Pharmacological evaluation, anti-oxidant and ant-ulcer activity of Uncaria tomentosa extract in experimental rats

*Corresponding author: Brajlata Prajapati Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Pharmacological evaluation, anti-oxidant and ant-ulcer activity of Uncaria tomentosa extract in experimental rats Brajlata Prajapati 1, *, Pradeep Kumar Mohanty 2 and Akhlesh Kumar Singhai 3 1 Research Scholar, School of Pharmacy, LNCT University, Bhopal (M.P), India. 2 HOD, department of Pharmacology, School of Pharmacy, LNCT University, Bhopal (M.P), India. 3 Director, School of Pharmacy, LNCT University, Bhopal (M.P), India. World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 Publication history: Received on 01 April 2025; revised on 01June 2025; accepted on 04June 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.3.0497 Abstract The present study aimed to evaluate the pharmacological, antioxidant, and anti-ulcer activities of Uncaria tomentosa extracts using both in vitro and in vivo experimental models. The plant material was extracted successively using petroleum ether and methanol via Soxhlet extraction. The methanolic extract exhibited a higher percentage yield (2.23%) compared to the petroleum ether extract (0.55%). Phytochemical screening indicated the presence of alkaloids, glycosides, flavonoids, tannins, and carbohydrates predominantly in the methanolic extract. Quantitative analysis showed significant levels of total phenolic content (61.7 mg GAE/g) and total flavonoid content (16.25 mg RE/g). Antioxidant activity was confirmed through DPPH radical scavenging assay, with the methanolic extract demonstrating an IC₅₀ of 48.68 µg/mL, compared to 22.04 µg/mL for standard ascorbic acid.In vivo anti-ulcer studies in ethanolinduced ulcer models in rats revealed that Uncaria tomentosa extract at 400 mg/kg significantly reduced the ulcer index (3.001 ± 0.255), gastric juice volume (2.221 ± 1.006 mL), and free acidity (17.986 ± 1.470 mEq/L), while mildly increasing gastric pH (3.008 ± 0.650). These effects were comparable to the reference drug ranitidine. The findings validate the traditional use of Uncaria tomentosa and highlight its potential as a natural source for antioxidant and antiulcer therapies. Keywords: Uncaria tomentosa; Antioxidant activity; Anti-ulcer; Phytochemical screening; DPPH assay; Ethanolinduced ulcers; Gastric protection; Medicinal plants 1. Introduction Medicinal plants have been utilized for therapeutic purposes since ancient times and continue to be a significant source for drug discovery. A substantial portion of the population in developing regions of Asia and Africa relies on plant-based traditional medicines for primary healthcare. The widespread use of these plants is primarily due to their easy accessibility and affordability. Herbal therapies typically involve parts of plants or unrefined herbal extracts containing a variety of phytochemicals, which are believed to act synergistically. These complex mixtures can serve as lead compounds for the development of numerous drugs currently used in the treatment of various diseases.[1] Ulcers are an open sore of the skin or mucus membrane characterized by sloughing of inflamed dead tissue. Ulcers are lesions on the surface of the skin or a mucous membrane characterized by a superficial loss of tissue. Ulcers are most common on the skin of the lower extremities and in the gastrointestinal tract, although they may be encountered at almost any site. There are many types of ulcers such as mouth ulcer, esophagus ulcer, peptic ulcer, and genital ulcer. Of these peptic ulcers is seen among many people. The peptic ulcers are erosion of lining of stomach or the duodenum. [2] Antiulcer medications are crucial for managing different forms of ulcers, notably peptic ulcers, which can cause serious World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 212 pain and perhaps serious consequences if left untreated. Here are some crucial details emphasising the significance of antiulcer medications: Pain and discomfort relief Antiulcer medications aid in reducing ulcer-related symptoms such heartburn, indigestion, and burning belly discomfort. These medications ease discomfort and raise the standard of living for ulcer sufferers by lowering stomach acid production and/or preserving the stomach lining. [3] The therapeutic potential of Uncaria tomentosa (cat’s claw) as an anti-ulcer agent was supported by both traditional use and modern pharmacological research. Its diverse chemical constituents, including alkaloids and proanthocyanidins, demonstrated promising biological activities contributing to its effectiveness in managing ulcerative