|Year : 2019 | Volume
| Issue : 2 | Page : 107-117
Attenuation of Complete Freund's Adjuvant-induced arthritis by different dilutions of Eupatorium perfoliatum and Crotalus horridus and their safety evaluation
Surender Singh1, Ritu Karwasra1, Prerna Kalra1, Debadatta Nayak2, Anil K Khurana2, Raj K Manchanda2
1 Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India
2 Central Council for Research in Homoeopathy, Ministry of AYUSH, Government of India, New Delhi, India
|Date of Submission||13-Mar-2019|
|Date of Acceptance||20-May-2019|
|Date of Web Publication||27-Jun-2019|
Dr. Surender Singh
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
Objective: The aim of this study was to investigate, the inhibitory effect of the homoeopathic drugs – Eupatorium perfoliatum and Crotalus horridus in experimental models of inflammation and Complete Freund's Adjuvant (CFA)-induced arthritis with evaluation of their safety aspects by acute and subacute toxicity studies. Materials and Methods: Animals were divided into eight groups (n = 6). Eupatorium perfoliatum and Crotalus horridus in different dilutions (6CH, 12CH, 30CH and 200CH) were administered orally, daily during the study period of 21 days, and their effect on joint dysfunction was evaluated by measuring joint diameter in CFA-induced arthritis model. In addition, inflammatory profiles of these homoeopathic drugs were screened in carrageenan-induced paw oedema model. Acute and subacute studies were carried out according to Organization for Economic Cooperation and Development 425, 407 guidelines. The subacute toxicity study was carried out for a duration of 28 days, and all the animals were observed for behavioural abnormalities. At the end of 28th day, animals were sacrificed to carry out the biochemical, haematological and histopathological estimations. Results: Findings of the study revealed that on CFA administration, there is a significant (P < 0.01) increase in joint diameter in all the tested animals. Maximum increase in joint diameter was observed on day 3 in all the treatment groups. Eupatorium perfoliatum 200CH showed significant decrease in joint diameter on day 21. In carrageenan study, the homoeopathic drugs produced a significant reduction in paw oedema at 5-h post-carrageenan administration. Study noted that the anti-inflammatory activity of Eupatorium perfoliatum was found to be superior to Crotalus horridus. Conclusion: Study inferred that Eupatorium perfoliatum and Crotalus horridus are safe at dilutions 6CH, 12CH, 30CH and 200CH and effective in minimising inflammation and arthritis in CFA-induced model.
Keywords: Crotalus horridus, Eupatorium perfoliatum, Histopathology, homoeopathic drugs, Inflammation, Rheumatoid arthritis
|How to cite this article:|
Singh S, Karwasra R, Kalra P, Nayak D, Khurana AK, Manchanda RK. Attenuation of Complete Freund's Adjuvant-induced arthritis by different dilutions of Eupatorium perfoliatum and Crotalus horridus and their safety evaluation. Indian J Res Homoeopathy 2019;13:107-17
|How to cite this URL:|
Singh S, Karwasra R, Kalra P, Nayak D, Khurana AK, Manchanda RK. Attenuation of Complete Freund's Adjuvant-induced arthritis by different dilutions of Eupatorium perfoliatum and Crotalus horridus and their safety evaluation. Indian J Res Homoeopathy [serial online] 2019 [cited 2022 May 17];13:107-17. Available from: https://www.ijrh.org/text.asp?2019/13/2/107/261634
| Introduction|| |
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune-mediated disorder characterised by cellular infiltration and proliferation of the synovial membrane, pannus formation, cartilage and bone erosion, leading to the progressive destruction of the joints through the interaction between infiltrating cells and mediators such as cytokines, prostanoids and proteolytic enzymes. In India, the prevalence of RA is found to be around 0.75% of the adult population. The pathogenesis of RA is associated with accumulation of macrophages, neutrophils and production of free radical-producing enzymes. Conventional treatment options available are disease-modifying antirheumatic drugs, Nonsteroidal Anti-inflammatory (NSAIDs) and glucocorticoids. Although these treatment options are effective in improving disease conditions, chronic and long-term use of these agents may lead to serious adverse effects such as gastric ulceration, cardiovascular abnormalities and emergence of opportunistic infections. Thousands of patients with inflammatory disorders face the problem of gastric ulcers and gastrointestinal bleeding due to prolonged use of NSAIDs, and also considering the chronic nature of these disorders, compliance became an another issue associated with these costly and toxic drugs. It is also seen that the large number of patients with these disorders rely on complementary and alternative medicines either alone or in combination with conventional treatment for better outcome. Hence, discovering new compounds of natural origin in homoeopathic system of medicine with potential therapeutic effect and minimal side effects have been pursued vigorously. Medicinal plants are being used in the form of homoeopathic drugs in the prevention and management of inflammatory disorders in prophylactic way.
