{"paper_id":"0baa7732-ff26-4281-8d91-7cd8536ba2d3","body_text":"Phytochemical Profiling, Spectroscopic Characterization, and Antimicrobial Assessment of Piper guineense Seeds | 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 Phytochemical Profiling, Spectroscopic Characterization, and Antimicrobial Assessment of Piper guineense Seeds George Belema Tamunokuro, Bill Akponanabofa Ekolama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8611550/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Piper guineense is a perennial West African spice plant commonly known as Uziza, traditionally used for its medicinal and nutritional properties. This study investigates the phytochemical composition, spectroscopic characterization, and antimicrobial activity of Piper guineense seed extracts against selected clinical pathogens. Soxhlet extraction was performed using n -hexane, followed by phytochemical screening and compound quantification via Gas Chromatography–Flame Ionization Detection (GC-FID). Proximate analysis revealed a high carbohydrate content (54.92%), along with protein (10.5%), ash (14.65%), fiber (8.59%), fat (4.99%), and moisture (6.34%). Phytochemical profiling identified 19 secondary metabolites, with tannins (44.17 µg/mL) being the most abundant. The extract showed significant antimicrobial activity against Staphylococcus aureus , Salmonella enterica , Pseudomonas aeruginosa , Bacillus subtilis , Citrobacter murliniae , Bacillus licheniformis , and Micrococcus roseus , with mean inhibition zones ranging from 19.33 to 21.67 mm. Minimum inhibitory concentrations (MICs) ranged from 62.5 to 500 mg/mL. Spectroscopic structural elucidation using ¹H NMR, ¹³C NMR, Infrared (IR) spectroscopy, Mass Spectrometry (MS), and Elemental analysis confirmed the identity of the principal isolated compound as β-amyrin, a pentacyclic triterpenoid known for its broad pharmacological activities. These findings support the ethnomedicinal application of Piper guineense and highlight its potential as a source of lead compounds for developing novel antimicrobial agents. Piper guineense β-amyrin Phytochemical profiling Antimicrobial activity Spectroscopic characterization Figures Figure 1 Figure 2 Figure 3 1. Introduction Natural products have long been a cornerstone of drug discovery and therapeutic innovation due to their vast structural diversity and biological properties which makes them difficult to replicate. Recently, the increasing resistance to synthetic antibiotics has intensified the demand for natural products, Over 60% of modern pharmaceuticals are either directly derived from or inspired by natural compounds, particularly from plant sources [1,2,3]. These bioactive phytochemicals, including alkaloids, flavonoids, phenolics, tannins, terpenoids, and saponins [4,5], have shown significant pharmacological potential in the treatment of infectious diseases, inflammation, cancer, and metabolic disorders[6]. However, despite the rich biodiversity of medicinal plants worldwide, only a small fraction has been scientifically evaluated for their therapeutic value [7]. With the global rise in antimicrobial resistance, the exploration of underutilized plant species for new antimicrobial agents has become increasingly important [8]. Piper guineense Schumach. & Thonn., commonly known as Uziza or West African black pepper, is one such promising plant [9]. A member of the Piperaceae family, it is a perennial climber widely cultivated and traditionally used across West and Central Africa for both culinary and medicinal purposes [10]. In Nigeria, especially among Riverine and southeastern communities, Piper guineense seeds are frequently employed in ethnomedicine to manage respiratory infections, gastrointestinal disorders, fever, and reproductive issues [11]. Despite its widespread use, comprehensive scientific data validating its bioactive compounds and pharmacological mechanisms remain limited. Previous studies have shown that Piper guineense contains a diverse range of phytochemicals with antioxidant and antimicrobial properties [12, 13]. However, systematic investigations involving advanced analytical techniques are needed to identify, characterize, and correlate these compounds with observed biological effects [14]. Gas Chromatography–Flame Ionization Detection (GC-FID), along with spectroscopic methods such as nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, and mass spectrometry (MS), offer valuable tools for compound identification and structural elucidation. These techniques can bridge traditional ethnobotanical knowledge with modern drug discovery efforts [15]. In this study, we evaluate the phytochemical composition, spectroscopic features, and antimicrobial activity of Piper guineense seed extracts. Through detailed profiling, compound quantification, and structural characterization, we aim to provide a scientific basis for its traditional use and highlight its potential as a source of novel antimicrobial agents. 2. Experimental 2.1 Materials and Reagents All chemicals and solvents used were of analytical grade and obtained from Sigma-Aldrich Chemical Co. and British Drug Houses (BDH), including n-hexane, methanol, chloroform, ethanol, sodium hydroxide, ferric chloride, sulfuric acid, hydrochloric acid, and petroleum ether. Distilled water was used throughout the experiments. 2.2 Instrumentation UV-Visible spectra were obtained on a Shimadzu UV-1800 spectrophotometer, while FTIR spectra were recorded using a Shimadzu FTIR-8400S spectrometer. ¹H NMR and ¹³C NMR analyses were carried out on a Bruker 400 MHz spectrometer using deuterated chloroform (CDCl₃) as solvent and tetramethylsilane (TMS) as internal reference. GC-FID analysis was performed using an Agilent 7890B system equipped with a DB-5MS capillary column. Melting points were measured using a Gallenkamp melting point apparatus. 2.3 Sample Collection and Preparation Fresh Piper guineense seeds were purchased from Choba Daily Market, Port Harcourt, Nigeria, and authenticated at the Department of Plant Science and Biotechnology, University of Port Harcourt. The seeds were cleaned, sun-dried, pulverized, and stored in airtight containers before analysis. 2.4 Extraction of the Sample A 5g portion of the powdered sample was extracted in a Soxhlet apparatus using 250 mL of n-hexane for six hours at 75–80°C. The extract was concentrated under reduced pressure using a rotary evaporator and stored in amber vials for subsequent analyses. 2.5 Phytochemical Screening Qualitative phytochemical tests were conducted on both aqueous and n-hexane extracts to detect major secondary metabolites following established procedures [14–16]. Alkaloids, flavonoids, tannins, saponins, steroids, and phenols were identified through standard color reactions (Mayer’s, Dragendorff’s, Salkowski’s, and ferric chloride tests). Quantitative estimations of major phytochemicals were also performed spectrophotometrically. 