conditions, particularly peptic ulcers. With increasing demand for natural and affordable treatments, U. tomentosa served as a valuable source for the development of novel plant-based anti-ulcer therapies. However, further clinical investigations were required to fully understand its mechanisms of action and therapeutic efficacy. [4] 2. Material and methods 2.1. Chemicals All chemicals and reagents used in the experimental procedures were of analytical grade and sourced from reputable suppliers to ensure reliability and accuracy of results. Glacial acetic acid, nitroprusside, sodium hydroxide, and ammonia were procured from Merck, known for its high-quality laboratory reagents. Petroleum ether was obtained from Research lab, and ethanol was supplied by Molychem, both recognized for their consistent reagent-grade products. Concentrated sulfuric acid was sourced from Fizmerck, while 95% alcohol, concentrated hydrochloric acid, and chloroform were provided by Clorofiltind, a well-established supplier of laboratory chemicals. Magnesium, necessary for various qualitative tests, was acquired from Himedia, a trusted name in microbiological and chemical supplies. The use of these reagents supported the precision and reproducibility of the experimental analyses carried out in this study. 2.2. Plant collection Uncaria tomentosa , a medicinal plant, was collected around 350gm in the localised area of Bhopal. After cleaning, the plant was dried under shade at room temperature for three days before being oven dried at 45°C until completely dry. To avoid contamination and deterioration, dried plant parts (leaves) were stored in airtight glass containers in a dry, cool location. 2.3. Extraction In the present study, the extraction of plant material was carried out using the continuous hot percolation technique with a Soxhlet apparatus. Finely powdered Uncaria tomentosa was loaded into the thimble of the Soxhlet extractor. The extraction process was conducted at a controlled temperature of 60°C, employing petroleum ether and methanol as non-polar solvents. The Soxhlation was continued separately for each solvent until the siphon tube exhibited no further change in color, indicating exhaustive extraction. To ensure complete removal of the solvents, the extracts were concentrated under reduced pressure using a rotary vacuum evaporator (Buchi type) at 40°C. The dried residues were weighed, and the percentage yield of each extract was determined using the following formula: [5] 100 usedMaterialPlantofWeight extractofWeight Yield% = 2.4. Phytochemical investigation An experiment was carried out to determine the presence or absence of several phytoconstituents using thorough qualitative phytochemical analysis. Medical reactions to testing were based on colour intensity or precipitate formation. The following standard methods were used. [6] 2.5. Quantitative Phytochemical Estimation 2.5.1. TPC The total phenolic content of Uncaria tomentosa extract was estimated using the Folin-Ciocalteu assay. A 0.2 mL aliquot of the extract was mixed with 2.5 mL of Folin-Ciocalteu reagent and 2 mL of 7.5% sodium carbonate, then diluted with distilled water to a final volume of 7 mL. The mixture was incubated at room temperature for 2 hours. Absorbance was measured at 760 nm using a UV-Vis spectrophotometer. Gallic acid was used as the standard, and a calibration curve World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 213 was prepared with concentrations ranging from 20 to 100 µg/mL. The assay is based on the reduction of the FolinCiocalteu reagent by phenolic compounds, producing a blue chromophore measurable spectrophotometrically. [7] 2.5.2. TFC The flavonoid content was measured using the aluminium chloride technique. 0.5 mL of Uncaria tomentosa extract solution was mixed with 2 mL of distilled water. Then, 0.15 ml of sodium nitrite (5%) was added and stirred thoroughly. After that, wait 6 minutes before adding 0.15 mL of 10% aluminium chloride and allowing standing for 6 minutes. Then, 2 millilitres of 4% sodium hydroxide were added. The mixture was shaken and properly combined. The absorbance of the combination was measured at 510 nm using a UV spectrophotometer. Calibration curves were created with standard solutions of Rutin Equivalent (RE) mg/gm. Rutin was concentrated to 20, 40, 60, 80, and 100 µg/mL. The calibration curve was used to determine the total flavonoid concentration, which was expressed as mg Rutin equivalent per gram of dry extract weight. [8] 2.6. DPPH Uncaria tomentosa extract's antioxidant activity was assessed utilizing the DPPH free radical scavenging test. A methanol solution containing 1 mg/ml extracts/standard