Homoeopathic drugs – Eupatorium perfoliatum and Crotalus horridus are available in different formulations or preparations for the treatment of different ailments. Eupatorium and Crotalus genera have been studied in some depth and several compounds with varying effects identified. Traditionally, Eupatorium perfoliatum was used for the treatment of fever and as a gastrointestinal aid for pains in the stomach. Leaves and flowering parts of the plant 'common boneset' have been extensively used within traditional medicine of North America's native inhabitants against fever and as diaphoretic agent., Previous literature on Eupatorium perfoliatum depicts its use as mother tincture in homoeopathic system of medicine and used against in vitro human monocyte cells for controlling inflammation and oxidative stress. It possesses significant anti-inflammatory, antioxidant, immunomodulatory, cytotoxic and antibacterial activities. Crotalus horridus is also known as timber rattlesnake; its venom is used in homoeopathic preparations. It is a venomous pit viper found in eastern parts of United States and produces high venom yield. The venom comprises of Type A-neurotoxic, Type B-haemorrhagic. It possesses antiretroviral action and is potent against Ehrlichiosis infections in different homoeopathic preparations. In this context, the health benefits of Eupatorium perfoliatum and Crotalus horridus in preventing cancers and infectious diseases have been widely focused. However, very few studies have targeted the eventual benefit of Eupatorium perfoliatum and Crotalus horridus in attenuation of inflammation.
| Materials and Methods|| |
Adult Wistar albino rats (150–180 g) from our institutional breeding stock were used in the study. Animals were housed at 25°C ± 2°C inside clean polypropylene cages in groups of three. They had access to food and water ad libitum throughout the duration of the study. The experimental protocols were duly approved by the Institutional Animal Ethics Committee, All India Institute of Medical Sciences, New Delhi, India (Animal Ethics Approval No-935/IAEC/16). All experiments were carried out in accordance with 'Guidelines for care and use of animals in scientific research (Indian National Science Academy 1998, Revised 2000)'.
Chemicals and drugs
Complete Freund's Adjuvant (CFA) was purchased from Difco, CA, USA. Indomethacin was purchased from Sigma Chemical Co., USA. All the other chemicals used were of analytical grade.
Homoeopathic preparations of Eupatorium perfoliatum and Crotalus horridus at different dilutions, i.e., 6CH, 12CH, 30CH and 200CH were procured from Central Council for Research in Homoeopathy (CCRH).
Dose calculation of study drugs for experimental animals
The route of administration is per oral, and the vehicle for administration is deionised water. The standard dose of 20 μl/100 g body weight of rat was administered using a micropipette and solution was mixed with 450 μl of distilled water administered orally with the help of oral catheter and flushed with 500 μl of distilled water.
Experimental design: Toxicity studies
Acute and subacute toxicity studies of these homeopathic drugs were also evaluated according to Organization for Economic Cooperation and Development (OECD) guidelines.
Acute toxicity study
Evaluation of acute oral toxicity of Eupatorium perfoliatum and Crotalus horridus at different dilutions 6CH, 12CH, 30CH and 200CH was carried out according to the OECD guidelines for testing of chemicals-425. A limit test (2000 μl/kg body weight) was performed using five male Wistar rats (150–180 g) from our breeding stock. All the animals were observed for behavioural changes and mortality till 14 days after administration of the dose [Table 1].
|Table 1: Mortality in normal and homoeopathic groups (at different dilutions) for a period of 10 days|
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Subacute toxicity studies
Evaluation of 28-day oral toxicity study of Eupatorium perfoliatum and Crotalus horridus at different dilutions 6CH, 12CH, 30CH and 200CH was carried out according to the OECD guidelines for testing of chemicals-407. Ninety Wistar rats (150–180 g) of both sexes from our breeding stock were allocated into nine groups (n = 5/sex/group). Group I received the vehicle (1 ml/kg body weight, 1% saline) and served as normal control and Group II–V received Eupatorium perfoliatum in different dilutions, i.e., 6CH, 12CH, 30CH and 200CH and Group VI–IX Crotalus horridus in different dilutions. Drug/vehicle was administered daily for 28 days [Table 2] and [Table 3].
