the synthesis and potential antibacterial, antioxidant and cytotoxic property of the plant-derived gold nanoparticles. 2 Material and methods 1.1. Chemicals and instrumentation Gold (III) chloride trihydrate (HAuCl 4. 3H 2 O-Himedia), Ascorbic acid (Himedia), DPPH (Himedia) double distilled water etc. UV-vis spectrophotometer, Transmission electron microscopy (TEM), Dynamic light scattering (DLS), Furrier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD) etc. 1.2. Dillenia indica sample collection and extract preparation The Dillenia indica extracts (DE) were prepared using methods followed by Unal (2020 ) with minor modifications. The leaves were collected from the Ayurvedic garden of the department of Dravyguna, Institute of Medical Sciences, Banaras Hindu University, Varanasi (Accession No. Dillenia 2023/01 ). And cut into tiny pieces. 100gm of leaves were soaked into 500ml of double distilled water in a glass beaker. The beaker was placed on a hot plate for 30 min at 50°C. After that extracts were filtered through Whatman’s filter paper. Filtrate were concentrate by lyophilizer (NSW-275 Lyophilizer -40) till all the double distilled water were evaporated. The extracts were stored at 4°C for further analysis. 1.3. Synthesis of Gold Nanoparticles (GNPs) 5ml of HAuCl 4 (1mM) were added to the 5ml of 1mgml -1 DE in 15ml glass vial. Formation of GNPs were observed after 1hr in random shaking. Their color was changed to dark pink color from pale yellow color, this is the first indication of formation of GNPs. For the confirmation based on the appearance localized surface plasmon resonance (LSPR) was done through UV-vis spectrometry. After the synthesis of GNPs, the reaction mixture were centrifuged at 10000rpm for 10min. GNPs were characterized by different techniques like UV-vis Spectroscopy, FTIR, DLS, XRD, TEM, etc. 1.4. Characterization of Gold Nanoparticles A. UV-vis Spectroscopy analysis UV-Vis spectroscopy is a commonly used technique to characterize the synthesized gold nanoparticles. The result of the synthesis can be analyzed by measuring the absorption spectrum of the sample using a UV-Vis spectrophotometer. The nanoparticle formation was monitored by recording the UV–vis spectra in the wavelength range 200–800 nm employing Shimadzu UV-1800 double beam UV–visible spectrophotometers operated at 1 nm resolution. The formation of gold nanoparticles was also confirm by changes in color from pale yellow to dark pink color. B. Fourier Transform Infrared Spectroscopy (FTIR) analysis Functional moieties associated with the conversion of gold ions into gold nanoparticles have been confirmed by FTIR (Perkin Elmer Spectrum Two FTIR spectrophotometer). Bio-synthesized gold nanoparticles were centrifuged at 7000 rpm for 15 min, and the pellets were cleaned with distilled water. The materials were dried and examined in a 400–4000nm range of wavelength. C. X-ray diffraction (XRD) analysis The structures and chemical properties of the synthesized gold nanoparticles have been investigated using an XRD (Bruker D8 advance Eco XRD) using Cu-Kα (λ = 1.54 Å) source in the region of 2θ from 20° to 80°. If sharp peaks of GNPs are present in XRD, then crystalline size of GNPs were calculated by using Debye-Scherrer formula. The Debye–Scherrer formula is given as-Dp = (0.94 X λ) / (β X Cosθ) Where, Dp = Average crystallite size, β = Line broadening in radians, θ = Bragg angle, λ = X-Ray wavelength D. Dynamic light scattering (DLS) and Zeta potential (ZP) analysis The size distribution and zeta potential of the particles in the Gold nanoparticles were measured using a Nano ZS zetasizer instrument (Malvern Instruments). A laser with a 633 nm wavelength, filtered double-distilled water as the medium solvent, 1.330 for the medium refractive index and 1.59 for the material, 25°C for the measurement temperature, and 0.8872 for the medium viscosity (cP) were some of the different measurement parameters employed. Prior to DLS measurement, the colloid was passed