Phytochemical screening of Cocos nucifera And Vernonia amygdalina extract and antibiogram of the mixture (Cocos Nucifera + Vernonia Amygdalina) on Salmonella typhi and Streptococcus pyogenes

Phytochemical screening of Cocos nucifera And Vernonia amygdalina extract and antibiogram of the mixture (Cocos Nucifera + Vernonia Amygdalina) on Salmonella typhi and Streptococcus pyogenes | 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 screening of Cocos nucifera And Vernonia amygdalina extract and antibiogram of the mixture ( Cocos Nucifera + Vernonia Amygdalina ) on Salmonella typhi and Streptococcus pyogenes Emmanuel Oluwatimileyin Matthew, Dr. Ibrahim Abdulkarim Ozigi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4458581/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background: Nigeria, a significant player in the global economy, has a rich agricultural heritage that includes diverse crops with potential medicinal properties. Among these, coconut ( Cocos nucifera ) and bitter leaf ( Vernonia amygdalina ) are notable for their traditional medicinal uses. This study aimed to evaluate the phytochemical composition and antibacterial efficacy of extracts from these plants, individually and in combination, against Salmonella typhi and Streptococcus pyogenes, addressing the need for alternative antibacterial therapies in the face of rising antibiotic resistance. Methods: Bitter leaf and coconut were sourced from Bakindogo market, Kaduna, and authenticated by a botanist. Extracts were prepared and subjected to phytochemical screening using standard methods. Antibacterial activity was evaluated through agar disk diffusion to measure zones of inhibition at different extract concentrations. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for both Salmonella typhi and Streptococcus pyogenes .Top of FormBottom of Form Results: Phytochemical analysis revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, and carbohydrates in both extracts, with anthraquinones present only in Vernonia amygdalina. The extracts showed significant antibacterial activity, particularly the mixture of Vernonia amygdalina and Cocos nucifera water. The mixture exhibited higher efficacy against Salmonella typhi, with zones of inhibition ranging from 9.5 mm to 17 mm, compared to Streptococcus pyogenes, which showed inhibition zones from 9.0 mm to 13 mm. The MIC for Salmonella typhi was 25 mg/mL, with an MBC of 100 mg/mL, indicating bactericidal properties at higher concentrations. Streptococcus pyogenes showed an MIC of 100mg/ml, but the MBC was not applicable, suggesting bacteriostatic effects. Conclusions: The study confirms the significant antibacterial potential of Vernonia amygdalina and Cocos nucifera water extracts, particularly against Salmonella typhi. These findings support their traditional use and highlight their potential as alternative antibacterial agents. Further research and pharmaceutical exploration are recommended to fully harness these plants' medicinal properties, particularly for developing new treatments for bacterial infections. Drug Discovery, Design, & Development General Microbiology Vernonia amygdalina Cocos nucifera Phytochemical screening Antibacterial activity Salmonella typhi Streptococcus pyogenes Herbal remedies and Antibiotic resistance BACKGROUND Nigeria stands as a significant player in the global economy, recognized as the largest black nation. With a robust economic growth trajectory, Nigeria's gross domestic product (GDP) soared from $170 billion in 2000 to $451 billion in 2012, albeit unofficial estimations suggest figures closer to $630 billion, inclusive of the informal sector [1][2]. The agricultural sector has consistently been a pivotal contributor to Nigeria's economy, with a noteworthy 26.15% contribution to the GDP in 2017 (NBS, 2019). This sector serves as the cornerstone for economic advancement, development, and poverty alleviation, as emphasized by Sertoglu, Ugural, and Bekun [3], positioning agriculture as the catalyst for sustained economic prosperity. In Nigeria, agriculture encompasses a diverse array of staple and cash crops, with significant cultivation dedicated to crops such as cassava, yam, maize, sorghum, rice, palm kernel, and coconut, among others [2]. Notably, coconut, revered as the "tree of life," holds immense economic potential, albeit receiving limited attention within Nigeria's agricultural landscape [4]. Originating from Southeast Asia, coconut exhibits remarkable adaptability, thriving across various soil types and climates. Its utility extends beyond traditional consumption, offering a plethora of industrial applications that could bolster Nigeria's industrial development and elevate living standards. The historical narratives surrounding the origin of coconut are diverse and often steeped in mythology. While Indian mythology attributes the creation of the coconut palm to the sage Vishwamitra, botanical evidence suggests its origin in Papua New Guinea or Malesia, with subsequent dispersal facilitated by natural phenomena and Polynesian voyagers. This dispersion underscores coconut's resilience and capacity for widespread proliferation across diverse geographical regions [5]. Alongside coconut, bitter leaf ( Vernonia amygdalina ) emerges as a botanical asset in Nigeria's agricultural bounty. This perennial shrub, renowned for its bitter-tasting leaves, harbors extensive medicinal properties, earning it recognition as a staple herb in traditional medicine across West Africa. Indigenous to various ecological zones, bitter leaf thrives in diverse environments, adapting to habitats ranging from forest margins to grasslands. The morphological characteristics of bitter leaf underscore its resilience and adaptability, with features such as soft woodiness, lanceolate leaves, and bisexual flowers contributing to its ecological versatility. Beyond its ecological significance, bitter leaf holds immense socio-economic value, particularly within Nigerian communities, where it is revered for its medicinal prowess and nutritional benefits a [6]. Amidst the backdrop of Nigeria's agricultural abundance, concerns surrounding food safety and security persist. Bacterial infections continue to pose significant challenges, necessitating innovative approaches to antimicrobial research. The emergence of multidrug-resistant pathogens underscores the urgency for alternative therapeutic interventions, including the exploration of traditional herbal remedies [7][8]. In light of these considerations, this study aims to conduct phytochemical screening of coconut water and bitter leaf extract, evaluating their antibacterial efficacy against Salmonella typhi and Streptococcus spp. By elucidating the phytochemical composition and antibiogram of this botanical mixture, we seek to contribute valuable insights into the potential therapeutic applications of traditional herbal remedies in combating bacterial infections. In summary, this research endeavors to bridge the gap between traditional botanical knowledge and contemporary biomedical research, offering a pathway towards sustainable healthcare solutions within the context of Nigeria's rich agricultural heritage. METHODS Collection, Identification, and Authentication of the Plants Bitter leaf and coconut were procured from Bakindogo market in Kaduna. Their identification and authentication were carried out by a botanist from the Department of Biological Science at Kaduna State University. The plants were washed using sterile water and dried at room temperature in the Department of Applied Biology at Kaduna Polytechnic. Preparation and Testing of the Mixture (Combination) of Bitter Leaf and Coconut Water The bitter leaf was washed and then squeezed into