HPTLC-Based Quantitative Analysis of Scopoletin and In vitro Antioxidant and Antimicrobial Evaluation of Hymenodictyon orixense Roxb. Bark

HPTLC-Based Quantitative Analysis of Scopoletin and In vitro Antioxidant and Antimicrobial Evaluation of Hymenodictyon orixense Roxb. Bark | 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 HPTLC-Based Quantitative Analysis of Scopoletin and In vitro Antioxidant and Antimicrobial Evaluation of Hymenodictyon orixense Roxb. Bark Anil Badnale, Satish Meshram, Nandaji Chalak, Ashwini Ghagare, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8549856/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 Hymenodictyon orixense Roxb. is a medically valuable evergreen tree that has been traditionally used worldwide to treat conditions such as inflammation, infection, wounds, and chronic diseases. This study was conducted to evaluate the phytochemical analysis, in vitro antioxidant properties, antimicrobial activity, and HPTLC characterization of the ethanol extracts from the bark of H. orixense . Roxb. by using scopoletin as a standard. The bark of H. orixense Roxb. was collected and identified as a medicinally valuable evergreen tree. This was dried under shade and Soxhlet-extracted using ethanol as the solvent. The preliminary phytochemical study of the ethanol bark extracts of H. orixense Roxb. identified the presence of alkaloids, flavonoids, phenols, tannins, saponins, steroids, glycosides, and fixed oils. The total content of the antioxidant compounds, like total phenols (249.3 ± 1.25µg GAE/mg), total flavonoid content (81.29 ± 0.56µg RE/mg), total tannin content (47.85 ± 0.44µg TAE/mg), and total alkaloid content (0.4% w/w) of the ethanol bark extracts of H. orixense Roxb. was determined. This study evaluated the antioxidant activity of ethanol extracts of H. orixense Roxb. using various in vitro assays, including the DPPH, ABTS, CUPRAC, superoxide scavenging, hydroxyl scavenging, and nitric oxide scavenging assays. The ethanol extracts of H. orixense Roxb. possessed key antioxidant activity in all the in vitro antioxidant assays. The studies revealed high DPPH scavenging activity and the least in ABTS scavenging activity, indicating higher electron donation and stronger scavenging of free radicals. The ethanol extracts of H. orixense Roxb. showed appreciable inhibition of S.aureus and E. coli with IC 50 comparable to the standard chloramphenicol in disc diffusion methods. Hymenodictyon orixense Roxb. HPTLC Scopoletin Antioxidant assay Antimicrobial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction H excelsum Roxb.Wall. ex-Mabb., also referred to as H. excelsum , is a deciduous tree that grows in the Rubiaceae family. It is known by local names such as Dendra Bandaru and Kala-bachnag in various areas of India and Thailand, where it is also referred to as U Lok and Som Kop, respectively. H. excelsum Roxb. grows in subtropical to tropical climates. It grows with a rounded top and a straight, cylindrical trunk that measures up to 10–12 meters in height. H. excelsum Roxb. has garnered attention in the fields of ethnomedicine and phytochemical studies due to its wide range of medicinal uses. The leaves, stems, roots, and flowers of H. excelum have been used by traditional healers for the treatment of inflammation, wounds, infections, and chronic diseases such as arthritis and peptic ulcers. Well known in traditional medicinal systems of native societies for a considerable number of years, this species contains variable bioactive compounds, which may lead to beneficial uses in treating many ailments [ 1 ]. In traditional medicinal use, the bark extracts of this species serve as astringents, febrifuges, and antimyesthetics. Its wood is most effectively used in treating herpes. Its leaves have been used in the treatment of chickenpox, excessive sweating due to fever, toothaches, throat inflammation, mouth ulcers, prostate cancer [ 2 ], and tonsillitis [ 3 ]. Its antibacterial, anticoagulant, anti-inflammatory, and protective properties against UV radiation, known as sun-screening properties, have been well identified [ 4 ]. Its roots contain bioactive compounds like rubiadin, rubiadin methyl ether, lucidin, nordamnacanthal, damnacanthal, and 2-benzyl. The stem bark exudes hymexelsin, which is an apiglucoside compound derived from scopoletin. The plant has been shown to contain other chemical constituents, including the iridoid glycoside loganin and four coumarins: scopoletin, scopolin, hymexelsin, and scopoletin 7-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside. The plant extracts have been used in traditional medicine The present investigation is to carry out a comprehensive phytochemical profiling of H. orixense Roxb. with quantitative estimation of Major secondary metabolites. In addition to evaluating the antioxidant efficacy against selected free radical scavenging assays and antimicrobial activity against selected Gram-positive and Gram-negative bacterial strains, thereby validating its ethnomedicine relevance. HPTLC will be used for quantification and fingerprints, ensuring standardization of the extract. The aim is to bridge traditional knowledge with modern analytical validation, providing a scientific basis for its therapeutic application and potential. 2. Materials and Methods 2.1 Plant collection and authentication The bark of H. orixense Roxb. was collected from a local field in Chatgaon District, Gadchiroli, Maharashtra. The plant sample was scientifically identified and authenticated by Prof. Nitin Dongarwar from the Department of Botany at Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur. A voucher specimen was created and stored in the Herbarium at Nagpur University, designated as sheet number 10071. 2.2 Chemicals and reagents The reagents used in the study included 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid (GA), Cupric chloride (CuCl₂), ABTS, and trichloroacetic acid, all of which were purchased from SRL Chem Laboratories. Hydrogen peroxide (H₂O₂) was obtained from Neurochem Laboratories. Trichloroacetic acid (TCA), ferric chloride, and sodium nitroprusside were sourced from Fisher Scientific. Additionally, Ascorbic acid (AA), DMSO, EDTA, and trio-barbituric acid (TBA) were purchased from HiMedia. 2.3 Extraction of Plant Material The plant bark was collected, decorticated, and cleaned of foreign particles. It was then air-dried in the shade to preserve its phytoconstituents and ground into a coarse powder. Once dried, the powder was stored in an amber glass container for further extraction. A hundred grams of this powdered bark was extracted with absolute ethanol using a Soxhlet apparatus. After extraction, the ethanolic solution was filtered, and the solvent was concentrated using a rotary evaporator. 3. Qualitative and Quantitative Assessment of Secondary Metabolites The ethanolic bark extract of H. orixense Roxb. was analysed using standard qualitative phytochemical tests to identify its secondary metabolites. The results showed the presence of alkaloids (indicated by a creamy precipitate in Mayer’s test), glycosides (red color in Borntrager test), flavonoids (red to pink in Shinoda test), phenolic compounds (white precipitate in Gelatin test), steroids (red in Salkowski’s test), tannins (reddish-yellow with nitric acid), fixed oils and fats (translucent stain in spot test), and saponins (formed foam in test) [ 5 ]. 3.1 Total Phenolic Content The total phenolic content (TPC) was measured using the Folin-Ciocalteu (FC) reagent, with gallic acid as the reference standard. A calibration curve was prepared with gallic acid (10–50µg/mL). A stock solution of the extract (1 mg/mL) was made in Methanol and diluted for testing. Then, 1 mL of this solution was mixed with 0.5 mL of 2N FC reagent and 1.5 mL of 20% sodium carbonate. After 2 hours of dark incubation, absorbance was read at 765 nm using a Shimadzu UV-Visible spectrophotometer. TPC was expressed as mg of gallic acid equivalents per gram of extract, with all tests performed in triplicate [ 6 ],[ 7 ]. 3.2 Total Flavonoid Content Total flavonoids (TFC) were determined by the aluminium chloride colorimetric method. A 10 mg sample was dissolved in Methanol, then 2 mL of this solution was mixed with 20% methanolic aluminium chloride and acetic acid in a volumetric flask. After 40 minutes of incubation, absorbance was measured at 415 nm using a Shimadzu UV spectrophotometer. Rutin (0.5 mg/mL) served as the standard. TFC was expressed as mg rutin equivalents per gram of extract, with triplicate measurements [ 8 ]. 3.3 Total Tannin Content The total tannin content (TTC) was estimated using the FC reagent. A stock solution of ethanolic extract (1 mg/mL) was prepared and diluted to different concentrations (100, 200, 300, 400, 500 ppm). To each, 1 mL was mixed with 0.5 mL of FC reagent, followed by the addition of 1.5 mL of 20% sodium carbonate solution. Absorbance was measured at 775 nm after 15 minutes of incubation. Tannins were expressed in mg tannic acid equivalents per 100 g of extract (mg TAE/100g). All experiments were done in triplicate [ 9 ]. 3.4 Total Alkaloid Content Total alkaloids were determined gravimetrically. 