Phytochemical investigation and assessment of anti-arthritic activity of Dicanthium annulatum herbal plant extract in Wistar rats

*Corresponding author: Naudishtha Khandare Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Phytochemical investigation and assessment of anti-arthritic activity of Dicanthium annulatum herbal plant extract in Wistar rats Naudishtha Khandare 1, *, Manju Prajapati 2 and Akhlesh Kumar Singhai 3 1 Research Scholar, School of Pharmacy, LNCT University, Bhopal (M.P), India. 2 Professor, 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), 201-210 Publication history: Received on 01 April 2025; revised on 01 June 2025; accepted on 04 June 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.22.3.0496 Abstract This study focuses on the phytochemical screening, antioxidant evaluation, and anti-arthritic activity of the methanolic extract of Dichanthium annulatum (whole plant). The extraction process yielded 4.16%, and preliminary phytochemical analysis confirmed the presence of alkaloids, glycosides, flavonoids, tannins, saponins, and carbohydrates. Quantitative estimation revealed a total phenolic content of 36.88 mg/g gallic acid equivalent and a total flavonoid content of 23.3 mg/g rutin equivalent. Antioxidant potential was assessed via the DPPH free radical scavenging assay, indicating moderate activity with an IC₅₀ value of 50.10 µg/mL. The anti-arthritic effect was evaluated in Wistar rats using the Freund’s Complete Adjuvant (FCA)-induced arthritis model. Oral administration of the extract (200 mg/kg and 400 mg/kg) significantly reduced paw edema and improved hematological (RBC, WBC, Hb) and biochemical (ALP, AST, ALT, CRP) parameters. The results were comparable to the standard drug diclofenac sodium. These findings suggest that Dichanthium annulatum possesses significant anti-inflammatory and anti-arthritic potential due to its bioactive constituents. Keywords: Dichanthium annulatum; Phytochemical Analysis; Antioxidant Activity; Anti-Arthritic Effect; Wistar Rats; FCA Model; Flavonoids; Phenolic Compounds 1. Introduction The traditional herbal medicines (HM) and their preparations have been widely used for thousands of years in many oriental countries, like in China, Korea, Japan, etc. Nonetheless, a feature of oriental herbal medicine preparations is that, during the decoction process, boiling water is used to extract all herbal medicines, whether they appear as individual herbs or as mixtures of herbs in composite formulas. This could be the primary cause of the difficulty in ensuring the quality of oriental herbal medications compared to western medications. as stated in the World Health Organization's 2000 "General Guidelines for Methodologies on Research and Evaluation of Traditional Medicines." Traditional medicine has not received formal recognition in the majority of nations, despite its long history and continuous usage over several centuries, as well as its recent popularity and widespread use. [1] Arthritis is inflammation of one or more joints. The degeneration of cartilage is a feature of arthritis. A joint is often protected by cartilage, which permits smooth motion. Additionally, cartilage absorbs shock when the joint is under pressure, such when you walk. When there is insufficient cartilage, the bones grind against one another, resulting in stiffness, pain, and inflammation. The individuals of any age can be affected with Arthritis; the usual age of onset is between 25 and 50 with a peak in the 40s and 50s [4].The key risk factors of arthritis include age, gender, excess weight, injury, dietary pattern, consumption of excess alcohol, life style, heredity, hormonal factors, environmental factors and lack of physical activity. Four major categories of medications are used to treat arthritis: Corticosteroids (steroids), disease-modifying anti-rheumatic World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 202 medications (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), and painkillers (analgesics). [2] Dichanthium annulatum (family: Poaceae) is a perennial grass widely distributed in tropical and subtropical regions. Traditionally, it has been used as a fodder plant and in soil conservation, but emerging ethnobotanical knowledge suggests potential medicinal applications. While not commonly used in modern herbal medicine, local communities have attributed antiinflammatory and wound-healing properties to the plant. Phytochemicals such as flavonoids, particularly quercetin, and phenolic compounds present in D. annulatum are known to exhibit strong antioxidant and anti-inflammatory effects in the management of arthritis. Despite its promising profile, the plant remains scientifically underexplored for its pharmacological activities. [3] 2. Material and methods 2.1. Chemicals All chemicals and reagents used in this study were of analytical grade. Glacial acetic acid, sodium hydroxide, ammonia, formalin, and magnesium were procured from Merck. Concentrated sulfuric acid was obtained from Fizmerck, while ethanol was sourced from Molychem. Diclofenac sodium, used as the standard anti-arthritic drug, was supplied by Reddy’s Laboratories. Chloroform, concentrated hydrochloric acid (HCl), and 95% alcohol were purchased from Clorofiltind. Magnesium was acquired from Himedia. The Folin-Ciocalteu reagent, essential for phenolic content determination, was obtained from Sigma-Aldrich, and sodium carbonate was procured from GHCL Limited. 2.2. Plant collection Dichanthium annulatum (300 g) was collected from a specific region in Bhopal and authenticated by a botanist. The whole plant was washed, shade-dried for three days, and then oven-dried at 45 °C. The dried material was coarsely powdered and stored in airtight containers for further use. 2.3. Extraction Soxhlet extraction was employed to phytoconstituents from Dichanthium annulatum . A known quantity of dried, powdered plant material was placed in a thimble and extracted using methanol as the solvent. The continuous reflux process allowed repeated washing of the plant material, facilitating efficient extraction. The process continued until complete extraction was achieved. The percentage yield was calculated using the formula: 100 usedMaterialPlantofWeight extractofWeight Yield% = After evaluating the organoleptic characteristics such as color, odor, and percentage yield, the prepared extracts were labelled and stored in airtight containers for subsequent use.[4] 2.4. Phytochemical investigation Phytochemical investigation of Dichanthium annulatum was carried out to identify the bioactive compounds present in the plant. The methanolic extract was subjected to various qualitative tests, revealing the presence of alkaloids, glycosides, flavonoids, tannins, and saponins. These compounds are known for their medicinal properties, including anti-inflammatory and antioxidant effects. The results suggest that Dichanthium annulatum contains a range of phytochemicals that may contribute to its therapeutic potential.[5] 2.5. Quantitative Phytochemical Estimation 2.5.1. TPC To ascertain the Dichanthium annulatum methanolic extract's overall phenolic content. In a test tube, 200 μL of FolinCiocalteu reagent, 3.16 mL of distilled water, and 40 μL (1 mg per 1 mL of methanol) of the plant extract (or standard gallic acid solution) were combined and gently shaken. After an incubation of 8 min, 600 μL sodium carbonate solutions were combined and added. The blend was incubated for 40 °C for 30 min before recording its absorbance in a spectrophotometer at 760 nm against a blank. The curve for calibration was prepared with standard solution of gallic acid equivalent using 10, 30, 50, 70, 90 µg/mLsolutions. [6] World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 203 2.5.2. TFC The flavonoid content of Dichanthium annulatum extract was quantified using a colorimetric method with aluminium chloride. A 0.5 mL extract was mixed with distilled water, followed by the addition of sodium nitrite, aluminium chloride, and sodium hydroxide. After a brief incubation, the absorbance was measured at 510 nm using a UV-Vis spectrophotometer. The total flavonoid content was expressed as rutin equivalents (mg/g) using a standard calibration curve with rutin concentrations of 10, 30, 50, 70, and 90 µg/mL.