In-vivoAnalgesic, Diuretic, Anti-diarrheal and in-vitroAnti-microbial activity study of the ethanolic extract of Phyllanthus reticulatus fruits

In-vivoAnalgesic, Diuretic, Anti-diarrheal and in-vitroAnti-microbial activity study of the ethanolic extract of Phyllanthus reticulatus fruits | 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 In-vivo Analgesic, Diuretic, Anti-diarrheal and in-vitro Anti-microbial activity study of the ethanolic extract of Phyllanthus reticulatus fruits Palash Chandra Roy, Md. Khalequeuzzaman, Md. Jahidul Kabir Nahid, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8462001/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Phyllanthus reticulatus commonly known as Pancoli. It is used as a traditional medicinal with properties such as antioxidant, antibacterial and anti-HIV-1. Methods Analgesic activity was determined by the acetic acid writhing test and the diuretic activity was assessed in albino mice by measuring urine volume and electrolyte excretion over 24 hours following oral administration. The anti-diarrheal effect was evaluated using the castor oil-induced model, and antimicrobial activity was tested through the disc diffusion method. Results In the analgesic assay, the PRFEE at a dose of 300 mg/kg showed the highest inhibition (89%, 1.50 ± 0.50 writhes, P < 0.001). The diuretic activity assessment indicated a substantial increase in urine output with a dose of 500 mg/kg producing 2.50 ± 0.289 ml of urine (P < 0.001). Additionally, this dose significantly enhanced sodium (133.16 ± 8.186 mmol/L, P < 0.001), Potassium (87.25 ± 3.792 mmol/L, P < 0.001), and chloride (106.79 ± 4.49 mmol/L, P < 0.001) excretion. In the anti-diarrheal study, PRFEE 500 mg/kg achieved 81.82% inhibition reducing stool count to 1 ± 0.408 (P < 0.001), demonstrating strong efficacy. The antimicrobial activity showed a Potent inhibitory effect with a dose of 750 mg/mL, exhibiting inhibition zones of 17 mm ( Escherichia coli ), 15 mm ( Pseudomonas aeruginosa ) and 19 mm ( Staphylococcus aureus ), comparable to those of kanamycin (16–19 mm). Conclusion The PRFEE exhibited significant Pharmacological effects across all evaluated Parameters. Phyllanthus reticulatus fruit Analgesic Anti-diarrheal Diuretics Anti-microbial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Medicinal plants have long played a significant role in healthcare systems across various regions of the world. These plants contain a wide range of bioactive compounds with therapeutic properties that are used in the treatment of numerous health conditions in both humans and animals [ 1 , 2 ]. Among these, Phyllanthus reticulatus (commonly known as Pancoli) is a shrub or small tree belonging to the Euphorbiaceae family. It has been traditionally used throughout Asia and Africa to treat liver disorders, gastrointestinal issues, and various infections. The plant typically grows to a height of 1 to 5 meters [ 3 ] with its flowering, and fruiting season extending from July to March [ 4 ]. Phytochemical analyses show that flavonoids, alkaloids, saponins, phenols, cardiac glycosides, tannins, anthocyanins, and terpenoid compounds are found in plant [ 5 ]. These constituents are believed to contribute to the plant’s wide range of pharmacological effects [ 6 ]. Previous studies have documented the traditional and pharmacological uses of P. reticulatus . This plant has demonstrated antioxidant, antibacterial, anti-HIV-1, antidiabetic, antifungal, antiplasmodial and antidiarrheal effects [ 7 – 10 ]. Additionally, an herbal hair dye formulation has been developed using an aqueous leaf extract of P. reticulatus in combination with other natural ingredients [ 11 ]. Methanolic extracts of the stem bark have shown antimicrobial activity [ 12 ]. Its fruits have been used traditionally for their anti-inflammatory effects [ 13 ]. Furthermore, traditional practices include the preparation of a pill made from leaf juice mixed with camphor and cubebs, which is dissolved in the mouth to treat bleeding gums, while the roots are used as a natural red dye [ 14 ]. Phyllanthus species is known for its diuretic effects, contributing to fluid and electrolyte balance [ 15 ]. Methanolic and ethyl acetate extracts from the plant have shown promising antimicrobial activity against both bacterial and fungal pathogens [ 16 , 17 ]. Pain can be measured using tests like the acetic acid-induced writhing and hot-plate methods, which show how drugs work in both the brain and the nerves. These drugs also help with diarrhea by controlling how the intestines move and produce liquid, which stops too much water from being lost, as seen in tests using castor oil-induced diarrhea [ 18 , 19 ]. Additionally, these drugs have a diuretic effect, meaning they increase urine and salt loss, showing their effect on kidney function and the body's water balance [ 20 ]. Both Gram-positive and Gram-negative bacteria are believed to be a big issue for public health [ 21 ]. While extensive research has confirmed the pharmacological potential of the plant’s leaves, bark, and stem, the fruit of P. reticulatus remains significantly underexplored. Despite its traditional medicinal use and potential as a source of novel bioactive compounds, there is a lack of comprehensive phytochemical and pharmacological data specifically focused on the fruit. The chemical constituents, biological properties, and therapeutic potential of the fruit have not yet been systematically investigated. Given this knowledge gap, further scientific investigation is essential. The fruit of P. reticulatus may contain unique active compounds that could offer therapeutic benefits in the treatment of pain, gastrointestinal disorders, metabolic diseases and microbial infections. A thorough study of the fruit could contribute to the development of affordable, accessible, and safe natural therapeutics, particularly important for populations in developing countries that rely heavily on herbal medicine. Therefore, the present study was undertaken to investigate the phytochemical composition and pharmacological properties of the ethanolic extract of Phyllanthus reticulatus fruits (PRFEE) with a specific focus on in vivo analgesic, diuretic, antidiarrheal, and in vitro antimicrobial activities. This research aims to bridge traditional knowledge and modern pharmacological validation, potentially identifying new bioactive compounds of therapeutic value. MATERIALS AND METHODS Plant Materials and Extraction Process The fruits of P. reticulatus were gathered from Savar, Dhaka, Bangladesh between October to November 2023. They were washed carefully to remove any unwanted stuff. The plant was confirmed by the Bangladesh National Herbarium in Mirpur, Dhaka, Bangladesh with the accession number DACB 87899. After washing, the fruits were dried in the shade for seven days and then dried in an oven at 40°C for 24 hours. The dried fruits were then turned into a rough powder using a disc mill grinder machine. Before storing, the powder was cleaned again and dried properly. The powder was kept in a sealed container in a dark and dry area. 341 g of dry powder of P. reticulatus fruits was taken by the extractor apparatus, and 2.5 liters of 98% ethanol were used in the Soxhlet apparatus, model-509.303. Chemicals Aspirin (Albion Laboratories Limited), Furosemide (Square Pharmaceutical Limited), and Loperamide (ACME Laboratories Limited) were purchased from the market. The bacterial strain organisms were collected from the Microbiology Research Laboratory, Gono Bishwabidyala. All the chemicals are sourced from the Department of Pharmacy Laboratory, Gono Bishwabidyalay, Savar, Dhaka. Experimental Animals Swiss albino mice both male and female aged between 6 and 7 weeks and weighing 20 to 30 grams were acquired from the Animal Research Laboratory at the Department of Pharmacy, Jahangirnagar University located in Savar, Dhaka. They were maintained under standard laboratory conditions including a temperature of 25 ± 1°C, a humidity level of 55–65% and a 12-hour light-dark cycle. The mice had free access to food and water and were allowed to acclimate to the laboratory environment for one week before the start of the experiments. The mice were divided into five groups for analgesic and anti-diarrheal test, and four groups for diuretics test with each group containing five mice. All experiments conducted on animals were approved by the Research Ethical Committee under the Center for Multidisciplinary Research of Gono Bishwabidyalay, Dhaka 1344, Bangladesh (Permission number: CMR/EC/023). Phytochemical activitys The PRFEE was checked for plant-based chemical components through several simple tests. Different chemicals were used according to standard methods, and the results showed clear color changes that showed the presence of carbohydrates, saponins, tannins, alkaloids, flavonoids, steroids, glycosides, and phenolic compounds, and the absence of steroid compounds [ 22 ]. Antioxidant activitys Reducing Power Capacity Assay (RPC) Reducing power capacity of the fruit extract was evaluated using the ferric–ferricyanide reduction method [ 23 ]. 1.0 ml of Plant extract or standard of different concentration mixed with 2.5ml of phosphate buffer (pH 6.6) & 2.5 ml potassium ferricyanide (1%). The reaction mixture is incubated for 20 min at 50 0 C to complete the reaction. 2.5ml of trichloro acetic acid (10%) solution is added into the test tube and the total mixture was centrifuged at 3000 rpm for 10 min. 2.5ml of supernatant solution is withdrawn from the mixture and mixed with 2.5 ml of distilled water. 0.5ml of ferric chloride (0.1%) solution was added to the diluted reaction mixture. Then the absorbance of the solution is measured at 700 nm using a spectrophotometer against blank. An increase in absorbance indicated enhanced reducing power. DPPH Free Radical Scavenging Assay The in vitro antioxidant activity of the fruit extract was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay, which measures the ability of antioxidants to quench DPPH radicals through hydrogen atom or electron donation [ 24 , 25 ], which measures the ability of antioxidants to quench DPPH radicals through hydrogen atom or electron donation. A 0.004% (w/v) DPPH solution was freshly prepared by dissolving 0.004 g DPPH in 100 mL ethanol. Stock solutions of the fruit extract and ascorbic acid (5 mg/mL) were prepared in ethanol, and serial dilutions yielded concentrations of 6.25 to 800 µg/mL. Aliquots (1 mL) of each concentration were mixed with 2 mL of DPPH solution and incubated in the dark at room temperature for 30 min. Absorbance was measured at 517 nm using a UV-Vis spectrophotometer. All tests were conducted in triplicate. The percentage of DPPH radical inhibition was calculated as: \(\:\%\:Inhibition=(1-\frac{Absorbance\:of\:sample}{Absorbance\:of\:control}\) ) × 100 The IC₅₀ value (µg/mL) was determined from a plot of % inhibition versus concentration by linear regression. Acute Toxicity Study An acute oral toxicity study was conducted on Swiss albino mice in accordance with OECD Guideline 423 [ 26 ]. Twelve mice were randomly allocated into four groups (n = 3) and administered a single oral dose of PRFEE at 250, 500, 1000 and 2000 mg/kg, respectively. The animals were monitored for 14 days for signs of toxicity, behavioral alterations, and mortality. In-vivo Evaluation of Analgesic Activity. Acetic acid induced writhing test The abdominal writhing test was done using acetic acid following the method by Saha et al. [ 27 ] with some changes. The animals were randomly divided into five groups. The control group got normal saline at a dose of 10 ml/kg of body weight. The standard group was given aspirin at 75 mg/kg, and the test groups were given PRFEE at doses of 100, 200 and 300 mg/kg, all by mouth. Thirty minutes after drug administration, all animals were injected intraperitoneal with 0.6% acetic acid to induce pain. After a latency period of 5 minutes, the number of abdominal