Pharmacological Investigation of Cassia hirsuta Seed Extract for its Anti-inflammatory Activity in Acute and Chronic Inflammatory Animal Models

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory illness which causes joint swelling, discomfort, and synovial joint degeneration, as well as a variety of extra-articular manifestations that can lead to severe disability and premature death. With a prevalence of about 1%, RA is a very common disorder.¹ In RA, joint inflammation is immunologically mediated with a complicated interplay between genes, environmental triggers, but the initiating agents are unknown.² 

Traditional Non-Steroidal Anti-Inflammatory Drug (NSAIDs), disease-modifying anti-rheumatic medications, glucocorticoids, and biologics are used to treat RA, but they only provide limited therapeutic benefit and are associated with severe toxicity.^3,4 Patients suffering RA frequently seek alternate means for symptomatic relief owing to the negative effects and high cost of immunomodulatory medications, and they are among the most frequent consumers of such approaches.^1,5 Cassia hirsuta, a member of the Fabaceae family is one of the most widely available Cassia species in tropical America, and is currently found in Indo-China, Thailand, California, New Mexico, and India. In India, it is extensively available in the Deccan in Mysore’s Babubudaii hills, Ramdurg, Bellary, and near Madras. It has traditionally been used to treat stomach problems, diarrhoea, abscesses, rheumatism, hematuria, fever, and other ailments. Powdered seeds are used as an antibiotic, in the treatment of herpes, and as a medication for Parkinson’s disease.⁶

Plant components or extracts are used to cure sickness in humans via phytomedicine.⁷ Herbal sources have been utilized in ancient traditional medical systems. The immune mediated disease development that resembles rheumatoid arthritis occurs only after 13 days in the Complete Freund’s adjuvant (CFA) paradigm.⁸ The fundamental rationale for the persistence of arachidonic acid-derived autacoids-induced articular and periarticular inflammation is an immunological hyper-reactive state in the synovium, which is primarily driven by TNF-α, IL-6, and IL-1b. As a result, while NSAIDs reduce inflammation, they have minimal effect on disease progression.⁹ As a result, the current research aimed to assess the anti-inflammatory and anti-arthritic efficacy of ethanol extract of Cassia hirsuta seeds in two distinct animal models, with the focus on pro-inflammatory cytokines linked to arthritis.

Materials and Methods

Extract preparation

The seeds were dried in the shade. Using a mechanical grinder, the dried material was reduced to a coarse powder and then extracted using the Soxhletor with ethanol as a solvent.

Animals

Healthy female mice weighing 25-30 g were used for acute toxicity studies. Healthy Wistar male rats of approximately 12 to 13 weeks of age, weighing between 200- 250 g were taken for investigating anti-inflammatory and anti-arthritic effects. All the animals were procured from drug testing laboratory, Bangalore.

The animals were acclimatized by keeping them in the animal home facility at Government College of Pharmacy in Bangalore. They were fed commercial pelleted rat food with unlimited water. The animals were cared for in compliance with CPCSEA regulations. The acute toxicity and anti-inflammatory activity investigations were conducted after receiving approval from the Institutional Animal Ethical Committee on 29/08/2020 with reference no. GCP/IAEC/DOP/20/E.C/ ADM/2020-2021/34.

Toxicity of the extracts

Acute toxicity (OECD 423 guidelines) was investigated using female mice (25- 30 g) maintained under standard husbandry conditions. Three female mice were given a maximum upper limit dose of 2000 mg/kg of ethanolic extract of Cassia hirsuta seeds (EECH) orally. Animals were studied for 48 hours to evaluate their general behaviour, indication of distress, and nervous manifestations. All the animals were observed for 14 days after the dose was administered.

Carrageenan-induced paw edema

This study employed five rat groups (n=6). They were starved overnight before the study and given unrestricted water access. Paw volumes were then measured with a plethysmometer on the day of the experiment (Ugo Basile 7140, Italy). Following this, group I was treated with normal saline as a control, group II was given Diclofenac (10 mg/kg)^10,11 and groups III, IV, & V were given EECH via gavage at doses of 100, 200, & 400 mg/kg, respectively. One hour following vehicle/drug treatment, 1% carrageenan (0.1 mL) was instilled in the sub-plantar portion of rat’s left hind paw to cause edema.^12,13 The increment in paw volume was assessed at 0.5, 1, 2, 3, 4, and 5 hours after the instillation of carrageenan.