[7] 2.6. DPPH The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay measures the antioxidant activity of plant extracts by evaluating their ability to scavenge the DPPH radical, which is purple in color. Antioxidants reduce the DPPH radical, leading to a decrease in absorbance at 517 nm. The results are expressed as IC50, the concentration required to inhibit 50% of the DPPH radical, providing a simple method to assess antioxidant potential. [8] The percentage (I%) of inhibition of free radical DPPH was computed as follows: % Scavenging Activity = 100[(Ac – As)/Ac] Where, Ac and as are absorbances of negative control and sample, respectively 2.7. FT-IR FTIR spectroscopy is a technique used to obtain the absorption or emission infrared spectrum of a Dichanthium annulatum extract. FTIR examination to create translucent sample discs, 10 mg of KBr pellet was used to enclose dried powder (methanolic extract). The pellet's powdered sample was placed onto an FTIR spectroscope with a resolution of 4 cm-1 and a scan range of 400 to 4000 cm-1. [9] 2.8. Acute Toxicity Study In the acute oral toxicity study, three animals were used in each phase, following the OECD 423 guideline. The study received approval from the Faculty Ethical Committee. Each treatment group consisted of five rats (n=3), which were allowed access to food and tap water. Rats were randomly assigned to their respective groups. The treatment groups were administered varying doses of methanol extract of Dichanthium annulatum , while the control group was given distilled water as the vehicle. A starting dose of one of four preset dose levels—5, 50, 300, or 2000 mg/kg body weight— must bechosen. [10] 2.9. Freund’s Adjuvant-Induced Arthritic Model: Rats were injected with 0.2 mL of Freund's adjuvant and PBS (1:1) into the left hind and forepaws. Paw volumes were measured using a plethysmometer on days 0, 7, 14, 21, and 28. The percentage inhibition of paw volume was calculated using the formula: (Vc - Vt) / Vc × 100 where Vc is the control paw volume and Vt is the treated paw volume. [11] 2.10. Experimental work 2.10.1. Animals required Animals were randomly selected from the Pinnacle Biomedical Research Institute (PBRI), Bhopal, India, and assigned to treatment groups. They were housed in propylene cages with sterile husk bedding, maintained at 22±2°C, 30.7% humidity, and a 12:12 light-dark cycle. The rats were fed standard pellets (Golden Feeds, New Delhi) and had unlimited access to water. They acclimatized to the lab environment for seven days before testing. Each experiment used a different group of six rats, and the study was approved by the Institutional Animal Ethics Committee (IAEC) of PBRI. 2.10.2. Experimental protocol Five groups of male Wistar albino rats (n = 6) were used in this study • Group I: served as control (without treatment) World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 204 • Group II: Negative control (rats with arthritis that receive no therapy) • Group III: Treated with Diclofenac sodium 20 mg/kg (positive control) the standard anti - arthritic drug • Group IV: Treated with Dichanthium annulatum received 200 mg/kg orally • Group V: Treated with Dichanthium annulatum received 400 mg/kg orally At the conclusion of the experiment, all animals were killed by cervical decapitation, and blood was drawn for plasma/serum separation in tubes containing EDTA and plain. The plasma/serum and homogenized samples were subjected to biochemical examination like total protein and albumin, globulin. [12] 2.11. Analysis of general parameters • Paw Volume: The left hind paw volume of each rat was measured using a paleothermometer on day 0 before FCA injection and at intervals until day 