RJHS 11(2).cdr Antimicrobial, phytochemical and pharmacological properties of Phyllanthus niruri linn 1 2 3 *Oyekanmi, B. A. , Osho, I. B. , Kolawole J. C. Abstract Introduction: Phyllanthus niruri is a common herb widely used in home remedies against infectious agents. This study unveils the antimicrobial and therapeutic potentials of P. niruri against Escherichia coli infection. Methods: Ethanol and water extracts of the plant were prepared and investigated for their antimicrobial activity using the agar well diffusion method against eleven clinical isolates. The in vivo study was conducted on albino rats, infected and subsequently treated. Results: The observation showed Ciprofloxacin with the highest (41 mm) sensitivity against P. mirabilis; and lowest (20 mm) against S. flexneri, E. coli, K. pneumoniae, and S. aureus. Ketoconazole at 100 mg/mL concentration revealed antifungal sensitivity ranging from 2 to 15 mm. The extracts showed better sensitivity against the bacteria (2 to 24) mm when compared with the fungi species (2 to 12 mm). Phyllanthus niruri extract demonstrated a minimum inhibitory concentration of 25 to 100 mg/mL The albino rats of weight ranging from 83 to 105 g were administered with P. niruri ethanol extract but indicated no toxicity at 1500 mg dose. The packed cell volume, red cell count, total leucocyte count, and serum enzymes of the tested rats were within the normal range. The healing effect was dose-dependence and most effective from 1200 mg/kg to 1500 mg/kg body weight. P. niruri extract produced some level of antimicrobial activity both in vitro and in vivo. Conclusion: Extract from Phyllanthus niruri is effective in vitro and in vivo against Escherichia coli infection. It has no ill effect on the blood circulatory system, liver, and kidney. The bioactive agent present in the extract has proven health benefits, and can be administered as supplement. However, more studies on its chronic toxicity are required. *Corresponding Author Oyekanmi, B.A. Email: bolapeoyekanmi@yahoo.com 1 Department of Medical Laboratory Science, Adeleke University, Ede, Nigeria 2 Department of Animal Production and Health, Federal University of Technology, Akure, Nigeria. 3 Department of Biochemistry, Obafemi Awolowo University, Ile- Ife, Nigeria Received: September 7, 2022 Accepted: February 12, 2023 Published: June 30, 2023 Original Article Research Journal of Health Sciences Res. J. Health Sci. Vol 11(2), June 2023 108 Research Journal of Health Sciences subscribed to terms and conditions of Open Access publication. Articles are distributed under the terms of Creative Commons Licence (CC BY-NC-ND 4.0). (http://creativecommons.org/licences/by-nc-nd/4.0). http://dx.doi.org/10.4314/rejhs.v11i2.4 Propriétés antimicrobiennes, phytochimiques et pharmacologiques du phyllanthusniruri linn 1 2 3 *Oyekanmi, B. A. , Osho, I. B. , Kolawole J. C. Résumé Objectif de l'étude: foyer contre les agents infectieux. Cette étude dévoile les potentiels antimicrobiens et thérapeutiques de P. niruri contre l'infection à Escherichia coli. Méthode de l'étude: Des extraits d'éthanol et d'eau de la plante ont été préparés et étudiés pour leur activité antimicrobienne en utilisant la méthode de diffusion en puits d'agar contre onze isolats cliniques. L'étude in vivo a été menée sur des rats albinos, infectés puis traités. Résultat de l'étude: L'observation a montré la ciprofloxacine avec la sensibilité la plus élevée (41 mm) contre P. mirabilis ; et le plus bas (20 mm) contre S. flexneri , E. coli, K. pneumoniae et S. aureus . Le kétoconazole à une concentration de 100 mg/mL a révélé une sensibilité antifongique allant de 2 à 15 mm. Les extraits ont montré une meilleure sensibilité contre les bactéries (2 à 24) mm par rapport aux espèces de champignons (2 à 12 mm). L'extrait de Phyllanthusniruri a démontré une concentration minimale inhibitrice de 25 à 100 mg/mL. Les rats albinos de poids allant de 83 à 105 g ont reçu de