was produced. Uncaria tomentosa extracts/standards (20100μg/ml) were produced from a 1mg/mL stock solution with 2mL of 0.1mM DPPH solution added. The resulting mixture was vortexed, incubated for 30 minutes at room temperature in a relatively dark environment, and measured at 517 nm with a UV spectrophotometer. For the control, add 3 ml of 0.1mM DPPH solution and incubate for 30 minutes at room temperature in the dark. The absorbance of the control was measured against methanol (as a blank) at 517 nm. [9] Percentage antioxidant activity of sample/standard was calculated by using formula: % Inhibition = [(Ab of controlAb of sample)/ Ab of control x 100] 2.7. Acute Toxicity Study The acute toxic class approach outlined in the guideline is a stepwise procedure that uses three animals of the same sex per phase. Depending on the animals' mortality and/or moribund stage, 2-4 steps may be required to determine the acute toxicity of the test chemical. The drug is given orally to a group of experimental animals at one of the prescribed dosages. The chemical is evaluated in stages, with each step including three animals of the same sex. The absence or presence of compound-related mortality in animals dosed at one stage determines the next phase, i.e., no further testing is required, dosing of three further animals with the same dose, and dosing of three other animals at the next higher or lower dose level. Each phase requires three animals. The beginning dose is set from one of four fixed levels: 5, 50, 300, and 2000 mg/kg body weight of Uncaria tomentosa . [10] 2.8. Experimental work 2.8.1. Animals Protocol • IAEC Approval All animal experiments were approved by Institutional Animal Ethics Committee (IAEC). • Animal used • Weight 270±20 gm • Strain Wistar rat • Sex: Wistar Male Housing ConditionThe animals were kept in six separate cages at a controlled temperature of 22 ± 2°C. All animals were fed a conventional food (golden feed, New Delhi) and provided with water on a regular basis. 2.8.2. Induction of ulcer in rats: Male Wistar rats weighing 270±20 g was fasted for 24 hours with free access to water and randomly divided into 5 groups. The control group was given a vehicle (5 ml of distilled water orally), and the inducer group was given 20 mg/kg of ethanol orally. Treatment groups III and IV were given ethanol 20 mg/kg and a test sample (Uncaria tomentosa extract200, 400 mg/kg), whereas the standard group (V) was given the standard antiulcer medicine (Ranitidine 20 mg/kg via oral route). The rats were slaughtered after an hour, and the stomach was removed and opened along the larger curvature. [11] World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 214 2.8.3. Experimental design • Group 1Normal control • Group 2Inducer group Ethanol 20 mg/kg orally • Group 3Treated with Uncaria tomentosa extract 200 mg/kg orally • Group 4Treated with Uncaria tomentosa extract 400 mg/kg orally • Group 5Treated with standard drug (Ranitidine) 20 mg/kg orally 2.9. Parameters assessed for anti-ulcer activity 2.9.1. Ulcer index The incidence and severity of the lesion were graded using the arbitrary scoring method listed below. The stomachs were then dissected along their greater curvature, cleaned with normal saline to remove gastric contents, and examined under a 10x magnifying lens for ulcer formation. Ulcers were counted and evaluated using the Kulkarni technique (0 = no ulcer, 0.5 = red coloration, 1 = spot ulcers, 2 = haemorrhagic streaks, 3 = ulcers > 3 but < 5, and 5 = ulcers > 5). The ulcer Index and percentage of ulcer inhibition were determined as follows: Ulcer index (UI) = UN + US + UP × 10−1 Where, UN = Average number of ulcers per animal, US = Average of severity score, UP = Percentage of animals with ulcers 2.9.2. Volume of gastric juice Each animal's gastric juice volume was determined after centrifugation at 1000 rpm for 10 minutes and evaluated. The volume of the centrifuged sample was calculated as mL per 100g body weight. 2.9.3. pH of gastric juice To determine pH, dilute 1 mL of gastric juice with 1 mL of distilled water and use a pH meter. 2.9.4. Determination of free acidity Dilute 1 ml of gastric juice with distilled water and transfer to a 50 ml conical flask. Add 2 drops of phenolphthalein indicator. 0.01 N NaOH was used for titration until a permanent pink color was obtained; the consumed volume was calculated. The free acidity was estimated with the following formula: 𝑨𝒄𝒊𝒅𝒊𝒕𝒚 = 𝑽𝒐𝒍𝒖𝒎𝒆𝒐𝒇𝑵𝒂𝑶𝑯 × 𝑵𝒐𝒓𝒎𝒂𝒍𝒊𝒕𝒚𝒐𝒇𝑵𝒂𝑶𝑯 × 𝟏𝟎𝟎 𝟎. 