5 g of powdered crude drug was extracted ultrasonically in three 50 mL portions with 0.1 M sulfuric acid. The filtered solution and washings were performed four times, discarding the supernatant each time, using 25 mL of dilute chloroform. Alkaloids were then extracted by making the solution basic with dilute ammonia. The basic solution was extracted with diethyl ether, filtered, dried, and weighed. Results were expressed as % w/w of the crude drug [ 6 ]. 3.5 High Performance Thin Layer Chromatography A stock solution of scopoletin at a concentration of 0.1 mg/mL was prepared using HPTLC-grade Methanol. An ethanolic extract of H. orixense Roxb. at a concentration of 5 mg/mL was also prepared by dissolving in MeOH and filtering through a whatman No. 01 filter. The above-mentioned solutions were spotted uniformly on silica gel 60 F₂₅₄ thin-layer chromatography plates, which measured 200 × 100 mm. The scopoletin solution, at concentrations ranging from 2 to 16 µL, and the ethanolic extract, at concentrations ranging from 1 to 7 µL, were spotted using the CAMAG Linomat 5 applicator (vision CATS software) at a program rate of 150 nL/s, with every spot programmed to be 8.0 mm in length [ 10 ]. A mobile phase consisting of ethyl acetate, formic acid, and dH 2 0. in the ratio of 6.7:0.5:2.6 V/V/V was used [ 11 ]. A 20 x 10 cm TLC plate was placed in a pre-saturated twin-tract glass chamber, along with the chosen mobile phase, for 20 minutes at room temperature. The development was done up to 70 mm solvent front using an automatic chamber. After that, the TLC plate was dried in air at room temperature for 10 minutes. The Rf value and spots of the separation were recorded. The dried plate was viewed under UV light at both 254 nm and 366 nm in a UV cabinet. All of the visible bands were recorded. Spectral scans were performed using a CAMAG TLC Scanner 4, equipped with deuterium and tungsten lamps that scanned wavelengths from 190 to 450 nm at a speed of 20 nm/s. Scanning was done track by track, and the Rf value of both substances was determined [ 10 ]. 4. In Vitro Investigation of Antibacterial Potential Against S. aureus and E. coli The antimicrobial activity of H. orixense Roxb. bark extract was tested using the Disk diffusion method against S. aureus (ATCC 23235) and E. coli (ATCC 25922). Mueller-Hinton Agar (MHA) was prepared and sterilized by autoclaving at 121°C and 15 psi for 10 minutes. Bacterial suspensions were adjusted to a 0.5 McFarland standard (approximately 1.5 × 10 8 CFU/mL). Wells with a 6 mm diameter were punched into the agar, and 10 µL of each sample—test samples, Chloramphenicol as a positive control, and Blank and distilled water as negative controls—were added. Plates were incubated at 35–37°C for 20–24 hours. Zones of inhibition were measured in triplicate, and average values were calculated [ 12 ] 5. In Vitro Antioxidant Assays 5.1 DPPH scavenging assay 5 µL of various concentrations of the test compound was added to 100 µL of a 0.1 mM DPPH solution in a 96-well plate. The reaction mixtures were prepared in triplicate for each concentration, with duplicate blanks created by adding 200 µL of DMSO/Methanol and 5 µL of the corresponding compound concentrations. The plate was incubated in the dark at room temperature for 30 minutes. Absorbance was measured at 517 nm using a microplate reader (iMark, BioRad). Methanol served as the blank. A control reaction mixture containing 20 µL of deionized water was included. The radical scavenging activity (% inhibition) was calculated relative to the control, and IC 50 values were determined using GraphPad Prism 6 [ 13 ]. %Inhibition = Abs.(control) − Abs. (Sample) /Abs. (control) × 100 5.2 ABTS scavenging assay In a 96-well plate, 10 µL of test samples or ascorbic acid (standard) at different concentrations were added to 200 µL of ABTS reagent. After incubating in the dark at room temperature for 10 minutes, the absorbance was measured at 750 nm using a microplate reader (iMark, Bio-Rad). Control wells without treatment served as a baseline. Antioxidant activity was expressed relative to the negative control, and IC₅₀ values were determined using GraphPad Prism 6 [ 14 ] %Inhibition = Abs.(control) − Abs. (Sample) /Abs. (control) × 100 5.3 Hydroxy Free Radical Scavenging Assay A reagent mixture was created by combining 10 µL of 0.5 M EDTA (HiMedia), 24.14 mg of Deoxyribose (SRL), 88 µL of FeCl₃ (10 mg/mL) and 28 µL of H₂O₂ (6%) (Neurochem Laboratories), then the volume was adjusted to 33 mL with water. Each well of a 96-well microplate received 10 µL of plant extract, 24 µL of phosphate buffer (50 mM, pH 7.4), and 10 µL of ascorbic acid. The plate was incubated at 37°C for 1 hour. Wells without treatment served as the negative control, with Gallic Acid (SRL) as the positive standard. After incubation, 50 µL of 10% TCA (Fisher Scientific) and 50 µL of 1% TBA (HiMedia) were added to each well. The formation of a pink chromogen indicated the degradation of deoxyribose, and the absorbance was read at 540 nm. IC 50 was calculated using GraphPad Prism 6 software [ 15 ],[ 16 ]. 5.4 Super Oxide Anion Radical Scavenging Assay Serial dilutions of the H. Orixense Roxb. extract was prepared at concentrations from 1 to 1000 µg/mL. Riboflavin, used as the reference standard, was similarly diluted from 1 to 50 µg/mL. The reaction mixtures were incubated in a 96-well microplate under ambient illumination for 30 minutes. After preparing the serial dilutions of the H. orixense Roxb. ethanolic extract (1 to 1000 µg/mL), Riboflavin was diluted to 1–50 µg/mL as a standard. The mixtures were then added to pre-treated samples, mixed thoroughly, and incubated under the same conditions. Untreated wells served as negative controls. Absorbance was measured at 560 nm with an ELISA microplate reader (iMark, BioRad, USA). The IC₅₀ was calculated using GraphPad Prism version 6 software [ 17 ]. %Inhibition = Abs.(control) − Abs. (Sample) /Abs. (control) × 100 5.5 Nitric Oxide Scavenging Assay The assay involved preparing a reaction mixture with 50 µL of 10 mM sodium nitroprusside (Fisher Scientific), 40 µL of distilled water, and 10 µL of gallic acid (SRL). Wells with the reaction mixture but without gallic acid acted as controls. The mixtures were pre-incubated under light at room temperature for 15 minutes to promote nitric oxide production. Then, 100 µL of Griess reagent was added to each well, including controls. The plate was incubated for an additional 5–10 minutes at room temperature to allow the chromophore to develop and stabilize. Absorbance was recorded at 540 nm and 660 nm using a microplate reader (iMark, BioRad, USA). The IC₅₀ was determined using GraphPad Prism version 6 software [ 18 ]. %Inhibition = (Abs.control − Abs.sample) / Abs.control × 100 5.6 CUPRAC Assay In this assay, 10 µL of the sample at various concentrations was added to specific wells of a 96-well plate, followed by the introduction of 200 µL of the reagent mixture. Reactions were performed in triplicate, with duplicate blanks containing either Methanol and the sample or the Trolox standard, all of which were incubated in the dark for 30 minutes. Control wells remained untreated. After incubation, absorbance was measured at 490 nm using an iMark microplate reader (Bio-Rad, USA), and IC₅₀ values were calculated using GraphPad Prism 6 [ 19 ] %Inhibition = Abs.(control) − Abs. (Sample) /Abs. (control) × 100 6. Results and Discussion The phytochemical screening of the bark extract of H. orixense Roxb. showed the presence of various active compounds. The presence of alkaloids was revealed through positive Wager’s and Mayer’s reactions. The flavonoids gave positive reactions with the Shinoda and lead acetate tests. The presence of tannic acid in the sample was detected using various reagents, including ferric chloride, lead acetate, acetic acid, and dilute iodine. On the other hand, the glycosides (Keller-Killian test), steroids (Salkowski test), carbohydrates (Molisch’s test), and proteins (Biuret test) resulted in negative reactions; therefore, these components are not present in the sample. The total phenolic content (TPC) in the crude extract was found to be 249.3 ± 1.25 µg GAE/mg, where GAE stands for gallic acid equivalents per milligram of extract. Total flavonoid content (TFC) came out to be 81.29 ± 0.56 µg RT/mg of extract in value, where RT stands for rutin equivalents per mg of extract. The tannin content (TTC) was found to be 47.85 ± 0.44 µg TA/mg of extract, where TA represents tannic acid equivalents per milligram. The total alkaloid content was found to be 0.4% w/w of the total extract yield. Ethanolic extract of H. orixense Roxb. (5 mg/ml) was subjected to phytochemical screening, which showed abundant polyphenolic compounds. Thereafter, HPTLC was performed. Scopoletin (0.1 mg/mL in Methanol) was used as the standard. Chromatographic fingerprinting was carried out on the chromatographic matrix using a mobile phase mixture of ethyl acetate, formic acid, and distilled water (6.7:0.5:2.6 v/v/v). Densitometric scanning was performed at wavelengths of 254 nm and 366 nm. Scopoletin yielded characteristic chromatograms from densitometric scanning at wavelengths of 254 nm and 366 nm. The compound had an Rf value of 0.802, which was identical to that of the standard. Quantitative estimation revealed that the extract contained quantifiable amounts of scopoletin, 