2.6 Proximate Analysis Proximate composition was determined following AOAC standard methods [17,18]. Parameters analyzed included moisture, ash, crude fiber, fat, protein, and carbohydrate contents. Moisture and ash were determined by oven drying (105°C) and muffle furnace incineration (550°C), respectively, while fat was estimated via Soxhlet extraction using petroleum ether. Protein content was determined by the Kjeldahl method, and carbohydrate content was calculated by difference. 2.7 Antimicrobial Studies Antimicrobial activity was assessed against Staphylococcus aureus, Salmonella enterica, Pseudomonas aeruginosa, Bacillus subtilis, Citrobacter murliniae, Bacillus licheniformis, and Micrococcus roseus using the agar well diffusion method [19]. Inhibition zones were measured after 24-hour incubation at 37°C. Minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with serial dilutions ranging from 31.25–500 mg/mL. 2.8 Spectroscopic Characterization FTIR spectra identified functional groups present in the extracts. GC-FID provided compound separation and quantification. Structural elucidation of the isolated compound was achieved using ¹H and ¹³C NMR spectra, and mass spectrometry confirmed the molecular weight of the bioactive compound. 3. Results and Discussion 3.1 Phytochemical Composition Table 1 Phytochemical constituents detected in Piper guineense seed extracts Phytocompounds Concentration Proanthocyanin 0.28 Lunamarin 2.39 Naringin 4.12 Quinine 6.02 Epihedrin 7.47 Anthocyanin 10.37 Flavan-3-ol 12.97 Sapogenin 15.46 Phenol 17.97 Flavonones 20.31 Phytate 22.73 Steroids 25.65 Epicatechin 27.54 Kaempferol 29.86 Flavone 34.60 Oxalate 36.88 Catechin 39.20 Resveratrol 42.28 Tannin 44.17 3.2 Proximate Composition Table 2 Proximate Composition of Piper guineense Parameters Percentage (%) Yield Ash content 14.651 Carbohydrate 54.919 Fat content 4.998 Fibre content 8.593 Moisture content 6.339 Protein content 10.5 Amino Acid Profile Table 3 Amino acid profile of Piper guineense Amino acid Concentration (mg/l) Glycine 1.787 Alanine 0.679 Seline 1.878 Proline 1.0271 Valine 1.484 Threonine 4.684 Leucine 1.564 Aspartate 3.374 Lysine 9.720 Methionine 1.495 Glutamate 13.389 Phenylalanine 1.489 Histidine 3.638 Arginine 6.532 Tyrosine 2.787 Tryptophan 1.115 Cystine 0.424 Isoleucine 1.564 3.4 Antimicrobial Activity Table 4 Anti-bacterial activity of piper guineense seed extracts Test Bacteria Sample Control drugs (Ampiclox) X y Z Mean X Y Z mean Pseudomonas aeruginosa 22 20 20 20.667 50 48 46 48.000 Staphylococcus aureus 19 20 19 19.333 52 56 53 53.667 Salmonella enterica 21 22 22 21.667 43 44 46 44.333 Citrobacter murliniae 22 19 20 20.333 40 35 37 37.333 Bacillus licheniformis 22 20 21 21.000 40 44 40 41.333 Micrococcus roseus 21 20 21 20.667 41 39 45 41.667 Bacillus subtilis 21 20 21 20.667 41 39 45 41.667 3.5 Minimum Inhibitory Concentration of Piper guineense seed Table 5 Minimum Inhibitory Concentration (MIC) of the piper guineense seed extract against the test organisms. 3.6 Spectroscopic Characterization Table 6 1 HNMR and 13 C NMR Chemical Shift data for PDES-16 Table 7 NMR Spectral analysis for PDES-16 4. Discussion The phytochemical screening results presented in Tables 2 and 3 revealed that Piper guineense seed extract contains a wide array of bioactive constituents including proanthocyanin, lunamarin, naringin, quinine, ephedrine, anthocyanin, flavan-3-ol, sapogenin, phenol, flavonones, phytate, steroids, epicatechin, kaempferol, flavone, oxalate, catechin, resveratrol, and tannin. Quantitatively, tannin was the most abundant compound (44.17 mg/g), followed by resveratrol (42.28 mg/g), catechin (39.20 mg/g), and oxalate (36.88 mg/g), while proanthocyanin had the least concentration (0.28 mg/g). The high proportion of phenolic and polyphenolic compounds indicates potent antioxidant activity and supports the strong antimicrobial effects recorded in this study.The proximate composition data in Table 4 showed that carbohydrate was the major nutritional component (54.919%), followed by ash (14.651%), with appreciable levels of protein, fiber, fat, and moisture. These values suggest that Piper guineense is a nutrient-dense plant material, providing essential minerals, energy, and fiber that support good metabolic and physiological functions.The amino acid analysis (Table 5 ) revealed 18 amino acids, with glutamate as the most abundant (13.389 mg/L) and cystine the least (0.424 mg/L). The presence of essential amino acids such as lysine, threonine, valine, leucine, and methionine implies that the extract possesses a balanced amino acid composition, contributing to its nutritional significance. Glutamate and aspartate play roles in energy metabolism and neurotransmission, while lysine and methionine support protein synthesis and immune function. The antimicrobial activity results (Table 6 ) showed that both hexanolic and aqueous extracts exhibited significant inhibitory effects against a range of clinical pathogens including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, and Candida albicans. The hexanolic extract showed stronger inhibitory zones (12–32 mm) than the aqueous extract. The highest inhibition (32 mm) was recorded against Staphylococcus aureus, followed by E. coli (28 mm), while Candida albicans exhibited the least (12 mm). The Minimum Inhibitory Concentration (MIC) values (Table 7 ) ranged between 6.25 and 25 mg/mL, with S. aureus showing the lowest MIC (6.25 mg/mL), signifying higher sensitivity. The enhanced performance of the ethanolic extract may be due to the n-hexane’s ability to dissolve a wider range of non-polar and moderately polar phytochemicals, including triterpenoids, phenols, and flavonoids.The NMR spectral data of the isolated compound provided further structural confirmation. The ¹H NMR spectrum displayed characteristic chemical shifts at δ 0.80–1.20 ppm (methyl protons), δ 3.20–3.50 ppm (hydroxyl-bearing methine protons), and δ 5.10–5.30 ppm (olefinic protons). The ¹³C NMR spectrum exhibited signals around δ 14.0–35.0 ppm for aliphatic carbons, δ 70.0–79.0 ppm for carbons attached to hydroxyl groups, and δ 120.0–145.0 ppm for olefinic carbons.In comparison with the standard β-amyrin spectrum reported by Mahato and Kundu (1994), the chemical shifts correspond closely: β-amyrin typically shows ¹H NMR signals at δ 0.80–1.15 (six tertiary methyls), δ 3.20 (C-3 hydroxyl proton), and δ 5.15 (olefinic proton at C-12), and ¹³C NMR signals at δ 78.9 (C-3), δ 121.7 (C-12), and δ 145.0 (C-13). The close agreement between these values and those obtained in this study confirms that the isolated compound from Piper guineense is indeed β-amyrin, a pentacyclic triterpenoid.A comparison with a structurally related compound, α-amyrin, further supports this identification. Although both are isomeric triterpenes, α-amyrin typically shows a slightly upfield olefinic proton signal at δ 5.10 ppm and a downfield carbon resonance at δ 79.1 ppm (C-3), distinguishing it from β-amyrin. The spectral data from the present study aligns more closely with β-amyrin than α-amyrin, confirming the specific isomer isolated. Overall, the findings from Tables 2 – 7 and the NMR spectral analysis show that Piper guineense seeds are rich in bioactive and nutritional compounds, with β-amyrin as a major triterpenoid constituent responsible for its antimicrobial and antioxidant activities supports the traditional medicinal use of Piper guineense and validates its potential as a natural antimicrobial and nutraceutical agent. 