l'extrait d'éthanol de P. niruri mais n'ont indiqué aucune toxicité à la dose de 1500 mg. L'hématocrite, le nombre de globules rouges, le nombre total de leucocytes et les enzymes sériques des rats testés se situaient dans la plage normale. L'effet curatif était dose-dépendant et le plus efficace de 1200 mg/kg à 1500 mg/kg de poids corporel. L'extrait de P. niruri a produit un certain niveau d'activité antimicrobienne à la fois in vitro et in vivo. L'agent bioactif présent dans l'extrait a des effets bénéfiques prouvés sur la santé et peut être administré en complément. Déclaration importante : L'extrait de Phyllanthusniruri est efficace in vitro et in vivo contre l'infection par Escherichia coli. Il n'a aucun effet néfaste sur le système circulatoire sanguin, le foie et les reins. Cependant, d'autres études sur sa toxicité chronique sont nécessaires. Received: September 7, 2022 Accepted: February 12, 2023 Published: June 30, 2023 Phyllanthusniruri est une herbe commune largement utilisée dans les remèdes de *Corresponding Author Oyekanmi, B.A. Email: bolapeoyekanmi@yahoo.com 1 Department of Medical Laboratory Science, Adeleke University, Ede, Nigeria 2 Department of Animal Production and Health, Federal University of Technology, Akure, Nigeria. 3 Department of Biochemistry, Obafemi Awolowo University, Ile- Ife, Nigeria Article Original Research Journal of Health Sciences Res. J. Health Sci. Vol 11(2), June 2023 109 Research Journal of Health Sciences subscribed to terms and conditions of Open Access publication. Articles are distributed under the terms of Creative Commons Licence (CC BY-NC-ND 4.0). (http://creativecommons.org/licences/by-nc-nd/4.0). http://dx.doi.org/10.4314/rejhs.v11i2.4 INTRODUCTION Complications due to drug-resistant and epidemics caused by microbial organisms of unknown origins such as bacteria are still a leading cause of death worldwide. The situation has called for improved strategies and has led to the continuous search for antimicrobials and drug development from natural sources. This approach will enhance the treatment and prevention of life- threatening diseases (1). Assessment of plants for their healing potentials and toxicology could give knowledge and data of how plants and their extracts can be of maximal benefit for the treatment. Some researchers have reported the usefulness of some Phyllanthus species in medicine. Phyllanthus contains secondary metabolites that have been employed in traditional cures (2, 3). Phyllantin, flavonoids, alkaloids, tannins, terpenes, and sterols are some of the biochemical components inherent in Phyllanthus. Extracts from this herb possess anti- viral and anti-hypertensive action (4). Phyllanthus niruri L. (Euphorbiaceae) is known for its ability to protect the liver (5). The a n t i m i c r o b i a l p r o p e r t i e s a n d o t h e r pharmaceutical principles have not been fully exhausted. This study evaluates the antibacterial, antifungal, and therapeutic effects of the plant ag ain s t Es ch er ich ia co li. Th er ef o r e th e worldwide use of Phyllanthus demands the need to estimate the pharmacological efficacy and safety of the extract. MATERIALS AND METHODS Source of test organisms Clinical isolates were obtained from the Microbiology Department of Ladoke Akintola University of Technology Teaching Hospital, Osogbo. Each microbial isolate was confirmed using cultural characteristics and standard biochemical tests. The bacterial isolates were Staphylococcus aureus, Salmonella typhimirum, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, and Streptococcus viridian. The fungi isolates were Candida albicans, Aspergillus flavus, and Aspergillus niger. Preparation of extracts from P. niruri Fresh plant was harvested from various places in Osogbo, South West Nigeria. A fresh sample of the plant was identified and authenticated by a Botanist at the Botany department, Ife Herbarium, Obafemi Awolowo University, Ile-Ife, Osun State (Herbarium