535.9 µg/5 mg. 6.2 Antimicrobial Activity The agar disc diffusion assay was used to evaluate the antibacterial properties of the H. orixense Roxb. plant extract against S. aureus and E. coli. The discs prepared with the plant extract (A) created visible inhibition zones. The positive control, chloramphenicol (Ab), actively inhibited the growth. The distilled water (D) and the discs labelled B had no inhibition zones, thus making the negative controls devoid of antimicrobial properties. The results from the antimicrobial study shown in Fig. 2 indicated an average inhibition zone of 21 mm for E. coli, similar to that of chloramphenicol, with an inhibition zone of 21.67 mm. For S. aureus, the average inhibition zone formed was 22.66 mm, which is close to the inhibition zone of the positive control, at 23.33 mm. The results of the blank and solvent controls ensured that the antimicrobial activity originated entirely from the bioactive agents in the H. orixense Roxb. extracts, as no activity was found in the blanks and solvents, indicating that the components of the extracts were responsible for their activities. In Vitro Antioxidant Assay The ethanolic extract of the H. orixense Roxb. exhibited diverse levels of antioxidant activity in various in vitro methods, as shown in Fig. 1 . The importance depicted in the table focuses on the fact that ABTS and CUPRAC exhibited the highest level of antioxidant activity, with low IC50 values of 3.656 and 5.490 µg/ml, respectively, indicating a higher free radical scavenging potential. While DPPH at 45.690 µg/ml and superoxide at 13.700 µg/ml exhibited good levels of efficiency, Hydroxyl at 143.890 µg/ml and Nitric oxide at 207.200 µg/ml showed significantly lower levels of antioxidant activity. It can also be perceived that the sensitivity levels of the various assays vary, and the most sensitive assay among them is the ABTS. This reflects the fact that the extract possesses a higher level of electron-donating capacity and free radical scavenging capacity. Discussion The current research study investigated the phytochemical diversity and biological potential of the ethanolic bark extract of H.orixense Roxb. The Phytochemical screening revealed the presence of both primary and secondary metabolites, with notable abundance of Phenolics, Flavonoids, tannins and Alkaloids. In contrast, carbohydrates, proteins, glycosides, and steroids were absent in the ethanolic extract. Quantitative analysis showed a significant content of total phenolics, flavonoids and tannin. Phenolic compounds are widely recognised for their antioxidant potential as they effectively scavenge free radicals and inhibit oxidative stress through an electron or hydrogen donation mechanism. Flavonoids also contribute to cellular protection by scavenging reactive oxygen species and reducing free radical formation. The HPTLC fingerprinting and analysis of scopoletin were valuable in providing the chemical constituents of the extract. The presence of scopoletin, a coumarin derivative, was established through successful analysis. The established HPTLC analysis method was efficient and accurate and can be applied for quality control analysis. The high concentration of scopoletin was directly responsible for the antioxidant and antimicrobial properties established in this research, since scopoletin has been shown to have significant free radical scavenging and antimicrobial properties. The ethanolic bark extract exhibited remarkable antimicrobial activity against E.coli and S.aureus , with inhibition comparable to the standard antibiotic chloramphenicol. The observed broad-spectrum efficacy of both Gram-positive and Gram-negative bacteria suggests the presence of bioactive constituents capable of disrupting bacterial cell walls or interfering with nucleic acid synthesis, causing the antibacterial activity. The in vitro antioxidant assays demonstrated that the extract possesses a strong capacity to scavenge free radicals, with variation observed among different assays. The Marked activity in the ABTS and CUPRAC assay reflects a high electron-donating and reducing potential, whereas the moderate scavenging observed in the DPPH and superoxide assays further substantiates the antioxidant efficacy of the extract. The comparatively lower activity against hydroxyl and nitric oxide radicals may be attributed to differences in assay sensitivity and specific reactivity of the phytoconstituents toward these radical species. Overall, the findings of this study establish H. orixense Roxb. bark as a rich source of biologically active compounds with significant antioxidant and antimicrobial properties. The presence of Scopoletin, along with abundant polyphenolic constituents, provides a scientific basis for its traditional medical use and highlights its potential application in the development of natural therapeutic agents. Conclusion The bark extract of H. orixense Roxb. has been comprehensively validated as a rich source of bioactive secondary metabolites, notably phenolics, flavonoids, tannins and alkaloids, which collectively have potent antioxidant and antimicrobial properties. The strong free radical scavenging activity is shown in ABTS and CUPRAC assays. It also showed significant antimicrobial inhibitory effects against S. aureus and E. coli in agar disc diffusion assay. HPTLC fingerprinting confirmed and quantified Scopoletin, ensuring the standardisation and quality control. Collectively, these results validate the traditional use of H. orixense bark and identify it as a promising source for phytopharmaceutical development. Further work should involve bioassay-guided fractionation, in vivo efficacy and safety studies, and formulation optimisation. Abbreviations DPPH: 2,2-diphenyl-1-picrylhydrazyl ABTS: 2, 2′-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid IC 50 : 50% Inhibitory concentration TPC : Total polyphenol content TFC : Total flavonoid content GAE: Gallic acid equivalent RE: Rutin equivalent TA : Tannic acid equivalent HPTLC : High Performance Thin Layer Chromatography FCR : Folin-Ciocalteu Reagent Rf: Retention factor RT: Room temperature Declarations Conflict of Interest No Author Contribution P.I. and S.P. conceptualised and designed the study. N.C., S.M., A.G., and S.P. carried out the experimental work and data collection. M.M. performed data analysis and interpretation. A.B. and P.I. drafted the original manuscript. S.P. and S.M. critically reviewed and revised the manuscript for important intellectual content. All authors read and approved the final manuscript. Acknowledgement The author expresses sincere gratitude to the Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, for providing the necessary facilities and support throughout the study. Special thanks are extended to my research guide, co-guide, and colleagues for their invaluable guidance and assistance during the research process. The author also gratefully acknowledges the Chhatrapati Shahu Maharaj National Research Fellowship–2022 (CSMNRF–2022) for providing financial support during this research study. References Chakraborty P, Sasi S, Nair AA, Anjum N, Tripathi YC. Medicinal applications, phytochemistry, and pharmacology of Hymenodictyon excelsum (Roxb.) Wall: a review. Org. Med. Chem. Int. J. 2017;2(3). Rahman MM. Evaluation of the therapeutic value of Hymenodictyon excelsum phytochemicals against prostate cancer using molecular docking studies. Jundishapur Journal of Natural Pharmaceutical Products. 2015;10(1):e18216. Soun-Udom M, Choowongkomon K, Vajrodaya S, Ratanabunyong S, Suksungworn R, Srisombat N, Bapia S, Duangsrisai S. Antioxidant and anti-HIV properties and isolation of bioactive compound of Hymenodictyon orixense (Roxb.) Mabb. Agriculture and Natural Resources. 2019;53(6):621–8. Reddy M, Chaturvedi AA. Pharmacognostical studies of Hymenodictyon orixence (Roxb.) Mabb. Leaf. International journal of Ayurveda research. 2010;1(2):103. Khandelwal, K R. (2007). Practical Pharmacognosy, Techniques and Experiments. 17th Edition, Nirali Prakashan, Pune, India. Badnale A, Itankar P, Prasad S, Chalak N, Dhaswadikar S, Meshram S, Ghagare A, Nikam Y. Phytochemical Profile, in vitro Antioxidant Potential and HPTLC Fingerprinting of Acalypha indica Linn. Leaves. Barbouchi M, Benzidia B, Elamrani K, Sabiri M, El Idrissi M. Phytochemical screening, quantitative analysis and antioxidant properties of crude extracts from stems, leaves, and flowers of three Ruta species. Kuwait Journal of Science. 2024;51(4):100287. Ordonez AA, Gomez JD, Vattuone MA. Antioxidant activities of Sechium edule (Jacq.) Swartz extracts. Food chemistry. 2006;97(3):452–8. Hagerman, A.E., Riedl, K.M., Jones, G.A., Sovik, K.N., Ritchard, N.T., Hartzfeld, P.W., High molecular weight plant polyphenolics (tannins) as biological antioxidants.Journal of Agricultural and Food Chemistry (1998)46, 1887–1892. 23. Ghagare A, Itankar P, Prasad S, Meshram S, Badnale A. High-performance Thin Layer Chromatographic quantification of hesperidin and naringenin from Citrus sinensis peel extract. Swe NN. Scopoletin from Hymenodictyon orixense (Roxb.) Mabb. 2008 Mar 1. Schuetz AN, Ferrell A, Hindler JA, Humphries R, Bobenchik AM. Overview of changes in the Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing: M100 32nd and 33rd editions. Journal of Clinical Microbiology. 