|Year : 2017 | Volume
| Issue : 1 | Page : 34-40
Preclinical evaluation of antiarthritic activity of ultra-diluted preparations of capsaicin alkaloids (CP-10), tumor necrosis factor-alpha, and Magnesium phosphoricum in wistar rats
Rajesh Shah1, Sadhana Sathaye2
1 Department of Research and Development, Life Force, Mumbai, Maharashtra, India
2 Department of Pharmaceutical Sciences and Technology, ICT, Mumbai, Maharashtra, India
|Date of Web Publication||23-Feb-2017|
Life Force, 411 Krushal Commercial Complex, GM Road, Chembur, Mumbai - 400 089, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: The use of animal models in the development of new medicine and research is common in the conventional medicine. Animal model for new drug discovery and efficacy testing for preclinical research is explored in Homoeopathy only by a few. This study explores the possibility to test in a controlled way the effects of homoeopathic remedies on experimental model of acute inflammation in rats. Methods: Wistar rats were divided into seven groups (seventy rats of 6–8 weeks' age); medicines were evaluated by oral administration in the Complete Freund's adjuvant (CFA)-induced arthritis. Two new homoeopathic preparations, a combination of Capsaicin and Dihydrocapsaicin (CP-10), tumor necrosis factor-alpha (TNF-α), and one existing homoeopathic medicine, Magnesium phosphoricum (0.6 ml oral), were evaluated against vehicle control using Diclofenac as a standard. Edema was measured using a water-based plethysmometer, before and at different times after arthritis induction. Results: Magnesium phosphoricum showed good results, almost similar to Diclofenac at days 7 and 21, whereas CP-10 and TNF-α showed nonsignificant results. The body processes reversed the inflammatory condition on day 7 onward indicated by similar paw volume of all the treatments. Arthritic index was higher with negative control, which was decreased by CP-10 although nonsignificantly on days 7 and 21. Diclofenac and Magnesium phosphoricum showed significant reduction in arthritic index on days 7 and 21. Conclusion: Ultra-diluted homoeopathic preparations of Magnesium phosphoricum exhibited definite antiarthritic activity. The same could have been confirmed studying the levels of inflammatory biomarkers in a study with longer treatment period.
Keywords: Arthritic index, Capsaicin, Complete Freund's adjuvant, Magnesium Phosphoricum, Tumor necrosis factor-alpha, Homoeopathy, Ultra-dilute potency
|How to cite this article:|
Shah R, Sathaye S. Preclinical evaluation of antiarthritic activity of ultra-diluted preparations of capsaicin alkaloids (CP-10), tumor necrosis factor-alpha, and Magnesium phosphoricum in wistar rats. Indian J Res Homoeopathy 2017;11:34-40
|How to cite this URL:|
Shah R, Sathaye S. Preclinical evaluation of antiarthritic activity of ultra-diluted preparations of capsaicin alkaloids (CP-10), tumor necrosis factor-alpha, and Magnesium phosphoricum in wistar rats. Indian J Res Homoeopathy [serial online] 2017 [cited 2019 Sep 23];11:34-40. Available from: http://www.ijrh.org/text.asp?2017/11/1/34/200848
| Introduction|| |
Homoeopathy is a system of alternative medicine based on the principle of “like cures like” Homoeopathy rests on the premise of treating diseases with extremely ultra-diluted medicinal agents that, in undiluted doses are deemed to produce similar disease symptoms in healthy individuals. Acceptance in the effectiveness of Homoeopathy in general is widespread and growing among the physicians and public.
The present study was undertaken to investigate the antiarthritic, anti-inflammatory effect of three homoeopathic preparations in ultra-dilute 30C potencies in animal model: (1) a combination of Capsaicin and Dihydrocapsaicin, (2) tumor necrosis factor-alpha (TNF-α), and (3) Magnesium phosphoricum. For blinding, the formulations were coded as formulation I, II, and III, respectively. Capsaicin and Dihydrocapsaicin are the plant alkaloids, potentized up to 30c potency using a standardized potentizer. TNF-α (cachexin or cachectin) is a cell signaling protein (cytokine) involved in systemic inflammation. This particular preparation is sourced from human TNF-α procured from Sigma Aldrich. Homoeopathic potencies were prepared by a serial dilution of 1:99, where one part of the drug substance is mixed with 99 parts of vehicle (alcohol) and exposed to ten mechanical strokes, to make 1c potency. Again, one part of 1c potency is mixed with 99 parts of vehicle to undergo the same process to arrive at 2C potency. Likewise, 30C, 200C, and more potencies were prepared.