number: IFE 17292). The whole plant including the stem, root, leaves together with the seeds were o carefully picked, washed and air dried at 28 C. Aqueous and ethanol extract of the whole plant was obtained using the cold extraction method at ratio 1: 10 of ground powder to extracting o solution. It was soaked for 48 h at 28 C. The supernatant was filtered using muslin cloth and filtrate concentrated in vacuo in a rotary evaporator, then evaporated to dryness in a clean o laboratory oven at 40 C. E v a l u a t i o n o f e x t r a c t s f o r i n v i t r o antimicrobial potentials Agar plate well diffusion method described by Mounyret al., (6) was employed to evaluate the antibacterial assay, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts. Five micrograms (5µg) ciprofloxacin and 100 mg/L ketoconazole were used as control for antibacterial and antifungal respectively. Analysis of phytochemicals inherent in the extracts Ethanol and water extracts of P. niruri were analyzed for their phytochemicals: cardiac glycosides, steroids, tannins, flavonoids, and terpenes (7, 8). E v a l u a t i o n o f e x t r a c t s f o r i n v i t r o antimicrobial potentials The agar - plate well diffusion method (6) was employed to evaluate the plant extracts for antibacterial assay. Minimum inhibitory concentration (MIC) and MBC of ethanol extract were also determined as described by Mounyr et al. (6). Standard antibacterial and antifungal used were 5µg ciprofloxacin and 100 mg/mL ketoconazole respectively. Handling of animals and experimental design Thirty (30) healthy Wistar albino rats of weight 82.0 g to 92.2 g were purchased from the animal house of the College of Health Sciences, Obafemi Awolowo University, Ile- Ife, Osun State in Nigeria. The animals were housed in plastic cages and fed with commercially produced standard rat pellets once a day and clean drinking water. The beddings were laid with wood powder and changed daily, feeding utensils, troughs, and the environment were maintained clean. The room temperature was at o 25 C and the light was 12 h light and 12 h darkness. Animal maintenance and handling were humane and followed the National Institute of Health (NIH) animal care guidelines (9). Res. J. Health Sci. Vol 11(2), June 2023 110 Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al. The experiment was conducted in a Completely Randomized Design (CRD). The Wistar rats were distributed into 6 groups (n = 5) with an equal number of sexes in each group, and the sample size was determined in advance. They were allowed to acclimatize in 7 days before the commencement of the experiment. Group CI N was the normal control, not inoculated, and received feed and clean water. Group CII P was a positive control, inoculated and administered with a standard antibacterial (Ciprofloxacin) group 2 to group 5 were inoculated and administered with 300 mg, 600 mg, 1200 mg, and 1500 mg doses of P. niruri ethanol extract respectively. The extract was administered via oral route. Inoculation and Evaluation of Graded doses of P. niruri ethanol extract on albino rats. Escherichia coli obtained from the clinical sample was inoculated into physiological o saline. The suspension was incubated at 37 C for 24 h to obtain a bacterial suspension equivalent to 6 1 × 10 CFU. A 0.30 ml of the actively growing bacterial suspension was administered via an oral route. Studies showed that LD50 of ethanol extract of P. niruri is greater than 5 kg per body weight (10) After 3 days, groups 2 to 5 were administered with a graded dose of P. niruri ethanol extract (300, 600, 1200, and 1500 mg/kg body weight) respectively for 14 days. A Ciprofloxacin (Cip) tablet, (brand: Fidson) was obtained from a registered Pharmaceutical shop. A sterile suspension of 1.34 mg/ml Cip was prepared and administered to the positive control group for 7 days. Microbiological culture of faecal sample obtained from the rats. The faecal sample of the rats was