Adjuvant-induced arthritis

The study employed six groups of rats (n=6). During the study, baseline paw volume measurements were taken with a plethysmometer. Except for group I that was treated with a single subplantar injection of CFA (0.1 mL) to induce rheumatoid arthritis 1 hour after administration of vehicle/drug, Group III was treated with Diclofenac (10 mg/kg) and Group IV, Group V, Group IV were treated with low, middle, and high doses of EECH orally from the first to the 28th day. On the 28th day, all animals were anaesthetized, and blood samples were obtained through retro orbital puncture for haematological examination. Separated serum was then analysed for rheumatoid factor (RF), serum glutamic pyruvic transaminase (SGPT), and serum glutamic oxaloacetic transaminase (SGOT). Animals were slaughtered, and their limbs were isolated, cleaned, and stored in 10% formalin. Histopathological examinations were performed on the isolated limbs. In brief, serum RF was estimated using a commercial serum RF latex kit, while SGPT and SGOT were calculated using diagnostic test kits. Hematology parameters were examined using a haematology analyzer.

Statistical method

Graphpad prism; Version 9.2.0 (332) was used for statistical analysis, which included one-way ANOVA carried by Dunnett’s test. p <0.05 was deemed significant.

Results

Toxicity profile

At a dose of 2000 mg/kg, EECH was proven to be safe for animals. As a result, this dose was designated as the lethal dose, and 100 mg/kg, 200 mg/kg, and 400 mg/ kg were chosen as the therapeutic dose (TD) for testing anti-inflammatory activity on Carrageenan-induced paw (CIP) edema and CFA-induced arthritis.

Effect of EECH on CIP edema

In this investigation, the greatest phlogistic reaction of carrageenan was found in control animals after 3-5 hours. In reference to control group, the EECH group exhibited a dose-dependent decrease in paw volume. EECH at dose of 200 & 400 mg/kg reduced edema compared to the control animals (Figure 1).

Effect of EECH on CFA-induced arthritis in rats

At all doses, the conventional medication Diclofenac sodium reduced paw volume much more than the EECH group. The highest swelling of joints occured on day 7, following which there was a progressive decline in all the groups except in control group on day 28. Throughout the research, EECH resulted in a dose-dependent decrease in joint swelling. Except for EECH 100 mg/kg, all have shown significant anti-arthritic effect when compared to control (Figure 2). 

Effect of EECH treatment on serum RF, SGPT and SGPT levels in CFA induced arthritis

The serum samples obtained on 28th day of the CFA research were used to estimate RF, SGPT, and SGOT levels. Figures 4, 5 and 6 show the serum RF, SGPT, and SGOT results, respectively.

In comparison to control group rats, EECH administration resulted in a dose-dependent decrease in serum RF, SGPT, and SGOT. There was a significant deduction in serum RF, SGPT, and SGOT in the EECH treated group.  

Effect of EECH treatment on hematological parameters in CFA induced arthritis

The blood obtained on 28th day of the CFA research was used to estimate RBC, WBC, Hb, and ESR values. Figures 7, 8, 9, and 10 show the results of the haematological parameters.

In comparison to control group rats, EECH administration caused a dose-dependent decrease in serum RBC, WBC, Hb, and ESR values . There was a reduction in RBC, WBC, Hb, and ESR values in the EECH treated group.

Effect of EECH treatment on histopathology in CFA induced arthritis

The normal control animals showed normal architecture of bone and synovium with normal histology as observed in Figures 11 & 12. The disease control animals injected with CFA (0.1 mL) showed lymphoid follicular fibrosis, extensive inflammation and arterial fibrosis (Figures 13 & 14). The standard group animals showed normal architecture of bone and normal (areolar) synovial cells with follicles as observed in Figures 15 and 16. The lower group animals showed normal architecture of bone and focal inflammation (Figures 17 and 18). The moderate group animals showed normal architecture of bone and normal (areolar) synovial lining cells with follicles as observed in Figures 19 and 20. The higher group animals showed normal architecture of bone and normal (areolar) synovial lining cells with follicles (Figures 21 and 22) because of the reduction in the release of chemical mediators of the inflammatory process.