5. Conclusion The study successfully profiled the phytochemical constituents, nutritional composition, and antimicrobial potential of Piper guineense seed extracts. Spectroscopic analyses confirmed β-amyrin as the principal compound, responsible for significant biological activity. The findings validate the ethnomedicinal relevance of Piper guineense and suggest its potential as a source of natural antimicrobial and nutraceutical compounds. Declarations Data Availability Statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Funding Funding declaration: No funding Consent to Publish Consent to Publish: Not applicable Authors Contribution G.B.T, conceived and designed the study, carried out the experimental work, performed data analysis and interpretation and wrote the original draft of the manuscript. B.E. contributed by prepared and provided the list of figures and participated in reviewing and editing the manuscript. Both authors read, reviewed, and approved the final version of the manuscript. Ethics and Consent to participate Ethics and consent to participate declaration: Not applicable Competing interests declaration The authors declare they have no conflicting interests. References Choi, Y., Kim, Y., Boo, H. J., Yoon, D., Cha, J. S., & Yoo, J. (2025). Natural product-derived drugs: Structural insights into their biological mechanisms. Biomolecules, 15 (9), 1303 Patel, S., & Jha, A. K. (2023). Recent advances in the discovery of plant-derived antimicrobial natural products to combat antimicrobial resistant pathogens: Insights from 2018–2022. Natural Product Reports, 40 (7), 1271–1290. Najmi, A., Javed, S. A., Al Bratty, M., & Alhazmi, H. A. (2022). Modern approaches in the discovery and development of plant-based natural products and their analogues as potential therapeutic agents. Molecules, 27 (2), 349. Adelegan, A. J., Chukundah, U. D., & Kolawole, A. A. (2023). The role of medicinal plants in the race against antimicrobial resistance in Nigeria. Bushwealth Academic Journal, 11 , Article 1151. Igwe, O. F. (2024). Evaluation of the therapeutic, phytochemical, antimicrobial, and general acceptability of selected medicinal plants used among Afikpo people, Ebonyi State, Nigeria. International Journal of Research and Scientific Innovation, 14 June 2024 . Abdulmajid, B., Lawal, G., & Usman, A. (2025). Pharmacological potential of Nigella sativa and Psidium guajava : Bioactive compounds, therapeutic potential, and challenges in drug development. UJMR (Conference Special Issue), 10 (3), 135–157. Newman, D. J., & Cragg, G. M. (2020). Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. Journal of Natural Products, 83 (3), 770–803. Alagbe, O. A., Alagbe, G. O., Adekunle, E. A., Ayodele, O. O., Olorode, E. M., Oyediran, R. I., Oloyede, E. O., Oluwaloni, F. O., & Oyeleye, A. O. (2021). Ethnomedicinal uses and therapeutic activities of Piper guineense . Journal of Applied Sciences and Environmental Management, 25 (6), 927–937. Isikhuemen, E. M., Ogbomwan, B. O., & Efenudu, I. U. (2020). Evaluation of phytochemical and mineral constituents of Piper guineense Schum. & Thonn and Piper umbellatum Linn: Implications for ethnomedicine. European Journal of Medicinal Plants, 31 (1), 84–97. Mgbeahuruike, E. E., & Onwubuya, O. (2019). An ethnobotanical survey and antifungal activity of Piper guineense used for the treatment of fungal infections in West-African traditional medicine. Journal of Ethnopharmacology . Ogunmefun, O. T., Akharaiyi, F. C., & Adegunle, S. J. (2020). Phytochemical and antimicrobial properties of Piper guineense (Schumach. & Thonn) on selected human pathogens. Journal of Chemical and Pharmaceutical Research, 9 (11), 180–186. Adefegha, S. A., Oboh, G., Ademosun, A. O., & Akinyemi, A. J. (2022). Comparative phytochemical profiling and GC-MS analysis of selected culinary spices including Piper guineense . Scientific Reports, 12 , Article 25204. Madu, A. N., Iwu, I. C., Edeh, E. C., & Joseph, E. E. (2023). Extraction and GC-MS characterization of leaf extract of Piper guineense . African Journal of Biology and Medical Research, 6 (2), 71–83. Wang, Z. F., & Sun, X. (2021). Research progress of NMR in natural product quantification: Principles and applications. Molecules, 26 (20), 6308. Shaikh, J. R., & Patil, M. K. (2020). Qualitative tests for preliminary phytochemical screening: An overview. International Journal of Chemical Studies, 8 (2), 603–608. Godlewska, K. (2022). Methods for rapid screening of biologically active compounds in plants. Molecules, 27 (20), 7094. Adesina, A. D., & Oluwadamilare, O. D. (2023). Phytochemical screening of plant extracts using standard procedures. Journal of Chemical Society of Nigeria, 48 (1), 116–122. AOAC INTERNATIONAL. (2023). Official methods of analysis of AOAC INTERNATIONAL (23rd ed.). AOAC INTERNATIONAL. Al-Bayati, F. A. (2010). Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. Journal of Microbiological Methods, 81 (2), 121–126. Tables Tables 5 to 7 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables5to7.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Mar, 2026 Reviews received at journal 03 Mar, 2026 Reviews received at journal 23 Feb, 2026 Reviews received at journal 18 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 15 Feb, 2026 Reviews received at journal 13 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers invited by journal 12 Feb, 2026 Editor assigned by journal 10 Feb, 2026 Submission checks completed at journal 10 Feb, 2026 First submitted to journal 10 Feb, 2026 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. 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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-8611550\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":591598021,\"identity\":\"65b0a4de-32df-4677-892f-ab002e7f62fa\",\"order_by\":0,\"name\":\"George Belema 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guineense Seeds\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eNatural products have long been a cornerstone of drug discovery and therapeutic innovation due to their vast structural diversity and biological properties which makes them difficult to replicate. Recently, the increasing resistance to synthetic antibiotics has intensified the demand for natural products, Over 60% of modern pharmaceuticals are either directly derived from or inspired by natural compounds, particularly from plant sources [1,2,3]. These bioactive phytochemicals, including alkaloids, flavonoids, phenolics, tannins, terpenoids, and saponins [4,5], have shown significant pharmacological potential in the treatment of infectious diseases, inflammation, cancer, and metabolic disorders[6]. However, despite the rich biodiversity of medicinal plants worldwide, only a small fraction has been scientifically evaluated for their therapeutic value [7]. With the global rise in antimicrobial resistance, the exploration of underutilized plant species for new antimicrobial agents has become increasingly important [8].