𝟏 3. Results 3.1. Percentage Yield Table 1 Percentage Yield of crude extracts of Uncaria tomentosa extract S.no Plant name Solvent Theoretical weight Yield(gm) % yield 1 Uncaria tomentosa Pet ether 350 1.67 0.55% 2 Methanol 295.23 6.59 2.23% World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 215 3.2. Preliminary Phytochemical study Table 2 Phytochemical testing of extract S. No. Experiment Presence or absence of phytochemical test Pet. Ether extract Methanolic extract 1. Alkaloids Dragendroff’s test Present Present Mayer’s reagent test Present Present Wagner’s reagent test Present Present Hager’s reagent test Present Present 2. Glycoside Borntrager test Absent Present Legal’s test Absent Present Killer-Killiani test Absent Present 3. Carbohydrates Molish’s test Present Present Fehling’s test Present Present Benedict’s test Present Present Barfoed’s test Present Present 4. Proteins and Amino Acids Biuret test Absent Absent Ninhydrin test Absent Absent 5. Flavonoids Alkaline reagent test Present Present Lead Acetate test Present Present 6. Tannin and Phenolic Compounds Ferric Chloride test Present Present Lead Acetate test Present Present Gelatin test Present Present 7. Saponin Foam test Present Absent 8. Test for Triterpenoids and Steroids Salkowski’s test Absent Absent Libbermann-Burchard’s test Absent Absent World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 216 3.3. Quantitative Analysis 3.3.1. Total Phenolic content (TPC) and Total Flavonoids content (TFC) estimation Table 3 Standard table for Gallic acid S. No. Concentration (µg/ml) Absorbance 1. 20 0.174 2. 40 0.202 3. 60 0.278 4. 80 0.313 5. 100 0.330 Table 4 Standard table for Rutin S. No. Concentration (µg/ml) Absorbance 1. 20 0.140 2. 40 0.176 3. 60 0.191 4. 80 0.235 5. 100 0.271 Figure 1 Represent standard curve of Gallic acid and Rutin Total Phenolic Content and Total Flavonoid Content in extract Table 5 Total Phenolic Content in extract Extracts Total Phenolic content (mg/gm equivalent of Gallic acid) Methanol 61.7 mg/gm Table 6 Total Flavonoid Content in extract Extracts Total Flavonoid content (mg/gm equivalent of rutin) Methanol 16.25 mg/gm World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 217 3.4. In vitro Antioxidant Assays 3.4.1. DPPH 1, 1diphenyl-2-picryl hydrazyl Assay Table 7 DPPH radical scavenging activity of Std. Ascorbic acid Concentration (μg/ml) Absorbance % Inhibition 20 0.489 50.854 40 0.430 56.783 60 0.346 65.226 80 0.286 71.256 100 0.143 85.628 Control 0.995 IC50 22.04 Table 8 DPPH radical scavenging activity of methanol extract of Uncaria tomentosa Concentration (µg/ml) Absorbance % Inhibition 20 0.518 44.120 40 0.466 49.730 60 0.453 51.132 80 0.414 55.339 100 0.365 60.625 Control 0.927 IC50 48.68 Figure 2 DPPH radical scavenging activity of Std. Ascorbic acid and extract of Uncaria tomentosa World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 218 3.5. Analysis of general parameters 3.5.1. Determination of Ulcer Index Table 9Observation of Ulcer Index Groups Ulcer Index Mean Group INormal Control 0 Group II Inducer Ethanol (20 mg/kg bw) 10.789±0.784 Group III Uncaria tomentosa Extract treated (200mg/kg) group 9.942±0.153 Group IV Uncaria tomentosa Extract treated (400mg/kg) group 3.001±0.255 Group V Standard (Ranitidine 20 mg/kg bw) 1.654±0.467 Figure 3 Bar chart represents ulcer index in Ethanol induced ulcer in rats 3.5.2. Determination of Volume of gastric juice Table 10 Observation of volume of gastric juice Treatment Group Volume of gastric juice Group INormal Control 1.752±0.447 Group II Inducer Ethanol (20 mg/kg bw) 6.890±0.863 Group III Uncaria tomentosa Extract treated (200mg/kg) group 5.110±0.845 Group IV Uncaria tomentosa Extract treated (400mg/kg) group 2.221±1.006 Group V Standard (Ranitidine 20 mg/kg bw) 1.992±0.741 World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 211-221 219 Figure 4 Bar chart represents gastric volume in Ethanol induced ulcer in rats 3.5.3. Determination of pH of gastric juice: Table 11 Observation of pH of gastric juice Treatment Group pH of gastric juice Group INormal Control 2.117±0.73 Group II Inducer Ethanol (20 mg/kg bw) 3.950±0.580 Group III Uncaria tomentosa Extract treated (200mg/kg) group 2.966±0.560 Group IV Uncaria tomentosa Extract treated (400mg/kg) group 3.008±0.650 Group V Standard (Ranitidine 20 mg/kg bw) 2.876±0.850 Figure 5 Bar chart represents pH in Ethanol induced ulcer in rats 3.5.4. Free acidity determination: Table 12 Observation of free acidity in Ethanol induced peptic ulcer in rats Treatment Group Free acidity determination (mE/L) Group INormal Control 15.280±3.543 Group II Inducer Ethanol (20 mg/kg bw) 26.648±3.640 Group III Uncaria tomentosa Extract treated (200mg/kg) group 25.012±2.561 Group IV Uncaria tomentosa Extract treated (400mg/kg) group 17.986±1.470 Group V Standard (Ranitidine 20 mg/kg bw) 16.023±1.750