coconut water. The mixture was left to soak for three days to allow maceration. After soaking for three days, the mixture of bitter leaf and coconut water was periodically shaken to ensure thorough extraction. The macerated mixture was then filtered to separate the micelle from the marc. The micelle was subsequently separated from the menstruum by evaporation using a water bath. The Mixture Extract Concentration Various concentrations of the mixture extract were prepared. A stock solution of 100 mg/mL was made by dissolving 1g of the crude extract in 10 mL of sterile distilled water. From this stock solution, different concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, and 12.5 mg/mL) were prepared accordingly. Phytochemical Screening of Crude Extracts The presence of bioactive compounds in the methanol extracts was determined by phytochemical tests using standard methods described by [9] and [10]. Test for Carbohydrates (Molisch's Test) A few drops of Molisch's reagent were added to 2 mL of the extract solution, followed by the addition of 1 mL of concentrated sulfuric acid down the side of the test tube. Changes in coloration at the interface of the two liquids were noted. Test for Tannins A few drops of 0.1% ferric chloride solution were added to the extract solutions, and changes were observed. Additionally, a few drops of lead acetate solution were added to the extract solutions, and changes were observed. Test for Alkaloids Meyer's reagent and Two drops of dilute sulfuric acid were added to 1 mL of the extract solution, and observed changes were noted. Another 1 mL portion of the extract was reacted with Dragendorff's reagent, and changes in the product formed were observed and recorded. An affirmation test for the presence of alkaloids was performed by adding two drops of Wagner's reagent, and observed changes were recorded. Test for Saponins A frothing test was performed by adding 5 mL of de-ionized water to the filtrate of the extracts and shaking vigorously to observe the formation of stable persistent frothing. This frothing was observed for up to 45 minutes. Test for Flavonoids Shinoda Test: Filtrates of the extracts (3 mL) were obtained, and four pieces of magnesium fillings were added, followed by a few drops of concentrated hydrochloric acid. Observed color changes were noted. Sodium Hydroxide Test: 5 mL of 10% sodium hydroxide was added to an equal volume of the extracts. Observed color changes were noted accordingly. Test for Terpenoids Liebermann-Burchard Test: Chloroform was added to the extract, mixed with 1 mL of acetic anhydride, and followed by the addition of 1 mL of concentrated sulfuric acid down the wall of the test tube. Observed changes were noted. Test for Anthraquinones Bornträger’s Test: 0.5 g of crude extracts was added to 10 ml of chloroform, shaken, and then filtered. Ten ml of the filtrate was reacted with an equal volume of ammonia (10 mL) and shaken gently. Observed changes were recorded. Characterization and Authentication of Test Organisms Clinical isolates of Salmonella typhi and Streptococcus spp. were obtained from 44 Army Reference Hospital in Kaduna State, Nigeria. Storage and Maintenance of Cultures The cultures were streaked onto Nutrient Agar slants and incubated at 37°C for 48 hours. These cultures were stored in a refrigerator at 4°C for subsequent use. Stock cultures were prepared and kept until needed. Broth cultures were prepared from these organisms. Preparation and Standardization of Inocula Nutrient broth was prepared according to the manufacturer’s specifications. The bacterial isolates were tested for sterility on Nutrient Agar, then re-grown in Nutrient broth at 37°C for 24 hours. McFarland's standard method was used to standardize the organisms to 1x10⁴ cfu/mL by preparing four sets of dilution tubes with 9 mL of normal saline and one tube with 4 mL of normal saline, labeled 10⁻¹ to 10⁻⁴. 1 mL of the overnight culture was added to the 10⁻¹ dilution tube, and serial dilutions were prepared to arrive at a dilution of 10⁻⁴ cfu/mL. Dilution with sterile Nutrient broth was done until the optical density matched that of McFarland's standard. Antimicrobial Susceptibility Test Nutrient agar was prepared according to the manufacturer’s specifications, and the molten agar was poured into sterile disposable plates and allowed to cool to about 45°C. The plates were swabbed with diluted standardized overnight cultures. Six mm Whatmann No. 1 paper discs, each soaked in the extract concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, and 12.5 mg/mL), coconut water, and bitter leaf juice, were placed on the agar plates. A standard antibiotic (200 mg/10 mL) was used as a positive control, and sterile distilled water was used as a negative control on a separate plate. Diameters of zones of inhibition were measured after incubating the plates at 25°C for 48 hours. The plates were replicated in triplicate, and the mean zones of inhibition for each organism at each extract concentration were measured using a ruler and recorded. Determination of Minimum Inhibitory Concentration (MIC) 2 ml of all the four-fold serial dilutions were used to impregnate sterile disks with a diameter of 5 mm. These impregnated disks were used to determine the MIC by placing them on the already streaked isolates of Salmonella typhi and Streptococcus pyogenes and incubated at 37°C for 24 hours. The zone of inhibition was measured using a ruler and recorded according to the concentration incorporated on the disk. Determination of Minimum Bactericidal Concentration (MBC) The MBC was determined by sub-culturing the prepared dilutions onto fresh Nutrient agar and further incubating for 24 hours at 37°C. These plates were observed for growth. The plates with the least concentration showing no growth were considered the MBC. RESULTS Phytochemical Constituents of Vernonia amygdalina, Cocos nucifera Water, and Their Mixture Comparative phytochemical screening of Vernonia amygdalina, Cocos nucifera water, and their mixture revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, and carbohydrates in all samples. Anthraquinones were present only in Vernonia amygdalina. Table 1: Phytochemical Composition of Bioactive Compounds Phytochemical Components Cocos nucifera Water Vernonia amygdalina Vernonia amygdalina and Cocos nucifera Water Mixture Alkaloids Present Present Present Saponins Present Present Present Tannins Present Present Present Flavonoids Present Present Present Terpenoids Present Absent Present Anthraquinones Absent Present Present Carbohydrates Present Present Present Bacterial Susceptibility Standard of Amoxil Table 2: Susceptibility Standard of Amoxil R I S ≤13 14-16 ≥17 R : Resistant I : Intermediate S : Susceptible ≤ : less than or equal to ≥ : greater than or equal to Antimicrobial Activity of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes The antibiogram of the mixture of Vernonia amygdalina and Cocos nucifera water was found to be effective against Salmonella typhi and Streptococcus pyogenes, showing zones of inhibition as summarized in Table 3. Table 3: Antimicrobial Activity of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes Test Organisms Concentration (mg/ml) Zone of Inhibition (mm) Positive control (500 mg/mL) Amoxicillin Reaction 100 50 25 12.5 1st isolate of S. typhi 17±0.1 12.5±0.5 12±0.1 9.5±0.5 18 S 2nd isolate of S. typhi 17±0.1 12.5±0.5 12±0.1 9.5±0.5 18 S 3rd isolate of S. typhi 17±0.1 12.5±0.5 12±0.1 9.5±0.5 18 S 4th isolate of S. typhi 17±0.1 12.5±0.5 12±0.1 9.5±0.5 18 S 1st isolate of S. pyogenes 13±0.1 13±0.1 11±0.5 9±0.1 17 S 2nd isolate of S. pyogenes 13±0.1 13±0.1 11±0.5 9±0.1 17 S 3rd isolate of S. pyogenes 13±0.1 13±0.1 11±0.5 9±0.1 17 S 4th isolate