writhes was counted over the next 30 minutes. The pain-relieving effect was shown as the percentage of writhing that was prevented. % Inhibition = Number of writhes in control group - Number of writhes in treated group ÷ Number of writhes in control group × 100 In vivo Evaluation of Diuretic Activity. Diuretic activity was assessed using the method of Geleta et al. [ 28 ] with slight modifications. Swiss albino mice (20–30 g) were ensured to have similar body weights. All the mice were not allowed to eat for the whole night but could drink water freely, and were acclimatized in individual metabolic cages for 24 hours before the experiment. The control group received 0.9% NaCl, the standard group was administered furosemide at a dose of 10 mg/kg and the test groups received PRFEE at 250 and 500 mg/kg by mouth. After dosing, urine was collected over 24 hours in individual metabolic cages. Collected urine was filtered and stored at -20°C for analysis of sodium, potassium, and chloride levels. The following parameters were calculated. Diuretic action (%) = Total urinary output ÷ Total liquid administered ×100 (1) Urinary excretion = Urinary excretion of test group ÷ Urinary excretion of control group (2) Diuretic activity = Diuretic action of the test group ÷ Diuretic action of standard drug (3) Analytical Procedure: The levels of sodium, potassium, and chloride in both the urine samples and the plant extract were checked. These measurements were done with an ion-selective electrode analyzer called the 9180 Electrolyte Analyzer Reagent, which was made by Roche in Germany. Evaluation of the Antidiarrheal Activity Castor Oil-Induced Diarrheal Test The diarrhea model caused by castor oil as described by Afroz et al. [ 29 ] was used with slight modifications. Mice were not given food for 18 hours. All mice were given 1.0 ml of castor oil by mouth. After 30 minutes, the control group was given normal saline at a dose of 10 ml/kg of body weight. The standard group received loperamide at 2 mg/kg. The test groups were given PRFEE at doses of 150, 300, and 500 mg/kg. Each mice were placed in a separate cage with paper lining. The paper was changed every hour for 4 hours. The number and weight of feces, along with stool consistency were recorded. The results were shown as a percentage of how much diarrhea was prevented [ 30 ]. % Inhibition of defecation = Mean of defecation control group - Mean of defecation treated group ÷ Mean of defecation control group × 100 In vitro Evaluation of Antimicrobial Activity Media Preparation: Mueller-Hinton agar and broth were prepared and sterilized according to the manufacturer’s instructions and standard protocols [ 31 ]. About 20–25 ml of agar was added to sterile petri dishes and let dry at room temperature. The dishes were kept in a refrigerator at 4°C until they were needed [ 32 ]. The bacteria used in this study were Staphylococcus aureus, which is Gram-positive and Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa, which are all Gram-negative from the Microbiology Research Laboratory, Gono Bishwabidyalay, Savar, Dhaka. Disc diffusion method: Antimicrobial activity of PRFEE was tested using a modified disc diffusion (Hole-Plate) method [ 33 , 34 ]. Bacterial suspensions were placed on nutrient agar plates and small wells, each about 8 millimeters in size were created using a clean cork borer. The extract was dissolved in a 1:1 mixture of DMSO and water for injection and applied at concentrations of 250, 500 and 750 µg/ml (100 µl Per well). Plates were kept at a temperature of 37°C for 24 hours. The areas where bacteria did not grow were measured in millimeters to assess the effectiveness of the substance in inhibiting bacterial growth. Water for injection and the solvent mixture served as negative controls, while Kanamycin-K (30 µg/disc) was used as the positive standard. All tests were performed in duplicate for accuracy. Statistical analysis Statistical analysis was done using SPSS version 30.0 for Windows [ 35 ]. The results are shown as S.E.M. A one-way ANOVA was used along with Dunnett’s post hoc test to compare multiple groups. Differences were considered statistically significant when the P value was below P < 0.05 - P < 0.001. RESULTS Screening Phytochemical Components The PRFEE was found to contain carbohydrates, tannins, saponins and phenolic constituents upon phytochemical analysis. However, steroids were absent. Antioxidant activity Determination of Reducing Power capacity (RPC) The reducing properties are generally associated with the presence of reluctance which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom). Absorbance Concentration (µg/ml) reducing power (Table 1 ). In the present study ferric reducing power activity of the extract of fruits of P. reticulatus plants are for (118.39 ± 4.30 mg/g) as Gallic acid (Table 2 ). The experimental results indicate lower ferric (y = 0.0029x + 0.039, R² = 0.9842) (Fig. 1 ). Table 1 Absorbance found with different concentration of Gallic Acid. Concentration (µg/ml) Absorbance 6.25 0.032 12.5 0.147 25 0.223 50 0.269 100 0.484 200 0.788 Table 2 Determination of Reducing Power capacity of the ethanolic extract of fruits of P. reticulatus. Sample Name Wt. of plant extract (µg/ml) Abs. GAE conc.(C) (µg/ml) GAE conc.(C) (mg/ml) V (ml) C × V (mg) TPC as GAE, A=(c×V)/m (mg/gm) Mean value ±SD P. reticulatus 200 0.107 23.445 0.023 1 0.023 117.241 118.39 ± 4.30 mg/g 200 0.105 22.756 0.022 1 0.022 113.793 200 0.111 24.828 0.024 1 0.024 124.128 DPPH free radical scavenging assay When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance. DPPH free radical scavenging study of the extract of fruits of P. reticulatus determine as % of inhibition and IC 50 (inhibition concentration 50%) value. Lower IC 50 values indicates good scavenging capacity and antioxidant property. The results of the present study showed that ethanolic extract have IC 50 value 639.033 µg/mL (Table 4 ) (Fig. 3 ), exhibited poor antioxidant property compared to standard 3.725 µg/ml (Table 3 ) (Fig. 2 ). Table 3 The percentage (%) scavenging Activity of Ascorbic Acid. Concentration (µg/ml) Control Absorbance sample % Inhibition IC 50 value 6.25 1.748 1.347 22.940 3.725 12.5 1.748 1.089 37.700 25 1.748 0.957 45.251 50 1.748 0.729 58.295 100 1.748 0.641 63.329 200 1.748 0.578 66.933 400 1.748 0.489 72.025 800 1.748 0.317 81.864 Table 4 The results and IC 50 ethanol extract of fruits of P. reticulatus is presented in the Table. The percentage (%) scavenging Activity of P. reticulatus fruits extract. Concentration (µg/ml) Control Absorbance sample % Inhibition IC 50 value 6.25 1.748 1.514 13.39% 639.033 12.5 1.748 1.467 16.08% 25 1.748 1.268 27.46% 50 1.748 1.025 41.36% 100 1.748 0.991 43.31% 200 1.748 0.953 45.48% 400 1.748 0.679 61.16% 800 1.748 0.575 67.11% Acute Toxicity No mortality was observed in any of the groups. The body weight remained normal and no significant toxic symptoms were observed. Analgesic activity PRFEE showed a strong pain-relieving effect that increases with higher doses in the acetic acid-induced writhing test. Aspirin at 75 mg/kg produced 80% inhibition (P < 0.001). PRFEE at 100, 200 and 300 mg/kg showed 20%, 71% and 89% inhibition, respectively. Significant reductions in writhing were observed at 200 mg/kg (P < 0.01) and 300 mg/kg (P < 0.001), while 100 mg/kg was not statistically significant (Table 5 ) (Fig. 4 ). Table 5 Effect of Aspirin and PRFEE on acetic acid-induced writhing in mice. Group Dose Mean of Writhing ± S.E.M % Inhibition Control 10 ml/kg 13.75 ± 0.48 - Aspirin 75 mg/kg 2.75 ± 0.48 *** 80% PRFEE 100 mg/kg 11.00 ± 1.00 20% 200 mg/kg 4.00 ± 0.50 ** 71% 300 mg/kg 1.50 ± 0.50 *** 89% Values are represented as S.E.M, (n = 5); P < 0.05 – P < 0.001. Diuretic activity Effect of PRFEE on 24-hour urine output and diuretic indices in mice. The control group produced 1.00 ± 0.00 ml of urine, while furosemide raised output to 2.75 ± 0.25 ml (P < 0.001). PRFEE at 250 and 500 mg/kg increased urine volume to 2.00 ± 0.001 ml (P < 0.01) and 2.50 ± 0.289 ml (P < 0.001), respectively. The diuretic actions were 2.00 and 2.50 with corresponding diuretic activities of 0.72 and 0.91, indicating that the 500 mg/kg dose achieved the standard drug effect (Table 6 ) (Fig. 5 ). Table 6 Diuretic effect of PRFEE by increasing urine volume. Group Dose Total urine volume (ml) ± S.E.M Diuretic action (%) Diuretics activity Control 25 ml/kg 1 ± 0.00 - Furosemide 10 mg/kg 2.75 ± 0.250 *** 2.75 - PRFEE 250 mg/kg 2 ± 0.001 ** 2 0.72 500 mg/kg 2.50 ± 0.289 *** 2.50 0.91 Values are represented as S.E.M, (n = 5); P < 0.05- P < 0.001 Electrolyte Excretion PRFEE significantly increased urinary excretion of Na⁺, K⁺ and Cl⁻ levels. In the control group urine levels were 87.85 ± 2.87 (Na⁺), 55.58 ± 4.73 (K⁺) and 61.84 ± 2.20 (Cl⁻). Furosemide at 10 mg/kg markedly increased electrolyte excretion Na⁺ to 125.16 ± 4.54, K⁺ to 80.45 ± 1.82 and Cl⁻ to 95.89 ± 3.10 mmol/l (P < 0.01-P < 0.001). PRFEE at 250 mg/kg significantly raised Na⁺, K⁺ and Cl⁻ to 106.28, 76.32 and 82.51 mmol/l, respectively (P < 0.05 - P < 0.001). At 500 mg/kg excretion levels further increased to 133.16 (Na⁺), 87.25 (K⁺) and 106.79 mmol/l (Cl⁻), all highly significant (P < 0.001), exceeding the effect of furosemide (Table 7 ) (Fig. 6 ). Table 7 Effect of PRFEE on urinary electrolyte excretion. Group Dose Total Urinary electrolyte excretion Na + mmol/l ± mean K + mmol/l ± mean Cl − mmol/l ± mean Control 25 ml/kg 87.85 ± 2.866 55.58 ± 4.729 61.84 ± 2.197 Furosemide 10 mg/kg 125.16 ± 4.535 *** 80.45 ± 1.821 ** 95.89 ± 3.095 *** PRFEE 250 mg/kg 106.28 ± 3.361 * 76.32 ± 5.434 * 82.51 ± 2.437 *** 500 mg/kg 133.16 ± 8.186 *** 87.25 ± 3.792 *** 106.79 ± 4.490 *** Values are represented as S.E.M, (n = 5); P < 0.05 - P < 0.001 Antidiarrheal Activity PRFEE has dose-dependent antidiarrheal effects in the control group had the mean of 5.50 ± 0.65 watery stools were observed, while loperamide at 2 mg/kg significantly reduced this to 2.25 ± 0.63 (P < 0.01) showing 60% inhibition. PRFEE at 150 mg/kg caused a mild reduction of 3.75 ± 0.48 (27% inhibition) while 300 mg/kg showed significant activity (P < 0.01) with 2.75 ± 0.48 stools (50% inhibition). The 500 mg/kg dose produced a highly significant reduction to 1.00 ± 0.41 stools (P < 0.001), achieving 81.82% inhibition exceeding that of Loperamide as shown (Table 8 ) and (Fig. 7 ). Table 8 Showing the percentage reduction in volume of small intestinal content. Group Dose Mean number of watery stools ± S.E.M Inhibition of diarrhea (%) Control 10 ml/kg - - Loperamide HCl 2 mg/kg 2.25 ± 0.629 ** 60% PRFEE 150 mg/kg 3.75 ± 0.479 27% 300 mg/kg 2.75 ± 0.479 ** 50% 500 mg/kg 1 ± 0.408 *** 81.82% Values are represented as S.E.M, (n = 5); P < 0.05 - P < 0.001 Antimicrobial Activity The PRFEE exhibits the capability to combat both Gram-positive and Gram-negative bacteria. No inhibition was observed at 250 µg/ml. The dose at 500 µg/ml showed moderate zones of inhibition were seen against Pseudomonas aeruginosa (13 mm) and Staphylococcus aureus (17 mm), while Escherichia coli showed no response. At 750 µg/ml, PRFEE showed strong antibacterial activity with inhibition zones of 17 mm ( E. coli ), 15 mm ( P. aeruginosa ) and 19 mm ( S. aureus ), comparable to the standard antibiotic kanamycin (30 µg). These results are summarized and visually represented in (Table 9 ) (Fig. 8 ). Table 9 Inhibitory effect of PRFEE on (1) Escherichia coli (2) Pseudomonas aeruginosa (3) Staphylococcus aureus (4) Negative control (5) Kanamycin. Group Zone of inhibition (mm) Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Control 0 0 0 Kanamycin 19 mm 16 mm 19 mm PRFEE 250 µg/ml 0 0 mm 0 PRFEE 500 µg/ml 0 13 mm 17 mm PRFEE 750 µg/ml 17 mm 15 mm 19 mm DISCUSSION The Present study evaluated the dose-dependent pharmacological activities. This research is the first to report on the pharmacological potential of the fruits clearly demonstrates that the PRFEE exhibits significant pharmacological properties