through a 0.22 μ polyvinylidene fluoride (PVDF) membrane syringe filter. The loaded sample was used for thrice in a quartz micro cuvette, and the mean result was reported. E. Transmission Electron Microscopy (TEM) and Energy Dispersive X-ray (EDX) analysis On a copper grid (200 mesh size), 5 μL of sonicated samples were loaded. Particles were given five minutes to settle and dried for ten minutes on a hot plate at fifty degrees Celsius. Images were taken with a 120 CX TEM (JEOL JEM) at 66,000X while samples were evaluated at 100 kV. Iridium Ultra version 1.4 ( www.ixrfsystems.com ) was used to perform morphological analysis on scanned pictures (1200 dpi) of the particles. The grid was rotated at different angles while photographs were taken at 100,000X to reveal the three-dimensional characteristics of the particles. The EDX analysis were done to examine the presence of gold element in the GNPs. 1.5. Applications of Gold Nanoparticles A. Antibacterial Activity Different test bacteria such as Bacillus subtilis (MTCC 619), Staphylococcus epidermidis (MTCC 435), and Staphylococcus aureus (MTCC 96) were procured from Microbial Type Culture Collection (MTCC) and Gene Bank, CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. All the bacterial cultures were maintained on nutrient agar slant in the refrigerator at 4°C. The antibacterial screening was performed by a modified method ( Pandey, 2024 ). In brief, the bacterial suspension of bacterial cultures was cultivated for about 24 hrs. Which later made to 0.5 McFarland turbidity standard. From the above-prepared suspension, about 100 μL were spread onto Mueller-Hinton agar plates using sterile swabs followed by drying at room temperature. Make wells of 6 mm in size were punctured with a sterile cork borer. After the addition of D. indica leaf extract, synthesized gold nanoparticles (GNPs) solution (20 μL), were loaded into the wells at 1mgml -1 concentration. DMSO as the negative control, streptomycin 10 μg/disc is used as a positive control. Plates were kept in the refrigerator for 1hr for the diffusion of samples followed by incubation at 37°C for 24 hrs. The results were observed in terms of zones of inhibition (mm) around the wells. B. DNA Nicking Assay This method was used to evaluate the potential of synthesized gold nanoparticles (GNPs) to protect supercoiled pBR322 plasmid DNA from destroying the effects of hydroxyl radicals produced by Fenton’s reagent. The reaction mixture contained 1μL plasmid DNA (50 μg/100 μL), 10 μL Fenton’s reagent (30 mM H 2 O 2 , 50 μM ascorbic acid, and 80 μM FeCl 3 ) and different concentrations of leaf extract and synthesized gold nanoparticles GNPs (5, 10, 15, 20 and 25 μg/mL) and double distilled water to make the final volume up to 20 μL. An equal volume of distilled water was added in place of Fenton’s reagent in the negative control. It was followed by incubation at 37°C for 30 min. The analysis of DNA was done on 0.8% agarose gel electrophoresis. The positive control is used as rutin. Densitometric analysis was done to examine the DNA damage quantitatively with the help of GelQuant software. The percentage of different forms of DNA i.e., supercoiled (Form I), double-stranded nicked (Form II) and linear (Form III) was calculated. C. Analysis of Antioxidant Activity by DPPH The antioxidant activity were done by DPPH method according to Nayak, 2020 with some modifications. Absorbance of plant extract and GNPs were recorded at 517nm. Ascorbic acid were taken as a standard. The absorbance of radical scavenging activity of GNPs were measured by following equation. Percent inhibition = [(OD of control – OD of Sample) / OD of control] x 100 The inhibition of 50% of the free radical of GNPs is known as IC 50 value that is calculated by linear regression analysis against concentration and % inhibition. D. MTT Assay Cell culture Murine Dalton’s lymphoma (DL) cells were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 μg/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA), henceforth