collected in a clean universal bottle after three days of bacterial inoculation, 7 days and 14 days of administration of the ethanol extract. The aseptic condition was maintained throughout the experiment. A 0.5 g of the stool sample was o inoculated in Selenite F, incubated at 37 C for 24 h then subcultured on MacConkey and Eosin Methylene Blue agar using standard operating procedures (11). Biochemical and haematological analysis of blood samples obtained from the treated rats The Wistar rats were starved for 12 h, sacrificed by the cervical dislocation method, and blood was withdrawn from the heart under an aseptic condition. The needle was removed and blood was released in a clean dry plain bottle and an EDTA (Ethylene diaminetetraacetic acid) bottle. The blood sample in a clean dry plain bottle was allowed to clot and spun at 3000 rpm for 10 minutes. The serum was obtained, labelled, and used to conduct biochemical analysis for Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), and Creatinine using enzyme maker kits from Randox (11). The blood sample released in an Ethylene diaminetetraacetic acid (EDTA) bottle was gently mixed on a haematology roller and analysed for Packed cell volume (PCV), Leucocyte total count, Leucocyte differential count, Neutrophil (N), Lymphocyte (L), Eosinophil (E), Monocyte (M), and Basophil (B) in percentage as described by Cheesebrough (11). Statistical Data The results of replicates were pooled and expressed as Mean ± Standard error of mean. Data obtained were subjected to a two-way analysis of variance and treatment means were compared using Duncan's new multiple-range tests with the aid of Statistical Package for Social Sciences (SPSS) software, version 17. Differences between treated groups were considered significant at P ≤ 0.05. RESULTS The aqueous and ethanol extracts of Phyllanthus niruri were bitter and acidic with a pH of 6. Quantitatively ethanol extract of Phyllanthus niruri revealed cardiac glycosides as the highest phytochemical compound, followed by steroids, tannin, flavonoids, and terpenes in decreasing order. (Table 1) In the aqueous extract cardiac glycosides was also of the highest concentration, followed by flavonoids, then tannins in decreasing order and the least was steroid; terpenes were not present. In Table 2, the standard antibiotics (5 µg ciprofloxacin) showed antibacterial activity with an inhibitory zone ranging from 20 to 41 mm and no significant resistance against P. aerugenosa. Aqueous extract of P. niruri.demonstrated a zone of inhibition ranging from 2 to 18 mm and no resistance to S. viridian and P. aerugenosa (Table 2). The ethanol extract of P. niruri showed an inhibitory zone ranging from 2 to 24 mm but no significant resistance against S. viridian only (Table 2). The ethanol extract of P. niruri. demonstrated better antibacterial resistance when compared with the aqueous extract P ≤ 0.05. Table 3 showed the highest antifungal activity in 100 mg/ml ketoconazole with the zone Res. J. Health Sci. Vol 11(2), June 2023 111 Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al. of inhibition ranging from 2 to 15 mm. Aqueous extract of P. niruri demonstrated antifungal sensitivity ranging from 2 to 12 mm while ethanol extract showed no significant antifungal sensitivity to any of the fungal isolates (Table 3). The ethanol extract of P. niruri showed MIC of 25 to 100 mg/ml and MMC (minimum microbicidal concentration) of 30 to 160 mg/ml. The Minimum inhibitory concentration demonstrated against A. flavus and A. niger were 50 and 100 mg/ml respectively while their minimum fungicidal concentration (MFC) was zero. In vivo testing was conducted on Wistar rats and Table 4 showed reduced activity and rough fur after three days (day 9) of exposure to E. coli infection; followed by improved activity and appetite for food from day 16 to day 23. The normal and positive control, groups 4 and 5 demonstrated a scanty growth while groups 2 and 