of S. pyogenes 13±0.1 13±0.1 11±0.5 9±0.1 17 S S : Susceptible Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Mixture on Salmonella typhi and Streptococcus pyogenes The MIC was determined using the agar disk diffusion and macro tube dilution methods, while the MBC was determined by sub-culturing the prepared dilutions into fresh media. The results in Table 4 show that the mixture of Vernonia amygdalina and Cocos nucifera water has significant antibacterial effects on Salmonella typhi at various concentrations. Streptococcus pyogenes showed partial sensitivity at 100 mg/ml. Table 4: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes Test Organisms MIC (mg/ml) MBC (mg/ml) 1st isolate of S. typhi 25 100 2nd isolate of S. typhi 25 100 3rd isolate of S. typhi 25 100 4th isolate of S. typhi 25 100 1st isolate of S. pyogenes 100 NA 2nd isolate of S. pyogenes 100 NA 3rd isolate of S. pyogenes 100 NA 4th isolate of S. pyogenes 100 NA NA : Not applicable DISCUSSION Phytochemical screening of Vernonia amygdalina leaf extract and Cocos nucifera water (Table 1) revealed the presence of alkaloids, flavonoids, tannins, anthraquinones, saponins, terpenoids, and carbohydrates, while anthraquinones were present only in Vernonia amygdalina. Studies have shown that alkaloids and flavonoids are key compounds responsible for the antibacterial activities in higher plants [11]. Saponins, tannins, and alkaloids are well-documented plant metabolites known for their antimicrobial properties [12]. The presence of these phytochemicals in the mixture may contribute to its antibacterial activity, supporting their traditional use in treating various bacterial and fungal infections. The antibacterial susceptibility test showed that all strains of the two clinical isolates, Salmonella typhi and Streptococcus pyogenes (Table 2), were highly susceptible to Amoxicillin (control). This observation aligns with the findings of Kassim et al. [13]. Salmonella typhi exhibited higher sensitivity to Amoxicillin with a zone of inhibition ranging from 18mm to 23mm, compared to Streptococcus pyogenes, which showed a range of 17mm to 20mm. The mixture of Vernonia amygdalina and Cocos nucifera water showed a broad-spectrum antibacterial activity, with efficacy increasing with higher concentrations. Salmonella typhi strains were highly sensitive to the mixture, with zones of inhibition ranging from 9.5mm to 17mm at concentrations of 12.5 mg/ml to 100 mg/ml. Streptococcus pyogenes strains were less sensitive, showing zones of inhibition from 9.0mm to 13mm at the same concentrations. The MIC and MBC tests revealed that the mixture had a good antibacterial effect on Salmonella typhi, with MIC and MBC values of 25 mg/ml and 100 mg/ml, respectively. For Streptococcus pyogenes, the MIC was 100 mg/ml, but the MBC was not applicable. These results suggest that the mixture is bacteriostatic at lower concentrations and bactericidal at higher concentrations. This supports the traditional use of large quantities of extracts by traditional practitioners for treating infections [15]. CONCLUSION The study investigated the antibacterial activity and minimum inhibitory concentration of the antibiogram mixture of Vernonia amygdalina and Cocos nucifera water. Phytochemical analysis confirmed the presence of alkaloids, flavonoids, tannins, anthraquinones, saponins, terpenoids, and carbohydrates in both plants, with anthraquinones found only in Vernonia amygdalina. The mixture exhibited a high potential to inhibit Salmonella typhi but was less effective against Streptococcus pyogenes, supporting its traditional use in therapy. This study highlights the potential application of these plants as alternative antibacterial agents. Based on the findings of this research, the following recommendations are made for the development of new antibacterial drugs: 1. Exploration by Pharmaceutical Companies: Pharmaceutical companies should investigate the antibacterial properties of the mixture of Vernonia amygdalina and Cocos nucifera water as a potential source of new antibacterial drugs. 2. Further Studies: Additional research should be conducted on these plants to uncover more of their potential in addressing gastrointestinal infections and other bacterial diseases. REFERENCES National Bureau of Statistics (NBS). (2019). Nigerian Gross Domestic Product Report. Retrieved from https://www.nigerianstat.gov.ng/ Aragon, C. M. (2000). Informal economy, informal sector, and legal rights. UNU/WIDER, Working Paper No. 2000/127. Sertoglu, K., Ugural, S., & Bekun, F. V. (2017). The causal nexus between economic growth and agricultural development: Evidence from BRICS and MINT countries. Eurasian Economic Review, 7 (3), 437-450. Lopes, H. (2010). Adoption of Improved Maize and Common Bean Varieties in Mozambique. Dissertation for Award of MARD degree at Sokoine University of Agriculture, Morogoro. Gandhi, M., & Singh, Y. (1989). On the Mythology of Indian Plants . Rupa & Co., New Delhi, India. Oseni, K., & Babatunde, O. (2016). Vernonia amygdalina: An Underutilized Vegetable with Nutraceutical Potentials – A Review. Turkish Journal of Agriculture – Food Science and Technology, 4 (9), 763-768. https://doi.org/10.24925/turjaf.v4i9.763-768.570 Nascimento, G. G. F., Locatelli, J., Freitas, P. C., & Silva, G. L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology, 31 , 247-256. Tongson, C. P. M., Davidson, P. M., Mahakarnchanakul, W., & Vibulsresth, P. (2005). Antimicrobial Effect of Thai Spices against Listeria monocytogenes and Salmonella typhimurium DTTO4. Journal of Food Protection, 68 , 2054-2058. Trease, G. E., & Evans, W. C. (1989). Pharmacognosy (11th ed.). Bailliere Tindall, London, 45-50. Harborne, J. B. (1984). Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis . Chapman and Hall. Cordell, G. A., Quinn-Beattie, M. L., & Farnsworth, N. R. (2001). The potential of alkaloids in drug discovery. Phytotherapy Research, 15 , 183-205. Tschesche, R. (1971). Advances in the Chemistry of Antibiotic Substances from Higher Plants. Journal of Pharmacognosy and Phytochemistry (pp. 274-289). Springer, Berlin, Heidelberg. Kassim, A., Omuse, G., Premji, Z., & Revathi, G. (2016). Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a University teaching hospital in Nairobi, Kenya: A cross-sectional study. African Journal of Laboratory Medicine, 5 (1), 15-21. Sihombing, D., Purba, R. H., & Sirait, R. S. (2012). The diversity of coconut in Indonesia: Collection, characterization, and utilization. Journal of Plantation Crops, 40 (3), 213-220. Akinyemi, K. O., Oluwa, O. K., & Omomigbehin, E. O. (2006). Antimicrobial Activity of Crude Extracts of Three Medicinal Plants Used in South-West Nigerian Folk Medicine on Some Food Borne Bacterial Pathogens. African Journal of Traditional, Complementary and Alternative Medicines, 3 (4), 13-22. Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE: In compliance with local guidelines, the collection of Vernonia amygdalina (Bitter leaf) and Cocos nucifera (Coconut) used in this study followed ethical standards and regulations. The plant material was collected from Bakindogo market in Kaduna, Nigeria. The identification and authentication of the plant were performed by a botanist in the Department of Biological Sciences, Kaduna State University. Furthermore, it's important to note that the plant material used in this study was cultivated rather than wild. No endangered or protected species were involved in this research. CONSENT FOR PUBLICATION: Not applicable AVAILABILITY OF DATA AND MATERIALS: As for the data availability statement, I confirm that all data generated or analyzed during this study are included in the published article. This consists of the phytochemical composition of the Vernonia amygdalina (Bitter leaf), Cocos nucifera (Coconut) water and the combination of the two, antibacterial activity results, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) values. COMPETING INTERESTS: The authors declare that they have no competing interests FUNDING: This research was funded by the first author; no any organization or society fund the research. AUTHORS' CONTRIBUTIONS: EOM carry out the experimental aspect of the research and also contributed to the write up, IAO supervised the experimental aspect and also contribute to the data presentation and the write up, AIU designed the study and also the major contributor in writing the manuscript data presentation and AAD also contributed by supervising the experimental aspect, analyzing the data and the write up. ACKNOWLEDGEMENTS: Not applicable Additional Declarations The authors declare no competing interests. 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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-4458581","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311322807,"identity":"6cd6e080-fbe0-4341-97f2-a5133dddeba0","order_by":0,"name":"Emmanuel Oluwatimileyin Matthew","email":"","orcid":"","institution":"Kaduna Polytechnic","correspondingAuthor":false,"prefix":"","firstName":"Emmanuel","middleName":"Oluwatimileyin","lastName":"Matthew","suffix":""},{"id":311322808,"identity":"5a513702-4787-49bd-9bbb-8bb8e2da2458","order_by":1,"name":"Dr. Ibrahim Abdulkarim Ozigi","email":"","orcid":"","institution":"Kaduna Polytechnic","correspondingAuthor":false,"prefix":"Dr.","firstName":"Ibrahim","middleName":"Abdulkarim","lastName":"Ozigi","suffix":""},{"id":311322809,"identity":"ad6f358b-9bc8-43f3-a94d-b68eb4c96881","order_by":2,"name":"Abdulgafar Ishola Uthman","email":"data:image/png;base64,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","orcid":"","institution":"Kaduna Polytechnic","correspondingAuthor":true,"prefix":"","firstName":"Abdulgafar","middleName":"Ishola","lastName":"Uthman","suffix":""},{"id":311322810,"identity":"abc6ef00-d805-4f08-bd15-29396c01db7a","order_by":3,"name":"ADEYERI DAVID ADEYELE","email":"","orcid":"","institution":"Kaduna Polytechnic","correspondingAuthor":false,"prefix":"","firstName":"ADEYERI","middleName":"DAVID","lastName":"ADEYELE","suffix":""}],"badges":[],"createdAt":"2024-05-22 06:18:48","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4458581/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-4458581/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61972089,"identity":"a1140fdc-1eda-43c1-b532-55032ab444eb","added_by":"auto","created_at":"2024-08-07 17:14:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":835562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458581/v2/2b69c814-64c2-4b9d-9040-f8dab7d27568.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePhytochemical screening of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCocos nucifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e And \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVernonia amygdalina\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e extract and antibiogram of the mixture (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCocos Nucifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e + \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVernonia Amygdalina\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSalmonella typhi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptococcus pyogenes\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eNigeria stands as a significant player in the global economy, recognized as the largest black nation. With a robust economic growth trajectory, Nigeria\u0026apos;s gross domestic product (GDP) soared from $170 billion in 2000 to $451 billion in 2012, albeit unofficial estimations suggest figures closer to $630 billion, inclusive of the informal sector [1][2]. The agricultural sector has consistently been a pivotal contributor to Nigeria\u0026apos;s economy, with a noteworthy 26.15% contribution to the GDP in 2017 (NBS, 2019). This sector serves as the cornerstone for economic advancement, development, and poverty alleviation, as emphasized by Sertoglu, Ugural, and Bekun [3], positioning agriculture as the catalyst for sustained economic prosperity.\u003c/p\u003e\n\u003cp\u003eIn Nigeria, agriculture encompasses a diverse array of staple and cash crops, with significant cultivation dedicated to crops such as cassava, yam, maize, sorghum, rice, palm kernel, and coconut, among others [2]. Notably, coconut, revered as the \u0026quot;tree of life,\u0026quot; holds immense economic potential, albeit receiving limited attention within Nigeria\u0026apos;s agricultural landscape [4]. Originating from Southeast Asia, coconut exhibits remarkable adaptability, thriving across various soil types and climates. Its utility extends beyond traditional consumption, offering a plethora of industrial applications that could bolster Nigeria\u0026apos;s industrial development and elevate living standards.\u003c/p\u003e\n\u003cp\u003eThe historical narratives surrounding the origin of coconut are diverse and often steeped in mythology. While Indian mythology attributes the creation of the coconut palm to the sage Vishwamitra, botanical evidence suggests its origin in Papua New Guinea or Malesia, with subsequent dispersal facilitated by natural phenomena and Polynesian voyagers. This dispersion underscores coconut\u0026apos;s resilience and capacity for widespread proliferation across diverse geographical regions [5].\u003c/p\u003e\n\u003cp\u003eAlongside coconut, bitter leaf (\u003cem\u003eVernonia amygdalina\u003c/em\u003e) emerges as a botanical asset in Nigeria\u0026apos;s agricultural bounty. This perennial shrub, renowned for its bitter-tasting leaves, harbors extensive medicinal properties, earning it recognition as a staple herb in traditional medicine across West Africa. Indigenous to various ecological zones, bitter leaf thrives in diverse environments, adapting to habitats ranging from forest margins to grasslands.\u003c/p\u003e\n\u003cp\u003eThe morphological characteristics of bitter leaf underscore its resilience and adaptability, with features such as soft woodiness, lanceolate leaves, and bisexual flowers contributing to its ecological versatility. Beyond its ecological significance, bitter leaf holds immense socio-economic value, particularly within Nigerian communities, where it is revered for its medicinal prowess and nutritional benefits a [6].\u003c/p\u003e\n\u003cp\u003eAmidst the backdrop of Nigeria\u0026apos;s agricultural abundance, concerns surrounding food safety and security persist. Bacterial infections continue to pose significant challenges, necessitating innovative approaches to antimicrobial research. The emergence of multidrug-resistant pathogens underscores the urgency for alternative therapeutic interventions, including the exploration of traditional herbal remedies [7][8].\u003c/p\u003e\n\u003cp\u003eIn light of these considerations, this study aims to conduct phytochemical screening of coconut water and bitter leaf extract, evaluating their antibacterial efficacy against Salmonella typhi and Streptococcus spp. By elucidating the phytochemical composition and antibiogram of this botanical mixture, we seek to contribute valuable insights into the potential therapeutic applications of traditional herbal remedies in combating bacterial infections.\u003c/p\u003e\n\u003cp\u003eIn summary, this research endeavors to bridge the gap between traditional botanical knowledge and contemporary biomedical research, offering a pathway towards sustainable healthcare solutions within the context of Nigeria\u0026apos;s rich agricultural heritage.