including analgesic, diuretic, antidiarrheal and antimicrobial activities. These effects can be attributed to its rich content of bioactive phytochemicals compounds, which are well-known for their diverse therapeutic effects. The results indicate that the fruits of P. reticulatus extract exhibited low DPPH radical scavenging activity and limited reducing power capacity, indicating weak chemical antioxidant potential. However, these assays mainly assess in vitro electron-donating ability and do not necessarily correlate with pharmacological effects. The observed biological activities may be attributed to the presence of alkaloids, flavonoids and phenolic compounds, which exert analgesic, diuretic and antidiarrheal effects through mechanism-specific pathways independent of antioxidant capacity [ 36 – 38 ]. PRFEE showed a strong analgesic effect in the acetic acid-induced writhing test with 300 mg/kg producing 89% inhibition, which was significantly greater than aspirin (75 mg/kg, 80%). Acetic acid-induced pain is mediated by the release of endogenous substances such as prostaglandins particularly PGE₂ and PGF₂α, which stimulate nociceptors [ 39 ]. Flavonoids and alkaloids reported are known to inhibit cyclooxygenase and lipoxygenase enzymes thereby suppressing prostaglandin synthesis and producing analgesia [ 40 , 41 ]. Further supporting the role of polyphenolic compounds in analgesic activity [ 42 ]. PRFEE significantly increased urine output with the 500 mg/kg dose (2.50 ml) achieving a response comparable to furosemide (2.75 ml). The extract also markedly enhanced urinary electrolyte excretion (Na⁺, K⁺, Cl⁻) with the higher dose surpassing the standard drug. Flavonoids and saponins have been reported to exert diuretic effects by altering renal tubular ion transport and increasing renal blood flow [ 43 ]. The observed saluretic effect (Na⁺ and Cl⁻ excretion) and kaliuretic activity (K⁺ excretion) suggest that PRFEE may act through a mechanism similar to loop diuretics possibly mediated by prostaglandins [ 44 ]. These findings indicate that PRFEE could be a promising natural diuretic. In the castor oil-induced diarrheal model, PRFEE exhibited protection with the 500 mg/kg dose producing 81.82% inhibition surpassing loperamide (60%). Castor oil causes diarrhea by releasing ricinoleic acid, which stimulates intestinal peristalsis and secretion of electrolytes and water via prostaglandin pathways [ 45 ]. Tannins, flavonoids and steroids are known to reduce intestinal motility and secretion by inhibiting prostaglandin synthesis and relaxing intestinal smooth muscles [ 46 ]. The significant effect of PRFEE can therefore be linked to its tannin and flavonoid content consistent with earlier findings in other Phyllanthus species [ 47 ]. The fruit extract also demonstrated broad-spectrum antibacterial activity with strong inhibition zones against Staphylococcus aureus (19 mm), Pseudomonas aeruginosa (15 mm) and Escherichia coli (17 mm) at 750 µg/ml comparable to the standard antibiotic kanamycin. Phenolic, flavonoids and alkaloids compounds are well known for their ability to disrupt bacterial cell walls inhibit nucleic acid synthesis and interfere with microbial enzymes, thereby exerting antimicrobial effects [ 48 , 49 ]. These findings justify the ethnomedicinal use of P. reticulatus in infectious diseases. CONCLUSION The PRFEE exhibits potent analgesic, diuretic, antidiarrheal and antimicrobial activities. The highest doses of PRFEE often surpassed standard drugs in efficacy, indicating the fruit as a Promising source of bioactive compounds. The study highlights the relevance of plant part selection in pharmacological research, as the fruit may contain higher levels of active constituents than the leaves and bark. These results support the traditional medicinal use of P. reticulatus and suggest that PRFEE could serve as a foundation for developing natural therapeutic agents. Further studies focusing on molecular docking, isolation of bioactive compounds, mechanistic evaluation, toxicity profiling and clinical trials are necessary to establish its safety and therapeutic applicability. Abbreviations PRFEE, Phyllanthus reticulatus Fruit Ethanolic Extract; S.E.M., Standard Error of Mean. Declarations Author contributions PR assisted in doing the project (experiments) for her thesis and analyzed the data and contributed to the writing of the paper. KZ supervised the project. He was involved in all parts of the experiments. JN analyzed the data and contributed to the writing of the paper. SM advised on the project and performed some experiments. TR and AM advised on the project. All authors read and approved the final manuscript. Acknowledgement The authors acknowledge the contribution of the Department of Pharmacy, Gono Bishwabidyalay, for laboratory facility and ancillary support to complete this work. animal rights Animals were used in the studies that are the basis of this research. Disclosure statement: No potential conflict of interest was reported by the author(s). Data availability statement: All raw data from original research papers and on-line databases are summarized in this review article, and the data sources are cited in the Reference section. Funding: The author(s) reported there is no funding associated with the work featured in this article. Animal Ethics approval and consent to participate Applicable. Consent for publication All authors have read and agreed to the published version of the manuscript. Competing interests The authors declare no competing interests. Human Clinical trial number Not applicable. References Rates SMK. Plants as source of drugs. Toxicon. 2001;39:603–13. Chaachouay N, Zidane L. Plant-derived natural products: a source for drug discovery and development. Drugs Drug Candidates. 2024;3:184–207. Izhar H, Shabbir A, Shahzad M et al. Phyllanthus reticulatus Prevents Ethanol-Induced Gastric Ulcer via Downregulation of IL‐8 and TNF‐α Levels. Evidence‐Based Complementary and Alternative Medicine 2021; 2021: 1734752. Sharma S, Kumar S. Phyllanthus reticulatus Poir.-an important medicinal plant: a review of its phytochemistry, traditional uses and pharmacological properties. Int J Pharm Sci Res. 2013;4:2528. Ominyi MC, Ali FU, Ebenyi LN, et al. Identification and Characterization of Phytochemicals in Ethanol Leaf-extracts of Phyllanthus reticulatus and Anthocleista djalonesis. Nigerian J Biochem Mol Biology. 2022;37:314–9. Sinan KI, de la Luz Cádiz-Gurrea M, Leyva-Jiménez FJ, et al. New insights on Phyllanthus reticulatus Poir. leaves and stem bark extracts: UPLC-ESI-TOF-MS profiles, and biopharmaceutical and in silico analysis. New J Chem. 2021;45:21049–65. Eldeen IMS, Seow EM, Abdullah R, et al. In vitro antibacterial, antioxidant, total phenolic contents and anti-HIV-1 reverse transcriptase activities of extracts of seven Phyllanthus sp. South Afr J Bot. 2011;77:75–9. Sahana A, Gupta KK, Chakraborty M et al. Pre-Clinical Evaluation of Anti-Diabetic Activity of Phyllanthus Reticulatus Fruit Extract. Journal of Advanced Zoology ; 44. Chellappandian M, Saravanan M, Pandikumar P, et al. Traditionally practiced medicinal plant extracts inhibit the ergosterol biosynthesis of clinically isolated dermatophytic pathogens. J de Mycol medicale. 2018;28:143–9. Khatun MH, Nesa ML, Islam R, et al. Antidiabetic and antidiarrheal effects of the methanolic extract of Phyllanthus reticulatus leaves in mice. Asian Pac J Reprod. 2014;3:121–7. Kiran kumar G, Hariprasath GM, Mukesh R, et al. Formulation of herbal based hair colour from Phyllanthus reticulatus. Nat Prod Res. 2025;39:2114–20. Begum T, Rahman MS, Rashid MA. Phytochemical and biological investigations of Phyllanthus reticulatus. Dhaka Univ J Pharm Sci. 2006;5:21–3. Kumar S, Sharma S, Kumar D, et al. Pharmacognostic study and anti–inflammatory activity of Phyllanthus reticulatus Poir. fruit. Asian Pac J Trop Disease. 2012;2:S332–5. Ram HNA, Shreedhara CS, Gajera FP et al. Phamacognostical Evaluation of Phyllanthus reticulatus Poir. Pharmacognosy Magazine ; 5. Yao AN, Kamagaté M, Amonkan AK, et al. The acute diuretic effect of an ethanolic fraction of Phyllanthus amarus (Euphorbiaceae) in rats involves prostaglandins. BMC Complement Altern Med. 2018;18:94. Haque T, Muhsin MDA, Akhter T, et al. Antimicrobial and analgesic activity of leaf extracts of Phyllanthus reticulatus Poir.(Family-Euphorbiaceae). Jahangirnagar Univ J Biol Sci. 2016;5:81–5. Hiremath KY, Shinde S, Jambale UK, et al. Anticancer effect of Phyllanthus reticulatus methanolic leaf extract on HCT-116 colon cancer cell line using GC-MS and FTIR analysis. Arch Razi Inst. 2024;79:1013. Nesa ML, Karim SMS, Api K, et al. Screening of Baccaurea ramiflora (Lour.) extracts for cytotoxic, analgesic, anti-inflammatory, neuropharmacological and antidiarrheal activities. BMC Complement Altern Med. 2018;18:35. Shah SF, Baig SG, Abidi S et al. PHARMACOLOGICAL EVALUATION OF Silybum marianum (L.) SEED EXTRACT: ANALGESIC, ANTI-INFLAMMATORY, DIURETIC, AND ANTIDIARRHEAL ACTIVITIES. Journal of Medical & Health Sciences Review ; 2. Sardar PK, Devnath HS, Biswas NN et al. Evaluation of Diuretic Property of Argemone mexicana along with Molecular Docking Study. Jordan Journal of Pharmaceutical Sciences ; 16. Azam A, Ahmed AS, Oves M et al. Antimicrobial activity of metal oxide nanoparticles against Gram-positive and Gram-negative bacteria: a comparative study. Int J Nanomed 2012; 6003–9. Ghani A. Medicinal plants of Bangladesh with chemical constituents and uses. Asiatic society of Bangladesh; 2003. Vijayalakshmi M, Ruckmani K. Ferric reducing antioxidant power assay in plant extract. ||| Bangladesh J Pharmacology|||. 2016;11:570–2. Hagaggi NSA, Abdul-Raouf UM, Radwan TAA. Variation of antibacterial and antioxidant secondary metabolites and volatiles in leaf and callus extracts of Phulai (Acacia Modesta Wall). BMC Plant Biol. 2024;24:93. Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement Altern Med. 2012;12:221. Guideline O, Testing FOR, Chemicals OF. Acute Oral Toxicity – Acute Toxic Class Method. Biswas NN, Saha S, Ali MK. Antioxidant, antimicrobial, cytotoxic and analgesic activities of ethanolic extract of Mentha arvensis L. Asian Pac J Trop Biomed. 2014;4:792–7. Geleta B, Eyasu M, Fekadu N, et al. Evaluation of diuretic activity of hydro-ethanolic extract of Moringa stenopetala leaves in Swiss albino mice. Clin Exp Pharmacol. 2015;5:2161. Afroz S, Alamgir M, Khan MTH, et al. Antidiarrhoeal activity of the ethanol extract of Paederia foetida Linn.(Rubiaceae). J Ethnopharmacol. 2006;105:125–30. Dey SK, Hira A, Howlader MSI, et al. Antioxidant and antidiarrheal activities of ethanol extract of Ardisia elliptica fruits. Pharm Biol. 2014;52:213–20. Martina SM, Cabalquinto GP, Lumogdang L. TAXONOMIC DESCRIPTION AND ANTIBACTERIAL ACTIVITY OF DILLENIA sp. AGAINST ESCHERICHIA coli. Biotropia. 2024;31:422–31. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal. 2016;6:71–9. Khan ZA, Siddiqui MF, Park S. Current and emerging methods of antibiotic susceptibility testing. Diagnostics. 2019;9:49. Khalil A. Antimicrobial activity of ethanolic extracts of Ocimum basilicum leaf from Saudi Arabia. Biotechnology. 2013;12:61–4. Abdel Aziz SM, Ahmed OM, Abd EL-Twab SM et al. Antihyperglycemic effects and mode of actions of Musa paradisiaca leaf and fruit peel hydroethanolic extracts in nicotinamide/streptozotocin-induced diabetic rats. Evidence‐Based Complementary and Alternative Medicine 2020; 2020: 9276343. Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: an overview. The scientific world journal 2013; 2013: 162750. Jothy SL, Zuraini Z, Sasidharan S. Phytochemicals screening, DPPH free radical scavenging and xanthine oxidase inhibitiory activities of Cassia fistula seeds extract. J Med Plants Res. 2011;5:1941–7. Azab A, Nassar A, Azab AN. Anti-inflammatory activity of natural products. Molecules. 2016;21:1321. Ahmad FM, Zafar A, Ahmed M, et al. Quercus floribunda Lindl. Ex A. Camus; a tremendous remedy against inflammation and associated symptoms. Fitoterapia. 