ESTIMATION OF ANTIARTHRITIC ACTIVITY OF A POLYHERBAL FORMULATION AGAINST ADJUVANT INDUCED ARTHRITIS IN RATS

ESTIMATION OF ANTIARTHRITIC ACTIVITY OF A POLYHERBAL FORMULATION AGAINST ADJUVANT INDUCED ARTHRITIS IN RATS

J. Mohan Rao *, Margret Chandira and B. S. Venkateswarlu

Vinayaka Mission's Research Foundation - Deemed to be University (VMRFDU), Sankari Main Road, Ariyanur, Tamil Nadu, India.

ABSTRACT: Objective: Rheumatoid arthritis (RA) is a systemic disorder which involves the activation of immune system against the self-tissues. The main targets of this disease are the joints. Being systemic, the development of this disease involves different mechanisms, and thus, the exact cause of this disease remains unknown. Although different drugs have been developed, none has been found to be the cure for this disease. The present study was commenced to evaluate the in-vivo anti-arthritic effect of polyherbal formulation of selected plants Polygonum glabrum, Canthium dicoccum, Ochna obtusata and Argyreia nervosa. Materials and Methods: In-vivo anti-arthritic activity of the ethanolic extract of different portions capsule formulation F4 investigated orally was assessed using complete Freund’s adjuvant-induced arthritis. Results: In complete Freund’s adjuvant-induced arthritis models, the polyherbal extract formulations significantly (P < 0.001) reduced joint and paw swelling and markedly improved body weight, hematology profile, and parameters in complete Freund’s adjuvant model. Conclusion: It could be concluded that the ethanolic extract of two different formulations holds anti-arthritic potential, supporting its traditional use in the treatment of RA.

Keywords: Argyreia nervosa, Canthium dicoccum, Ethanolic extract, Freund’s adjuvant-induced arthritis, In-vivo rheumatoid, Ochna obtusata, Polygonum glabrum, Polyherbal

INTRODUCTION: Herbal medicine is the oldest form of health care known to humankind. It is an integral part of the development of modern civilization. In herbal medicine, plant-based formulation is used to alleviate diseases. However, the most important challenges faced by these formulations arise due to their lack of complete evaluation. Hence, evaluation is necessary to ensure the quality and purity of the herbal product. It is very important to establish a system of evaluation for every plant medicine in the market since the scope for variation in different batches of medicine is enormous ¹. Inflammation is a normal protective response to tissue injury, which involves a complex array of enzyme activation, mediator release, fluid extravasations, cell migration, tissue breakdown, and repair ². It is characterized by redness, swelling, pain, joint stiffness, and loss of joint function.

Inflammation is associated with membrane alterations, an increase in vascular permeability, and protein denaturation ³. Arthritis is a chronic, inflammatory, systemic autoimmune disorder. It is an inflammation of synovial joint due to immune-mediated response ⁴. One-fifth of the world’s elderly suffer from arthritis ⁵. The current treatment of arthritis includes minimization of this associated pain and inflammation using nonsteroidal anti-inflammatory drugs (NSAIDs) and deceleration of disease progression using anti-rheumatic drugs ⁶. Due to adverse reactions to the NSAIDs and disease-modifying antirheumatic drugs, the arthritic patients tend to search for other treatments that are effective and less toxic. Therefore, complementary and alternative medicines are commonly preferred by such patients ⁷.

1. Polygonum glabrum: The tribes of Chhattisgarh use the root paste as medicine for snakebite ⁸ and also have different uses such as jaundice and piles ⁹ antimalarial agent in Sudan ¹⁰ dysentery ¹¹ and Anthelmintic ¹². The whole plant decoction is used as a remedy for colic pain, pneumonia, and the boiled paste is applied in cuts and wounds ¹³. Peels from the stem are used for treating rheumatism ¹⁴. Ochna obtusata DC. (Family Ochnaceae) is a small tree up to 8 m tall. The family is characterized by the presence of secondary metabolites such as flavonoids and terpenoids ¹⁵.

Moreover, it is extensively used in Indian traditional medicine to treat epilepsy, menstrual complaints, lumbago, asthma, ulcers, and as an antidote to snake bites ¹⁶ and has glycosides, saponins, steroids, flavones and fatty acids ¹⁷. It is also ulcer, asthma and bronchitis and also possesses antiulcer genic activity ¹⁸. Canthium dicoccum ethanolic extract of whole extract showed almost equipotent antidiabetic activity compared to standard drug glibenclamide ¹⁹. Moreover, also, egg albumin-induced arthritis model ²⁰.