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eCollection, Identification, and Authentication of the Plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBitter leaf and coconut were procured from Bakindogo market in Kaduna. Their identification and authentication were carried out by a botanist from the Department of Biological Science at Kaduna State University. The plants were washed using sterile water and dried at room temperature in the Department of Applied Biology at Kaduna Polytechnic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation and Testing of the Mixture (Combination) of Bitter Leaf and Coconut Water\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bitter leaf was washed and then squeezed into coconut water. The mixture was left to soak for three days to allow maceration. After soaking for three days, the mixture of bitter leaf and coconut water was periodically shaken to ensure thorough extraction. The macerated mixture was then filtered to separate the micelle from the marc. The micelle was subsequently separated from the menstruum by evaporation using a water bath.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Mixture Extract Concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVarious concentrations of the mixture extract were prepared. A stock solution of 100 mg/mL was made by dissolving 1g of the crude extract in 10 mL of sterile distilled water. From this stock solution, different concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, and 12.5 mg/mL) were prepared accordingly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhytochemical Screening of Crude Extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of bioactive compounds in the methanol extracts was determined by phytochemical tests using standard methods described by [9] and [10].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest for Carbohydrates (Molisch\u0026apos;s Test)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA few drops of Molisch\u0026apos;s reagent were added to 2 mL of the extract solution, followed by the addition of 1 mL of concentrated sulfuric acid down the side of the test tube. Changes in coloration at the interface of the two liquids were noted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest for Tannins\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eA few drops of 0.1% ferric chloride solution were added to the extract solutions, and changes were observed.\u003c/li\u003e\n \u003cli\u003eAdditionally, a few drops of lead acetate solution were added to the extract solutions, and changes were observed.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eTest for Alkaloids\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eMeyer\u0026apos;s reagent and Two drops of dilute sulfuric acid were added to 1 mL of the extract solution, and observed changes were noted.\u003c/li\u003e\n \u003cli\u003eAnother 1 mL portion of the extract was reacted with Dragendorff\u0026apos;s reagent, and changes in the product formed were observed and recorded.\u003c/li\u003e\n \u003cli\u003eAn affirmation test for the presence of alkaloids was performed by adding two drops of Wagner\u0026apos;s reagent, and observed changes were recorded.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eTest for Saponins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA frothing test was performed by adding 5 mL of de-ionized water to the filtrate of the extracts and shaking vigorously to observe the formation of stable persistent frothing. This frothing was observed for up to 45 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest for Flavonoids\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eShinoda Test: Filtrates of the extracts (3 mL) were obtained, and four pieces of magnesium fillings were added, followed by a few drops of concentrated hydrochloric acid. Observed color changes were noted.\u003c/li\u003e\n \u003cli\u003eSodium Hydroxide Test: 5 mL of 10% sodium hydroxide was added to an equal volume of the extracts. Observed color changes were noted accordingly.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eTest for Terpenoids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiebermann-Burchard Test: Chloroform was added to the extract, mixed with 1 mL of acetic anhydride, and followed by the addition of 1 mL of concentrated sulfuric acid down the wall of the test tube. Observed changes were noted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest for Anthraquinones\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBorntr\u0026auml;ger\u0026rsquo;s Test: 0.5 g of crude extracts was added to 10 ml of chloroform, shaken, and then filtered. Ten ml of the filtrate was reacted with an equal volume of ammonia (10 mL) and shaken gently. Observed changes were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization and Authentication of Test Organisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical isolates of \u003cem\u003eSalmonella\u003c/em\u003e typhi and \u003cem\u003eStreptococcus\u003c/em\u003e spp. were obtained from 44 Army Reference Hospital in Kaduna State, Nigeria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStorage and Maintenance of Cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cultures were streaked onto Nutrient Agar slants and incubated at 37\u0026deg;C for 48 hours. These cultures were stored in a refrigerator at 4\u0026deg;C for subsequent use. Stock cultures were prepared and kept until needed. Broth cultures were prepared from these organisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation and Standardization of Inocula\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNutrient broth was prepared according to the manufacturer\u0026rsquo;s specifications. The bacterial isolates were tested for sterility on Nutrient Agar, then re-grown in Nutrient broth at 37\u0026deg;C for 24 hours. McFarland\u0026apos;s standard method was used to standardize the organisms to 1x10⁴ cfu/mL by preparing four sets of dilution tubes with 9 mL of normal saline and one tube with 4 mL of normal saline, labeled 10⁻\u0026sup1; to 10⁻⁴. 1 mL of the overnight culture was added to the 10⁻\u0026sup1; dilution tube, and serial dilutions were prepared to arrive at a dilution of 10⁻⁴ cfu/mL. Dilution with sterile Nutrient broth was done until the optical density matched that of McFarland\u0026apos;s standard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial Susceptibility Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNutrient agar was prepared according to the manufacturer\u0026rsquo;s specifications, and the molten agar was poured into sterile disposable plates and allowed to cool to about 45\u0026deg;C. The plates were swabbed with diluted standardized overnight cultures. Six mm Whatmann No. 1 paper discs, each soaked in the extract concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, and 12.5 mg/mL), coconut water, and bitter leaf juice, were placed on the agar plates. A standard antibiotic (200 mg/10 mL) was used as a positive control, and sterile distilled water was used as a negative control on a separate plate. Diameters of zones of inhibition were measured after incubating the plates at 25\u0026deg;C for 48 hours. The plates were replicated in triplicate, and the mean zones of inhibition for each organism at each extract concentration were measured using a ruler and recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Minimum Inhibitory Concentration (MIC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2 ml of all the four-fold serial dilutions were used to impregnate sterile disks with a diameter of 5 mm. These impregnated disks were used to determine the MIC by placing them on the already streaked isolates of \u003cem\u003eSalmonella\u003c/em\u003e typhi and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e and incubated at 37\u0026deg;C for 24 hours. The zone of inhibition was measured using a ruler and recorded according to the concentration incorporated on the disk.