2023;170:105628. Zhu C, Liu N, Tian M, et al. Effects of alkaloids on peripheral neuropathic pain: a review. Chin Med. 2020;15:106. Ferraz CR, Carvalho TT, Manchope MF, et al. Therapeutic potential of flavonoids in pain and inflammation: mechanisms of action, pre-clinical and clinical data, and pharmaceutical development. Molecules. 2020;25:762. Atawodi SE, Pfundstein B, Haubner R, et al. Content of polyphenolic compounds in the Nigerian stimulants Cola nitida ssp. alba, Cola nitida ssp. rubra A. Chev, and Cola acuminata Schott & Endl and their antioxidant capacity. J Agric Food Chem. 2007;55:9824–8. Hailu W, Engidawork E. Evaluation of the diuretic activity of the aqueous and 80% methanol extracts of Ajuga remota Benth (Lamiaceae) leaves in mice. BMC Complement Altern Med. 2014;14:135. Liguori A, Casini A, Di Loreto M, et al. Loop diuretics enhance the secretion of prostacyclin in vitro, in healthy persons, and in patients with chronic heart failure. Eur J Clin Pharmacol. 1999;55:117–24. Chakraborty S, Roy B, Sen S et al. Fermented Polyherbal Formulation Restored Ricinoleic Acid-Induced Diarrhea in Sprague Dawley Rats and Exhibited In Vitro Antibacterial Effect on Multiple Antibiotic‐Resistant Gastrointestinal Pathogens. Journal of Pathogens 2024; 2024: 1997064. Palombo EA. Phytochemicals from traditional medicinal plants used in the treatment of diarrhoea: modes of action and effects on intestinal function. Phytotherapy Research: Int J Devoted Pharmacol Toxicol Evaluation Nat Prod Derivatives. 2006;20:717–24. Tadesse E, Engidawork E, Nedi T, et al. Evaluation of the anti-diarrheal activity of the aqueous stem extract of Lantana camara Linn (Verbenaceae) in mice. BMC Complement Altern Med. 2017;17:190. Abdul Qadir M, Shahzadi SK, Bashir A et al. Evaluation of phenolic compounds and antioxidant and antimicrobial activities of some common herbs. International journal of analytical chemistry 2017; 2017: 3475738. Erdemoglu N, Ozkan S, Tosun F. Alkaloid profile and antimicrobial activity of Lupinus angustifolius L. alkaloid extract. Phytochem Rev. 2007;6:197–201. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 13 Feb, 2026 Editor assigned by journal 29 Dec, 2025 Submission checks completed at journal 29 Dec, 2025 First submitted to journal 27 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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05:59:58","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171258,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/0f85f52e6f56463059485c2a.html"},{"id":99790433,"identity":"a53fdefd-412c-4675-98cd-900bf4114f5c","added_by":"auto","created_at":"2026-01-08 12:58:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37475,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve of Gallic Acid of reducing power capacity.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/81c4ed79882b1249dd8ca989.png"},{"id":99494413,"identity":"c822b33c-35bb-496a-a693-ee2d0f20a312","added_by":"auto","created_at":"2026-01-05 05:59:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36455,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve of DPPH of Ascorbic Acid.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/503e9e0edf7833bae085c30e.png"},{"id":99790264,"identity":"2bac91ab-143c-4fc2-8d6f-c781ced67ca0","added_by":"auto","created_at":"2026-01-08 12:57:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39485,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curve of DPPH of Extract.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/9d44f04d04501aae11c04381.png"},{"id":99494415,"identity":"9e29f105-2478-43f1-a04c-f9b15cffeb3b","added_by":"auto","created_at":"2026-01-05 05:59:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18820,"visible":true,"origin":"","legend":"\u003cp\u003eAcetic acid-induced writhing test of PRFEE\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/aa28cab76c1e25b041c319b0.png"},{"id":99494422,"identity":"4e201f86-92c4-4ee3-8b23-42fab2e955b2","added_by":"auto","created_at":"2026-01-05 05:59:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16987,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of furosemide and PRFEE on urine volume in albino mice.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/606c197f5285f06423040607.png"},{"id":99790107,"identity":"ac1ea995-7cb2-4c56-9208-e3295bde6a91","added_by":"auto","created_at":"2026-01-08 12:55:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":26120,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of PRFEE on urinary sodium, potassium and chloride (mmol/L) ions concentration in albino mice.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/8bab33af810bae7e20b5f375.png"},{"id":99790682,"identity":"4491acc0-e147-4620-8408-3e42c2e2d212","added_by":"auto","created_at":"2026-01-08 12:58:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17728,"visible":true,"origin":"","legend":"\u003cp\u003eAntidiarrheal activity of the PRFEE.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/d38c36e150c6b2afd84da7b9.png"},{"id":99790171,"identity":"81ace904-edfb-40ab-a256-d3e6b5804cb6","added_by":"auto","created_at":"2026-01-08 12:57:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":636317,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-microbial activity in (Hole-Plate) disk diffusion\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/cb8df03253e074049e41880e.png"},{"id":100356398,"identity":"99894f21-c2ac-449e-871d-addb03e91f6a","added_by":"auto","created_at":"2026-01-16 07:08:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2106909,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8462001/v1/68a8119e-a636-43e5-8b60-c2c1075e77fc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cem\u003eIn-vivo\u003c/em\u003eAnalgesic, Diuretic, Anti-diarrheal and \u003cem\u003ein-vitro\u003c/em\u003eAnti-microbial activity study of the ethanolic extract of \u003cem\u003ePhyllanthus reticulatus\u003c/em\u003e fruits\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMedicinal plants have long played a significant role in healthcare systems across various regions of the world. These plants contain a wide range of bioactive compounds with therapeutic properties that are used in the treatment of numerous health conditions in both humans and animals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among these, \u003cem\u003ePhyllanthus reticulatus\u003c/em\u003e (commonly known as Pancoli) is a shrub or small tree belonging to the Euphorbiaceae family. It has been traditionally used throughout Asia and Africa to treat liver disorders, gastrointestinal issues, and various infections. The plant typically grows to a height of 1 to 5 meters [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] with its flowering, and fruiting season extending from July to March [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Phytochemical analyses show that flavonoids, alkaloids, saponins, phenols, cardiac glycosides, tannins, anthocyanins, and terpenoid compounds are found in plant [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These constituents are believed to contribute to the plant\u0026rsquo;s wide range of pharmacological effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previous studies have documented the traditional and pharmacological uses of \u003cem\u003eP. reticulatus\u003c/em\u003e. This plant has demonstrated antioxidant, antibacterial, anti-HIV-1, antidiabetic, antifungal, antiplasmodial and antidiarrheal effects [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, an herbal hair dye formulation has been developed using an aqueous leaf extract of \u003cem\u003eP. reticulatus\u003c/em\u003e in combination with other natural ingredients [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Methanolic extracts of the stem bark have shown antimicrobial activity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Its fruits have been used traditionally for their anti-inflammatory effects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, traditional practices include the preparation of a pill made from leaf juice mixed with camphor and cubebs, which is dissolved in the mouth to treat bleeding gums, while the roots are used as a natural red dye [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Phyllanthus species is known for its diuretic effects, contributing to fluid and electrolyte balance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Methanolic and ethyl acetate extracts from the plant have shown promising antimicrobial activity against both bacterial and fungal pathogens [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Pain can be measured using tests like the acetic acid-induced writhing and hot-plate methods, which show how drugs work in both the brain and the nerves. These drugs also help with diarrhea by controlling how the intestines move and produce liquid, which stops too much water from being lost, as seen in tests using castor oil-induced diarrhea [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, these drugs have a diuretic effect, meaning they increase urine and salt loss, showing their effect on kidney function and the body's water balance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Both Gram-positive and Gram-negative bacteria are believed to be a big issue for public health [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While extensive research has confirmed the pharmacological potential of the plant\u0026rsquo;s leaves, bark, and stem, the fruit of \u003cem\u003eP. reticulatus\u003c/em\u003e remains significantly underexplored. Despite its traditional medicinal use and potential as a source of novel bioactive compounds, there is a lack of comprehensive phytochemical and pharmacological data specifically focused on the fruit. The chemical constituents, biological properties, and therapeutic potential of the fruit have not yet been systematically investigated. Given this knowledge gap, further scientific investigation is essential. The fruit of \u003cem\u003eP. reticulatus\u003c/em\u003e may contain unique active compounds that could offer therapeutic benefits in the treatment of pain, gastrointestinal disorders, metabolic diseases and microbial infections. A thorough study of the fruit could contribute to the development of affordable, accessible, and safe natural therapeutics, particularly important for populations in developing countries that rely heavily on herbal medicine. Therefore, the present study was undertaken to investigate the phytochemical composition and pharmacological properties of the ethanolic extract of \u003cem\u003ePhyllanthus reticulatus\u003c/em\u003e fruits (PRFEE) with a specific focus on in vivo analgesic, diuretic, antidiarrheal, and in vitro antimicrobial activities. This research aims to bridge traditional knowledge and modern pharmacological validation, potentially identifying new bioactive compounds of therapeutic value.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials and Extraction Process\u003c/h2\u003e \u003cp\u003eThe fruits of \u003cem\u003eP. reticulatus\u003c/em\u003e were gathered from Savar, Dhaka, Bangladesh between October to November 2023. They were washed carefully to remove any unwanted stuff. The plant was confirmed by the Bangladesh National Herbarium in Mirpur, Dhaka, Bangladesh with the accession number DACB 87899. After washing, the fruits were dried in the shade for seven days and then dried in an oven at 40\u0026deg;C for 24 hours. The dried fruits were then turned into a rough powder using a disc mill grinder machine. Before storing, the powder was cleaned again and dried properly. The powder was kept in a sealed container in a dark and dry area. 341 g of dry powder of \u003cem\u003eP. reticulatus\u003c/em\u003e fruits was taken by the extractor apparatus, and 2.5 liters of 98% ethanol were used in the Soxhlet apparatus, model-509.303.