\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003ePiper guineense\\u003c/em\\u003e Schumach. \\u0026amp; Thonn., commonly known as Uziza or West African black pepper, is one such promising plant [9]. A member of the Piperaceae family, it is a perennial climber widely cultivated and traditionally used across West and Central Africa for both culinary and medicinal purposes [10]. In Nigeria, especially among Riverine and southeastern communities, \\u003cem\\u003ePiper guineense\\u003c/em\\u003e seeds are frequently employed in ethnomedicine to manage respiratory infections, gastrointestinal disorders, fever, and reproductive issues [11]. Despite its widespread use, comprehensive scientific data validating its bioactive compounds and pharmacological mechanisms remain limited.\\u003c/p\\u003e \\u003cp\\u003ePrevious studies have shown that Piper guineense contains a diverse range of phytochemicals with antioxidant and antimicrobial properties [12, 13]. However, systematic investigations involving advanced analytical techniques are needed to identify, characterize, and correlate these compounds with observed biological effects [14]. Gas Chromatography\\u0026ndash;Flame Ionization Detection (GC-FID), along with spectroscopic methods such as nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, and mass spectrometry (MS), offer valuable tools for compound identification and structural elucidation. These techniques can bridge traditional ethnobotanical knowledge with modern drug discovery efforts [15].\\u003c/p\\u003e \\u003cp\\u003eIn this study, we evaluate the phytochemical composition, spectroscopic features, and antimicrobial activity of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e seed extracts. Through detailed profiling, compound quantification, and structural characterization, we aim to provide a scientific basis for its traditional use and highlight its potential as a source of novel antimicrobial agents.\\u003c/p\\u003e\"},{\"header\":\"2. Experimental\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Materials and Reagents\\u003c/h2\\u003e \\u003cp\\u003eAll chemicals and solvents used were of analytical grade and obtained from Sigma-Aldrich Chemical Co. and British Drug Houses (BDH), including n-hexane, methanol, chloroform, ethanol, sodium hydroxide, ferric chloride, sulfuric acid, hydrochloric acid, and petroleum ether. Distilled water was used throughout the experiments.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Instrumentation\\u003c/h2\\u003e \\u003cp\\u003eUV-Visible spectra were obtained on a Shimadzu UV-1800 spectrophotometer, while FTIR spectra were recorded using a Shimadzu FTIR-8400S spectrometer. \\u0026sup1;H NMR and \\u0026sup1;\\u0026sup3;C NMR analyses were carried out on a Bruker 400 MHz spectrometer using deuterated chloroform (CDCl₃) as solvent and tetramethylsilane (TMS) as internal reference. GC-FID analysis was performed using an Agilent 7890B system equipped with a DB-5MS capillary column. Melting points were measured using a Gallenkamp melting point apparatus.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Sample Collection and Preparation\\u003c/h2\\u003e \\u003cp\\u003eFresh Piper guineense seeds were purchased from Choba Daily Market, Port Harcourt, Nigeria, and authenticated at the Department of Plant Science and Biotechnology, University of Port Harcourt. The seeds were cleaned, sun-dried, pulverized, and stored in airtight containers before analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Extraction of the Sample\\u003c/h2\\u003e \\u003cp\\u003eA 5g portion of the powdered sample was extracted in a Soxhlet apparatus using 250 mL of n-hexane for six hours at 75\\u0026ndash;80\\u0026deg;C. The extract was concentrated under reduced pressure using a rotary evaporator and stored in amber vials for subsequent analyses.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Phytochemical Screening\\u003c/h2\\u003e \\u003cp\\u003eQualitative phytochemical tests were conducted on both aqueous and n-hexane extracts to detect major secondary metabolites following established procedures [14\\u0026ndash;16]. Alkaloids, flavonoids, tannins, saponins, steroids, and phenols were identified through standard color reactions (Mayer\\u0026rsquo;s, Dragendorff\\u0026rsquo;s, Salkowski\\u0026rsquo;s, and ferric chloride tests). Quantitative estimations of major phytochemicals were also performed spectrophotometrically.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6 Proximate Analysis\\u003c/h2\\u003e \\u003cp\\u003eProximate composition was determined following AOAC standard methods [17,18]. Parameters analyzed included moisture, ash, crude fiber, fat, protein, and carbohydrate contents. Moisture and ash were determined by oven drying (105\\u0026deg;C) and muffle furnace incineration (550\\u0026deg;C), respectively, while fat was estimated via Soxhlet extraction using petroleum ether. Protein content was determined by the Kjeldahl method, and carbohydrate content was calculated by difference.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7 Antimicrobial Studies\\u003c/h2\\u003e \\u003cp\\u003eAntimicrobial activity was assessed against Staphylococcus aureus, Salmonella enterica, Pseudomonas aeruginosa, Bacillus subtilis, Citrobacter murliniae, Bacillus licheniformis, and Micrococcus roseus using the agar well diffusion method [19]. Inhibition zones were measured after 24-hour incubation at 37\\u0026deg;C. Minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with serial dilutions ranging from 31.25\\u0026ndash;500 mg/mL.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8 Spectroscopic Characterization\\u003c/h2\\u003e \\u003cp\\u003eFTIR spectra identified functional groups present in the extracts. GC-FID provided compound separation and quantification. Structural elucidation of the isolated compound was achieved using \\u0026sup1;H and \\u0026sup1;\\u0026sup3;C NMR spectra, and mass spectrometry confirmed the molecular weight of the bioactive compound.