Discussion

Inflammation is a critical physiological response that occurs in response to a wide range of harmful substances. Sustained, excessive, or inappropriate inflammation, on the other hand is the root cause of a variety of disorders, including RA, psoriasis, and inflammatory bowel disease.¹⁴

Several medicinal plants have been used in the millennia to treat problems related to inflammatory states or to regulate the inflammatory elements of diseases. The presence of phytoconstituents in these medicinal plants may exert anti-inflammatory effects by interfering with inflammatory pathways such as discharge of pro-inflammatory mediators and leukocyte migration due to inflammatory stimulus.

In CIP edema and CFA-induced arthritis models, EECH demonstrated anti-inflammatory and anti-arthritic effects.

CIP edema is most commonly used for assessing anti-inflammatory medications because it is substantially associated with early exudative stages of inflammation in rats. Following subplantar instillation, there is a rapid rise in paw volume that can be linked to vascular permeability generated by histamine and serotonin activity. Due to the synthesis of prostaglandins, paw edema progressively grows to a peak over the first 4-6 hours of the induction period.¹⁵ A similar pattern was seen in control group in our investigation. Diclofenac inhibited inflammation only after three and six hours of carrageenan treatment, indicating that Diclofenac’s action on prostaglandins occurs during second phase of CIP edema. During all the observation periods, the EECH group showed dose-dependent suppression of inflammation, indicating that EECH has inhibitory effect against numerous autacoid mediators.

The efficacy of EECH in diminishing joint inflammation in the CFA-induced arthritis method was investigated to confirm the test drug’s anti-arthritic activity as this model exhibits significant parallels with human disease.^8,16

Along with measuring joint swelling, we also evaluated blood levels of RF, SGPT, and SGOT in arthritic mice, which have a significant role in disease development.

EECH minimized joint edema in a dose-dependent manner during the evaluation period. Although the drop in joint swelling was never statistically relevant at a low dose (100 mg/kg), dosing with a moderate dose (200 mg/kg) and higher dose (400 mg/kg) resulted in a substantial suppression on all assessment days when compared to control.

In support to this view, EECH resulted in a relevant drop in blood levels of RF, SGPT, and SGOT when compared to control mice. All the CFA inoculated animals had higher serum RF, SGPT, and SGOT levels. EECH caused a dose-dependent decrease in the increased levels of RF, SGPT, and SGOT, which was significant at high dose (400 mg/kg). EECH caused a considerable drop in WBC and ESR and a significant increment in RBC and Hb levels.

In the current investigation, histological analysis indicated that extract dosages of 200 mg/kg and 400 mg/kg reduced severe soft tissue edema and joint space constriction following the CFA challenge in arthritic control animals.

In acute inflammation, the EECH may have acted by suppresing the production of ROS and NO by nitric oxide synthase or by neutrophilic infiltration, thereby inhibiting the generation or release of inflammatory mediators such as IL, TNF-α, and leukotrienes. In arthritis, it might have operated by lowering the synthesis/release of T cell mediators such as IL, TNF-α, as seen by reduced cartilage and bone degradation in histological examinations.

Flavanoids, phenols, triterpenoids, and other phytoconstituents are primarily responsible for antioxidant activities in EECH. As a result, this seed extract EECH can be utilised as an anti-inflammatory and anti-arthritic medication to reduce free radicals.

Conclusion

In both acute and chronic animal studies, EECH showed significant anti-inflammatory action. The extract prevented inflammation caused by carrageenan and CFA, and it also had stronger antioxidant properties. One or more of these phytochemicals discovered in the EECH might be responsible for the action. These findings imply that EECH might be used to treat inflammation. This trait must be established in other different models, and further research can be conducted in human beings.

Conflicts of Interest

None

Acknowledgements

The study was funded by Government college of pharmacy, Bangalore. India.