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals\u003c/h3\u003e\n\u003cp\u003eAspirin (Albion Laboratories Limited), Furosemide (Square Pharmaceutical Limited), and Loperamide (ACME Laboratories Limited) were purchased from the market. The bacterial strain organisms were collected from the Microbiology Research Laboratory, Gono Bishwabidyala. All the chemicals are sourced from the Department of Pharmacy Laboratory, Gono Bishwabidyalay, Savar, Dhaka.\u003c/p\u003e\n\u003ch3\u003eExperimental Animals\u003c/h3\u003e\n\u003cp\u003eSwiss albino mice both male and female aged between 6 and 7 weeks and weighing 20 to 30 grams were acquired from the Animal Research Laboratory at the Department of Pharmacy, Jahangirnagar University located in Savar, Dhaka. They were maintained under standard laboratory conditions including a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, a humidity level of 55\u0026ndash;65% and a 12-hour light-dark cycle. The mice had free access to food and water and were allowed to acclimate to the laboratory environment for one week before the start of the experiments. The mice were divided into five groups for analgesic and anti-diarrheal test, and four groups for diuretics test with each group containing five mice. All experiments conducted on animals were approved by the Research Ethical Committee under the Center for Multidisciplinary Research of Gono Bishwabidyalay, Dhaka 1344, Bangladesh (Permission number: CMR/EC/023).\u003c/p\u003e\n\u003ch3\u003ePhytochemical activitys\u003c/h3\u003e\n\u003cp\u003eThe PRFEE was checked for plant-based chemical components through several simple tests. Different chemicals were used according to standard methods, and the results showed clear color changes that showed the presence of carbohydrates, saponins, tannins, alkaloids, flavonoids, steroids, glycosides, and phenolic compounds, and the absence of steroid compounds [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAntioxidant activitys\u003c/h3\u003e\n\u003cp\u003eReducing Power Capacity Assay (RPC)\u003c/p\u003e \u003cp\u003eReducing power capacity of the fruit extract was evaluated using the ferric\u0026ndash;ferricyanide reduction method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 1.0 ml of Plant extract or standard of different concentration mixed with 2.5ml of phosphate buffer (pH 6.6) \u0026amp; 2.5 ml potassium ferricyanide (1%). The reaction mixture is incubated for 20 min at 50\u003csup\u003e0\u003c/sup\u003e C to complete the reaction. 2.5ml of trichloro acetic acid (10%) solution is added into the test tube and the total mixture was centrifuged at 3000 rpm for 10 min. 2.5ml of supernatant solution is withdrawn from the mixture and mixed with 2.5 ml of distilled water. 0.5ml of ferric chloride (0.1%) solution was added to the diluted reaction mixture. Then the absorbance of the solution is measured at 700 nm using a spectrophotometer against blank. An increase in absorbance indicated enhanced reducing power.\u003c/p\u003e \u003cp\u003eDPPH Free Radical Scavenging Assay\u003c/p\u003e \u003cp\u003eThe in vitro antioxidant activity of the fruit extract was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay, which measures the ability of antioxidants to quench DPPH radicals through hydrogen atom or electron donation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which measures the ability of antioxidants to quench DPPH radicals through hydrogen atom or electron donation. A 0.004% (w/v) DPPH solution was freshly prepared by dissolving 0.004 g DPPH in 100 mL ethanol. Stock solutions of the fruit extract and ascorbic acid (5 mg/mL) were prepared in ethanol, and serial dilutions yielded concentrations of 6.25 to 800 \u0026micro;g/mL. Aliquots (1 mL) of each concentration were mixed with 2 mL of DPPH solution and incubated in the dark at room temperature for 30 min. Absorbance was measured at 517 nm using a UV-Vis spectrophotometer. All tests were conducted in triplicate. The percentage of DPPH radical inhibition was calculated as:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\%\\:Inhibition=(1-\\frac{Absorbance\\:of\\:sample}{Absorbance\\:of\\:control}\\)\u003c/span\u003e \u003c/span\u003e ) \u0026times; 100\u003c/p\u003e \u003cp\u003eThe IC₅₀ value (\u0026micro;g/mL) was determined from a plot of % inhibition versus concentration by linear regression.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAcute Toxicity Study\u003c/h2\u003e \u003cp\u003eAn acute oral toxicity study was conducted on Swiss albino mice in accordance with OECD Guideline 423 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Twelve mice were randomly allocated into four groups (n\u0026thinsp;=\u0026thinsp;3) and administered a single oral dose of PRFEE at 250, 500, 1000 and 2000 mg/kg, respectively. The animals were monitored for 14 days for signs of toxicity, behavioral alterations, and mortality.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-vivo Evaluation of Analgesic Activity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAcetic acid induced writhing test\u003c/p\u003e \u003cp\u003eThe abdominal writhing test was done using acetic acid following the method by Saha et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] with some changes. The animals were randomly divided into five groups. The control group got normal saline at a dose of 10 ml/kg of body weight. The standard group was given aspirin at 75 mg/kg, and the test groups were given PRFEE at doses of 100, 200 and 300 mg/kg, all by mouth. Thirty minutes after drug administration, all animals were injected intraperitoneal with 0.6% acetic acid to induce pain. After a latency period of 5 minutes, the number of abdominal writhes was counted over the next 30 minutes. The pain-relieving effect was shown as the percentage of writhing that was prevented.\u003c/p\u003e \u003cp\u003e% Inhibition\u0026thinsp;=\u0026thinsp;Number of writhes in control group - Number of writhes in treated group\u0026thinsp;\u0026divide;\u0026thinsp;Number of writhes in control group \u0026times; 100\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo Evaluation of Diuretic Activity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDiuretic activity was assessed using the method of Geleta et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with slight modifications. Swiss albino mice (20\u0026ndash;30 g) were ensured to have similar body weights. All the mice were not allowed to eat for the whole night but could drink water freely, and were acclimatized in individual metabolic cages for 24 hours before the experiment. The control group received 0.9% NaCl, the standard group was administered furosemide at a dose of 10 mg/kg and the test groups received PRFEE at 250 and 500 mg/kg by mouth. After dosing, urine was collected over 24 hours in individual metabolic cages. Collected urine was filtered and stored at -20\u0026deg;C for analysis of sodium, potassium, and chloride levels. The following parameters were calculated.\u003c/p\u003e \u003cp\u003eDiuretic action (%)\u0026thinsp;=\u0026thinsp;Total urinary output\u0026thinsp;\u0026divide;\u0026thinsp;Total liquid administered \u0026times;100 (1)\u003c/p\u003e \u003cp\u003eUrinary excretion\u0026thinsp;=\u0026thinsp;Urinary excretion of test group\u0026thinsp;\u0026divide;\u0026thinsp;Urinary excretion of control group (2)\u003c/p\u003e \u003cp\u003eDiuretic activity\u0026thinsp;=\u0026thinsp;Diuretic action of the test group\u0026thinsp;\u0026divide;\u0026thinsp;Diuretic action of standard drug (3)\u003c/p\u003e \u003cp\u003eAnalytical Procedure:\u003c/p\u003e \u003cp\u003eThe levels of sodium, potassium, and chloride in both the urine samples and the plant extract were checked. These measurements were done with an ion-selective electrode analyzer called the 9180 Electrolyte Analyzer Reagent, which was made by Roche in Germany.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of the Antidiarrheal Activity\u003c/h3\u003e\n\u003cp\u003eCastor Oil-Induced Diarrheal Test\u003c/p\u003e \u003cp\u003eThe diarrhea model caused by castor oil as described by Afroz et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] was used with slight modifications. Mice were not given food for 18 hours. All mice were given 1.0 ml of castor oil by mouth. After 30 minutes, the control group was given normal saline at a dose of 10 ml/kg of body weight. The standard group received loperamide at 2 mg/kg. The test groups were given PRFEE at doses of 150, 300, and 500 mg/kg. Each mice were placed in a separate cage with paper lining. The paper was changed every hour for 4 hours. The number and weight of feces, along with stool consistency were recorded. The results were shown as a percentage of how much diarrhea was prevented [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e% Inhibition of defecation\u0026thinsp;=\u0026thinsp;Mean of defecation control group - Mean of defecation treated group\u0026thinsp;\u0026divide;\u0026thinsp;Mean of defecation control group \u0026times; 100\u003c/p\u003e\n\u003ch3\u003eIn vitro Evaluation of Antimicrobial Activity\u003c/h3\u003e\n\u003cp\u003eMedia Preparation:\u003c/p\u003e \u003cp\u003eMueller-Hinton agar and broth were prepared and sterilized according to the manufacturer\u0026rsquo;s instructions and standard protocols [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. About 20\u0026ndash;25 ml of agar was added to sterile petri dishes and let dry at room temperature. The dishes were kept in a refrigerator at 4\u0026deg;C until they were needed [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The bacteria used in this study were Staphylococcus aureus, which is Gram-positive and Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa, which are all Gram-negative from the Microbiology Research Laboratory, Gono Bishwabidyalay, Savar, Dhaka.\u003c/p\u003e \u003cp\u003eDisc diffusion method:\u003c/p\u003e \u003cp\u003eAntimicrobial activity of PRFEE was tested using a modified disc diffusion (Hole-Plate) method [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Bacterial suspensions were placed on nutrient agar plates and small wells, each about 8 millimeters in size were created using a clean cork borer. The extract was dissolved in a 1:1 mixture of DMSO and water for injection and applied at concentrations of 250, 500 and 750 \u0026micro;g/ml (100 \u0026micro;l Per well). Plates were kept at a temperature of 37\u0026deg;C for 24 hours. The areas where bacteria did not grow were measured in millimeters to assess the effectiveness of the substance in inhibiting bacterial growth. Water for injection and the solvent mixture served as negative controls, while Kanamycin-K (30 \u0026micro;g/disc) was used as the positive standard. All tests were performed in duplicate for accuracy.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was done using SPSS version 30.0 for Windows [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The results are shown as S.E.M. A one-way ANOVA was used along with Dunnett\u0026rsquo;s post hoc test to compare multiple groups. Differences were considered statistically significant when the P value was below P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eScreening Phytochemical Components\u003c/h2\u003e\n \u003cp\u003eThe PRFEE was found to contain carbohydrates, tannins, saponins and phenolic constituents upon phytochemical analysis. However, steroids were absent.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eAntioxidant activity\u003c/h2\u003e\n \u003cp\u003eDetermination of Reducing Power capacity (RPC)\u003c/p\u003e\n \u003cp\u003eThe reducing properties are generally associated with the presence of reluctance which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom). Absorbance Concentration (\u0026micro;g/ml) reducing power (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In the present study ferric reducing power activity of the extract of fruits of \u003cem\u003eP. reticulatus\u003c/em\u003e plants are for (118.