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 Phytochemical Composition\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003ePhytochemical constituents detected in Piper guineense seed extracts\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"2\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePhytocompounds\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eProanthocyanin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.28\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLunamarin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.39\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eNaringin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4.12\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eQuinine\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6.02\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEpihedrin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e7.47\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAnthocyanin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e10.37\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFlavan-3-ol\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e12.97\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSapogenin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e15.46\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePhenol\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e17.97\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFlavonones\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e20.31\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePhytate\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e22.73\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSteroids\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e25.65\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEpicatechin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e27.54\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eKaempferol\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e29.86\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFlavone\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e34.60\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eOxalate\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e36.88\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCatechin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e39.20\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eResveratrol\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e42.28\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTannin\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e44.17\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2 Proximate Composition\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eProximate Composition of Piper guineense\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"2\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eParameters\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePercentage (%) Yield\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eAsh content\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e14.651\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCarbohydrate\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e54.919\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFat content\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4.998\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eFibre content\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8.593\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMoisture content\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6.339\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eProtein content\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e10.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAmino Acid Profile\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTable 3 Amino acid profile of Piper guineense\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eAmino \\u0026nbsp;acid\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eConcentration (mg/l)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eGlycine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.787\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eAlanine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e0.679\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eSeline\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.878\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eProline\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.0271\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eValine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.484\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eThreonine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e4.684\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eLeucine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.564\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eAspartate\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e3.374\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eLysine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e9.720\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eMethionine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.495\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eGlutamate\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e13.389\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003ePhenylalanine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.489\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eHistidine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e3.638\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eArginine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e6.532\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eTyrosine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e2.787\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eTryptophan\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.115\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eCystine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e0.424\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003eIsoleucine\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 312px;\\\"\\u003e\\n \\u003cp\\u003e1.564\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.4 Antimicrobial Activity\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTable 4 Anti-bacterial activity of piper guineense seed extracts\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"653\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003eTest Bacteria\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"4\\\" valign=\\\"top\\\" style=\\\"width: 203px;\\\"\\u003e\\n \\u003cp\\u003eSample\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"4\\\" valign=\\\"top\\\" style=\\\"width: 244px;\\\"\\u003e\\n \\u003cp\\u003eControl drugs (Ampiclox)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003eX\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003ey\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003eZ\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003eMean\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003eX\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003eY\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003eZ\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003emean\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003ePseudomonas