|Table 2: Biochemical parameters analysed in normal control and homoeopathic medicines Eupatorium perfoliatum and Crotalus horridus at different dilutions by autoanalyzer|
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|Table 3: Haematological parameters (red blood cells, white blood cells and platelets) were measured by different methods in all groups|
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Experimental protocol: Carrageenan-induced paw oedema model
The experimental protocol and methodologies used were the same as described earlier. Animals were divided into 11 groups (n = 6) and fasted overnight with access to water ad libitum. Thereafter, Group I received normal control (1% normal saline, p.o), Group II received carrageenan alone (1% solution of carrageenan dissolved in normal saline), Group III received standard drug indomethacin (3 mg/kg), Groups IV, V, VI and VII received Eupatorium perfoliatum at different dilutions 6CH, 12CH, 30CH and 200CH and Group VIII–XI Crotalus horridus in different dilutions. Sixty minutes post-administration of the drug/vehicle, paw oedema was induced by subcutaneous administration of 0.1 mL of 1% λi-carrageenan (constituted in normal saline) into the left hind paw of the animal. Paw oedema was measured using a digital plethysmometer (Orchid Scientific, India) at 1-h, 3-h and 5-h post-carrageenan administration. Increase in paw volume and percentage inhibition of paw oedema of all the treatment groups were calculated.
Experimental protocol: Chronic model of inflammation
Experiment was conducted according to previous standardised methodology. Eleven groups of rats (n = 6) were used in the study and fasted overnight with access to water ad libitum.
Group 1 (normal control): normal saline (1 ml/kg/day; p.o.) was administered to rats for a period of 21 days.
Group 2 (CFA control): normal saline was administered to rats for a period of 21 days, and on 0th day, a single subplantar injection of 0.1 ml CFA (Difco: 0.05% [w/v] of Mycobacterium butyricum in mineral oil) in the left hind paw was given.
Group 3 (Indomethacin 3 mg/kg + CFA): indomethacin was administered to rats for a period of 21 days, and on day zero, a single subplantar injection of 0.1 ml CFA in the left hind paw was given.
Group 4–7 (Eupatorium perfoliatum + CFA): received Eupatorium perfoliatum at different dilutions 6CH, 12CH, 30CH and 200CH for a period of 21 days, and on day zero, a single subplantar injection of 0.1 ml CFA in the left hind paw was given.
Group 8–11 (Crotalus horridus + CFA): received Crotalus horridus at different dilutions 6CH, 12CH, 30CH and 200CH for a period of 21 days, and on day zero, a single subplantar injection of 0.1 ml CFA in the left hind paw was given.
On day 21, the animals were sacrificed, and terminal blood collection was performed by retro-orbital plexus and then centrifuged at 3000 rpm to separate the serum and stored at −20°C.
Measurement of increase in joint diameter
The joint diameter was measured by means of micrometre screw gauge placed on the left ankle joint transversely, and the diameter was measured on day 0 before injecting CFA and then on day 3, 7, 14 and 21 during the study. The increase in joint diameter was obtained by subtracting the day 0 values from the measurements taken on day 3, 7, 14 and 21 and expressed in mm.
All data were represented as mean ± standard error of mean (n = 6). Comparison between groups was done by one-way analysis of variance followed by Dunnett's multiple comparison tests using the Graphpad Prism version 5.03, San Diego, CA, USA. P <0.05 was considered as statistically significant.
| Results|| |
Effect of different dilutions of Eupatorium perfoliatum and Crotalus horridus on acute oral toxicity study
In acute toxicity study, no mortality or toxic signs were observed in any group at different dilutions which indicates that oral LD50 of Eupatorium perfoliatum and Crotalus horridus is >2000 μl All the five Wistar rats were observed at the end of study period in all treatment groups.
Effect of different dilutions of Eupatorium perfoliatum and Crotalus horridus on subacute oral toxicity study for 28 days
No significant changes in body weights of treated rats were observed when compared with normal control group. No significant change in mean organ weight of liver, kidney, brain, heart, testes and ovaries were observed at the end of 28th day, when compared to normal control group. The mean organ weight of liver of normal control group is 3.49 ± 0.05; Eupatorium perfoliatum at 6CH, 12CH, 30CH and 200CH was found to be 3.33, 3.32, 3.36 and 3.46, respectively. Crotalus horridus at 6CH, 12CH, 30CH and 200CH was found to be 3.50, 3.50, 3.44 and 3.49, respectively. The mean organ weight of kidney of normal control group is 0.71 ± 0.00; Eupatorium perfoliatum at 6CH, 12CH, 30CH and 200CH was found to be 0.72, 0.71, 0.71 and 0.72, respectively. Crotalus horridus at 6CH, 12CH, 30CH and 200CH was found to be 0.68, 0.69, 0.74 and 0.74, respectively.