2025;63(9):e01623-23. Kumar R, Shikha D, Sinha SK. DPPH radical scavenging assay: A tool for evaluating antioxidant activity in 3% cobalt–doped hydroxyapatite for orthopaedic implants. Ceramics International. 2024;50(8):13967–73. Meshram S, Itankar P, Prasad S, Ghagare A, Badnale A. Investigating the antioxidant and antiproliferative activity of Mimusops elengi Linn Extract against Hepatocellular Carcinoma. Imam MZ, Akter S, Mazumder ME, Rana MS. Antioxidant activities of different parts of Musa sapientum L. ssp. Sylvestris fruit. Journal of Applied Pharmaceutical Science. 2011 Dec 30(Issue):68–72. 27. Hazra B, Biswas S, Mandal N. Antioxidant and free radical scavenging activity of Spondias pinnata. BMC Complementary and Alternative Medicine. 2008; 8:1–0. Noda Y, Anzai K, Mori A, Kohno M, Shinmei M, Packer L. Hydroxyl and superoxide anion radical scavenging activities of natural source antioxidants using the computerized JES-FR30 ESR spectrometer system. Iubmb Life. 1997;42(1):35–44. Rao SB, Jayanthi M, Yogeetha R, Ramakrishnaiah H, Nataraj J. Free radical scavenging activity and reducing power of Gnidia glauca (Fresen.) Gilg. Journal of Applied Pharmaceutical Science. 2013;3(6):203–7 Apak R, Güçlü K, Özyürek M, Karademir SE. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. Journal of agricultural and food chemistry. 2004;52(26):7970–81. Additional Declarations No competing interests reported. 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-8549856","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578198894,"identity":"4f391da5-16b2-4457-96b4-705d39fe7ee2","order_by":0,"name":"Anil Badnale","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYFACxsbDDAzMDAzsDIwPgFwePiK0NEC0MDMwG4C0sBFjD0wLmwSIR1AL/+zmhsMFNdZ5/M3Mxyq/5tjJsDEwP3x0A48WiTsHGw7POJZeLHGYLe227LZkoMPYjI1z8FlzI7HhMA/bYRBpdltyGzNQCw+bND4t8mAt/w4nzj/M/61Ycls9YS0GIC28bYcTNwDtYvy47TBhLYYgLTP70hM3HmYzlmbcdpyHjZmAX+RupD98XPDNOnHe8eaHH39uq7bnZ29++Biv95EBMw+YJFY5CDD+IEX1KBgFo2AUjBgAAIrMSoOE/bX4AAAAAElFTkSuQmCC","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":true,"prefix":"","firstName":"Anil","middleName":"","lastName":"Badnale","suffix":""},{"id":578198895,"identity":"6728e407-583b-4b0b-b261-7a075517d4d5","order_by":1,"name":"Satish Meshram","email":"","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":false,"prefix":"","firstName":"Satish","middleName":"","lastName":"Meshram","suffix":""},{"id":578198898,"identity":"6647960c-84e2-4448-8b87-fbda1356c898","order_by":2,"name":"Nandaji Chalak","email":"","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":false,"prefix":"","firstName":"Nandaji","middleName":"","lastName":"Chalak","suffix":""},{"id":578198903,"identity":"d1b3f27c-e439-468b-a634-2e15e032b67a","order_by":3,"name":"Ashwini Ghagare","email":"","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":false,"prefix":"","firstName":"Ashwini","middleName":"","lastName":"Ghagare","suffix":""},{"id":578198910,"identity":"37761426-0873-415a-a532-c54fad6802e9","order_by":4,"name":"Snehal Patil","email":"","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":false,"prefix":"","firstName":"Snehal","middleName":"","lastName":"Patil","suffix":""},{"id":578198913,"identity":"04919804-ee6e-420b-86e8-8c90a2b58ed3","order_by":5,"name":"Mandar Muley","email":"","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":false,"prefix":"","firstName":"Mandar","middleName":"","lastName":"Muley","suffix":""},{"id":578198916,"identity":"762c75da-d226-4aad-a661-659e8c507f82","order_by":6,"name":"Satyendra Prasad","email":"","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":false,"prefix":"","firstName":"Satyendra","middleName":"","lastName":"Prasad","suffix":""},{"id":578198918,"identity":"0f08ab27-9074-46af-9fef-813d8122bc6e","order_by":7,"name":"Prakash Itankar","email":"","orcid":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University","correspondingAuthor":false,"prefix":"","firstName":"Prakash","middleName":"","lastName":"Itankar","suffix":""}],"badges":[],"createdAt":"2026-01-08 09:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8549856/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8549856/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100951996,"identity":"ec808cfb-2753-4486-b24a-b441e54703ad","added_by":"auto","created_at":"2026-01-23 07:11:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":951786,"visible":true,"origin":"","legend":"","description":"","filename":"HymenodictyonorixenseResearchpaper.docx","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/426c70a23d7f725f20aa4cc3.docx"},{"id":100943421,"identity":"a7d7c82f-735d-4fe4-bcc1-abfd113469b1","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9141,"visible":true,"origin":"","legend":"","description":"","filename":"fe86f03b89104834ad4f36966db45651.json","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/baeaa17d7b6a17e7c26d7a81.json"},{"id":100951980,"identity":"1038cfd0-fa4e-44b9-9009-dc162af2823c","added_by":"auto","created_at":"2026-01-23 07:11:38","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":62101,"visible":true,"origin":"","legend":"","description":"","filename":"fe86f03b89104834ad4f36966db456511enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/9b34ddb5c2f224791a3b0d86.xml"},{"id":100943429,"identity":"4a723482-148e-4a5c-8a2c-788f566eea67","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":376239,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/a8a5c50be77987f8eab0cfad.png"},{"id":100951994,"identity":"6355504b-6f15-4f6e-8a8f-797a0c7bb443","added_by":"auto","created_at":"2026-01-23 07:11:39","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41064,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/4fdaafbecb3ef9a868eeba1f.png"},{"id":100943430,"identity":"13d4768d-6106-41c1-8945-7c28f980d827","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":210190,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/5e22093814d60e4c9d42af63.png"},{"id":100951983,"identity":"a68a07ac-8610-447e-96e1-1232081ac989","added_by":"auto","created_at":"2026-01-23 07:11:38","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":261254,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/64866bf287f213758091c57a.png"},{"id":100952139,"identity":"54650662-9158-4aa4-8664-141ede779416","added_by":"auto","created_at":"2026-01-23 07:12:01","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23354,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/40be7dfd97bc9d810dd71b49.png"},{"id":100952019,"identity":"3ba829a8-9c93-49ec-a06d-509c64f1bab9","added_by":"auto","created_at":"2026-01-23 07:11:42","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41842,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/c2893c7a6e97f6754f9e3001.png"},{"id":100943437,"identity":"74f3ab5e-0786-47e5-affb-2be51cdc2aa8","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10805,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/ac8538ed39ff4c37f5bef710.png"},{"id":100943435,"identity":"a1020132-e220-424a-9268-5e898bc27faa","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29038,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/e026d65c49c88f9a985080cd.png"},{"id":100950867,"identity":"d389b139-869e-41a6-876e-411958206252","added_by":"auto","created_at":"2026-01-23 07:09:24","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":37332,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/7301c2da6e4cab551755e845.png"},{"id":100952303,"identity":"4b23dfc7-2657-4b18-8a62-c2096012e0dd","added_by":"auto","created_at":"2026-01-23 07:12:38","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5764,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/97f2b5d0265001e491c8d938.png"},{"id":100943438,"identity":"36da6d9d-eb2e-4f63-ab0c-1cd74fc56687","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60874,"visible":true,"origin":"","legend":"","description":"","filename":"fe86f03b89104834ad4f36966db456511structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/1275b4c6803bd98ec43ea105.xml"},{"id":100943439,"identity":"7fa18c77-1bf7-4b37-acbe-25aa2e17e1f2","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69353,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/156bd3b767aadab4a0337e31.html"},{"id":100952077,"identity":"0ef89828-8104-4ae6-8a7b-07b1b5f9c61c","added_by":"auto","created_at":"2026-01-23 07:11:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":532527,"visible":true,"origin":"","legend":"\u003cp\u003e(A) \u0026nbsp;HPTLC scanned plate at 254 nm and (B) HPTLC scanned plate at 366 nm\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/b8e3a1b44d3b9a2766b01496.png"},{"id":100943423,"identity":"80e769ce-cd3b-44e0-990a-b77147eb3f87","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110712,"visible":true,"origin":"","legend":"\u003cp\u003e(A): HPTLC chromatograms of scopoletin and (B): ): HPTLC chromatograms of H.orixense\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/7673f05040350d56cd4bbe08.png"},{"id":100943427,"identity":"fc7a644d-07e6-4871-a931-456170438490","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":303489,"visible":true,"origin":"","legend":"\u003cp\u003e(A): 3D Densitometric Surface Plot, (B): Calibration Curve of Scopoletin and \u0026nbsp;(C)UV-Visible Spectral Overlay\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/ba4b98a3f1f3fcf3aed82084.png"},{"id":101202555,"identity":"5d294e0e-d57e-4a20-b267-919607b3cc45","added_by":"auto","created_at":"2026-01-27 09:36:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":664997,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition observed in disc diffusion assay\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/1d35cd91f1cdda0cf4dd7e75.png"},{"id":100943426,"identity":"e777475b-e877-4e26-823f-86ea9db2920e","added_by":"auto","created_at":"2026-01-23 05:38:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":73786,"visible":true,"origin":"","legend":"\u003cp\u003eComparative Antioxidant Activity of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. Bark Extract\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/3ac01dd7dd7a8f829088691e.png"},{"id":103241621,"identity":"48d8c4ff-b0f8-44ff-821c-842f689532c9","added_by":"auto","created_at":"2026-02-23 14:12:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2674660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8549856/v1/8fb51230-e46c-4b2a-b19a-3c6a58f42e44.