considered as complete medium. DL cells were also maintained as semisolid tumor in the peritoneum of BALB/c mice following serial transplant in order to maintain the tumorigenicity potential. Cytotoxic assay The lytic activity of the GNPs and PE against the DL cells was measured by cytotoxicity assay (CytoTox 96 cytotoxicity assay kit, Promega, USA) ( Hira, 2015 ). Tumor target cells (5×10 3 ) were co-cultured in the presence of increasing concentrations of the indicated formulations in a 96 well culture dish. The cells were incubated for 18 hours at 37°C, 5% CO 2 . Specific lysis (percentage of cytotoxicity) was ascertained from the under mentioned formula: Cell viability assay Effect of GNPs and PE on the viability of DL tumor cells was estimated by a colorimetric XTT (sodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) assay (Roche, Indianapolis, IN). Tumor cells were seeded (5×10 3 cells/well) in a 96-well culture dish and incubated for 18 hours at 37°C, 5% CO 2 . In a plate reader, the OD was recorded at 450 nm (Synergy HT, BioTek, USA). The proportion of viable cells was computed using the formula below ( Hira, 2015 ). Cell growth inhibition assay Growth inhibitory potential of the above compounds against the DL cells were studied by MTT assay ( Hira, 2014 ). Tumor target cells (5×10 3 cells /well) in a 96 well culture dish were treated with serial concentrations of the compounds. Following incubation at 37°C, 5% CO 2 , for 48 hours, the proliferation of the tumor cells was assessed by MTT assay using CellTiter 96 kit (Promega, USA). The measurement of absorbance (OD values) was made at 570 nm in a plate reader (BioTek, USA) ( Hira, 2014 ). Percent inhibition of the tumor cells was calculated using the mentioned formula: The Experimental OD indicate the values of the tumor cells in the presence of the compounds and the Target OD indicate the corresponding values of the tumor cells alone, cultured in medium only. Apoptosis study Evaluation of apoptotic cell death in DL tumor cells was performed following treatment with the indicated formulations (50 μg) for 12 hours. Untreated cells were used as a positive control. The cells were washed in PBS and stained with PE conjugated Annexin V for 30 min. These cells were washed in the Annexin buffer. PE-conjugated Annexin V-positive cells were visualized under a fluorescence microscope (EVOS FL Cell Imaging System equipped with Plan Fluor, 40X, NA 0.75 objective, Life Technologies) as described earlier ( Roy, 2024 ). Statistical analysis Unpaired student’s t-test or one way ANOVA followed by Tukey’s post hoc test was performed while comparing between the groups. Each experiment was performed in triplicate and the data were presented as mean ± SD (standard deviation). Differences were considered significant for ‘p’ values < 0.01-0.05 (*), p<0.001-0.01 (**), p<0.0001-0.001 (***) and p< 0.0001 (****). ns= non-significant. 3 Result and Discussions A. UV-vis Spectroscopy Analysis In general, the synthesis of GNPs results in a red-shift of the SPR band compared to the bulk gold due to the quantum confinement effect. The intensity of the SPR band is proportional to the concentration and size of the nanoparticles. The green synthesized biogenic GNPs were examined using UV-vis spectroscopy. The D. indica leaves were used as capping and reducing agent for the synthesis of GNPs. After 24hr of incubation, the color of the mixture gets changed to dark pink color, this is the indication of formation of GNPs (Graph1). The GNPs were formed at 533nm wavelength. The previous reports also exhibited the gold nanoparticles SPR band with absorption peak ranges from 530-535nm ( Sett, 2016 , Huang, 2019 ). The comparative spectra of GNPs, DLE, and Chloroauric acid were shown in the Graph 1A. Their stability were also checked after one months of the formation of GNPs. Different concentration of the chloroauric acid were used to formed the formation of GNPs. The optimum concentration of chloroauric