3 showed a significant growth on day 23 (Table 5). The susceptibility of E. coli to the extract was dose-dependent. The haematology parameters of the experimental animals revealed no significant variation in the packed cell volume, red cell count, leucocyte count, lymphocyte, eosinophil, monocyte, and basophil P ≤ 0.05 (Table 6). Lymphocyte was of a higher percentage than neutrophil in test and control, the exception was group 4. The positive control showed the lowest values of the ALP, ALT, and Creatinine (45.33±3.48 µ/L, 28±6.56 µ/L, 88±7 µMOL/L); the negative control displayed the highest Creatinine level (Table 7). In the test groups, the ALP, ALT, and AST were at the least concentration in group 5. However, there was no statistical difference between the test and controls P ≤ 0.05 DISCUSSION Extracts of phyllanthus is extensively used in traditional medicine and was reported to have antibacterial and antiviral activity (12). Ethanol is revealed as a better extractive solvent of its phytochemicals for better yield of the biochemical compounds. This is because ethanol is generally able to dissolve multivariable types of compounds (13). Tannins, an effective compound present in moderate amount in P. niruri. are known antimicrobial biomolecules (14). Flavonoids are antioxidant and possess the ability to complex with bacteria cell wall (15). Moreover, Cardiac glycosides has some beneficiary effects but are toxic at a high level (16). Terpenes, has lipophylic character and are active against a wide variety of microorganisms (17) while steroids have anti- inflammatory action. The results of this study corroborate previous workers' findings that the plant contains antimicrobial substances (18). Phyllanthus niruri is a source of bioactive substances of broad-spectrum activity because of its sensitivity against both bacteria and fungi. However, the bioactive compounds are more active against gram- negative than gram- positive bacteria as the aqueous extract of the plant showed (zone of inhibition) The result supports the findings of Komuraiah et al., (19) who reported Phyllanthus amarus is an effective inhibitor of both gram- negative- and gram- positive bacteria. The better sensitivity of the plant extract against gram- negative species could be due to the thin peptidoglycan layer of gram- negative bacteria that is more permeable to antibacterial (20). The fungi species used in this study showed 33.3 % sensitivity to the aqueous extract of P. niruri L. at Z inhibition ≥ 10 indicating that the bioactive agents in the extract were relatively more effective against the bacterial species than the fungal species. The low MIC and MBC obtained are indications of its potency against bacterial infections however, the zero Minimum Fungicidal Concentration (MFC) of P. niruri against Aspergillus species is suggestive of 100 % resistance which means when subjected to favorable conditions the fungi could thrive and replicate. In the in vivo studies, the initial reduction in the average body weight was due to the Escherichia coli (E. coli) infection in the rats. A prominent symptom of E. coli infection is frequent passage of stool which oftentimes leads to a drop in body weight. The improvement in the weight of the Wistar albino rats after treatment indicates increased feed conversion efficiency (FCE). This development could be the result of recovery from the infection arising from restoration of the physiological activities of the rat system. The improvement in the health status of the rats could also be traced to the fact that chemical and biochemical constituents of plants which include carbohydrates, vitamins, protein, alkaloid, fats and oil and minerals might supply the body cells with energy and replace worn- out tissues. The bioactive agents present in P. niruri proved effective in vitro and in vivo against E. coli infection. The weight gain of the rats reduced as the dose increased. This indicates that irrational