No signs of gastric ulceration or erosion were observed. Change in mean organ weight of different organs in control and treated groups were presented.
Biochemical and haematological observations also did not produce any significant changes in haematological and biochemical parameters of male [Table 4] and female [Table 5] rats as compared to those in normal control group. All animals survived until the scheduled necropsy, and their physical and behavioural examinations did not reveal any treatment-related adverse effects. No pathological changes were observed in histological section of heart, kidney, liver, testis, ovaries and brain of homoeopathic drugs-treated male and female [Figure 1] and [Figure 2] rats as compared to normal control animals.
|Table 4: Inhibition of paw volume was measured using plethysmometer in carrageenan-induced paw oedema model in Wistar rats|
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|Table 5: Percentage inhibition inflammation was measured in carrageenan-induced paw oedema model in Wistar rats|
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|Figure 1: The histological analysis of haematoxylin and eosin in different organs (kidney, brain and liver) control and experimental groups of animals|
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|Figure 2: The histological analysis of haematoxylin and eosin in different organs (testis, heart and ovaries) control and experimental groups of animals|
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Effect of different dilutions of Eupatorium perfoliatum and Crotalus horridus on acute inflammation
The carrageenan-induced paw oedema model was done to evaluate the involvement of anti-inflammatory activity of homoeopathic drugs at the doses tested. Administration of carrageenan produced a significant (P < 0.05) increase in paw oedema that was persistent throughout the observation period. Maximum paw oedema was observed at 5-h post-carrageenan administration in all the tested animals [Table 6]. The standard drug, indomethacin, produced a significant reduction in paw oedema at 3-h and 5-h post-carrageenan administration. Percentage inhibition of paw oedema was calculated for all treatment groups [Table 7]. The percentage inhibition found in Eupatorium perfoliatum dilutions (6CH, 12CH, 30CH and 200CH) are 8.064%, 8.064%, 16.12% and 27.41%, respectively. In another homoeopathic medicine, i.e., Crotalus horridus, the percentage inhibition was found to be 4.838%, 11.29%, 11.29% and 22.58% respectively. However, indomethacin inhibits 43.54% paw oedema in carrageenan-induced paw oedema model.
|Table 6: Effect of administering different homoeopathic drugs on paw diameter in complete Freund's adjuvant induced arthritis in Wistar rats|
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|Table 7: The percentage inhibition of paw edema in carrageenan induced rat paw edema model|
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Effect of different dilutions of Eupatorium perfoliatum and Crotalus horridus on chronic inflammation
Injection of CFA into the left hind paw produced an increase in joint swelling, erythema and dysfunction in all the experimental groups. The first signs of arthritis development were visible on day 14, after immunisation with CFA. Treatment with homoeopathic drugs at different dilutions suppressed hind paw swelling to lesser extent. However, maximum protection was observed in Eupatorium perfoliatum 200CH group against CFA-related changes.
Effect of different dilutions of Eupatorium perfoliatum and Crotalus horridus on joint diameter
After immunisation with CFA, there was significant increase (P < 0.001) in the joint diameter of injected rats in all experimental groups as compared to normal control. Maximum increase in joint diameter was observed on day 3; after that, there was gradual decrease in joint inflammation in all groups except CFA control animals. There was small increase in ankle joint diameter in CFA control group, after day 14 which is the result of heightened cell-mediated immunity response. Significant inhibition (P < 0.001) in joint inflammation was observed by Eupatorium perfoliatum 200CH-treated group [Table 8].
|Table 8: The increase in joint diameter in CFA-induced rheumatoid arthritis on day 3, 7, 14 and 21|
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| Discussion|| |
The present study demonstrated that the homoeopathic drugs' (Eupatorium perfoliatum and Crotalus horridus) treatment is safe at chronic administration and was effective in controlling inflammation and joint dysfunction in RA.