acid is 1 mM, is the best for the synthesis of GNPs (Graph 1B). By measuring the UV-Vis spectrum of the colloidal suspension at different time intervals during the synthesis, the formation of the GNPs can be monitored. Initially, there may be no or weak absorption in the UV-Vis region. But after 10 min, the absorbance increases due to formation of GNPs. As the synthesis proceeds, the SPR band becomes more pronounced and shifts to longer wavelengths, indicating the formation of larger nanoparticles (Graph 1C). Temperature is also the main factor during formation of GNPs. As the temperature increases from 20°C to 100°C the GNPs formed were stable at the same absorbance. There are very less effect of temperature on it (Graph 1D). B. Fourier Transform Infrared Spectroscopy (FTIR) analysis FTIR spectra were conducted to determine the presence of phytochemical components and bioactive molecules in D. indica leaf extract. The FTIR spectra provide information about the different functional groups found in the organic compounds present in the extract that may cause the reduction of gold ions and stabilize the reduced GNPs. The results of this study FTIR analysis show different stretches of the bond in extract (Graph 2 A): 3401.50 for O-H stretching(alcohol), 1727.14 for Aldehyde, Ester, 1637.40 for Alkenyl C=C Stretching, 1514.62 for Aromatic Nitro Compounds, 1107.08 for Aromatic C-H in plane bending and 600.12 cm −1 for C-Br Stretching. For GNPs following bands are found: 3400.75 for O-H stretching (alcohol), 1609.61 for C=C-C Aromatic Ring Stretching, 1509.19 for Aromatic Nitro Compounds, 1424.75 for Carbonate ions, 1262.39 for O-H in Plane Bend, Aryl-O-Stretching, 1049.23 for C-O Stretching Coupled with C-O Bending of C-OH of Carbohydrates, 779.63 for C-Cl Stretching, and 613.73 for C-Br Stretching. Graph 2B shows the medium and sharp peaks near 3400.75 cm −1 and 3401.50 cm −1 indicate the presence of free alcoholic groups in both extract and GNPs, while the peak at 1384.93 cm −1 determined the presence of O-H Bending (Phenol) in compounds of extract. The peak near 2929.30 cm −1 corresponds to C-H Stretching, and the peaks at 1184.28 cm −1 are assigned to Aromatic C-H in Plane Bending in GNPs. The chemicals are also present in D. indica leaf, and all these bioactive compounds are accountable for reducing gold ions and the stability of GNPs. All the functional groups, as mentioned earlier, may tend to reduce Au + to Au 0 by providing the electrons to gold and might have the ability to stabilize the GNPs produced samples. C. X-ray diffraction (XRD) analysis The crystalline nature of GNPs synthesized by D. indica leaf extract was studied by XRD analysis in the range of 20–80° at 2θ angles (Graph 3). It is seeming that the characteristic peaks at 38.07, 44.44, 64.58, and 77.55 in the 2θ values resemble to the lattice planes (111), (200), (220), and (311). The strong and narrow diffraction peaks confirmed the crystalline structure of synthesized GNPs. All the different peaks confirm the face-centered-cubic (fcc) structure of GNPs. The unassigned peaks were occurred due to the impurities present in the sample or may be related to crystalline and amorphous organic phase ( Awwad, 2013 ). D. Dynamic light scattering (DLS) analysis The size of the GNPs in the colloidal solution was measured using DLS. The first and second peaks showed that the GNPs were 107.7 and 4840 nm in diameter and 53.18 and 704.9 nm, respectively, and that the peaks intensities were 97.6% and 1.9%, respectively (Graph 4A and 4B). The average size of GNPs was 83.84nm in diameter. The Polydispersity index (PdI) was found to be 0.275. The polydispersity index shows the proportion of different-sized particles to total particles. More the polydispersity index, less the particles are monodispersed (Rajathi, 2014).The 0.275 polydispersity index indicates that the particle distribution is almost monodispersed. The zeta potential of synthesized GNPs was found to be -8.46mV. E. Transmission