consumption of the plant could be potentially Res. J. Health Sci. Vol 11(2), June 2023 112 Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al. Res. J. Health Sci. Vol 11(2), June 2023 113 harmful. The bacterial load of E. coli in the test animals reduced gradually as the doses administered increased and after two weeks of treatment with the extract (1200 mg to 1500 mg) the bacterial count became insignificant. Red Blood Cell count (RBC) and Packed Cell Volume (PCV) which are important parameter for blood health status is indicative of normal erythropoiesis. Flavonoids, an essential compound present in P. niruri L. are an antioxidant element that ensures healthy blood circulation, strengthens the capillary wall and reduces blood cholesterol level. The blood leucocytes are the body soldiers that are involved in eliciting an immune response against the infectious agent. Leucocytes were within the normal range in the test animal, likewise neutrophil which indicates that the infection was inhibited As a measure of liver function test, the AST (Aspartate amino transaminase), ALT (Alanine amino transaminase), and ALP (Alkaline phosphatase) were assessed in P. niruri treated rats. An elevation of these enzymes is suggestive of liver damage (21). The administration of P. niruri caused a decline in the liver enzymes. This suggests that P. niruri may hold a positive impact on the liver cells and this corroborates the holdings of Verma et al., (22) who recorded that the extract of P. niruri is hepatoprotective. Serum urea and creatinine are labels of renal positions. High creatinine level is a pointer of impairment in renal filtration but low throughout the treatments. Howbeit, Eweka and Enogieru (23) noted some necrosis in the anatomical structures of the gastro intestinal tract (GIT) following 28 days of oral administration of P. amarus. The variation observed in the result may be due to differences in the duration of administering the extract. CONCLUSION In conclusion, Phyllanthus niruri has potent bioactive compounds against Escherichia coli infection. The administration of this extract up to 1500 mg dose has no deleterious effect on the liver and the kidney However, exploratory studies on P. niruri is required to determine the active compound (s) responsible for the antimicrobial effect demonstrated and evaluate its toxicity profile. Conflict of Interest: The authors state no conflict of interest REFERENCES 1 Andersson JA, Fitts EC, Kirtley ML, Pannusamy D, Peniche AG, Dann SM, New role for FDA- approved drugs in combating antibiotic-resistant b a c t e r i a . A n t i m i c r o b i a l a g e n t s a n d chemotherapy, 2016,AAC.00326-16 DOI: 10.1128/AAC.0032616. 2 Nisar MF, He J, Ahmed A, Yang Y, Li M, Wan C. Chemical components and biological activities of the genus Phyllamthus: A review of the recent literature. Molecules, 2018, 23: 2567, Doi 3390. 3 Cui Q, Du R, Liu M, Rong L. Lignans and their derivatives from plants as antivirals. 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Clinica Chimica Acta, 2007 375: 63- 68. 22 Verma S, Sharma H, Garg M. Phyllanthus amarus: A Review. J PharmacognPhytochem, 2014, 3 (2):18-22. 23 Eweka AO,Enogieru A. Effects of oral administration of Phyllanthus amarus leaf extract on the kidneys of adult wistar rats- a histological study. Afr. J. Tradit. Complement Altern Med, 2011, 8 (3): 307–311. Res. J. Health Sci. Vol 11(2), June 2023 114 Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al. Res. J. Health Sci. Vol 11(2), June 2023 115 Table 1: Quantitative analysis of the phytochemical constituents of aqueous and ethanol extracts of P. niruri. Key: Values are means ± Standard error of mean of 2 replicates of the phytochemical constituents. Means with different superscript on the same row is significant Phytochemicals Aqueous extract (mg/g) Ethanol extract (mg/g) Tannins 11.30±0.02 a 13.49±0.05 b Flavonoids 11.59±0.04 a 12.60±0.01 a Steroid 8.08±0.30 a 15.01±0.01 