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.30 mg/g) as Gallic acid (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The experimental results indicate lower ferric (y\u0026thinsp;=\u0026thinsp;0.0029x\u0026thinsp;+\u0026thinsp;0.039, R\u0026sup2; = 0.9842) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAbsorbance found with different concentration of Gallic Acid.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbsorbance\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.147\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.223\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.269\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.484\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.788\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetermination of Reducing Power capacity of the ethanolic extract of fruits of \u003cem\u003eP. reticulatus.\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample Name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWt. of plant extract (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbs.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAE conc.(C) (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAE conc.(C) (mg/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eV (ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC \u0026times; V (mg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTPC as GAE, A=(c\u0026times;V)/m (mg/gm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean value\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;SD\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ereticulatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.445\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e117.241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e118.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.30\u003c/p\u003e\n \u003cp\u003emg/g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.756\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.793\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.828\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124.128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eDPPH free radical scavenging assay\u003c/h2\u003e\n \u003cp\u003eWhen DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance. DPPH free radical scavenging study of the extract of fruits of \u003cem\u003eP. reticulatus\u003c/em\u003e determine as % of inhibition and IC\u003csub\u003e50\u003c/sub\u003e (inhibition concentration 50%) value. Lower IC\u003csub\u003e50\u003c/sub\u003e values indicates good scavenging capacity and antioxidant property. The results of the present study showed that ethanolic extract have IC\u003csub\u003e50\u003c/sub\u003e value 639.033 \u0026micro;g/mL (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), exhibited poor antioxidant property compared to standard 3.725 \u0026micro;g/ml (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe percentage (%) scavenging Activity of Ascorbic Acid.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbsorbance sample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Inhibition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.940\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\" align=\"left\"\u003e\n \u003cp\u003e3.725\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.700\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.251\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.729\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58.295\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.641\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.329\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e66.933\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.489\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e72.025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.864\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe results and IC\u003csub\u003e50\u003c/sub\u003e ethanol extract of fruits of \u003cem\u003eP. reticulatus\u003c/em\u003e is presented in the Table. The percentage (%) scavenging Activity of \u003cem\u003eP. reticulatus\u003c/em\u003e fruits extract.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbsorbance sample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Inhibition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.514\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.39%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\" align=\"left\"\u003e\n \u003cp\u003e639.033\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.08%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.46%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41.36%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.31%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.953\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.48%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67.11%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u0026nbsp;\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eAcute Toxicity\u003c/h2\u003e\n \u003cp\u003eNo mortality was observed in any of the groups. The body weight remained normal and no significant toxic symptoms were observed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eAnalgesic activity\u003c/h2\u003e\n \u003cp\u003ePRFEE showed a strong pain-relieving effect that increases with higher doses in the acetic acid-induced writhing test. Aspirin at 75 mg/kg produced 80% inhibition (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PRFEE at 100, 200 and 300 mg/kg showed 20%, 71% and 89% inhibition, respectively. Significant reductions in writhing were observed at 200 mg/kg (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 300 mg/kg (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while 100 mg/kg was not statistically significant (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of Aspirin and PRFEE on acetic acid-induced writhing in mice.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean of Writhing\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Inhibition\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 ml/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAspirin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRFEE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eValues are represented as S.E.M, (n\u0026thinsp;=\u0026thinsp;5); P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u0026ndash; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eDiuretic activity\u003c/h2\u003e\n \u003cp\u003eEffect of PRFEE on 24-hour urine output and diuretic indices in mice. The control group produced 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 ml of urine, while furosemide raised output to 2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 ml (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PRFEE at 250 and 500 mg/kg increased urine volume to 2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 ml (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.289 ml (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively. The diuretic actions were 2.00 and 2.50 with corresponding diuretic activities of 0.72 and 0.91, indicating that the 500 mg/kg dose achieved the standard drug effect (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDiuretic effect of PRFEE by increasing urine volume.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal urine volume (ml)\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDiuretic action (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDiuretics activity\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 ml/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFurosemide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.250 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRFEE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.289 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eValues are represented as S.E.M, (n\u0026thinsp;=\u0026thinsp;5); P\u0026thinsp;\u0026lt;\u0026thinsp;0.05- P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eElectrolyte Excretion\u003c/h2\u003e\n \u003cp\u003ePRFEE significantly increased urinary excretion of Na⁺, K⁺ and Cl⁻ levels. In the control group urine levels were 87.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87 (Na⁺), 55.58\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73 (K⁺) and 61.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20 (Cl⁻). Furosemide at 10 mg/kg markedly increased electrolyte excretion Na⁺ to 125.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.54, K⁺ to 80.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82 and Cl⁻ to 95.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10 mmol/l (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01-P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PRFEE at 250 mg/kg significantly raised Na⁺, K⁺ and Cl⁻ to 106.28, 76.32 and 82.51 mmol/l, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 500 mg/kg excretion levels further increased to 133.16 (Na⁺), 87.25 (K⁺) and 106.79 mmol/l (Cl⁻), all highly significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), exceeding the effect of furosemide (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of PRFEE on urinary electrolyte excretion.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eTotal Urinary electrolyte excretion\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003emmol/l\u0026thinsp;\u0026plusmn;\u0026thinsp;mean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003emmol/l\u0026thinsp;\u0026plusmn;\u0026thinsp;mean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCl\u003csup\u003e\u0026minus;\u003c/sup\u003emmol/l\u0026thinsp;\u0026plusmn;\u0026thinsp;mean\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 ml/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e55.58\u0026thinsp;\u0026plusmn;\u0026thinsp;4.729\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.197\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFurosemide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e125.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.535 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.821 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.095 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRFEE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.28\u0026thinsp;\u0026plusmn;\u0026thinsp;3.361 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76.32\u0026thinsp;\u0026plusmn;\u0026thinsp;5.434 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.437 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e133.16\u0026thinsp;\u0026plusmn;\u0026thinsp;8.186 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.792 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.79\u0026thinsp;\u0026plusmn;\u0026thinsp;4.490 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eValues are represented as S.E.M, (n\u0026thinsp;=\u0026thinsp;5); P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eAntidiarrheal Activity\u003c/h2\u003e\n \u003cp\u003ePRFEE has dose-dependent antidiarrheal effects in the control group had the mean of 5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 watery stools were observed, while loperamide at 2 mg/kg significantly reduced this to 2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) showing 60% inhibition. PRFEE at 150 mg/kg caused a mild reduction of 3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 (27% inhibition) while 300 mg/kg showed significant activity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) with 2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 stools (50% inhibition). The 500 mg/kg dose produced a highly significant reduction to 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 stools (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), achieving 81.82% inhibition exceeding that of Loperamide as shown (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) and (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eShowing the percentage reduction in volume of small intestinal content.