Electron Microscopy (TEM) and Energy Dispersive X-ray (EDX) analysis The previous study revealed that the silver nanoparticles synthesized by D. indica were spherical and their sizes were 16-95nm ( Bhakya, 2016 ). TEM is used to determine the morphology, shape, and size of GNPs. TEM images reveal that the particles are spherical, triangular and hexagonal in shape and are uniformly distributed without cluster. Average particle size is 83nm reported in synthesized GNPs. The selected area electron diffraction (SAED) pattern of GNPs prepared by D. indica extract suggests the crystalline nature of GNPs which is in good agreement with the X-ray diffraction (XRD) ( Fig 1 ). The lattice fringes between the two adjacent planes to be 2nm apart. The elemental analysis were also done by EDAX that confirms the gold element present in the GNPs with weight percent of 35.40 and 03.40 by atomic percent as shown in the graph 5, 6 and Table 1 . View this table: View inline View popup Download powerpoint Table 1: EDAX analysis Download figure Open in new tab Fig 1. Fig (A-E) showing the synthesized GNPs with different size and shape of synthesized GNPs. Fig F shows the SAED pattern of GNPs 4. Application of GNPs A. Antibacterial assay In vitro bioassay demonstrated strong antibacterial activity of D. indica leaf extract and, synthesized GNPs against test bacteria. It showed pronounced inhibition against pathogenic bacteria viz. Bacillus subtilis (MTCC 619), Staphylococcus epidermidis (MTCC 435), and Staphylococcus aureus (MTCC 96) with inhibition zones of 15–30 mm ( Fig 2 ). The leaf extract of D. indica in different solvents exhibits antimicrobial activities ( Apu, 2010 ). The plant’s phytochemicals, which are present in the extracts, are responsible for the antibacterial properties of GNPs, which are synthesized using plant extracts and have wide-ranging uses in the administration of medicines, tissue imaging, and clinical pathogen identification ( Akintelu, 2020 ). Due to the diversity and potential of plants in producing GNPs with various shapes, a great deal of research is still being done in this subject despite the abundance of literature on the synthesis, characterization, and uses of synthesized GNPs using plant extracts ( Francis, 2018 , Dubey, 2010 , Kumar, 2018 , Dang, 2019 ,) Download figure Open in new tab Fig 2. Antibacterial activity of D. indica leaf extract and, synthesized GNPs against (A) Staphylococcus epidermidis (MTCC 435), (B) Staphylococcus aureus (MTCC 96), and (C) Bacillus subtilis (MTCC 619) B. DNA nicking assay The DNA nicking assay revealed that the leaf extract and synthesized gold nanoparticles (GNPs) have a protective effect on the supercoiled DNA pBR322 from the destructive effects of hydroxyl radicals generated by Fenton’s reagent. It was observed that supercoiled form of plasmid DNA (Form I; Lane 1) was degraded to single-stranded, double-stranded nicked, and linear forms of DNA (Form III and II, respectively; Lane 2) due to hydroxyl radicals generated in Fenton’s reaction mixture. However, the addition of leaf extract and synthesized GNPs at different concentrations (from 5 to 25 μg/well) to the reaction mixture minimized the hydroxyl radical-mediated DNA damage i.e., conversion of supercoiled DNA (Form I) to the formation of single-stranded nicked DNA (Form III) and double-stranded nicked and linear DNA (Form II) as shown in lanes 4–8. Lane 3 shows the positive control rutin, which maintained the integrity of the DNA to Form I ( Fig. 3 ). The densitometric analysis determined the percentage of DNA present in three forms. Table 2 demonstrates that the amount of supercoiled DNA in the presence of leaf extract and Fenton’s reagent was found to be 52.69 % (25 μg), 46.61 % (20 μg), 39.26% (15μg), 32.79 % (10 μg) and 29.38 % (5μg), indicating that the increasing concentration of leaf extract protects the damaged DNA with higher intensity. The results were in comparison with the amount of supercoiled DNA present in the positive control (rutin + Fenton’s reagent) with 52.29%. In the presence of Fenton’s reagent only, the amount of Form I was degraded, and simultaneously, Form II increased from 57.34% to 58.46%. Similarly, in table 3 , treatment of plasmid DNA with Fenton’s reagent in the presence of synthesized gold nanoparticles resulted in 39.48-79.65% of DNA Form I, 41.22-69.74% of DNA Form II, respectively, and no DNA Form III was formed, indicating the protective nature of synthesized GNPs. View this table: View inline View popup Download powerpoint Table 2: Densitometric analysis of different forms of DNA (in percentage) after treatment with varying concentrations of leaf extract View this table: View inline View popup Download powerpoint Table 3: Densitometric analysis of different forms of DNA (in percentage) after treatment with varying concentrations of synthesized gold nanoparticles Download figure Open in new tab Figure 3. a) DNA Nicking assay revealed the protective potential of leaf extract with free radicals g enerated by Fenton’s reagent A) Lane 1: Negative control (only DNA); Lane 2: Fenton’s reagent; L ane 3: Positive control (rutin, 10 μg); and Lane 4–8: Fenton’s reagent +5, 10, 15, 20 and 25μg of the crude extract; Form I=Supercoiled; Form II=Linear; Form III=Single strand nicked DNA. Download figure Open in new tab Figure 4. a) DNA Nicking assay revealed the protective potential of synthesized gold nanoparticles (GNPs) with free radicals generated by Fenton’s reagent A) Lane 1: Negative control (only DNA); Lane 2: Fenton’s reagent; Lane 3: Positive control (rutin, 10 μg); and Lane 4–8: Fenton’s reagent +5, 10, 15, 20 and 25μg of the synthesized GNPs; Form I=Supercoiled; Form II=Linear; Form III=Single strand nicked DNA. C. Antioxidant Activity by DPPH Natural anti-oxidants, such as phenolics, terpenoids, and alkaloids, play an extraordinarily desired role in enhancing human health by lowering genetic mutation rates, preventing cell damage, and reducing or stopping a variety of diseases through the detoxification of oxygen free radicals ( García, 2020 ). A popular method to determine the antioxidant activities of plant extracts is the DPPH assay. It is an easy, quick, sensitive, and reproducible assay ( Ebrahimzadeh, 2008 ). In this study, the antioxidant activity of GNPs and extract was evaluated by DPPH method. For this experiment, seven different concentrations (0.05-1.0 ml) of sample were taken against the ascorbic acid as a standard. GNPs and extract both shows the antioxidant activity ( Figure 8 ). At low concentration the GNPs shows better antioxidant activity than extract but at high concentration the extract shows better than GNPs. The IC 50 value of extract and GNPs are 893.68 and 927.84 μg/ ml respectively against the standard IC 50 value as 8.4 μg/ ml ( Brighente, 2007 ). Download figure Open in new tab Figure 8. Graph A and B showing the free radical scavenging activity of Leaf extract and GNPs. D. MTT Assay We performed dose dependent tumoricidal effects of GNPs and compared with PE and assessed the loss of viability in DL tumor cells ( Gupta, 2025 ). Data suggests that percent cell viability loss in DL cells was observed following treatment with GNPs compared to PE in a dose dependent fashion ( Figure 8A ). DL cell viability in the presence of GNPs reduced to <25% compared to 77% in the presence of PE at a concentration of 100μg tested (p<0.01). Similar results were observed in other concentrations tested ( Figure 9A ). GNPs also augmented the cytotoxicity in the tumor cells compared to treatment with PE in a dose dependent manner ( Figure 9B ). In the highest concentration tested, GNPs was significantly more cytotoxic compared to PE indicating tumoricidal potential of GNPs against the DL cells (p<0.01) ( Figure 9B ). Long-term (48 hrs) co-culture of DL tumor cells with GNPs results in significant growth inhibition of tumor cells compared with PE indicating the efficacy of the construct over the PE ( Figure 9C ). Microscopic observation of