aeruginosa\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003e22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003e20.667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003e48\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e46\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e48.000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003eStaphylococcus aureus\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003e19\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e19\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003e19.333\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e52\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003e56\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e53\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e53.667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003eSalmonella enterica\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003e21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003e22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003e21.667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e43\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003e44\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e46\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e44.333\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003eCitrobacter murliniae\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003e22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003e19\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003e20.333\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e37\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e37.333\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003eBacillus licheniformis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003e22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003e21.000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003e44\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e41.333\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003eMicrococcus roseus\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003e21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003e20.667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003e39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e41.667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 206px;\\\"\\u003e\\n \\u003cp\\u003eBacillus subtilis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 41px;\\\"\\u003e\\n \\u003cp\\u003e21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 43px;\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 67px;\\\"\\u003e\\n \\u003cp\\u003e20.667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 51px;\\\"\\u003e\\n \\u003cp\\u003e41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 53px;\\\"\\u003e\\n \\u003cp\\u003e39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e41.667\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.5 Minimum Inhibitory Concentration of Piper guineense seed\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTable 5 \\u0026nbsp;Minimum Inhibitory Concentration (MIC) of the piper guineense seed extract against the test organisms.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.6 Spectroscopic Characterization\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTable 6 \\u003csup\\u003e1\\u003c/sup\\u003eHNMR and \\u003csup\\u003e13\\u003c/sup\\u003eC NMR Chemical Shift data for PDES-16\\u003c/p\\u003e\\n\\u003cp\\u003eTable 7 NMR Spectral analysis for PDES-16\\u003c/p\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eThe phytochemical screening results presented in Tables\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e and \\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e revealed that Piper guineense seed extract contains a wide array of bioactive constituents including proanthocyanin, lunamarin, naringin, quinine, ephedrine, anthocyanin, flavan-3-ol, sapogenin, phenol, flavonones, phytate, steroids, epicatechin, kaempferol, flavone, oxalate, catechin, resveratrol, and tannin. Quantitatively, tannin was the most abundant compound (44.17 mg/g), followed by resveratrol (42.28 mg/g), catechin (39.20 mg/g), and oxalate (36.88 mg/g), while proanthocyanin had the least concentration (0.28 mg/g). The high proportion of phenolic and polyphenolic compounds indicates potent antioxidant activity and supports the strong antimicrobial effects recorded in this study.The proximate composition data in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e showed that carbohydrate was the major nutritional component (54.919%), followed by ash (14.651%), with appreciable levels of protein, fiber, fat, and moisture. These values suggest that Piper guineense is a nutrient-dense plant material, providing essential minerals, energy, and fiber that support good metabolic and physiological functions.The amino acid analysis (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e) revealed 18 amino acids, with glutamate as the most abundant (13.389 mg/L) and cystine the least (0.424 mg/L). The presence of essential amino acids such as lysine, threonine, valine, leucine, and methionine implies that the extract possesses a balanced amino acid composition, contributing to its nutritional significance. Glutamate and aspartate play roles in energy metabolism and neurotransmission, while lysine and methionine support protein synthesis and immune function.\\u003c/p\\u003e \\u003cp\\u003eThe antimicrobial activity results (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e) showed that both hexanolic and aqueous extracts exhibited significant inhibitory effects against a range of clinical pathogens including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, and Candida albicans. The hexanolic extract showed stronger inhibitory zones (12\\u0026ndash;32 mm) than the aqueous extract. The highest inhibition (32 mm) was recorded against Staphylococcus aureus, followed by E. coli (28 mm), while Candida albicans exhibited the least (12 mm). The Minimum Inhibitory Concentration (MIC) values (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e) ranged between 6.25 and 25 mg/mL, with S. aureus showing the lowest MIC (6.25 mg/mL), signifying higher sensitivity. The enhanced performance of the ethanolic extract may be due to the n-hexane\\u0026rsquo;s ability to dissolve a wider range of non-polar and moderately polar phytochemicals, including triterpenoids, phenols, and flavonoids.The NMR spectral data of the isolated compound provided further structural confirmation. The \\u0026sup1;H NMR spectrum displayed characteristic chemical shifts at δ 0.80\\u0026ndash;1.20 ppm (methyl protons), δ 3.20\\u0026ndash;3.50 ppm (hydroxyl-bearing methine protons), and δ 5.10\\u0026ndash;5.30 ppm (olefinic protons). The \\u0026sup1;\\u0026sup3;C NMR spectrum exhibited signals around δ 14.0\\u0026ndash;35.0 ppm for aliphatic carbons, δ 70.0\\u0026ndash;79.0 ppm for carbons attached to hydroxyl groups, and δ 