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"HPTLC-Based Quantitative Analysis of Scopoletin and In vitro Antioxidant and Antimicrobial Evaluation of Hymenodictyon orixense Roxb. Bark","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eH excelsum\u003c/em\u003e Roxb.Wall. ex-Mabb., also referred to as \u003cem\u003eH. excelsum\u003c/em\u003e, is a deciduous tree that grows in the Rubiaceae family. It is known by local names such as Dendra Bandaru and Kala-bachnag in various areas of India and Thailand, where it is also referred to as U Lok and Som Kop, respectively. \u003cem\u003eH. excelsum\u003c/em\u003e Roxb. grows in subtropical to tropical climates. It grows with a rounded top and a straight, cylindrical trunk that measures up to 10\u0026ndash;12 meters in height. \u003cem\u003eH. excelsum\u003c/em\u003e Roxb. has garnered attention in the fields of ethnomedicine and phytochemical studies due to its wide range of medicinal uses. The leaves, stems, roots, and flowers of \u003cem\u003eH. excelum\u003c/em\u003e have been used by traditional healers for the treatment of inflammation, wounds, infections, and chronic diseases such as arthritis and peptic ulcers. Well known in traditional medicinal systems of native societies for a considerable number of years, this species contains variable bioactive compounds, which may lead to beneficial uses in treating many ailments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn traditional medicinal use, the bark extracts of this species serve as astringents, febrifuges, and antimyesthetics. Its wood is most effectively used in treating herpes. Its leaves have been used in the treatment of chickenpox, excessive sweating due to fever, toothaches, throat inflammation, mouth ulcers, prostate cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and tonsillitis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Its antibacterial, anticoagulant, anti-inflammatory, and protective properties against UV radiation, known as sun-screening properties, have been well identified [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Its roots contain bioactive compounds like rubiadin, rubiadin methyl ether, lucidin, nordamnacanthal, damnacanthal, and 2-benzyl. The stem bark exudes hymexelsin, which is an apiglucoside compound derived from scopoletin. The plant has been shown to contain other chemical constituents, including the iridoid glycoside loganin and four coumarins: scopoletin, scopolin, hymexelsin, and scopoletin 7-O-β-D-xylopyranosyl-(1\u0026rarr;6)-β-D-glucopyranoside. The plant extracts have been used in traditional medicine\u003c/p\u003e \u003cp\u003eThe present investigation is to carry out a comprehensive phytochemical profiling of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. with quantitative estimation of Major secondary metabolites. In addition to evaluating the antioxidant efficacy against selected free radical scavenging assays and antimicrobial activity against selected Gram-positive and Gram-negative bacterial strains, thereby validating its ethnomedicine relevance. HPTLC will be used for quantification and fingerprints, ensuring standardization of the extract. The aim is to bridge traditional knowledge with modern analytical validation, providing a scientific basis for its therapeutic application and potential.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant collection and authentication\u003c/h2\u003e \u003cp\u003eThe bark of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. was collected from a local field in Chatgaon District, Gadchiroli, Maharashtra. The plant sample was scientifically identified and authenticated by Prof. Nitin Dongarwar from the Department of Botany at Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur. A voucher specimen was created and stored in the Herbarium at Nagpur University, designated as sheet number 10071.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eThe reagents used in the study included 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid (GA), Cupric chloride (CuCl₂), ABTS, and trichloroacetic acid, all of which were purchased from SRL Chem Laboratories. Hydrogen peroxide (H₂O₂) was obtained from Neurochem Laboratories. Trichloroacetic acid (TCA), ferric chloride, and sodium nitroprusside were sourced from Fisher Scientific. Additionally, Ascorbic acid (AA), DMSO, EDTA, and trio-barbituric acid (TBA) were purchased from HiMedia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Extraction of Plant Material\u003c/h2\u003e \u003cp\u003eThe plant bark was collected, decorticated, and cleaned of foreign particles. It was then air-dried in the shade to preserve its phytoconstituents and ground into a coarse powder. Once dried, the powder was stored in an amber glass container for further extraction. A hundred grams of this powdered bark was extracted with absolute ethanol using a Soxhlet apparatus. After extraction, the ethanolic solution was filtered, and the solvent was concentrated using a rotary evaporator.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Qualitative and Quantitative Assessment of Secondary Metabolites","content":"\u003cp\u003eThe ethanolic bark extract of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. was analysed using standard qualitative phytochemical tests to identify its secondary metabolites. The results showed the presence of alkaloids (indicated by a creamy precipitate in Mayer\u0026rsquo;s test), glycosides (red color in Borntrager test), flavonoids (red to pink in Shinoda test), phenolic compounds (white precipitate in Gelatin test), steroids (red in Salkowski\u0026rsquo;s test), tannins (reddish-yellow with nitric acid), fixed oils and fats (translucent stain in spot test), and saponins (formed foam in test) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Total Phenolic Content\u003c/h2\u003e \u003cp\u003eThe total phenolic content (TPC) was measured using the Folin-Ciocalteu (FC) reagent, with gallic acid as the reference standard. A calibration curve was prepared with gallic acid (10\u0026ndash;50\u0026micro;g/mL). A stock solution of the extract (1 mg/mL) was made in Methanol and diluted for testing. Then, 1 mL of this solution was mixed with 0.5 mL of 2N FC reagent and 1.5 mL of 20% sodium carbonate. After 2 hours of dark incubation, absorbance was read at 765 nm using a Shimadzu UV-Visible spectrophotometer. TPC was expressed as mg of gallic acid equivalents per gram of extract, with all tests performed in triplicate [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e],[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Total Flavonoid Content\u003c/h2\u003e \u003cp\u003eTotal flavonoids (TFC) were determined by the aluminium chloride colorimetric method. A 10 mg sample was dissolved in Methanol, then 2 mL of this solution was mixed with 20% methanolic aluminium chloride and acetic acid in a volumetric flask. After 40 minutes of incubation, absorbance was measured at 415 nm using a Shimadzu UV spectrophotometer. Rutin (0.5 mg/mL) served as the standard. TFC was expressed as mg rutin equivalents per gram of extract, with triplicate measurements [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Total Tannin Content\u003c/h2\u003e \u003cp\u003eThe total tannin content (TTC) was estimated using the FC reagent. A stock solution of ethanolic extract (1 mg/mL) was prepared and diluted to different concentrations (100, 200, 300, 400, 500 ppm). To each, 1 mL was mixed with 0.5 mL of FC reagent, followed by the addition of 1.5 mL of 20% sodium carbonate solution. Absorbance was measured at 775 nm after 15 minutes of incubation. Tannins were expressed in mg tannic acid equivalents per 100 g of extract (mg TAE/100g). All experiments were done in triplicate [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Total Alkaloid Content\u003c/h2\u003e \u003cp\u003eTotal alkaloids were determined gravimetrically. 