In this study, the antiarthritic activity of the three potentized (30C) medicines was compared with Diclofenac (10 mg/kg, source- Sigma Aldrich). The selection of medicines was based on the fundamental principle of Homoeopathy “like cures like” and the phenomenon of hormesis, which state that the substance known to produce inflammation or pain may have built-in capacity to relieve the same, if administered in small dose. The author had done anecdotal work using the new homoeopathic preparations used in this study, where he had observed activity against inflammation and arthritis.
| Materials and Methods|| |
Male and female Albino Wistar rats (180–200 g) were procured from Haffkine Bio Pharmaceutical, Parel, Mumbai, and housed under a controlled environment (room temperature: 23 ± 2°C, relative humidity: 60 ± 5%, 12 h day/night cycle) with balanced diet and water ad libitum. The animals were acclimatized for a period of 1 week and were kept under pathogen-free conditions. All animal experiments were approved by the Institutional Animal Ethics Committee (IAEC), Department of Pharmaceutical Sciences and Technology, ICT, Mumbai (ICT/IAEC/2012/P-39).
Complete Freund's adjuvant-induced arthritis
Arthritis was induced in rats by injecting 0.1 ml of fine emulsion of complete Freund's adjuvant (CFA) in olive oil (1:2, v/v) (at a concentration of 0.25 mg heat-killed Mycobacterium tuberculosis per milliliter of emulsion) in the subplantar surface of rats' left hind foot.,, Equal amount of saline was injected at the same site of the right foot. Arthritis was induced in all the animals except normal control. A volume of 0.1 ml saline was injected in both paws of the normal control animals. All rats were randomly divided into seven groups each containing 5 males (M) and 5 females (F). Group I: normal control (distilled water), Group II: vehicle control (dispensing alcohol), Group III: untreated control (CFA), Group IV: homoeopathic formulation I Capsaicin and Dihydrocapsaicin 30C, Group V: homoeopathic formulation TNF-α 30C, Group VI: homoeopathic formulation Magnesium phosphoricum 30C, and Group VII: Diclofenac.
Dosage preparation and administration
Homoeopathic formulations Capsaicin and Dihydrocapsaicin and TNF-α in 30C potency and the vehicle control (Curative Power Lab Ltd., dispensing alcohol 91% v/v, Batch number: HD-18, Mfg. date: February 2013) were supplied by Life Force Homeopathy, Mumbai, India. Formulation Magnesium phosphoricum in 30C potency, liquid dilution, was sourced from Dr. Willmar Schwabe India Pvt Ltd. (Batch Number: 2981299, Mfg. date: November 2009) CFA was obtained from Haffkine Biopharmaceuticals, Parel, Mumbai (Lot number 098K8729). The rats received 0.6 ml of homoeopathic formulation or vehicle, in each respective group, and 10 mg/kg of Diclofenac every day for 21 days by oral route. Animals were kept nil by mouth for 15 minutes before the drug administration.
Evaluation of the severity of arthritis
Severity of arthritis was evaluated based on the following parameters, namely, body weight, paw volume, arthritic index, and mechanical pain threshold. All animals were treated with CFA except normal control animals, and evaluation parameters were assessed before adjuvant injection and then on days 7, 14, and 21 after adjuvant injection. The body weights of all the rats were recorded, and the paw volumes were measured using a Digital Plethysmometer (UGO Basile [SRL], biological research apparatus, 21025 Comerio VA Italy, Model 7141). For arthritic index, the physical symptoms of arthritis were evaluated by the Arthritic Index Grading System [Table 1]. The animals were sacrificed as per the approved method of euthanasia at the end of study.
Arthritis index  for each group was calculated by adding the scores for each rat. Mechanical pain threshold was assessed using external mechanical force. The mechanical pain threshold of hind paw was determined by compressing the paw using a custom-designed balance pressure apparatus consisting of conical tip of 1 mm diameter. The load on the conical tip was gradually increased and the load cell allowed visualization on the digital display of the force applied at each moment of the test in grams. The threshold force of rats squeaking or struggling was expressed in grams.
The results were expressed as the mean for the parametric data sets and as the median minimum, maximum for the nonparametric data sets. The significant difference between the means (parametric) was evaluated by one-way ANOVA followed by Dunnett's post hoc multiple comparison test for normal data.
| Results|| |
CFA-induced arthritis in rats is used as a model to evaluate the antiarthritic activity of drugs in preclinical research. CFA induces a chronic immune-mediated inflammation similar to arthritis characterized by elevation of pro-inflammatory mediators and development of swelling, pain, and deformity of joints. Inflammation was indicated by increase in paw volume. The reduction in inflammation due to treatment was indicated by decreased paw volume for evaluation of therapeutic efficacy of the drug, administered in the ultra-diluted form.