Argyreia nervosa seeds possess hypotensive and spasmolytic activity due to the mixture of ergot alkaloids, isolated and analyzed by ultraviolet. Due to instability only, one constituent was identified as ergometrine. Other constituents such as caffeic acid and ethyl caffeate were identified ^{21, 22}. Apart from ergoline alkaloids, N-formylloline alkaloids, flavonoid sulfates, steroids, and triterpenoids were isolated from other parts of A. nervosa ^{23, 24}. Para-hydroxycinnmate, scopeltin, and argyroside ^{25, 26} isolated oil from the seed of A. nervosa and evaluated the antibacterial effect ²⁷. In the previous studies, the author noticed ethanolic extract of the above plants and polyherbal formulations with different fractions of ethanolic extract showed good antioxidant activity as well as in-vitro antiarthritis activity ²⁸. By considering the above facts, the present study aims to develop formulations from crude plant extract of the above plants and several antirheumatoid constituents which act by several modes of action to influence multiple biological pathways and thereby produce more effective through oral route. The study was also designed to produce a safe, cheaper formulation that can reduce rheumatoid, thereby providing multifaceted benefits.

MATERIALS AND METHODS:

1.1 Plant Source and Authentication: P. glabrum, O. obtusata DC., C. dicoccum and A. nervosa were collected from Tirumala Hills, Tirupati, and Chittoor district of Andhra Pradesh, near Seshachalam and Tirumala Hills (Rayalaseema region, Andhra Pradesh, India), areas that are geographically located in the South Eastern Ghats, are recognized for their rich flora and fauna. The plant specimen was verified to be of the correct species by Dr. Madhava Setty, a botanist from the Department of Botany, S. V. University, Tirupati, Specimen Voucher no: 1972, 1220, 1012, 2162, preserved for further reference at our laboratory.

1.2 Drugs and Chemicals: Diclofenac sodium was obtained as a generous sample from Meditech Pharma Pvt. Ltd., Mumbai, ethanol (Sigma-Aldrich, USA) and complete Freund’s adjuvant (Sigma-Aldrich, USA).

1.3 Experimental Animals: Swiss Albino rats of either sex weighing from 200 to 300 g were used. The rats were housed under standard conditions of temperature (23–25°C), and relative humidity (55%) with 12 h light and 12 h dark cycle. They were fed with a standard pellet diet and tap water ad libitum. The experiment was designed and carried out according to the norms of the ethical committee (CPSCEA) and approved by the institutional animal ethical committee (1987/PO/Re/S/17/CPCSEA).

1.4 Preliminary Phytochemical Studies ^29-31: Previously, various preliminary phytochemical tests were performed for the extract used for capsule formulations using standard procedures, and the above formulations showed the presence of main carbohydrates, alkaloids, glycosides, phenols, tannins, flavonoids, and saponins which majorly responsible for the desired activity.

1.5 Preparation of Polyherbal Granules: Polyherbal granules were prepared using by wet granulation method. The polyherbal extract was mixed well with lactose monohydrate, added the required quantity of starch to obtain a smooth mass, and then passed through # 12 to produce granules. Prepared granules were gently subjected to drying (< 0.05 was considered statistically significant, and P < 0.001 was considered statistically highly significant, as compared to control group.

1.6 Preparation of Polyherbal Granules: Polyherbal granules were prepared by the wet granulation method. The polyherbal extract was mixed well with lactose monohydrate, added the required quantity of starch to obtain a smooth mass, and then passed through # 12 to produce granules. Prepared granules were gently subjected to drying.

1.7 Formulation of Polyherbal Capsules: Prepared granules were packed into a hard gelatin capsule (size 2) using a hand-operated capsule filling machine such that each capsule contains 300 mg of granules. Polyherbal capsules without CCS were labeled as F1, and capsules containing 3%, 4%, and 5% of CCS were labeled as F2, F3, and F4, respectively, and quantities for formulation trails are presented in Table 1. Animals were housed in polypropylene cages, maintained under standardized conditions (12 h light/dark cycle, 24°C, and 35–60% humidity), and provided free access to standard palate diet and purified drinking water libitum. The animals were deprived of food for 24 h before experimentation but allowed free access to water throughout.

TABLE 1: COMPOSITION OF DIFFERENT INGREDIENTS USED FOR FORMULATION

8 Acute Toxicity Study: For the acute toxicity study on mice, “Fixed-dose” method of the organization for economic cooperation and development guideline 420 was followed ^{38, 39}. The formulation was suspended in distilled water and administered by gavages (orally) at single doses of 2000 mg/kg. The animals had free access to water and food throughout the experiment, except for the fasting period before the oral administration of the single dose of the formulation. The general behavior of the rats was continuously monitored for 3 h and then every 30 min for the next 3 h till 24 h and then daily for a total of 14 days. Changes in the normal activity of rats, their body weights, sign and symptoms of toxicity, and mortality were monitored and recorded.