5 g of powdered crude drug was extracted ultrasonically in three 50 mL portions with 0.1 M sulfuric acid. The filtered solution and washings were performed four times, discarding the supernatant each time, using 25 mL of dilute chloroform. Alkaloids were then extracted by making the solution basic with dilute ammonia. The basic solution was extracted with diethyl ether, filtered, dried, and weighed. Results were expressed as % w/w of the crude drug [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.5 High Performance Thin Layer Chromatography\u003c/h2\u003e \u003cp\u003eA stock solution of scopoletin at a concentration of 0.1 mg/mL was prepared using HPTLC-grade Methanol. An ethanolic extract of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. at a concentration of 5 mg/mL was also prepared by dissolving in MeOH and filtering through a whatman No. 01 filter. The above-mentioned solutions were spotted uniformly on silica gel 60 F₂₅₄ thin-layer chromatography plates, which measured 200 \u0026times; 100 mm. The scopoletin solution, at concentrations ranging from 2 to 16 \u0026micro;L, and the ethanolic extract, at concentrations ranging from 1 to 7 \u0026micro;L, were spotted using the CAMAG Linomat 5 applicator (vision CATS software) at a program rate of 150 nL/s, with every spot programmed to be 8.0 mm in length [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A mobile phase consisting of ethyl acetate, formic acid, and dH\u003csub\u003e2\u003c/sub\u003e0. in the ratio of 6.7:0.5:2.6 V/V/V was used [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A 20 x 10 cm TLC plate was placed in a pre-saturated twin-tract glass chamber, along with the chosen mobile phase, for 20 minutes at room temperature. The development was done up to 70 mm solvent front using an automatic chamber. After that, the TLC plate was dried in air at room temperature for 10 minutes. The Rf value and spots of the separation were recorded. The dried plate was viewed under UV light at both 254 nm and 366 nm in a UV cabinet. All of the visible bands were recorded. Spectral scans were performed using a CAMAG TLC Scanner 4, equipped with deuterium and tungsten lamps that scanned wavelengths from 190 to 450 nm at a speed of 20 nm/s. Scanning was done track by track, and the Rf value of both substances was determined [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"4. In Vitro Investigation of Antibacterial Potential Against S. aureus and E. coli","content":"\u003cp\u003eThe antimicrobial activity of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. bark extract was tested using the Disk diffusion method against S. aureus (ATCC 23235) and E. coli (ATCC 25922). Mueller-Hinton Agar (MHA) was prepared and sterilized by autoclaving at 121\u0026deg;C and 15 psi for 10 minutes. Bacterial suspensions were adjusted to a 0.5 McFarland standard (approximately 1.5 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL). Wells with a 6 mm diameter were punched into the agar, and 10 \u0026micro;L of each sample\u0026mdash;test samples, Chloramphenicol as a positive control, and Blank and distilled water as negative controls\u0026mdash;were added. Plates were incubated at 35\u0026ndash;37\u0026deg;C for 20\u0026ndash;24 hours. Zones of inhibition were measured in triplicate, and average values were calculated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e"},{"header":"5. In Vitro Antioxidant Assays","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.1 DPPH scavenging assay\u003c/h2\u003e \u003cp\u003e5 \u0026micro;L of various concentrations of the test compound was added to 100 \u0026micro;L of a 0.1 mM DPPH solution in a 96-well plate. The reaction mixtures were prepared in triplicate for each concentration, with duplicate blanks created by adding 200 \u0026micro;L of DMSO/Methanol and 5 \u0026micro;L of the corresponding compound concentrations. The plate was incubated in the dark at room temperature for 30 minutes. Absorbance was measured at 517 nm using a microplate reader (iMark, BioRad). Methanol served as the blank. A control reaction mixture containing 20 \u0026micro;L of deionized water was included. The radical scavenging activity (% inhibition) was calculated relative to the control, and IC\u003csub\u003e50\u003c/sub\u003e values were determined using GraphPad Prism 6 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e%Inhibition\u0026thinsp;=\u0026thinsp;Abs.(control)\u0026thinsp;\u0026minus;\u0026thinsp;Abs. (Sample) /Abs. (control) \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.2 ABTS scavenging assay\u003c/h2\u003e \u003cp\u003eIn a 96-well plate, 10 \u0026micro;L of test samples or ascorbic acid (standard) at different concentrations were added to 200 \u0026micro;L of ABTS reagent. After incubating in the dark at room temperature for 10 minutes, the absorbance was measured at 750 nm using a microplate reader (iMark, Bio-Rad). Control wells without treatment served as a baseline. Antioxidant activity was expressed relative to the negative control, and IC₅₀ values were determined using GraphPad Prism 6 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e%Inhibition\u0026thinsp;=\u0026thinsp;Abs.(control)\u0026thinsp;\u0026minus;\u0026thinsp;Abs. (Sample) /Abs. (control) \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.3 Hydroxy Free Radical Scavenging Assay\u003c/h2\u003e \u003cp\u003eA reagent mixture was created by combining 10 \u0026micro;L of 0.5 M EDTA (HiMedia), 24.14 mg of Deoxyribose (SRL), 88 \u0026micro;L of FeCl₃ (10 mg/mL) and 28 \u0026micro;L of H₂O₂ (6%) (Neurochem Laboratories), then the volume was adjusted to 33 mL with water. Each well of a 96-well microplate received 10 \u0026micro;L of plant extract, 24 \u0026micro;L of phosphate buffer (50 mM, pH 7.4), and 10 \u0026micro;L of ascorbic acid. The plate was incubated at 37\u0026deg;C for 1 hour. Wells without treatment served as the negative control, with Gallic Acid (SRL) as the positive standard. After incubation, 50 \u0026micro;L of 10% TCA (Fisher Scientific) and 50 \u0026micro;L of 1% TBA (HiMedia) were added to each well. The formation of a pink chromogen indicated the degradation of deoxyribose, and the absorbance was read at 540 nm. IC\u003csub\u003e50\u003c/sub\u003e was calculated using GraphPad Prism 6 software [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e],[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Super Oxide Anion Radical Scavenging Assay\u003c/h2\u003e \u003cp\u003eSerial dilutions of the \u003cem\u003eH. Orixense\u003c/em\u003e Roxb. extract was prepared at concentrations from 1 to 1000 \u0026micro;g/mL. Riboflavin, used as the reference standard, was similarly diluted from 1 to 50 \u0026micro;g/mL. The reaction mixtures were incubated in a 96-well microplate under ambient illumination for 30 minutes. After preparing the serial dilutions of the \u003cem\u003eH. orixense\u003c/em\u003e Roxb. ethanolic extract (1 to 1000 \u0026micro;g/mL), Riboflavin was diluted to 1\u0026ndash;50 \u0026micro;g/mL as a standard. The mixtures were then added to pre-treated samples, mixed thoroughly, and incubated under the same conditions. Untreated wells served as negative controls. Absorbance was measured at 560 nm with an ELISA microplate reader (iMark, BioRad, USA). The IC₅₀ was calculated using GraphPad Prism version 6 software [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e%Inhibition\u0026thinsp;=\u0026thinsp;Abs.(control)\u0026thinsp;\u0026minus;\u0026thinsp;Abs. (Sample) /Abs. (control) \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5.5 Nitric Oxide Scavenging Assay\u003c/h2\u003e \u003cp\u003eThe assay involved preparing a reaction mixture with 50 \u0026micro;L of 10 mM sodium nitroprusside (Fisher Scientific), 40 \u0026micro;L of distilled water, and 10 \u0026micro;L of gallic acid (SRL). Wells with the reaction mixture but without gallic acid acted as controls. The mixtures were pre-incubated under light at room temperature for 15 minutes to promote nitric oxide production. Then, 100 \u0026micro;L of Griess reagent was added to each well, including controls. The plate was incubated for an additional 5\u0026ndash;10 minutes at room temperature to allow the chromophore to develop and stabilize. Absorbance was recorded at 540 nm and 660 nm using a microplate reader (iMark, BioRad, USA). The IC₅₀ was determined using GraphPad Prism version 6 software [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e%Inhibition = (Abs.control\u0026thinsp;\u0026minus;\u0026thinsp;Abs.sample) / Abs.control \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.6 CUPRAC Assay\u003c/h2\u003e \u003cp\u003eIn this assay, 10 \u0026micro;L of the sample at various concentrations was added to specific wells of a 96-well plate, followed by the introduction of 200 \u0026micro;L of the reagent mixture. Reactions were performed in triplicate, with duplicate blanks containing either Methanol and the sample or the Trolox standard, all of which were incubated in the dark for 30 minutes. Control wells remained untreated. After incubation, absorbance was measured at 490 nm using an iMark microplate reader (Bio-Rad, USA), and IC₅₀ values were calculated using GraphPad Prism 6 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e%Inhibition\u0026thinsp;=\u0026thinsp;Abs.