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Minimum Bactericidal Concentration (MBC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MBC was determined by sub-culturing the prepared dilutions onto fresh Nutrient agar and further incubating for 24 hours at 37\u0026deg;C. These plates were observed for growth. The plates with the least concentration showing no growth were considered the MBC.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003ePhytochemical Constituents of Vernonia amygdalina, Cocos nucifera Water, and Their Mixture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparative phytochemical screening of Vernonia amygdalina, Cocos nucifera water, and their mixture revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, and carbohydrates in all samples. Anthraquinones were present only in Vernonia amygdalina.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eTable 1: Phytochemical Composition of Bioactive Compounds\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable cellspacing=\"3\" style=\"border: none;width:440.3pt;background:white;\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0.75pt;height: 38.15pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePhytochemical Components\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0.75pt;height: 38.15pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eCocos nucifera Water\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0.75pt;height: 38.15pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eVernonia amygdalina\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0.75pt;height: 38.15pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eVernonia amygdalina and Cocos nucifera Water Mixture\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eAlkaloids\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eSaponins\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eTannins\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eFlavonoids\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eTerpenoids\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eAbsent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eAnthraquinones\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eAbsent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"padding: 0.75pt;height: 22.9pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;height: 23.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eCarbohydrates\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;height: 23.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;height: 23.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;height: 23.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePresent\u003c/span\u003e\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\u003eBacterial Susceptibility Standard of Amoxil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eTable 2: Susceptibility Standard of Amoxil\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable cellspacing=\"3\" style=\"width:260.6pt;margin-left:-.4pt;background:white;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.2pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;height: 19.5pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eR\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;height: 19.5pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eI\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69.75pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;height: 19.5pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 96.2pt;border: none;padding: 0.75pt;height: 19.5pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026le;13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88.65pt;border: none;padding: 0.75pt;height: 19.5pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e14-16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69.75pt;border: none;padding: 0.75pt;height: 19.5pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026ge;17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;R\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e: Resistant \u0026nbsp; \u0026nbsp;\u003cstrong\u003eI\u003c/strong\u003e: Intermediate \u0026nbsp; \u003cstrong\u003eS\u003c/strong\u003e: Susceptible\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e\u0026le;\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e: less than or equal to \u0026nbsp; \u0026nbsp; \u003cstrong\u003e\u0026ge;\u003c/strong\u003e: greater than or equal to\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial Activity of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibiogram of the mixture of Vernonia amygdalina and Cocos nucifera water was found to be effective against Salmonella typhi and Streptococcus pyogenes, showing zones of inhibition as summarized in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eTable 3: Antimicrobial Activity of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable cellspacing=\"3\" style=\"width:449.6pt;background:white;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eTest Organisms\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 209.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eConcentration (mg/ml)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eZone of Inhibition (mm)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003ePositive control (500 mg/mL)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eAmoxicillin\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eReaction\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e50\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e1st isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;height: 2.6pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e2nd isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;height: 2.6pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;height: 2.6pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;height: 2.6pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;height: 2.6pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;height: 2.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;height: 2.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e3rd isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e4th isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e12\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9.5\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e1st isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e11\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e2nd isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e11\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e3rd isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e11\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e4th isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.55pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e13\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e11\u0026plusmn;0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e9\u0026plusmn;0.