1. In-vivo Evaluation Selected Polyherbal Capsule Complete Freund’s adjuvant Induced Arthritis in Rats: The male Swiss albino rats were divided into five different groups of six animals, each as follows:

• Group I: Normal control.
• Group II: Arthritic control.
• Group III: Capsule formulation (F4).
• Group IV: Diclofenac sodium (10 mg/kg b.wt orally).

Before the experiment, each animal's paw volume (baseline) at 0 day was measured. In complete Freund’s adjuvant (5 mg of heat-killed, powdered Mycobacterium tuberculosis cell was suspended with liquid paraffin to get a 5 mg/ml suspension) was used to induce arthritis in rats. The rats were anesthetized with an intraperitoneal injection of 40 mg/kg of thiopentone sodium. Mineral oil was injected in the right ankle joint of a normal group of animals. Adjuvant arthritis was induced by subcutaneous injection of Freund’s complete adjuvant (FCA) (0.1 ml) into sub plantar tissue of the right hind paw of each rat. The test groups consisted of FCA injected rats challenged with the respective doses of the test drugs administered orally 24 h before FCA injection, while the vehicle control rats were injected with 0.1 ml of liquid paraffin (incomplete Freund’s adjuvant) only. The drug treatments were continued once daily at the same time after the challenge for 20 more days. The swelling in the rats' injected and contralateral hind paws was monitored daily using a liquid displacement plethysmometer. An increase in the extent of erythema and edema of the tissues shows the severity of the inflammation. The change in body weight and paw edema was recorded at desired frequent intervals ^{40, 41}. At the end of the study, blood samples were withdrawn from all groups through retro-orbital plexus puncture, and whole blood was used for hematological analysis and serum was used for biochemical analysis ⁴². Hematological parameters such as the hemoglobin (Hb) level, the red blood cell (RBC) count, the white blood cell (WBC) count and the erythrocyte sedimentation rate (ESR) were estimated manually using fresh blood. Serum samples were collected after centrifugation of whole blood at 3000 rpm for 20 min. Liver markers such as aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and creatinine were analyzed using an auto analyzer (Vital Scientific N.V., the Netherlands). The liver enzyme levels were estimated using Lab Kit enzymatic kits. The C-reactive protein (CRP) and serum copper CRP levels estimated using the enzyme-linked immunosorbent assay kit (obtained from Alpha Diagnostic Intl., USA) and the colorimetric bathocuproin disulfonate method of Zak and Landers, respectively ^{43, 44}.

3. Statistics: All values are shown as mean ± standard error of the mean. Statistical analysis was performed using a one-way analysis of variance followed by Dunnett’s test. P<0.05 was considered statistically significant and P<0.001 was considered statistically highly significant, as compared to control group.

RESULTS: Polyherbal granules were prepared from extract and formulation additives by moist granulation technique, and the composition for formulation trails is presented in Table 1. Prepared granules were subjected to various flow property measures such as determination of Carr’s index, Hausner ratio and angle of repose, and from the results of preformulation studies clear that all blends were possessed good flow characteristics prepared granules were packed in capsule shells (2) with the help of hand-operated capsule filling machine. All capsule formulations were subjected to various pharmacopoeial tests. Their results like weight variation were found to be within limits, drug content was founded to be within the range, and disintegration time was founded to be within the range. The in-vitro dissolution study was performed using the USP Type-II dissolution test apparatus. The operating conditions were 900 ml of phosphate buffer pH 6.8 as dissolution fluid, paddle rotates data speed of 100 RPMAT 37±0.5°C. The results of the in-vitro dissolution study reveal that marker component rut in was released from the capsules. Percentage cumulative drug release for rut in from formulation F1 and F4 was found to be within the range of 52.86±0.05–98.99±0.01at 12 h ⁴⁵. From the results, polyherbal capsule formulation F4 showed good physical properties such as disintegration, hardness, and dissolution rate. After the comparative study of different formulations having different excipients, CCS 15 mg (5%) is better suitable. Hence, F4 was selected and evaluated in in-vivo in this article.

4.1 Clinical Signs of Intoxication, Body Weight, and Mortality: In the preliminary acute toxicity study, the formulation seems to be safe at 2000mg/kg. There were no toxic or deleterious effects in Table 2 observed immediately within 24 h and up to 14 days of the observation period. There was no major change in body weight shown in Table 3 and no mortality recorded in Table 4.

TABLE 2: CAGE‑SIDE OBSERVATIONS OF ANIMALS (GENERAL BEHAVIOR)

DISCUSSION: In the preliminary acute toxicity study, the prepared capsule seems safe at 2000 mg/kg. There were no toxic or deleterious effects observe dimmediatelyin 24h and up to14 days of observation period. There was no major change in body weight and no mortality found in any animal Table 3 and 4. The preliminary phytochemical screening of polyherbal formulation the formulated capsules showed the presence of alkaloids, flavonoids and tannins ⁴⁶.