DL tumor cells following treatment with GNPs showed significant loss in cell membrane integrity suggesting significant impact of the compound on the viability and proliferative potential of the DL cells following treatment with GNPs. PE treatment did not produce similar effect as demonstrated in Figure S1 . Download figure Open in new tab Graph 1. Graph (A) showing the formation of GNPs, their Stability, Plant extract and Chloroauric acid, (B) showing the formation of GNPs at different concentration of Chloroauric acid, (C) showing peaks at different time intervals, (D) GNPs formation at different temperature Download figure Open in new tab Graph 2. Graph (A) showing the different functional groups present in leaf extract, (B) GNPs. Download figure Open in new tab Graph 3. Graph showing the XRD patterns of synthesized GNPs. Download figure Open in new tab Graph 4. Graph A showing the size distribution and B showing the zeta potential of GNPs. Download figure Open in new tab Graph 5. Graph showing the frequency of GNPs against length. Download figure Open in new tab Graph 6. Graph showing the EDAX analysis of GNPs. Download figure Open in new tab Supplementary (S) 1. Effects of GNPs or PE on DL tumor cells as observed under compound microscope. GNPs induces loss in membrane integrity in DL cells. Download figure Open in new tab Figure 9. Tumoricidal effects of GNPs against DL tumor cells. Concentration dependent studies on viability (A), cytotoxicity (B) and long-term growth inhibition (C) of DL tumor cells in the presence of GNPs and PE. Antitumor effect of GNPs was further assessed by the induction of significant apoptosis in DL tumor cells compared with treatment with PE. Both GNPs and PE fluoresce green following uptake by the DL tumor cells. Treatment of GNPs treated cells with Annexin-PE results in the staining of the apoptotic cells indicating the externalization of Annexin V following treatment ( Figure 10 ). Download figure Open in new tab Figure 10. Apoptosis in DL tumor cells in the presence of GNPs. DL tumor cells were treated with GNPs and PE (50 μg) for 12 hours at 37°C, 5%CO 2 . The cells were washed and treated with PE conjugated Annexin-V for 30 minutes. The images were captured in EVOS-FL. Treatment of DL tumor cells PE does not induce detectable levels of apoptosis which was indicated by the absence of apoptotic cells stained with Annexin PE ( Figure 10 ). GNPs induces significant apoptosis in DL cells compared with PE and appears to destabilize tumor cell integrity resulting in significant loss in cell viability and apoptosis. GNPs is highly effective in inhibition of long-term proliferation of tumor cells indicating its suitability as antitumor agent against lymphoma cells ( Figure 9 , & 10 ). 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Share Biofabrication of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves with their anticancer, antibacterial, and antioxidant activities Ashish Gupta , Brajesh Chandra Pandey , Mohd Yaseen , Renu Kushwaha , Jaya Verma , Pratima Chaudhary , Partha Pratim Manna , Rajesh Kumari Manhas , Ida Tiwari , Nishi Kumari bioRxiv 2025.06.05.653664; doi: https://doi.org/10.1101/2025.06.05.653664 Share This Article: Copy Citation Tools Biofabrication of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves with their anticancer, antibacterial, and antioxidant activities Ashish Gupta , Brajesh Chandra Pandey , Mohd Yaseen , Renu Kushwaha , Jaya Verma , Pratima Chaudhary , Partha Pratim Manna , Rajesh Kumari Manhas , Ida Tiwari , Nishi Kumari bioRxiv 2025.06.05.653664; doi: https://doi.org/10.1101/2025.06.05.653664 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17635) Bioengineering (13859) Bioinformatics (41846) Biophysics (21401) Cancer Biology (18534) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24285) Genetics (15582) Genomics (22463) Immunology (17700) Microbiology (40298) Molecular Biology (17141) Neuroscience (88424) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)