28. The change in paw volume was calculated by subtracting the initial volume from the final measurement. [13] • Biochemical Analysis: On day 28, blood was collected via retro-orbital puncture, and serum was analyzed for acid phosphatase and alkaline phosphatase levels. [14] • Haematological Analysis: Complete blood parameters, including red blood cell count, haemoglobin, platelet count, white blood cell count (total and differential), neutrophils, MCHC, MCV, MCH, and PCV, were measured using Erba test kits and the Star 21 Automated Analyzer. [15] • Body Weight Assessment: Body weight was recorded on days 0, 5, 15, and 25 and expressed as the percentage change relative to the baseline weight on day 0. [16] 3. Results and discussion 3.1. Percentage Yield Table 1 Percentage Yield of crude extracts of Dichanthium annulatum extract S. No Plant name Solvent Theoretical weight Yield(gm) % yield 1. Dichanthium annulatum Methanol 300 12.5 4.16% 3.2. Preliminary Phytochemical study Table 2 Phytochemical testing of extract S. No. Experiment Absence or Presence of phytochemical test Methanolic extract 1. Alkaloids 1.1 Dragendroff’s test Present (+ ve) 1.2 Mayer’s reagent test Present (+ ve) 1.3 Wagner’s reagent test Present (+ ve) 1.4 Hager’s reagent test Present (+ ve) 2. Glycoside 2.1 Borntrager test Present (+ ve) 2.2 Legal’s test Present (+ ve) 2.3 Killer-Killiani test Present (+ ve) 3. Carbohydrates 3.1 Molish’s test Present (+ ve) 3.2 Fehling’s test Present (+ ve) 3.3 Benedict’s test Present (+ ve) World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 205 3.4 Barfoed’s test Present (+ ve) 4. Proteins and Amino Acids 4.1 Biuret test Absent (- ve) 4.2 Ninhydrin test Absent (- ve) 5. Flavonoids 5.1 Alkaline reagent test Present (+ ve) 5.2 Lead Acetate test Present (+ ve) 6. Tannin and Phenolic Compounds 6.1 Ferric Chloride test Present (+ ve) 7. Saponin 7.1 Foam test Present (+ ve) 8. Test for Triterpenoids and Steroids 8.1 Salkowski’s test Absent (- ve) 8.2 Libbermann-Burchard’s test Absent (- ve) 3.3. Quantitative Analysis 3.3.1. Total Phenolic content (TPC) and Total Flavonoids content (TFC) estimation Table 3 Standard table for Gallic acid and Rutin S. No. Concentration (µg/ml) Absorbance 1. 10 0.089 2. 30 0.115 3. 50 0.130 4. 70 0.145 5. 90 0.160 S. No. Concentration (µg/ml) Absorbance 1. 10 0.110 2. 30 0.135 3. 50 0.158 4. 70 0.175 5. 90 0.193 World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 206 Figure 1 Represent standard curve of Gallic acid and Rutin 3.4. In vitro Antioxidant Assays 3.4.1. DPPH 1, 1diphenyl-2-picryl hydrazyl Assay Table 4 DPPH radical scavenging activity of Std. Ascorbic acid Concentration (μg/ml) Concentration (μg/ml) Concentration (μg/ml) 20 20 20 40 40 40 60 60 60 80 80 80 100 100 100 Control 0.996 IC50 40.10 Table 5 DPPH radical scavenging activity of methanol extract of Dichanthium annulatum Concentration (µg/ml) Absorbance % Inhibition 20 0.558 40.064 40 0.492 47.153 60 0.454 51.235 80 0.386 58.539 100 0.336 63.909 Control 0.931 IC50 52.68 y = 0.0021x + 0.1323 R² = 0.949 0 0.1 0.2 0.3 0.4 050 100 150 Absorbance Concentration World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 207 Figure 2 DPPH radical scavenging activity of Std. Ascorbic acid and extract of Dichanthium annulatum 3.5. Functional group identified by FTIR Study Figure 3 FTIR of Dichanthium annulatum 3.6. Paw volume Table 6 Effect of Dichanthium annulatum paw volume of Freund’s adjuvant induced arthritic rats Paw volume of the rats in mm Mean ± SD (% inhibition) Groups Day 0 Day 7 Day 14 Day 21 Day 28 Group I 3.83 ± 0.05 3.85 ± 0.08 3.84 ± 0.09 3.85 ± 0.15 3.85 ± 0.17 Group II 4.12 ± 0.07 4.44± 0.011 4.65± 0.03 4.79± 0.013 5.12 ± 0.07 Group III 3.92 ± 0.08 4.34 ± 0.011 4.51 ± 0.09 4.38 ± 0.06 4.11 ± 0.05 Group IV 4.15 ± 0.09 4.54 ± 0.08 5.12 ± 0.06 4.91 ± 0.10 4.78± 0.09 Group V 4.21 ± 0.07 4.68 ± 0.06 5.01 ± 0.04 4.55 ± 0.06 4.16± 0.09 World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 208 Figure 4 Paw volume in rats with Dichanthium