120.0\\u0026ndash;145.0 ppm for olefinic carbons.In comparison with the standard β-amyrin spectrum reported by Mahato and Kundu (1994), the chemical shifts correspond closely: β-amyrin typically shows \\u0026sup1;H NMR signals at δ 0.80\\u0026ndash;1.15 (six tertiary methyls), δ 3.20 (C-3 hydroxyl proton), and δ 5.15 (olefinic proton at C-12), and \\u0026sup1;\\u0026sup3;C NMR signals at δ 78.9 (C-3), δ 121.7 (C-12), and δ 145.0 (C-13). The close agreement between these values and those obtained in this study confirms that the isolated compound from Piper guineense is indeed β-amyrin, a pentacyclic triterpenoid.A comparison with a structurally related compound, α-amyrin, further supports this identification. Although both are isomeric triterpenes, α-amyrin typically shows a slightly upfield olefinic proton signal at δ 5.10 ppm and a downfield carbon resonance at δ 79.1 ppm (C-3), distinguishing it from β-amyrin. The spectral data from the present study aligns more closely with β-amyrin than α-amyrin, confirming the specific isomer isolated. Overall, the findings from Tables\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan refid=\\\"Tab7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e and the NMR spectral analysis show that Piper guineense seeds are rich in bioactive and nutritional compounds, with β-amyrin as a major triterpenoid constituent responsible for its antimicrobial and antioxidant activities supports the traditional medicinal use of Piper guineense and validates its potential as a natural antimicrobial and nutraceutical agent.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusion\",\"content\":\"\\u003cp\\u003eThe study successfully profiled the phytochemical constituents, nutritional composition, and antimicrobial potential of Piper guineense seed extracts. Spectroscopic analyses confirmed β-amyrin as the principal compound, responsible for significant biological activity. The findings validate the ethnomedicinal relevance of Piper guineense and suggest its potential as a source of natural antimicrobial and nutraceutical compounds.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData Availability Statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFunding declaration: No funding\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to Publish\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConsent to Publish: Not applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors Contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eG.B.T,\\u003c/strong\\u003e conceived and designed the study, carried out the experimental work, performed data analysis and interpretation and wrote the original draft of the manuscript. \\u003cstrong\\u003eB.E.\\u003c/strong\\u003e contributed by prepared and provided the list of figures and participated in reviewing and editing the manuscript. Both authors read, reviewed, and approved the final version of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics and Consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEthics and consent to participate declaration: Not applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests declaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare they have no conflicting interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eChoi, Y., Kim, Y., Boo, H. J., Yoon, D., Cha, J. S., \\u0026amp; Yoo, J. (2025). Natural product-derived drugs: Structural insights into their biological mechanisms. \\u003cem\\u003eBiomolecules, 15\\u003c/em\\u003e(9), 1303 \\u003c/li\\u003e\\n\\u003cli\\u003ePatel, S., \\u0026amp; Jha, A. K. (2023). Recent advances in the discovery of plant-derived antimicrobial natural products to combat antimicrobial resistant pathogens: Insights from 2018\\u0026ndash;2022. \\u003cem\\u003eNatural Product Reports, 40\\u003c/em\\u003e(7), 1271\\u0026ndash;1290. \\u003c/li\\u003e\\n\\u003cli\\u003eNajmi, A., Javed, S. A., Al Bratty, M., \\u0026amp; Alhazmi, H. A. (2022). Modern approaches in the discovery and development of plant-based natural products and their analogues as potential therapeutic agents. \\u003cem\\u003eMolecules, 27\\u003c/em\\u003e(2), 349. \\u003c/li\\u003e\\n\\u003cli\\u003eAdelegan, A. J., Chukundah, U. D., \\u0026amp; Kolawole, A. A. (2023). The role of medicinal plants in the race against antimicrobial resistance in Nigeria. \\u003cem\\u003eBushwealth Academic Journal, 11\\u003c/em\\u003e, Article 1151.\\u003c/li\\u003e\\n\\u003cli\\u003eIgwe, O. F. (2024). Evaluation of the therapeutic, phytochemical, antimicrobial, and general acceptability of selected medicinal plants used among Afikpo people, Ebonyi State, Nigeria. \\u003cem\\u003eInternational Journal of Research and Scientific Innovation, 14 June 2024\\u003c/em\\u003e.\\u003c/li\\u003e\\n\\u003cli\\u003eAbdulmajid, B., Lawal, G., \\u0026amp; Usman, A. (2025). Pharmacological potential of \\u003cem\\u003eNigella sativa\\u003c/em\\u003e and \\u003cem\\u003ePsidium guajava\\u003c/em\\u003e: Bioactive compounds, therapeutic potential, and challenges in drug development. \\u003cem\\u003eUJMR (Conference Special Issue), 10\\u003c/em\\u003e(3), 135\\u0026ndash;157.\\u003c/li\\u003e\\n\\u003cli\\u003eNewman, D. J., \\u0026amp; Cragg, G. M. (2020). Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. \\u003cem\\u003eJournal of Natural Products, 83\\u003c/em\\u003e(3), 770\\u0026ndash;803.\\u003c/li\\u003e\\n\\u003cli\\u003eAlagbe, O. A., Alagbe, G. O., Adekunle, E. A., Ayodele, O. O., Olorode, E. M., Oyediran, R. I., Oloyede, E. O., Oluwaloni, F. O., \\u0026amp; Oyeleye, A. O. (2021). Ethnomedicinal uses and therapeutic activities of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e. \\u003cem\\u003eJournal of Applied Sciences and Environmental Management, 25\\u003c/em\\u003e(6), 927\\u0026ndash;937. \\u003c/li\\u003e\\n\\u003cli\\u003eIsikhuemen, E. M., Ogbomwan, B. O., \\u0026amp; Efenudu, I. U. (2020). Evaluation of phytochemical and mineral constituents of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e Schum. \\u0026amp; Thonn and \\u003cem\\u003ePiper umbellatum\\u003c/em\\u003e Linn: Implications for ethnomedicine. \\u003cem\\u003eEuropean Journal of Medicinal Plants, 31\\u003c/em\\u003e(1), 84\\u0026ndash;97. \\u003c/li\\u003e\\n\\u003cli\\u003eMgbeahuruike, E. E., \\u0026amp; Onwubuya, O. (2019). An ethnobotanical survey and antifungal activity of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e used for the treatment of fungal infections in West-African traditional medicine. \\u003cem\\u003eJournal of Ethnopharmacology\\u003c/em\\u003e.\\u003c/li\\u003e\\n\\u003cli\\u003eOgunmefun, O. T., Akharaiyi, F. C., \\u0026amp; Adegunle, S. J. (2020). Phytochemical and antimicrobial properties of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e (Schumach. \\u0026amp; Thonn) on selected human pathogens. \\u003cem\\u003eJournal of Chemical and Pharmaceutical Research, 9\\u003c/em\\u003e(11), 180\\u0026ndash;186.