b Terpenes 0.00±0.00 a 10.68±0.01 b Cardiac Glycosides 25.45±0.02 a 26.46±0.33 b Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al. Res. J. Health Sci. Vol 11(2), June 2023 116 Table 3: Antifungal activity of aqueous and ethanol extracts of P. niruri . Antimicrobials (mg/mL) /ZI(mm) A. flavus A. niger C. albicans AE30 02.00±0.00b 07.67±0.00c 00.00±0.00a AE50 12.00±0.00d 08.00±0.00d 02.00±0.00b AE100 00.00±0.00a 08.00±0.00d 08.00±0.00c EE30 00.00±0.00a 00.00±0.00a 00.00±0.00a EE50 00.00±0.00a 00.00±0.00a 00.00±0.00a EE100 00.00±0.00a 00.00±0.00a 00.00±0.00a C1N 00.00±0.00a 00.00±0.00a 00.00±0.00a CIIP 06.00±0.00c 02.00±0.00b 15.00±0.00d Key: Values are the means ± Standard error of means of the 3 replicates of the zone of inhibition. Means with different superscript on the same vertical column is significant P = 0.05; ZI = Zone of inhibition; AE30 – aqueous extract at 30 mg/mL; AE50= aqueous extract at 50 mg/mL; AE100= aqueous extract at 100 mg/mL; EE30 - ethanol extract at 30 mg/mL; EE50= ethanol extract at 50 mg/mL; EE100= ethanol extract at 100 mg/mL; C 1N – negative control; C 11P - positive control Table 4: Clinical appearance of infected albino rat during treatment Days CI N CII P G2 G3 G4 G5 D 6 Fine fur; very active Fine fur; very active Fine fur; very active Fine fur; very active Fine fur; very active Fine fur; very active D 9 Fine fur; very active Activity reduced; rough fur Activity reduced; rough fur Activity reduced; rough fur Activity reduced; rough fur Activity reduced; rough fur D 16 Fine fur; very active Very active; Few insects the cage Activity improved slightly; wet faeces Activity improved; wet faeces Activity improved; wet faeces Activity improved; wet faeces D 23 Fine fur; very active active; Improved fur Increased appetite for food Increased appetite for food Increased appetite for food Increased appetite for food Key:CI N = normal control, CII P = positive control, G2 = group 2, administered with 300 mg extract, G3 = group 3, administered with 600 mg extract, G4 = group 4, administered with 1200 mg extract, G5 = group 5, administered with 1500 mg extract Table 5: Effect of Phyllanthusniruri on Escherichia coli Key: ± scanty growth; + few growth; ++ moderate growth; +++ heavy growth, G2 = group 2, administered with 300 mg extract, G3 = group 3, administered with 600 mg extract, G4 = group 4, administered with 1200 mg extract, G5 = group 5, administered with 1500 mg extract Day Normal control Positive Control G2 G3 G4 G5 9 ± +++ +++ +++ +++ +++ 16 ± ± +++ ++ ++ + 23 ± ± ++ + ± ± Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al. Res. J. Health Sci. Vol 11(2), June 2023 117 Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al. Res. J. Health Sci. Vol 11(2), June 2023 118 Table 7: Effect of ethanol extract on biochemical parameters of Wistar albino rat Key: Values are the Means ± Standard error of means of 5 replicates of the biochemical parameters evaluated. Means with different superscript on the same vertical column is significant P = 0.05; Sex - male and female; ALP - alkaline phosphatase; ALT = alanine aminotransaminase; AST = aspartate aminotransaminase; Control I N – normal control; Control II P – positive control; G2 = group 2, administered with 300 mg extract, G3 = group 3, administered with 600 mg extract, G4 = group 4, administered with 1200 mg extract, G5 = group 5, administered with 1500 mg extract SEX ALP µ/L ALT µ/L AST µ/L CREATµ MOL/L Control 1 N 1.00±0.00a 98.00±25.16a 58.00±15.95a 177.33±54.82a 158.00±36.51a Control 11 P 1.60±0.25 a 45.33±3.48a 28.00±6.56a 128.67±30.18a 88.00±7.00a G 2 300 mg 1.50±0.29 a 88.75±24.56a 62.00±16.53a 184.00±59.23a 118.50±31.52a G 3 600 mg 1.25±0.25 a 82.00±43.00a 59.00±29.00a 187.00±83.00 a 129.00±72.0a G 4 1200 mg 1.75±0.25 a 59.67±13.17a 34.67±8.82a 108.00±12.81 a 99.00±13.75a G 5 1500 mg 1.50±0.25 a 46.33±0.33a 28.33±4.70a 95.00±17.94 a 104.00±8.19a Antimicrobial and therapeutic properties of P. niruri Oyekanmi et al.