annulatum 3.7. Haematological parameters Table 7 Effect of Dichanthium annulatum haematological characteristics of rats with adjuvant-induced arthritis and control Groups RBC (millions/mm3) WBC (Thousands/mm3) Hb (gm/dL) Group I 7.23 ± 0.051 9.89 ± 0.021 14.21 ± 0.041 Group II 3.41 ± 0.046 19.91 ± 0.153 9.08 ± 0.042 Group III 6.08 ± 0.019 9.89 ± 0.011 13.15 ± 0.071 Group IV 5.48 ± 0.688 11.37 ± 0.059 10.99 ± 0.152 Group V 5.98 ± 0.023 9.101 ± 0.211 11.12 ± 0.051 Figure 5 Haematological parameters 3.8. Biochemical parameters Table 8 Effect of Dichanthium annulatum Adjuvant-induced arthritis and control rats' biochemical characteristics Groups ALP (Alkaline phosphatise) AST (Aspartate transaminase) ALT (Alanine transaminase) CRP (C-reactive protein) Group I 151.11 ± 0.015 60.47 ± 0.181 67.59 ± 0.249 106.7 ± 0.191 Group II 250.13 ± 1.378 118.04 ± 0.111 99.28 ± 0.080 137.08 ± 0.143 World Journal of Biology Pharmacy and Health Sciences, 2025, 22(03), 201-210 209 Group III 136.08 ± 0.473 69.44 ± 0.201 75.64 ± 0.161 110.06 ± 0.299 Group IV 192.07 ± 0.891 94.53 ± 2.059 87.23 ± 0.148 122.04 ± 0.145 Group V 183.09± 0.411 80.35 ± 0.196 81.03 ± 0.264 112.09 ± 0.214 Figure 6 Biochemical parameters The Dichanthium annulatum extract, when assessed for its therapeutic potential, exhibited significant results across various parameters. The methanol extract showed a 4.16% yield and contained bioactive compounds such as alkaloids, glycosides, flavonoids, and saponins, which are known for their medicinal properties. The antioxidant activity, measured by the DPPH assay, displayed a moderate IC50 value of 52.68 µg/mL, indicating the extract's capacity to scavenge free radicals. In the Freund's adjuvant-induced arthritis model, the extract significantly reduced paw volume, especially after 28 days, suggesting its anti-inflammatory properties. This reduction was confirmed by the calculation of paw volume inhibition, which was higher in treated groups than the control. Haematological analysis revealed that the extract positively influenced the red blood cell (RBC) count and haemoglobin levels, which were reduced in the untreated arthritic rats, indicating a recovery in blood parameters. Additionally, the biochemical markers such as alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), and C-reactive protein (CRP) were found to be reduced in the treatment groups, further supporting the extract's anti-inflammatory and tissue-protective effects. These promising results suggest that Dichanthium annulatum possesses anti-arthritic and antioxidant potential, making it a valuable candidate for the development of natural anti-inflammatory therapies. However, further research into its active compounds, dosage optimization, and long-term effects is necessary to fully validate its therapeutic applications. 4. Conclusion Strong evidence for the anti-arthritic properties of Dichanthium annulatum (whole plant) extract is presented in this study. The extract's notable anti-inflammatory properties were revealed by the arthritic rat model's decreased paw volume. Important bioactive substances with anti-inflammatory and antioxidant qualities, like flavonoids and phenolics, were found during the phytochemical analysis. These substances probably have a part in the anti-arthritic benefits that have been noted. The extract was also found to be safe, with no significant toxicity observed in acute oral toxicity studies and no adverse effects on haematological or biochemical parameters in rats. These results suggest that Dichanthium annulatum holds promise as a safe, natural, and efficient treatment for inflammatory diseases like arthritis. To investigate its methods of action, ideal dosage, and possible clinical uses, more research is necessary. With its rich phytochemical content and demonstrated pharmacological activity, Dichanthium annulatum might be a useful supplement to the treatment options available for arthritis, offering a natural alternative or adjunct to conventional therapies.