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean number of watery stools\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInhibition of diarrhea (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 ml/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLoperamide HCl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.629 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRFEE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.479\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.479 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500 mg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.408 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.82%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eValues are represented as S.E.M, (n\u0026thinsp;=\u0026thinsp;5); P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eAntimicrobial Activity\u003c/h2\u003e\n \u003cp\u003eThe PRFEE exhibits the capability to combat both Gram-positive and Gram-negative bacteria. No inhibition was observed at 250 \u0026micro;g/ml. The dose at 500 \u0026micro;g/ml showed moderate zones of inhibition were seen against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (13 mm) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (17 mm), while \u003cem\u003eEscherichia coli\u003c/em\u003e showed no response. At 750 \u0026micro;g/ml, PRFEE showed strong antibacterial activity with inhibition zones of 17 mm (\u003cem\u003eE. coli\u003c/em\u003e), 15 mm (\u003cem\u003eP. aeruginosa\u003c/em\u003e) and 19 mm (\u003cem\u003eS. aureus\u003c/em\u003e), comparable to the standard antibiotic kanamycin (30 \u0026micro;g). These results are summarized and visually represented in (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab9\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInhibitory effect of PRFEE on (1) Escherichia coli (2) Pseudomonas aeruginosa (3) Staphylococcus aureus (4) Negative control (5) Kanamycin.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eZone of inhibition (mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEscherichia coli\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePseudomonas aeruginosa\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStaphylococcus aureus\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eKanamycin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRFEE 250 \u0026micro;g/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRFEE 500 \u0026micro;g/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRFEE 750 \u0026micro;g/ml\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe Present study evaluated the dose-dependent pharmacological activities. This research is the first to report on the pharmacological potential of the fruits clearly demonstrates that the PRFEE exhibits significant pharmacological properties including analgesic, diuretic, antidiarrheal and antimicrobial activities. These effects can be attributed to its rich content of bioactive phytochemicals compounds, which are well-known for their diverse therapeutic effects. The results indicate that the fruits of \u003cem\u003eP. reticulatus\u003c/em\u003e extract exhibited low DPPH radical scavenging activity and limited reducing power capacity, indicating weak chemical antioxidant potential. However, these assays mainly assess in vitro electron-donating ability and do not necessarily correlate with pharmacological effects. The observed biological activities may be attributed to the presence of alkaloids, flavonoids and phenolic compounds, which exert analgesic, diuretic and antidiarrheal effects through mechanism-specific pathways independent of antioxidant capacity [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. PRFEE showed a strong analgesic effect in the acetic acid-induced writhing test with 300 mg/kg producing 89% inhibition, which was significantly greater than aspirin (75 mg/kg, 80%). Acetic acid-induced pain is mediated by the release of endogenous substances such as prostaglandins particularly PGE₂ and PGF₂α, which stimulate nociceptors [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Flavonoids and alkaloids reported are known to inhibit cyclooxygenase and lipoxygenase enzymes thereby suppressing prostaglandin synthesis and producing analgesia [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Further supporting the role of polyphenolic compounds in analgesic activity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. PRFEE significantly increased urine output with the 500 mg/kg dose (2.50 ml) achieving a response comparable to furosemide (2.75 ml). The extract also markedly enhanced urinary electrolyte excretion (Na⁺, K⁺, Cl⁻) with the higher dose surpassing the standard drug. Flavonoids and saponins have been reported to exert diuretic effects by altering renal tubular ion transport and increasing renal blood flow [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The observed saluretic effect (Na⁺ and Cl⁻ excretion) and kaliuretic activity (K⁺ excretion) suggest that PRFEE may act through a mechanism similar to loop diuretics possibly mediated by prostaglandins [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These findings indicate that PRFEE could be a promising natural diuretic.\u003c/p\u003e \u003cp\u003eIn the castor oil-induced diarrheal model, PRFEE exhibited protection with the 500 mg/kg dose producing 81.82% inhibition surpassing loperamide (60%). Castor oil causes diarrhea by releasing ricinoleic acid, which stimulates intestinal peristalsis and secretion of electrolytes and water via prostaglandin pathways [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Tannins, flavonoids and steroids are known to reduce intestinal motility and secretion by inhibiting prostaglandin synthesis and relaxing intestinal smooth muscles [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The significant effect of PRFEE can therefore be linked to its tannin and flavonoid content consistent with earlier findings in other \u003cem\u003ePhyllanthus\u003c/em\u003e species [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The fruit extract also demonstrated broad-spectrum antibacterial activity with strong inhibition zones against Staphylococcus aureus (19 mm), Pseudomonas aeruginosa (15 mm) and Escherichia coli (17 mm) at 750 \u0026micro;g/ml comparable to the standard antibiotic kanamycin. Phenolic, flavonoids and alkaloids compounds are well known for their ability to disrupt bacterial cell walls inhibit nucleic acid synthesis and interfere with microbial enzymes, thereby exerting antimicrobial effects [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These findings justify the ethnomedicinal use of \u003cem\u003eP. reticulatus\u003c/em\u003e in infectious diseases.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe PRFEE exhibits potent analgesic, diuretic, antidiarrheal and antimicrobial activities. The highest doses of PRFEE often surpassed standard drugs in efficacy, indicating the fruit as a Promising source of bioactive compounds. The study highlights the relevance of plant part selection in pharmacological research, as the fruit may contain higher levels of active constituents than the leaves and bark. These results support the traditional medicinal use of \u003cem\u003eP. reticulatus\u003c/em\u003e and suggest that PRFEE could serve as a foundation for developing natural therapeutic agents. Further studies focusing on molecular docking, isolation of bioactive compounds, mechanistic evaluation, toxicity profiling and clinical trials are necessary to establish its safety and therapeutic applicability.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePRFEE,\u0026nbsp;\u003cem\u003ePhyllanthus reticulatus\u003c/em\u003e Fruit Ethanolic Extract; S.E.M., Standard Error of Mean.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePR assisted in doing the project (experiments) for her thesis and analyzed the data and contributed to the writing of the paper. KZ supervised the project. He was involved in all parts of the experiments. JN analyzed the data and contributed to the writing of the paper. SM advised on the project and performed some experiments. TR and AM advised on the project. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the contribution of the Department of Pharmacy, Gono Bishwabidyalay, for laboratory facility and ancillary support to complete this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eanimal rights\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were used in the studies that are the basis of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement:\u003c/strong\u003e No potential conflict of interest was reported by the author(s).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw data from original research papers and on-line databases are summarized in this review article, and the data sources are cited in the Reference section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The author(s) reported there is no funding associated with the work featured in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Ethics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript. Competing interests The authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Clinical trial number\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRates SMK. Plants as source of drugs. Toxicon. 2001;39:603\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaachouay N, Zidane L. Plant-derived natural products: a source for drug discovery and development. Drugs Drug Candidates. 2024;3:184\u0026ndash;207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzhar H, Shabbir A, Shahzad M et al. Phyllanthus reticulatus Prevents Ethanol-Induced Gastric Ulcer via Downregulation of IL‐8 and TNF‐α Levels. \u003cem\u003eEvidence‐Based Complementary and Alternative Medicine\u003c/em\u003e 2021; 2021: 1734752.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma S, Kumar S. Phyllanthus reticulatus Poir.-an important medicinal plant: a review of its phytochemistry, traditional uses and pharmacological properties. Int J Pharm Sci Res. 2013;4:2528.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOminyi MC, Ali FU, Ebenyi LN, et al. Identification and Characterization of Phytochemicals in Ethanol Leaf-extracts of Phyllanthus reticulatus and Anthocleista djalonesis. Nigerian J Biochem Mol Biology. 2022;37:314\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinan KI, de la Luz C\u0026aacute;diz-Gurrea M, Leyva-Jim\u0026eacute;nez FJ, et al. New insights on Phyllanthus reticulatus Poir. leaves and stem bark extracts: UPLC-ESI-TOF-MS profiles, and biopharmaceutical and in silico analysis. New J Chem. 2021;45:21049\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEldeen IMS, Seow EM, Abdullah R, et al. In vitro antibacterial, antioxidant, total phenolic contents and anti-HIV-1 reverse transcriptase activities of extracts of seven Phyllanthus sp. South Afr J Bot. 2011;77:75\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahana A, Gupta KK, Chakraborty M et al. Pre-Clinical Evaluation of Anti-Diabetic Activity of Phyllanthus Reticulatus Fruit Extract. \u003cem\u003eJournal of Advanced Zoology\u003c/em\u003e; 44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChellappandian M, Saravanan M, Pandikumar P, et al. Traditionally practiced medicinal plant extracts inhibit the ergosterol biosynthesis of clinically isolated dermatophytic pathogens. J de Mycol medicale. 2018;28:143\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhatun MH, Nesa ML, Islam R, et al. Antidiabetic and antidiarrheal effects of the methanolic extract of Phyllanthus reticulatus leaves in mice. Asian Pac J Reprod. 