Compounds have well-known anti-inflammatory and antiarthritis activity. The effects observed with formulated capsules could be due to the synergistic actions of these compounds. In the present study, formulated capsules demonstrated a highly significant (P<0.001) antiarthritis activity at different formulations in rat model of antiarthritis activity results shown in Table 5.

The animal model used for in-vivo evaluation of antiarthritis activity completes Freund’s adjuvant-induced arthritis animal model in which clinical and pathological alterations are kin to those seen in human rheumatoid arthritis RA ^47,48. Complete Freund’s adjuvant is a mixture of heat-killed M. tuberculosis with liquid paraffin, which stimulates cell-mediated immunity, thus potentiating the production of certain immunoglobulins in the body ⁴⁹. Adjuvant-induced arthritis in the rat can be alienated into three distinctive phases; first, the induction phase without the manifestation of synovitis, followed by early synovitis, and finally, late synovitis accompanied by unremitting cartilage and joint tissue destruction.

In this method, the arthritis model offers an opportunity to examine the pathological changes in a variety of tissues other than joints ⁵⁰. Anemia is the most common extracellular manifestation in RA and may be caused by the decreased level of plasma iron due to sequestration of iron in the endothelial reticule system and synovial tissue, ultimately failure of bone marrow to counter anemia ⁵¹. IL-1in association with the acute phase response also decreases plasma iron content, and it is challenging to speculate that the sequestration of less deformable erythrocytes by endothelial.

TABLE 3: MEAN BODY WEIGHT AND PERCENTAGE BODY WEIGHT GAIN

Cells in the spleenlsoplaysa causative role in shortened half-life of erythrocytes, thus, resulting in anemia ⁵². Alternatively, a rise in both WBC and platelet counts might be due to the stimulation of the immune system against the invading pathogenic microorganism. It is evident by the influx of inflammatory mono nuclear cells in the joints arthriticrats ^{53, 54}.

In the present experimental study, the herbal formulation-treated groups had considerably increased levels of Hb and RBC, while the level of WBC and platelets was significantly reduced in contrast to arthritic control group but comparable to normal control group Table 6. Similarly, ESR is an imperative haematological index for the diagnosis as well as prognosis of infectious and inflammatory diseases. With reference to the standard drug and herbal treatment, its fractions remarkably decreased ESR count in arthritic rats, thus justifying its significant role in arthritic conditions. Rheumatoid factor (RF), a key serologic marker, is an autoantibody directed against the Fc (Fragment, crystallizable) portion of IgG and form immune complexes that contribute toward the success of RA. Noteworthy decrease in RF level in the serum of arthritic rats treated with polyherbal extract in specification veil the protective against RA Table 6.

From these haematological findings, it can be proposed that polyherbal formulation changes the alterations in blood parameters toward normal by inhibiting the inflammatory response, which might be due to its blocking action on pro-inflammatory cytokines and cyclooxygenase well as suppressing the immune response as supported by the previous studies.

TABLE 4: MORTALITY RECORD

TABLE 5: RESULTS FOR COMPLETE FREUND’S ADJUVANT‑INDUCED ARTHRITIS IN RATS FOR SELECTED CAPSULE FORMULATIONS (F4)

Values are expressed as mean ± standard error of the mean n=5; One‑way analysis of variance followed by Dunnett’s test. P<0.05 was considered statistically significant and P<0.001 was considered statistically highly significant, as compared to control group.

TABLE 6: RESULTS FOR HAEMATOLOGICAL PARAMETERS IN ARTHRITIC RATS

RBC: Red blood cell, WBC: White blood cell, ESR: Erythrocyte sedimentation rate, Hb: Hemoglobin, RF: Rheumatoid factor. Values are expressed as mean ± standard error of the mean n=5; One‑way analysis of variance followed by Dennett’s test. P<0.05 was considered statistically significant, and P<0.001 was considered statistically highly significant, as compared to the control group

CONCLUSION: The acute toxicity test for oral preparation of capsule formulation indicates that it is relatively safe and non-toxic to rats. The above polyherbal extract with different portions P. glabrum ethanolic extract, C. dicoccum ethanolic extract, O. obtusata ethanolic extract, and A. nervosa ethanolic extract (2:1:1:1) in F4 capsule is proven with a good in-vivo antiarthritis activity which is a medicinally valuable plant. Its antiarthritic effect might be due to its anti-inflammatory, antioxidant, and immunosuppressant actions, although the mechanism is unknown.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Rao TJM, Chandira M and Venkateswarlu BS: Estimation of antiarthritic activity of a polyherbal formulation against adjuvant induced arthritis in rats. Int J Pharm Sci & Res 2022; 13(11): 4712-18. doi: 10.13040/IJPSR.0975-8232.13(11).4712-18.

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