No significant difference was observed with respect to negative control in both male and female rats, and a good increase in weight on days 7, 14, and 21 indicated normal growth in all the groups.
In the acute phase of arthritic inflammation on day 1, homoeopathic formulation Magnesium phosphoricum indicated a significant reduction in inflammation by reduced paw volume, similar to standard Diclofenac. Homoeopathic formulations Capsaicin and Dihydrocapsaicin and II also showed a nonsignificant reduction in inflammation similar to negative control. The body processes reversed this inflammatory phase as seen by decrease in inflammation by the negative control on day 7. A nonsignificant rise in the inflammation was seen with homoeopathic formulations on day 21, especially with the formulation TNF-α, which was not seen with Diclofenac, suggestive of the continued primary action of the formulation. Such an increase is peculiar of ultra-dilute homoeopathic formulation, as per the homoeopathic principle of “like cures like,” where initial effect (inflammation index) caused by the medicinal substance gets reduced subsequently by secondary effect  (which could be body's defense mechanism), leading to anti-inflammatory effect [Figure 1] and [Figure 2]. This study has demonstrated the initial (primary) effect of formulation TNF-α by increasing the inflammation, which could have possibly reversed on further continuation of treatment.
Formulation Capsaicin and Dihydrocapsaicin 30C is an alkaloid which is known to produce pain and inflammation, not only in crude form but also in high dilution dose as seen in a Homoeopathic Pathogenetic Trial (HPT). The same compound has also demonstrated anti-pain and anti-inflammatory effects with 30C potency on a range of painful conditions in a clinical trial. The current study has produced similar effects in an animal model. Longer study to explore this phenomenon can be conducted in future.
Arthritic index was higher with negative control which was decreased by formulation Capsaicin and Dihydrocapsaicin on days 7 and 21 although not significantly. Diclofenac and formulation Magnesium phosphoricum showed a significant reduction in arthritic index on days 7 and 21 [Table 2]. Formulation Magnesium phosphoricum exhibited similar response as that of standard Diclofenac on day 7.
Similar to the paw volume, a trend in nonsignificant rise in the arthritic index was seen in all the groups, probably due to the immunological process systemically. It is extremely important to plan a further study with an increase in the duration of administration of the homoeopathic formulation to evaluate if on a longer administration the symptoms could reverse significantly.
P value was calculated using paired t-test for all the three formulations as compared to vehicle control and negative control. It was noted that the statistical significant results (P < 0.05) were obtained with formulation Magnesium phosphoricum as compared to vehicle control in male rats. In case of Diclofenac, statistical significant results (P < 0.05) were observed in female rats as compared to vehicle control and negative control.
A decreased pain threshold in negative control (CFA) on day 7 indicated induction of arthritic process, i.e., immune-mediated inflammation. No drug treatment could reverse this significantly as compared to negative control on day 7, indicating induction of arthritis and subsequent pain. Reversal of mechanical pain threshold in negative control group on day 14 indicated natural body process toward recovery although none of the drugs could alleviate the decreased pain threshold. On day 21, negative control groups almost reversed back the decreased pain threshold to normal. Diclofenac, too, increased the pain threshold, indicating good analgesic, anti-inflammatory activity. None of the homoeopathic formulations could alleviate the pain threshold to normal by day 21 [Figure 3] and [Figure 4]. Continuation of treatment further could probably alleviate the pain threshold, relieving the arthritic symptoms. This corresponds to the other parameters as discussed before, where a rise in paw volume and arthritic index was seen in the homoeopathic treatment groups.
| Discussion|| |
The medicinal substances sourced from different materials (herbs, minerals, and biological) are known to act differently, as observed in clinical homoeopathic practice. It is a common observation that medicines from herbs have quicker and shorter span of action; medicines from minerals and metals (depending on the exact ingredient) have slow or fast but longer duration of action; while those from animal source (many subvarieties) may take longer time to act and the action may last longer., Of course, there are many other factors that determine the duration of action. Usually, the medicines sourced from biological materials take longer duration to set in action, as compared to the mineral and plant materials. Different formulations having different actions need further exploration.
Our understanding about formulation Magnesium phosphoricum (a tissue salt) is based on clinical experience, where it is known to work quickly. It has been in use as an analgesic. It seems that the primary action of this magnesium compound may have been quick and short, quickly followed by the secondary action. Formulation Magnesium phosphoricum did not undergo HPT. Interesting to note that magnesium compounds showed efficacy in pain relief when administered in crude form., Intravenous MgSO4 infusion has shown analgesic effect. The magnesium compound showed anti-inflammatory efficacy in ultra-diluted form in this study. This calls for further study.