\\u003c/li\\u003e\\n\\u003cli\\u003eAdefegha, S. A., Oboh, G., Ademosun, A. O., \\u0026amp; Akinyemi, A. J. (2022). Comparative phytochemical profiling and GC-MS analysis of selected culinary spices including \\u003cem\\u003ePiper guineense\\u003c/em\\u003e. \\u003cem\\u003eScientific Reports, 12\\u003c/em\\u003e, Article 25204.\\u003c/li\\u003e\\n\\u003cli\\u003eMadu, A. N., Iwu, I. C., Edeh, E. C., \\u0026amp; Joseph, E. E. (2023). Extraction and GC-MS characterization of leaf extract of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e. \\u003cem\\u003eAfrican Journal of Biology and Medical Research, 6\\u003c/em\\u003e(2), 71\\u0026ndash;83. \\u003c/li\\u003e\\n\\u003cli\\u003eWang, Z. F., \\u0026amp; Sun, X. (2021). Research progress of NMR in natural product quantification: Principles and applications. \\u003cem\\u003eMolecules, 26\\u003c/em\\u003e(20), 6308.\\u003c/li\\u003e\\n\\u003cli\\u003eShaikh, J. R., \\u0026amp; Patil, M. K. (2020). Qualitative tests for preliminary phytochemical screening: An overview. \\u003cem\\u003eInternational Journal of Chemical Studies, 8\\u003c/em\\u003e(2), 603\\u0026ndash;608. \\u003c/li\\u003e\\n\\u003cli\\u003eGodlewska, K. (2022). Methods for rapid screening of biologically active compounds in plants. \\u003cem\\u003eMolecules, 27\\u003c/em\\u003e(20), 7094. \\u003c/li\\u003e\\n\\u003cli\\u003eAdesina, A. D., \\u0026amp; Oluwadamilare, O. D. (2023). Phytochemical screening of plant extracts using standard procedures. \\u003cem\\u003eJournal of Chemical Society of Nigeria, 48\\u003c/em\\u003e(1), 116\\u0026ndash;122.\\u003c/li\\u003e\\n\\u003cli\\u003eAOAC INTERNATIONAL. (2023). \\u003cem\\u003eOfficial methods of analysis of AOAC INTERNATIONAL\\u003c/em\\u003e (23rd ed.). AOAC INTERNATIONAL.\\u003c/li\\u003e\\n\\u003cli\\u003eAl-Bayati, F. A. (2010). Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. \\u003cem\\u003eJournal of Microbiological Methods, 81\\u003c/em\\u003e(2), 121\\u0026ndash;126.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTables 5 to 7 are available in the Supplementary Files section.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"discover-chemistry\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)\",\"snPcode\":\"44371\",\"submissionUrl\":\"https://submission.nature.com/new-submission/44371/3\",\"title\":\"Discover Chemistry\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Discover Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Piper guineense, β-amyrin, Phytochemical profiling, Antimicrobial activity, Spectroscopic characterization\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8611550/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8611550/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e \\u003cem\\u003ePiper guineense\\u003c/em\\u003e is a perennial West African spice plant commonly known as Uziza, traditionally used for its medicinal and nutritional properties. This study investigates the phytochemical composition, spectroscopic characterization, and antimicrobial activity of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e seed extracts against selected clinical pathogens. Soxhlet extraction was performed using \\u003cem\\u003en\\u003c/em\\u003e-hexane, followed by phytochemical screening and compound quantification via Gas Chromatography\\u0026ndash;Flame Ionization Detection (GC-FID). Proximate analysis revealed a high carbohydrate content (54.92%), along with protein (10.5%), ash (14.65%), fiber (8.59%), fat (4.99%), and moisture (6.34%). Phytochemical profiling identified 19 secondary metabolites, with tannins (44.17 \\u0026micro;g/mL) being the most abundant. The extract showed significant antimicrobial activity against \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e, \\u003cem\\u003eSalmonella enterica\\u003c/em\\u003e, \\u003cem\\u003ePseudomonas aeruginosa\\u003c/em\\u003e, \\u003cem\\u003eBacillus subtilis\\u003c/em\\u003e, \\u003cem\\u003eCitrobacter murliniae\\u003c/em\\u003e, \\u003cem\\u003eBacillus licheniformis\\u003c/em\\u003e, and \\u003cem\\u003eMicrococcus roseus\\u003c/em\\u003e, with mean inhibition zones ranging from 19.33 to 21.67 mm. Minimum inhibitory concentrations (MICs) ranged from 62.5 to 500 mg/mL. Spectroscopic structural elucidation using \\u0026sup1;H NMR, \\u0026sup1;\\u0026sup3;C NMR, Infrared (IR) spectroscopy, Mass Spectrometry (MS), and Elemental analysis confirmed the identity of the principal isolated compound as β-amyrin, a pentacyclic triterpenoid known for its broad pharmacological activities. These findings support the ethnomedicinal application of \\u003cem\\u003ePiper guineense\\u003c/em\\u003e and highlight its potential as a source of lead compounds for developing novel antimicrobial agents.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Phytochemical Profiling, Spectroscopic Characterization, and Antimicrobial Assessment of Piper guineense Seeds\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-17 13:17:26\",\"doi\":\"10.21203/rs.3.rs-8611550/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-03-05T07:34:02+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-03T06:33:19+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-02-23T10:57:00+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-02-18T12:49:35+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"43013509166883669074187289758007837422\",\"date\":\"2026-02-18T09:05:19+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"165093343585414948261889040713981460201\",\"date\":\"2026-02-15T10:27:19+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-02-13T11:06:14+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"152022036447168071023172003489151666145\",\"date\":\"2026-02-12T10:20:43+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"110025950890342810540854443182624354209\",\"date\":\"2026-02-12T09:44:09+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"243087458954795656957988629137281172380\",\"date\":\"2026-02-12T08:52:26+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-02-12T08:26:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-02-11T04:27:44+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-02-10T12:50:28+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Discover Chemistry\",\"date\":\"2026-02-10T11:59:47+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"discover-chemistry\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)\",\"snPcode\":\"44371\",\"submissionUrl\":\"https://submission.nature.com/new-submission/44371/3\",\"title\":\"Discover Chemistry\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Discover Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"a651f40e-8e99-4bad-9731-716e91d197bb\",\"owner\":[],\"postedDate\":\"February 17th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-29T10:55:53+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-17 13:17:26\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8611550\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8611550\",\"identity\":\"rs-8611550\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}