(control)\u0026thinsp;\u0026minus;\u0026thinsp;Abs. (Sample) /Abs. (control) \u0026times; 100\u003c/p\u003e \u003c/div\u003e"},{"header":"6. Results and Discussion","content":"\u003cp\u003eThe phytochemical screening of the bark extract of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. showed the presence of various active compounds. The presence of alkaloids was revealed through positive Wager\u0026rsquo;s and Mayer\u0026rsquo;s reactions. The flavonoids gave positive reactions with the Shinoda and lead acetate tests. The presence of tannic acid in the sample was detected using various reagents, including ferric chloride, lead acetate, acetic acid, and dilute iodine. On the other hand, the glycosides (Keller-Killian test), steroids (Salkowski test), carbohydrates (Molisch\u0026rsquo;s test), and proteins (Biuret test) resulted in negative reactions; therefore, these components are not present in the sample. The total phenolic content (TPC) in the crude extract was found to be 249.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 \u0026micro;g GAE/mg, where GAE stands for gallic acid equivalents per milligram of extract. Total flavonoid content (TFC) came out to be 81.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 \u0026micro;g RT/mg of extract in value, where RT stands for rutin equivalents per mg of extract. The tannin content (TTC) was found to be 47.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 \u0026micro;g TA/mg of extract, where TA represents tannic acid equivalents per milligram. The total alkaloid content was found to be 0.4% w/w of the total extract yield.\u003c/p\u003e \u003cp\u003eEthanolic extract of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. (5 mg/ml) was subjected to phytochemical screening, which showed abundant polyphenolic compounds. Thereafter, HPTLC was performed. Scopoletin (0.1 mg/mL in Methanol) was used as the standard. Chromatographic fingerprinting was carried out on the chromatographic matrix using a mobile phase mixture of ethyl acetate, formic acid, and distilled water (6.7:0.5:2.6 v/v/v). Densitometric scanning was performed at wavelengths of 254 nm and 366 nm. Scopoletin yielded characteristic chromatograms from densitometric scanning at wavelengths of 254 nm and 366 nm. The compound had an Rf value of 0.802, which was identical to that of the standard. Quantitative estimation revealed that the extract contained quantifiable amounts of scopoletin, 535.9 \u0026micro;g/5 mg.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e6.2 Antimicrobial Activity\u003c/h2\u003e \u003cp\u003eThe agar disc diffusion assay was used to evaluate the antibacterial properties of the \u003cem\u003eH. orixense\u003c/em\u003e Roxb. plant extract against S. aureus and E. coli. The discs prepared with the plant extract (A) created visible inhibition zones. The positive control, chloramphenicol (Ab), actively inhibited the growth. The distilled water (D) and the discs labelled B had no inhibition zones, thus making the negative controls devoid of antimicrobial properties. The results from the antimicrobial study shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicated an average inhibition zone of 21 mm for E. coli, similar to that of chloramphenicol, with an inhibition zone of 21.67 mm. For S. aureus, the average inhibition zone formed was 22.66 mm, which is close to the inhibition zone of the positive control, at 23.33 mm. The results of the blank and solvent controls ensured that the antimicrobial activity originated entirely from the bioactive agents in the \u003cem\u003eH. orixense\u003c/em\u003e Roxb. extracts, as no activity was found in the blanks and solvents, indicating that the components of the extracts were responsible for their activities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro Antioxidant Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe ethanolic extract of the \u003cem\u003eH. orixense\u003c/em\u003e Roxb. exhibited diverse levels of antioxidant activity in various in vitro methods, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The importance depicted in the table focuses on the fact that ABTS and CUPRAC exhibited the highest level of antioxidant activity, with low IC50 values of 3.656 and 5.490 \u0026micro;g/ml, respectively, indicating a higher free radical scavenging potential. While DPPH at 45.690 \u0026micro;g/ml and superoxide at 13.700 \u0026micro;g/ml exhibited good levels of efficiency, Hydroxyl at 143.890 \u0026micro;g/ml and Nitric oxide at 207.200 \u0026micro;g/ml showed significantly lower levels of antioxidant activity. It can also be perceived that the sensitivity levels of the various assays vary, and the most sensitive assay among them is the ABTS. This reflects the fact that the extract possesses a higher level of electron-donating capacity and free radical scavenging capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current research study investigated the phytochemical diversity and biological potential of the ethanolic bark extract of \u003cem\u003eH.orixense\u003c/em\u003e Roxb. The Phytochemical screening revealed the presence of both primary and secondary metabolites, with notable abundance of Phenolics, Flavonoids, tannins and Alkaloids. In contrast, carbohydrates, proteins, glycosides, and steroids were absent in the ethanolic extract. Quantitative analysis showed a significant content of total phenolics, flavonoids and tannin. Phenolic compounds are widely recognised for their antioxidant potential as they effectively scavenge free radicals and inhibit oxidative stress through an electron or hydrogen donation mechanism. Flavonoids also contribute to cellular protection by scavenging reactive oxygen species and reducing free radical formation. The HPTLC fingerprinting and analysis of scopoletin were valuable in providing the chemical constituents of the extract. The presence of scopoletin, a coumarin derivative, was established through successful analysis. The established HPTLC analysis method was efficient and accurate and can be applied for quality control analysis. The high concentration of scopoletin was directly responsible for the antioxidant and antimicrobial properties established in this research, since scopoletin has been shown to have significant free radical scavenging and antimicrobial properties. The ethanolic bark extract exhibited remarkable antimicrobial activity against \u003cem\u003eE.coli\u003c/em\u003e and \u003cem\u003eS.aureus\u003c/em\u003e, with inhibition comparable to the standard antibiotic chloramphenicol. The observed broad-spectrum efficacy of both Gram-positive and Gram-negative bacteria suggests the presence of bioactive constituents capable of disrupting bacterial cell walls or interfering with nucleic acid synthesis, causing the antibacterial activity. The in vitro antioxidant assays demonstrated that the extract possesses a strong capacity to scavenge free radicals, with variation observed among different assays. The Marked activity in the ABTS and CUPRAC assay reflects a high electron-donating and reducing potential, whereas the moderate scavenging observed in the DPPH and superoxide assays further substantiates the antioxidant efficacy of the extract. The comparatively lower activity against hydroxyl and nitric oxide radicals may be attributed to differences in assay sensitivity and specific reactivity of the phytoconstituents toward these radical species. Overall, the findings of this study establish \u003cem\u003eH. orixense\u003c/em\u003e Roxb. bark as a rich source of biologically active compounds with significant antioxidant and antimicrobial properties. The presence of Scopoletin, along with abundant polyphenolic constituents, provides a scientific basis for its traditional medical use and highlights its potential application in the development of natural therapeutic agents.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe bark extract of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. has been comprehensively validated as a rich source of bioactive secondary metabolites, notably phenolics, flavonoids, tannins and alkaloids, which collectively have potent antioxidant and antimicrobial properties. The strong free radical scavenging activity is shown in ABTS and CUPRAC assays. It also showed significant antimicrobial inhibitory effects against S. aureus and E. coli in agar disc diffusion assay. HPTLC fingerprinting confirmed and quantified Scopoletin, ensuring the standardisation and quality control. Collectively, these results validate the traditional use of \u003cem\u003eH. orixense\u003c/em\u003e bark and identify it as a promising source for phytopharmaceutical development. Further work should involve bioassay-guided fractionation, in vivo efficacy and safety studies, and formulation optimisation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eDPPH:\u0026nbsp;\u003c/strong\u003e2,2-diphenyl-1-picrylhydrazyl\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eABTS:\u003c/strong\u003e2, 2\u0026prime;-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIC 50\u003c/strong\u003e: 50% Inhibitory concentration\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTPC\u003c/strong\u003e: Total polyphenol content\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTFC\u003c/strong\u003e: Total flavonoid content\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGAE:\u0026nbsp;\u003c/strong\u003eGallic acid equivalent\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRE:\u0026nbsp;\u003c/strong\u003eRutin equivalent\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTA\u003c/strong\u003e: Tannic acid equivalent\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPTLC\u003c/strong\u003e: \u0026nbsp; High Performance Thin Layer Chromatography\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFCR\u003c/strong\u003e: Folin-Ciocalteu Reagent\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRf:\u0026nbsp;\u003c/strong\u003eRetention factor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT:\u0026nbsp;\u003c/strong\u003eRoom temperature\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eNo\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.I. and S.P. conceptualised and designed the study. N.C., S.M., A.G., and S.P. carried out the experimental work and data collection. M.M. performed data analysis and interpretation. A.B. and P.I. drafted the original manuscript. S.P. and S.M. critically reviewed and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe author expresses sincere gratitude to the Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, for providing the necessary facilities and support throughout the study. Special thanks are extended to my research guide, co-guide, and colleagues for their invaluable guidance and assistance during the research process. The author also gratefully acknowledges the Chhatrapati Shahu Maharaj National Research Fellowship\u0026ndash;2022 (CSMNRF\u0026ndash;2022) for providing financial support during this research study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChakraborty P, Sasi S, Nair AA, Anjum N, Tripathi YC. Medicinal applications, phytochemistry, and pharmacology of Hymenodictyon excelsum (Roxb.) Wall: a review. Org. Med. Chem. Int. J. 2017;2(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman MM. Evaluation of the therapeutic value of Hymenodictyon excelsum phytochemicals against prostate cancer using molecular docking studies. Jundishapur Journal of Natural Pharmaceutical Products. 2015;10(1):e18216.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoun-Udom M, Choowongkomon K, Vajrodaya S, Ratanabunyong S, Suksungworn R, Srisombat N, Bapia S, Duangsrisai S. Antioxidant and anti-HIV properties and isolation of bioactive compound of Hymenodictyon orixense (Roxb.) Mabb. Agriculture and Natural Resources. 2019;53(6):621\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy M, Chaturvedi AA. Pharmacognostical studies of Hymenodictyon orixence (Roxb.) Mabb. Leaf. International journal of Ayurveda research. 2010;1(2):103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhandelwal, K R. (2007). Practical Pharmacognosy, Techniques and Experiments. 17th Edition, Nirali Prakashan, Pune, India.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadnale A, Itankar P, Prasad S, Chalak N, Dhaswadikar S, Meshram S, Ghagare A, Nikam Y. Phytochemical Profile, in vitro Antioxidant Potential and HPTLC Fingerprinting of Acalypha indica Linn. Leaves.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbouchi M, Benzidia B, Elamrani K, Sabiri M, El Idrissi M. Phytochemical screening, quantitative analysis and antioxidant properties of crude extracts from stems, leaves, and flowers of three Ruta species. Kuwait Journal of Science. 2024;51(4):100287.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrdonez AA, Gomez JD, Vattuone MA. Antioxidant activities of Sechium edule (Jacq.) Swartz extracts. Food chemistry. 2006;97(3):452\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHagerman, A.E., Riedl, K.M., Jones, G.A., Sovik, K.N., Ritchard, N.T., Hartzfeld, P.W., High molecular weight plant polyphenolics (tannins) as biological antioxidants.Journal of Agricultural and Food Chemistry (1998)46, 1887\u0026ndash;1892. 23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhagare A, Itankar P, Prasad S, Meshram S, Badnale A. High-performance Thin Layer Chromatographic quantification of hesperidin and naringenin from Citrus sinensis peel extract.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSwe NN. Scopoletin from Hymenodictyon orixense (Roxb.) Mabb. 2008 Mar 1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuetz AN, Ferrell A, Hindler JA, Humphries R, Bobenchik AM. Overview of changes in the Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing: M100 32nd and 33rd editions. Journal of Clinical Microbiology. 2025;63(9):e01623-23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar R, Shikha D, Sinha SK. DPPH radical scavenging assay: A tool for evaluating antioxidant activity in 3% cobalt\u0026ndash;doped hydroxyapatite for orthopaedic implants. Ceramics International. 2024;50(8):13967\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeshram S, Itankar P, Prasad S, Ghagare A, Badnale A. Investigating the antioxidant and antiproliferative activity of Mimusops elengi Linn Extract against Hepatocellular Carcinoma.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImam MZ, Akter S, Mazumder ME, Rana MS. Antioxidant activities of different parts of Musa sapientum L. ssp. Sylvestris fruit. Journal of Applied Pharmaceutical Science. 2011 Dec 30(Issue):68\u0026ndash;72. 27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHazra B, Biswas S, Mandal N. Antioxidant and free radical scavenging activity of Spondias pinnata. BMC Complementary and Alternative Medicine. 2008; 8:1\u0026ndash;0.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoda Y, Anzai K, Mori A, Kohno M, Shinmei M, Packer L. Hydroxyl and superoxide anion radical scavenging activities of natural source antioxidants using the computerized JES-FR30 ESR spectrometer system. Iubmb Life. 1997;42(1):35\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao SB, Jayanthi M, Yogeetha R, Ramakrishnaiah H, Nataraj J. Free radical scavenging activity and reducing power of Gnidia glauca (Fresen.) Gilg. Journal of Applied Pharmaceutical Science. 2013;3(6):203\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApak R, G\u0026uuml;\u0026ccedil;l\u0026uuml; K, \u0026Ouml;zy\u0026uuml;rek M, Karademir SE. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. Journal of agricultural and food chemistry. 2004;52(26):7970\u0026ndash;81.\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":"Hymenodictyon orixense Roxb., HPTLC, Scopoletin, Antioxidant assay, Antimicrobial","lastPublishedDoi":"10.21203/rs.3.rs-8549856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8549856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eHymenodictyon orixense\u003c/em\u003e Roxb. is a medically valuable evergreen tree that has been traditionally used worldwide to treat conditions such as inflammation, infection, wounds, and chronic diseases. This study was conducted to evaluate the phytochemical analysis, in vitro antioxidant properties, antimicrobial activity, and HPTLC characterization of the ethanol extracts from the bark of \u003cem\u003eH. orixense\u003c/em\u003e. Roxb. by using scopoletin as a standard. The bark of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. was collected and identified as a medicinally valuable evergreen tree. This was dried under shade and Soxhlet-extracted using ethanol as the solvent. The preliminary phytochemical study of the ethanol bark extracts of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. identified the presence of alkaloids, flavonoids, phenols, tannins, saponins, steroids, glycosides, and fixed oils. The total content of the antioxidant compounds, like total phenols (249.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u0026micro;g GAE/mg), total flavonoid content (81.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u0026micro;g RE/mg), total tannin content (47.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u0026micro;g TAE/mg), and total alkaloid content (0.4% w/w) of the ethanol bark extracts of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. was determined. This study evaluated the antioxidant activity of ethanol extracts of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. using various in vitro assays, including the DPPH, ABTS, CUPRAC, superoxide scavenging, hydroxyl scavenging, and nitric oxide scavenging assays. The ethanol extracts of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. possessed key antioxidant activity in all the in vitro antioxidant assays. The studies revealed high DPPH scavenging activity and the least in ABTS scavenging activity, indicating higher electron donation and stronger scavenging of free radicals. The ethanol extracts of \u003cem\u003eH. orixense\u003c/em\u003e Roxb. showed appreciable inhibition of S.aureus and E. coli with IC\u003csub\u003e50\u003c/sub\u003e comparable to the standard chloramphenicol in disc diffusion methods.\u003c/p\u003e","manuscriptTitle":"HPTLC-Based Quantitative Analysis of Scopoletin and In vitro Antioxidant and Antimicrobial Evaluation of Hymenodictyon orixense Roxb. Bark","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 05:37:58","doi":"10.21203/rs.3.rs-8549856/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":"60a4c6fa-b35f-44f7-96e3-b79ff2a39797","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T14:12:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 05:37:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8549856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8549856","identity":"rs-8549856","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}