As mentioned earlier, inflammatory disorders such as RA is a chronic, deforming disease of joints of unknown aetiology. It is characterised by inflammation of synovial membrane, pain, destructive changes in cartilage and bone, restricting mobility of joints. The activated leucocytes and synovial fibroblasts in joint tissue secrete several pro-inflammatory mediators such as tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, IL-8 and interferon- γ (IFN-γ) that cause inflammation and joint damage. Several studies have proposed that the pro-inflammatory cytokines such as TNF-α and IL-1 play a central role in the pathogenesis of RA as they contribute to the synoviocyte self-proliferation and increase the production of tissue enzymes, resulting in cartilage destruction, while numerous other studies suggest that the transcription factor, i.e., NF-κB is also a pivotal regulator of inflammation and joint degradation by upregulating expression of many pro-inflammatory genes, such as, IL-1 β, IL-6, TNF-α, chemokines and matrix metalloproteinases. Some studies also demonstrated the increased NF-κB levels in the synovial joint of human and animal models. In short, we can conclude that in the process of bone erosion, the transcription factor (NF-κB) upregulate the expression of TNF-α, which further triggers the production of other cytokines, and furthermore, it exerts its arthritogenic potency through the induction of IL-1. Therefore, IL-1, NF-κB and TNF-α have been shown to be dominant players in the induction of inflammation and bone erosion. The concomitant presence of chronic pain and associated other symptoms is considered as suggestive underlying immune activity in RA. Thus, the reduction of pain, inflammation and joint damage is the focus of drug treatment of inflammatory disorders such as RA.
In our present investigation, we evaluated the effect of homoeopathic drugs on acute and chronic inflammation. These drugs or preparations inhibit the paw oedema, induced by carrageenan injection. Carrageenan-induced paw oedema model is an experimental model used for assessing the anti-inflammatory activity of test substance. Carrageenan has time-dependent triphasic response, in which the first and foremost phase (1–2 h after carrageenan administration) primarily attributed to release of histamine, the second phase (2–3 h) being attributed to release of serotonin and kinins and the third phase (3–5 h) primarily attributed to release of prostaglandins. Different dilutions of homoeopathic drugs act on all the three phases and produced significant reduction in paw oedema. The effect observed may be due to significant changes in the inflammatory response which are comparable with indomethacin. The response to anti-inflammatory activity is directly linked with the antiarthritic activity and to the progression of disease. CFA model is used to screen the newer compounds for its anti-inflammatory or antiarthritic activity. To assess the chronic anti-inflammatory activity of homoeopathic drugs – Eupatorium perfoliatum and Crotalus horridus, CFA model was used. It has been used in various studies as a model for evaluation of sub-chronic or chronic inflammation in rats and is of considerable relevance to the study of pathophysiological and pharmacological control of inflammatory and analgesic processes. CFA elicits joint swelling, synovial membrane inflammation and cartilage destruction. Treatment of CFA rats with Eupatorium perfoliatum 200CH inhibited paw swelling, stiffness and joint inflammation.
In acute toxicity study, there were no mortality or any signs of toxicity observed after oral administration of drugs up to the dose level of 2000 μl/kg in rats. In subacute toxicity study, the body and organ weights of experimental animals did not show any significant changes after administration of the different homoeopathic drugs for 28 days, when compared to the normal control group. Haematological parameters analysed included complete blood count of experimental and control group animals. Analysis of blood parameters in animal studies is relevant to evaluate the risk of alterations of the haematopoietic system in toxicity studies. In this study, administration of the different homoeopathic drugs after 28 days induces no significant change in all haematological parameters as compared to normal control group. Assay of biochemical parameters were performed in order to evaluate the liver, renal, lipid and glycaemic profiles of all experimental animals, In this study, assay of the liver profile parameters (serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase) revealed normal functioning of the liver after 28 days of administration of different homoeopathic drugs, with normal values in experimental animals as compared to normal control group. Histopathological examinations of the liver, kidney, heart, brain, testis and ovaries did not reveal any morphological changes after administration of test drugs. Thus, both homoeopathic drugs, i.e., Eupatorium perfoliatum and Crotalus horridus at different dilutions were found to be safe with no pathological abnormalities on any organ of treated animals.
| Conclusion|| |
Homoeopathic drugs – Eupatorium perfoliatum and Crotalus horridus are safe and effective in treating inflammation and arthritis in CFA-induced model. However, further studies are required to find the potential mechanism of these homoeopathic drugs and their other possible pharmacological activity.
Financial support and sponsorship
This research received grant from CCRH, Ministry of AYUSH, Government of India.
Conflicts of interest
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]