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.5pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46.85pt;border: none;padding: 0.75pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cul style=\"margin-bottom:0in;margin-top:0in;\" type=\"disc\"\u003e\n \u003cli style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eS\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e: Susceptible\u003c/span\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMinimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Mixture on Salmonella typhi and Streptococcus pyogenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MIC was determined using the agar disk diffusion and macro tube dilution methods, while the MBC was determined by sub-culturing the prepared dilutions into fresh media. The results in Table 4 show that the mixture of Vernonia amygdalina and Cocos nucifera water has significant antibacterial effects on Salmonella typhi at various concentrations. Streptococcus pyogenes showed partial sensitivity at 100 mg/ml.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eTable 4: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable cellspacing=\"3\" style=\"width:310.1pt;background:white;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eTest Organisms\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eMIC (mg/ml)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eMBC (mg/ml)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e1st isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e2nd isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e3rd isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e4th isolate of S. typhi\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e1st isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e2nd isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e3rd isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145.85pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e4th isolate of S. pyogenes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83.25pt;border: none;padding: 0.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:115%;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cul style=\"margin-bottom:0in;margin-top:0in;\" type=\"disc\"\u003e\n \u003cli style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:115%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eNA\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e: Not applicable\u003c/span\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePhytochemical screening of \u003cem\u003eVernonia amygdalina\u003c/em\u003e leaf extract and \u003cem\u003eCocos nucifera\u003c/em\u003e water (Table 1) revealed the presence of alkaloids, flavonoids, tannins, anthraquinones, saponins, terpenoids, and carbohydrates, while anthraquinones were present only in Vernonia amygdalina. Studies have shown that alkaloids and flavonoids are key compounds responsible for the antibacterial activities in higher plants [11]. Saponins, tannins, and alkaloids are well-documented plant metabolites known for their antimicrobial properties [12]. The presence of these phytochemicals in the mixture may contribute to its antibacterial activity, supporting their traditional use in treating various bacterial and fungal infections.\u003c/p\u003e\n\u003cp\u003eThe antibacterial susceptibility test showed that all strains of the two clinical isolates, Salmonella typhi and Streptococcus pyogenes (Table 2), were highly susceptible to Amoxicillin (control). This observation aligns with the findings of Kassim et al. [13]. Salmonella typhi exhibited higher sensitivity to Amoxicillin with a zone of inhibition ranging from 18mm to 23mm, compared to Streptococcus pyogenes, which showed a range of 17mm to 20mm.\u003c/p\u003e\n\u003cp\u003eThe mixture of Vernonia amygdalina and Cocos nucifera water showed a broad-spectrum antibacterial activity, with efficacy increasing with higher concentrations. Salmonella typhi strains were highly sensitive to the mixture, with zones of inhibition ranging from 9.5mm to 17mm at concentrations of 12.5 mg/ml to 100 mg/ml. Streptococcus pyogenes strains were less sensitive, showing zones of inhibition from 9.0mm to 13mm at the same concentrations.\u003c/p\u003e\n\u003cp\u003eThe MIC and MBC tests revealed that the mixture had a good antibacterial effect on Salmonella typhi, with MIC and MBC values of 25 mg/ml and 100 mg/ml, respectively. For Streptococcus pyogenes, the MIC was 100 mg/ml, but the MBC was not applicable. These results suggest that the mixture is bacteriostatic at lower concentrations and bactericidal at higher concentrations. This supports the traditional use of large quantities of extracts by traditional practitioners for treating infections [15].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe study investigated the antibacterial activity and minimum inhibitory concentration of the antibiogram mixture of Vernonia amygdalina and Cocos nucifera water. Phytochemical analysis confirmed the presence of alkaloids, flavonoids, tannins, anthraquinones, saponins, terpenoids, and carbohydrates in both plants, with anthraquinones found only in Vernonia amygdalina. The mixture exhibited a high potential to inhibit Salmonella typhi but was less effective against Streptococcus pyogenes, supporting its traditional use in therapy. This study highlights the potential application of these plants as alternative antibacterial agents.\u003c/p\u003e\n\u003cp\u003eBased on the findings of this research, the following recommendations are made for the development of new antibacterial drugs:\u003c/p\u003e\n\u003cp\u003e1. Exploration by Pharmaceutical Companies: Pharmaceutical companies should investigate the antibacterial properties of the mixture of Vernonia amygdalina and Cocos nucifera water as a potential source of new antibacterial drugs.\u003c/p\u003e\n\u003cp\u003e2. Further Studies: Additional research should be conducted on these plants to uncover more of their potential in addressing gastrointestinal infections and other bacterial diseases.\u003c/p\u003e"},{"header":"REFERENCES","content":"\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003eNational Bureau of Statistics (NBS). (2019). Nigerian Gross Domestic Product Report. Retrieved from\u0026nbsp;\u003ca href=\"https://www.nigerianstat.gov.ng/\" target=\"_new\"\u003ehttps://www.nigerianstat.gov.ng/\u003c/a\u003e\u003c/li\u003e\n \u003cli\u003eAragon, C. M. (2000). Informal economy, informal sector, and legal rights. UNU/WIDER, Working Paper No. 2000/127.\u003c/li\u003e\n \u003cli\u003eSertoglu, K., Ugural, S., \u0026amp; Bekun, F. V. (2017). The causal nexus between economic growth and agricultural development: Evidence from BRICS and MINT countries. \u003cem\u003eEurasian Economic Review, 7\u003c/em\u003e(3), 437-450.\u003c/li\u003e\n \u003cli\u003eLopes, H. (2010). Adoption of Improved Maize and Common Bean Varieties in Mozambique. Dissertation for Award of MARD degree at Sokoine University of Agriculture, Morogoro.\u003c/li\u003e\n \u003cli\u003eGandhi, M., \u0026amp; Singh, Y. (1989). \u003cem\u003eOn the Mythology of Indian Plants\u003c/em\u003e. Rupa \u0026amp; Co., New Delhi, India.\u003c/li\u003e\n \u003cli\u003eOseni, K., \u0026amp; Babatunde, O. (2016). Vernonia amygdalina: An Underutilized Vegetable with Nutraceutical Potentials \u0026ndash; A Review. \u003cem\u003eTurkish Journal of Agriculture \u0026ndash; Food Science and Technology, 4\u003c/em\u003e(9), 763-768.\u0026nbsp;\u003ca href=\"https://doi.org/10.24925/turjaf.v4i9.763-768.570\" target=\"_new\"\u003ehttps://doi.org/10.24925/turjaf.v4i9.763-768.570\u003c/a\u003e\u003c/li\u003e\n \u003cli\u003eNascimento, G. G. F., Locatelli, J., Freitas, P. C., \u0026amp; Silva, G. L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. \u003cem\u003eBrazilian Journal of Microbiology, 31\u003c/em\u003e, 247-256.\u003c/li\u003e\n \u003cli\u003eTongson, C. P. M., Davidson, P. M., Mahakarnchanakul, W., \u0026amp; Vibulsresth, P. (2005). Antimicrobial Effect of Thai Spices against Listeria monocytogenes and Salmonella typhimurium DTTO4. \u003cem\u003eJournal of Food Protection, 68\u003c/em\u003e, 2054-2058.