2014;3:121\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiran kumar G, Hariprasath GM, Mukesh R, et al. Formulation of herbal based hair colour from Phyllanthus reticulatus. Nat Prod Res. 2025;39:2114\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBegum T, Rahman MS, Rashid MA. Phytochemical and biological investigations of Phyllanthus reticulatus. Dhaka Univ J Pharm Sci. 2006;5:21\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Sharma S, Kumar D, et al. Pharmacognostic study and anti\u0026ndash;inflammatory activity of Phyllanthus reticulatus Poir. fruit. Asian Pac J Trop Disease. 2012;2:S332\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRam HNA, Shreedhara CS, Gajera FP et al. Phamacognostical Evaluation of Phyllanthus reticulatus Poir. \u003cem\u003ePharmacognosy Magazine\u003c/em\u003e; 5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao AN, Kamagat\u0026eacute; M, Amonkan AK, et al. The acute diuretic effect of an ethanolic fraction of Phyllanthus amarus (Euphorbiaceae) in rats involves prostaglandins. BMC Complement Altern Med. 2018;18:94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaque T, Muhsin MDA, Akhter T, et al. Antimicrobial and analgesic activity of leaf extracts of Phyllanthus reticulatus Poir.(Family-Euphorbiaceae). Jahangirnagar Univ J Biol Sci. 2016;5:81\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHiremath KY, Shinde S, Jambale UK, et al. Anticancer effect of Phyllanthus reticulatus methanolic leaf extract on HCT-116 colon cancer cell line using GC-MS and FTIR analysis. Arch Razi Inst. 2024;79:1013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNesa ML, Karim SMS, Api K, et al. Screening of Baccaurea ramiflora (Lour.) extracts for cytotoxic, analgesic, anti-inflammatory, neuropharmacological and antidiarrheal activities. BMC Complement Altern Med. 2018;18:35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah SF, Baig SG, Abidi S et al. PHARMACOLOGICAL EVALUATION OF Silybum marianum (L.) SEED EXTRACT: ANALGESIC, ANTI-INFLAMMATORY, DIURETIC, AND ANTIDIARRHEAL ACTIVITIES. \u003cem\u003eJournal of Medical \u0026amp; Health Sciences Review\u003c/em\u003e; 2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSardar PK, Devnath HS, Biswas NN et al. Evaluation of Diuretic Property of Argemone mexicana along with Molecular Docking Study. \u003cem\u003eJordan Journal of Pharmaceutical Sciences\u003c/em\u003e; 16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzam A, Ahmed AS, Oves M et al. Antimicrobial activity of metal oxide nanoparticles against Gram-positive and Gram-negative bacteria: a comparative study. Int J Nanomed 2012; 6003\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhani A. Medicinal plants of Bangladesh with chemical constituents and uses. Asiatic society of Bangladesh; 2003.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVijayalakshmi M, Ruckmani K. Ferric reducing antioxidant power assay in plant extract. ||| Bangladesh J Pharmacology|||. 2016;11:570\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHagaggi NSA, Abdul-Raouf UM, Radwan TAA. Variation of antibacterial and antioxidant secondary metabolites and volatiles in leaf and callus extracts of Phulai (Acacia Modesta Wall). BMC Plant Biol. 2024;24:93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement Altern Med. 2012;12:221.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuideline O, Testing FOR, Chemicals OF. Acute Oral Toxicity \u0026ndash; Acute Toxic Class Method.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiswas NN, Saha S, Ali MK. Antioxidant, antimicrobial, cytotoxic and analgesic activities of ethanolic extract of Mentha arvensis L. Asian Pac J Trop Biomed. 2014;4:792\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeleta B, Eyasu M, Fekadu N, et al. Evaluation of diuretic activity of hydro-ethanolic extract of Moringa stenopetala leaves in Swiss albino mice. Clin Exp Pharmacol. 2015;5:2161.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfroz S, Alamgir M, Khan MTH, et al. Antidiarrhoeal activity of the ethanol extract of Paederia foetida Linn.(Rubiaceae). J Ethnopharmacol. 2006;105:125\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDey SK, Hira A, Howlader MSI, et al. Antioxidant and antidiarrheal activities of ethanol extract of Ardisia elliptica fruits. Pharm Biol. 2014;52:213\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartina SM, Cabalquinto GP, Lumogdang L. TAXONOMIC DESCRIPTION AND ANTIBACTERIAL ACTIVITY OF DILLENIA sp. AGAINST ESCHERICHIA coli. Biotropia. 2024;31:422\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal. 2016;6:71\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan ZA, Siddiqui MF, Park S. Current and emerging methods of antibiotic susceptibility testing. Diagnostics. 2019;9:49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil A. Antimicrobial activity of ethanolic extracts of Ocimum basilicum leaf from Saudi Arabia. Biotechnology. 2013;12:61\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdel Aziz SM, Ahmed OM, Abd EL-Twab SM et al. Antihyperglycemic effects and mode of actions of Musa paradisiaca leaf and fruit peel hydroethanolic extracts in nicotinamide/streptozotocin-induced diabetic rats. \u003cem\u003eEvidence‐Based Complementary and Alternative Medicine\u003c/em\u003e 2020; 2020: 9276343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Pandey AK. Chemistry and biological activities of flavonoids: an overview. \u003cem\u003eThe scientific world journal\u003c/em\u003e 2013; 2013: 162750.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJothy SL, Zuraini Z, Sasidharan S. Phytochemicals screening, DPPH free radical scavenging and xanthine oxidase inhibitiory activities of Cassia fistula seeds extract. J Med Plants Res. 2011;5:1941\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzab A, Nassar A, Azab AN. Anti-inflammatory activity of natural products. Molecules. 2016;21:1321.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad FM, Zafar A, Ahmed M, et al. Quercus floribunda Lindl. Ex A. Camus; a tremendous remedy against inflammation and associated symptoms. Fitoterapia. 2023;170:105628.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu C, Liu N, Tian M, et al. Effects of alkaloids on peripheral neuropathic pain: a review. Chin Med. 2020;15:106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerraz CR, Carvalho TT, Manchope MF, et al. Therapeutic potential of flavonoids in pain and inflammation: mechanisms of action, pre-clinical and clinical data, and pharmaceutical development. Molecules. 2020;25:762.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtawodi SE, Pfundstein B, Haubner R, et al. Content of polyphenolic compounds in the Nigerian stimulants Cola nitida ssp. alba, Cola nitida ssp. rubra A. Chev, and Cola acuminata Schott \u0026amp; Endl and their antioxidant capacity. J Agric Food Chem. 2007;55:9824\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHailu W, Engidawork E. Evaluation of the diuretic activity of the aqueous and 80% methanol extracts of Ajuga remota Benth (Lamiaceae) leaves in mice. BMC Complement Altern Med. 2014;14:135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiguori A, Casini A, Di Loreto M, et al. Loop diuretics enhance the secretion of prostacyclin in vitro, in healthy persons, and in patients with chronic heart failure. Eur J Clin Pharmacol. 1999;55:117\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakraborty S, Roy B, Sen S et al. Fermented Polyherbal Formulation Restored Ricinoleic Acid-Induced Diarrhea in Sprague Dawley Rats and Exhibited In Vitro Antibacterial Effect on Multiple Antibiotic‐Resistant Gastrointestinal Pathogens. \u003cem\u003eJournal of Pathogens\u003c/em\u003e 2024; 2024: 1997064.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalombo EA. Phytochemicals from traditional medicinal plants used in the treatment of diarrhoea: modes of action and effects on intestinal function. Phytotherapy Research: Int J Devoted Pharmacol Toxicol Evaluation Nat Prod Derivatives. 2006;20:717\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadesse E, Engidawork E, Nedi T, et al. Evaluation of the anti-diarrheal activity of the aqueous stem extract of Lantana camara Linn (Verbenaceae) in mice. BMC Complement Altern Med. 2017;17:190.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdul Qadir M, Shahzadi SK, Bashir A et al. Evaluation of phenolic compounds and antioxidant and antimicrobial activities of some common herbs. \u003cem\u003eInternational journal of analytical chemistry\u003c/em\u003e 2017; 2017: 3475738.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErdemoglu N, Ozkan S, Tosun F. Alkaloid profile and antimicrobial activity of Lupinus angustifolius L. alkaloid extract. Phytochem Rev. 2007;6:197\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phyllanthus reticulatus fruit, Analgesic, Anti-diarrheal, Diuretics, Anti-microbial","lastPublishedDoi":"10.21203/rs.3.rs-8462001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8462001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePhyllanthus reticulatus\u003c/em\u003e commonly known as Pancoli. It is used as a traditional medicinal with properties such as antioxidant, antibacterial and anti-HIV-1.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAnalgesic activity was determined by the acetic acid writhing test and the diuretic activity was assessed in albino mice by measuring urine volume and electrolyte excretion over 24 hours following oral administration. The anti-diarrheal effect was evaluated using the castor oil-induced model, and antimicrobial activity was tested through the disc diffusion method.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the analgesic assay, the PRFEE at a dose of 300 mg/kg showed the highest inhibition (89%, 1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 writhes, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The diuretic activity assessment indicated a substantial increase in urine output with a dose of 500 mg/kg producing 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.289 ml of urine (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, this dose significantly enhanced sodium (133.16\u0026thinsp;\u0026plusmn;\u0026thinsp;8.186 mmol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), Potassium (87.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.792 mmol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and chloride (106.79\u0026thinsp;\u0026plusmn;\u0026thinsp;4.49 mmol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) excretion. In the anti-diarrheal study, PRFEE 500 mg/kg achieved 81.82% inhibition reducing stool count to 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.408 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), demonstrating strong efficacy. The antimicrobial activity showed a Potent inhibitory effect with a dose of 750 mg/mL, exhibiting inhibition zones of 17 mm (\u003cem\u003eEscherichia coli\u003c/em\u003e), 15 mm (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e) and 19 mm (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e), comparable to those of kanamycin (16\u0026ndash;19 mm).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe PRFEE exhibited significant Pharmacological effects across all evaluated Parameters.\u003c/p\u003e","manuscriptTitle":"In-vivoAnalgesic, Diuretic, Anti-diarrheal and in-vitroAnti-microbial activity study of the ethanolic extract of Phyllanthus reticulatus fruits","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 05:59:53","doi":"10.21203/rs.3.rs-8462001/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-13T10:17:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-29T07:02:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-29T07:01:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2025-12-27T15:20:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d872132-85a8-47be-a9b5-8bf0652336bc","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-09T15:53:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-05 05:59:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8462001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8462001","identity":"rs-8462001","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}