The significant reduction in paw volume by homoeopathic formulation Magnesium phosphoricum indicated its anti-inflammatory activity during the acute phase of inflammation. Administration of ultra-dilute potency of TNF-α, which is a pro-inflammatory cytokine, exhibited an increase in paw volume on day 21 [Figure 1]. Although this increase is not statistically significant, a trend toward rise in inflammation is seen as compared to the vehicle control and also to its own activity on days 7 and 14, indicating that the formulation in ultra-dilute potency was capable of producing inflammation showing that it acted and produced biological changes, as compared to the control.
High-dilution medicinal substances are known to induce biological changes and hence symptoms as primary action, which are observed in HPTs. As per the homoeopathic principle and hypothesis, the therapeutic effect of the medicinal substance is through the secondary action of the organism, which is observed once the primary stimulus (formulation) is withdrawn. The constant increase in inflammation in case of TNF-α till day 21, in comparison with the control group, could be the continued primary action of the formulation. It may be hypothesized that on discontinuing the stimulus, there could have been possible reduction in inflammation as a secondary action of the organism. Further studies in this direction alone could bring insight in this phenomenon.
It was noted that the homoeopathic formulations were administered three times a day, while Diclofenac was administered only once a day. Since the handling of animals is known to tire the animals, it might affect the results. In future studies doses may be maintained identical.
| Conclusion|| |
The study was undertaken to evaluate the antiarthritic activity of ultra-dilute potencies of homoeopathic formulations from different origins on CFA-induced arthritic model in rats. A significant decrease in inflammation as well as arthritic index was seen by high-dilution homoeopathic formulation Magnesium phosphoricum similar to Diclofenac, demonstrating evidence that small dose of substance having a capacity to produce inflammation could work against inflammation, as per like cures like, the fundamental principle of Homoeopathy.
No such effect was seen on the mechanical pain threshold by the homoeopathic formulations.
Further study needs to be designed with longer duration of administration of these formulations to study whether these formulations could reverse the arthritic index and resultant pain and inflammation on long-term administration.
We thank the members of IAEC, sponsor, and subject experts for their technical, ethical, legal, and medical inputs; and the department staff for their cooperation.
Financial support and sponsorship
Conflict of interest
| References|| |
Pinto S, Rao AV, Rao A. Lipid peroxidation, erythrocyte antioxidants and plasma antioxidants in osteoarthritis before and after homeopathic treatment. Homeopathy 2008;97:185-9.
Shah R. Standardization of the potentizing machine and quantification of impact of potentization. Indian J Res Homoeopathy 2016;10:126-32.
Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc 2007;2:1269-75.
Snekhalatha U, Anburajan M, Venkatraman B, Menaka M. Evaluation of complete Freund's adjuvant-induced arthritis in a Wistar rat model. Comparison of thermography and histopathology. Z Rheumatol 2013;72:375-82.
Liu YL, Lin HM, Zou R, Wu JC, Han R, Raymond LN, et al.
Suppression of complete Freund's adjuvant-induced adjuvant arthritis by cobratoxin. Acta Pharmacol Sin 2009;30:219-27.
Hahnemann S. Organon of Medicine. 6th
ed. New Delhi: B. Jain Publishers; 1979. p. 149.
Shah R. Hydroquinone: Homoeopathy pathogenetic trial. Indian J Res Homoeopathy 2013;7:47-61.
Shah R. Clinical trial for evaluation of a human immunodeficiency virus nosode in the treatment for human immunodeficiency virus-infected individuals. Indian J Res Homoeopathy 2015;9:25-33.
Allen HC. Materia Medica of Nosodes. Rep. ed. New Delhi: B. Jain Publisher; 2005. p. 258.
Dhawale ML. Principles and Practices of Homeopathy. Part 1. Mumbai: Institute of Clinical Research; 1967. p. 280-87
Hering C. The Guiding Symptoms of Our Materia Medica. Rep. ed. Vol. 10. New Delhi: B. Jain Publishers; 1989. p. 248.
Begon S, Pickering G, Eschalier A, Dubray C. Magnesium increases morphine analgesic effect in different experimental models of pain. Anesthesiology 2002;96:627-32.
Ferasatkish R, Dabbagh A, Alavi M, Mollasadeghi G, Hydarpur E, Moghadam AA, et al.
Effect of magnesium sulfate on extubation time and acute pain in coronary artery bypass surgery. Acta Anaesthesiol Scand 2008;52:1348-52.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]