\u003c/li\u003e\n \u003cli\u003eTrease, G. E., \u0026amp; Evans, W. C. (1989). \u003cem\u003ePharmacognosy\u003c/em\u003e (11th ed.). Bailliere Tindall, London, 45-50.\u003c/li\u003e\n \u003cli\u003eHarborne, J. B. (1984). \u003cem\u003ePhytochemical Methods: A Guide to Modern Techniques of Plant Analysis\u003c/em\u003e. Chapman and Hall.\u003c/li\u003e\n \u003cli\u003eCordell, G. A., Quinn-Beattie, M. L., \u0026amp; Farnsworth, N. R. (2001). The potential of alkaloids in drug discovery. \u003cem\u003ePhytotherapy Research, 15\u003c/em\u003e, 183-205.\u003c/li\u003e\n \u003cli\u003eTschesche, R. (1971). Advances in the Chemistry of Antibiotic Substances from Higher Plants. \u003cem\u003eJournal of Pharmacognosy and Phytochemistry\u003c/em\u003e (pp. 274-289). Springer, Berlin, Heidelberg.\u003c/li\u003e\n \u003cli\u003eKassim, A., Omuse, G., Premji, Z., \u0026amp; Revathi, G. (2016). Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic susceptibility at a University teaching hospital in Nairobi, Kenya: A cross-sectional study. \u003cem\u003eAfrican Journal of Laboratory Medicine, 5\u003c/em\u003e(1), 15-21.\u003c/li\u003e\n \u003cli\u003eSihombing, D., Purba, R. H., \u0026amp; Sirait, R. S. (2012). The diversity of coconut in Indonesia: Collection, characterization, and utilization. \u003cem\u003eJournal of Plantation Crops, 40\u003c/em\u003e(3), 213-220.\u003c/li\u003e\n \u003cli\u003eAkinyemi, K. O., Oluwa, O. K., \u0026amp; Omomigbehin, E. O. (2006). Antimicrobial Activity of Crude Extracts of Three Medicinal Plants Used in South-West Nigerian Folk Medicine on Some Food Borne Bacterial Pathogens. \u003cem\u003eAfrican Journal of Traditional, Complementary and Alternative Medicines, 3\u003c/em\u003e(4), 13-22.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE:\u0026nbsp;\u003c/strong\u003eIn compliance with local guidelines, the collection of\u0026nbsp;\u003cem\u003eVernonia amygdalina\u003c/em\u003e (Bitter leaf) and \u003cem\u003eCocos nucifera\u003c/em\u003e (Coconut) used in this study followed ethical standards and regulations. The plant material was collected from Bakindogo market in Kaduna, Nigeria. The identification and authentication of the plant were performed by a botanist in the Department of Biological Sciences, Kaduna State University.\u003c/p\u003e\n\u003cp\u003eFurthermore, it\u0026apos;s important to note that the plant material used in this study was cultivated rather than wild. No endangered or protected species were involved in this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS:\u0026nbsp;\u003c/strong\u003eAs for the data availability statement, I confirm that all data generated or analyzed during this study are included in the published article. This consists of the phytochemical composition of the\u0026nbsp;\u003cem\u003eVernonia amygdalina\u003c/em\u003e (Bitter leaf), \u003cem\u003eCocos nucifera\u003c/em\u003e (Coconut) water and the combination of the two, antibacterial activity results, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) values.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u003cstrong\u003eCOMPETING INTERESTS:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING:\u0026nbsp;\u003c/strong\u003eThis research was funded by the first author; no any organization or society fund the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026apos; CONTRIBUTIONS:\u0026nbsp;\u003c/strong\u003eEOM carry out the experimental aspect of the research and also contributed to the write up, IAO supervised the experimental aspect and also contribute to the data presentation and the write up, AIU designed the study and also the major contributor in writing the manuscript data presentation and AAD also contributed by supervising the experimental aspect, \u0026nbsp;analyzing the data and the write up.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\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":"Vernonia amygdalina, Cocos nucifera, Phytochemical screening, Antibacterial activity, Salmonella typhi, Streptococcus pyogenes, Herbal remedies and Antibiotic resistance","lastPublishedDoi":"10.21203/rs.3.rs-4458581/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4458581/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eNigeria, a significant player in the global economy, has a rich agricultural heritage that includes diverse crops with potential medicinal properties. Among these, coconut (\u003cem\u003eCocos nucifera\u003c/em\u003e) and bitter leaf (\u003cem\u003eVernonia amygdalina\u003c/em\u003e) are notable for their traditional medicinal uses. This study aimed to evaluate the phytochemical composition and antibacterial efficacy of extracts from these plants, individually and in combination, against Salmonella typhi and Streptococcus pyogenes, addressing the need for alternative antibacterial therapies in the face of rising antibiotic resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Bitter leaf and coconut were sourced from Bakindogo market, Kaduna, and authenticated by a botanist. Extracts were prepared and subjected to phytochemical screening using standard methods. Antibacterial activity was evaluated through agar disk diffusion to measure zones of inhibition at different extract concentrations. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for both \u003cem\u003eSalmonella typhi\u003c/em\u003e and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e.Top of FormBottom of Form\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003ePhytochemical analysis revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, and carbohydrates in both extracts, with anthraquinones present only in Vernonia amygdalina. The extracts showed significant antibacterial activity, particularly the mixture of Vernonia amygdalina and Cocos nucifera water. The mixture exhibited higher efficacy against Salmonella typhi, with zones of inhibition ranging from 9.5 mm to 17 mm, compared to Streptococcus pyogenes, which showed inhibition zones from 9.0 mm to 13 mm. The MIC for Salmonella typhi was 25 mg/mL, with an MBC of 100 mg/mL, indicating bactericidal properties at higher concentrations. Streptococcus pyogenes showed an MIC of 100mg/ml, but the MBC was not applicable, suggesting bacteriostatic effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eThe study confirms the significant antibacterial potential of Vernonia amygdalina and Cocos nucifera water extracts, particularly against Salmonella typhi. These findings support their traditional use and highlight their potential as alternative antibacterial agents. Further research and pharmaceutical exploration are recommended to fully harness these plants' medicinal properties, particularly for developing new treatments for bacterial infections.\u003c/p\u003e","manuscriptTitle":"Phytochemical screening of Cocos nucifera And Vernonia amygdalina extract and antibiogram of the mixture (Cocos Nucifera + Vernonia Amygdalina) on Salmonella typhi and Streptococcus pyogenes","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-08-07 17:06:38","doi":"10.21203/rs.3.rs-4458581/v2","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}},{"code":1,"date":"2024-06-06 11:24:45","doi":"10.21203/rs.3.rs-4458581/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":"f512a5fc-ff89-4c6d-aa91-09af892c69f3","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35121379,"name":"Drug Discovery, Design, \u0026 Development"},{"id":35121380,"name":"General Microbiology"}],"tags":[],"updatedAt":"2024-07-23T04:39:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-07 17:06:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-4458581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4458581","identity":"rs-4458581","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}