Evaluation of an Ayurvedic preparation-‘Ekangaveera Rasa' for possible metal toxicity: Behavioral, Biochemical and Histopathological Analysis
Suhani Sumalatha1, Gururaj Tantri2, Jessica Shreshta1, Ashwija Shetty1, Vanishree S Nayak1, Sushma Prabhath1*
1Department of Anatomy, Kasturba Medical College Manipal,
Manipal Academy of Higher Education, Manipal, Karnataka, India – 576104.
2Department of Shalya Tantra, Muniyal College of Ayurveda, Manipal, Karnataka, India- 576104.
*Corresponding Author E-mail: sushma.rk@manipal.edu
ABSTRACT:
Ayurveda, an ancient Indian medicinal system, is highly regarded as complementary and alternative medicine in recent years. However, there are reports to show the toxic effects of specific Ayurvedic preparations, which use metals as an ingredient in them. One such medicine is 'Ekangaveera Rasa' This herbo-minero-metallic preparation is used to treat the pain of neurological origin. Therefore, the present study aimed to evaluate the possible alteration caused by Ekangaveera Rasa on the brain, liver, and kidneys by histological, biochemical, and behavioral analysis. For this evaluation, Wistar rats were divided into four groups: Group 1- normal control; group 2, 3 and 4 were treated with Ekangaveera Rasa at doses of 120, 240 and 600 mg/kg bodyweight for 30 days. Behavioral parameters were assessed on the 15th and 30th day by using the passive avoidance test. At the end of the treatment period, blood was collected and subjected to the basophilic stripling and biochemical analysis. Tissues were processed for histopathological evaluation. The histopathological, biochemical, and behavioral analysis did not show any harmful effect, thereby rendering Ekangaveera Rasa nontoxic and further glorifying the healing benefits of Ayurveda.
KEYWORDS: Ekangaveera Rasa, Ayurveda, Traditional medicine, Toxicity, Organs, Analysis.
INTRODUCTION:
Ayurveda, an Indian system of medicine that is rooted over 5000 years ago, is now considered as complementary and alternative medicine1. Traditional medicine is the knowledge, skills, and practices of holistic healthcare recognized and accepted for its role in the maintenance of health and the treatment of diseases. It is based on indigenous theories, beliefs, and experiences that are passed on from generation to generation 2-,4. Ayurveda is globally recognized and accepted at present under its qualitative strength and its focus on the consistent functioning of life. Despite the constant effort in promoting the therapies of this vibrant ancient medicine, some areas still require further research and scientific approach and extensive evidence.
Expensive new drugs, increasing side effects, the need for remedial treatment for several chronic diseases, are some of the reasons that have created public interest in adopting and accepting complementary and alternative medicines5.
Medicinal plants are believed to possess powerful constituents that are life sustaining6. It has compelled scientists to examine these plants to determine potential healing benefits. Such plants are used in various medicinal preparations in Ayurveda7-10.
Although these traditional medicines contain valuable elements necessarily, they also carry certain metals as an ingredient. From the therapeutic point of view, metals are used for the maintenance of health and curing of diseases in Ayurveda. Preparation and use of metallic bhashma in the Indian System of Medicine have also been documented previously11. However, monitoring the safety of herbal medications become essential, and there is a need for pharmaco-vigilant practices for Ayurvedic medicine12. One such herbo-metallic-preparation is 'Ekangaveera Rasa.' It is used to treat the pain of neurological origin and conditions such as facial palsy, hemiplegia, sciatica, cervical spondylosis, brachial neuralgia, etc. It contains equal parts of pepper, fresh ginger, Indian gooseberry fruit, Bhringaraja as herbal component and mercury, sulfur, manganese, tin, lead, copper, mica, and iron as its metal component 13.
However, the metals are used in the Ayurvedic medicinal preparations, only after following a meticulous process of purification, i.e., Shodhana, thereby extracting a nontoxic compound14,15. But, in recent years, specific metallic preparations used in the Indian medicinal system are alleged to cause adverse effects of the hepatic, renal, and nervous system along with various other side effects12.
The available work in the field of science, which reports the toxic effects of metals in the ayurvedic preparation of Ekangaveera Rasa, are few. Hence we aimed to study and evaluate the possible toxicity and the alteration caused by the Ekangaveera Rasa on biochemical., behavioral analysis and histopathological of brain, liver, and kidneys.
MATERIAL AND METHODS:
Experimental animals:
Healthy adult male Wistar albino rats weighing about 150-200 g were used for the study. The animals were housed under standard environmental conditions of temperature and humidity (25±0.5°C) with 12 hours light/ dark cycle. They were fed with a diet of standard pellets and water ad libitum. The experiments were performed in the Central Animal House Facility, Kasturba Medical College, Manipal, after the approval from the Institutional animal ethics committee (Ref. No: IAEC/KMC/49/2013).
Procurement and preparation of Test Material:
Ekangaveera Rasa was procured from Shree Dhootapapeshwar Ltd. Khetwadi, Mumbai, India. It was then fed to the animals after calculating and measuring the desired dose and dissolving it in distilled water.
Experimental model and Treatment groups:
The animals grouped into four sets with six animals in each group, i.e., Group 1: control (treated only with distilled water), Group 2:120 mg/kg body weight (BW), Group 3: 240mg/kg BW and Group 4: 600 mg/kg BW treated groups. The groups 2, 3, and 4 were treated with the recommended human equivalent dose, double the recommended dose, and five times the human equivalent dose, respectively.
Treatment Plan:
The doses of Ekangaveera Rasa (120, 240, and 600 mg/kg) in the rat were calculated by extrapolating the human equivalent dose and were orally administered between 10 and 11 a.m. daily for 30days, in a volume not exceeding one ml/100 g rat weight. Rats were subjected to neurobehavioural analysis. At the end of the experiment, i.e., on the 30th day, blood samples were withdrawn from the retro-orbital sinus and processed for biochemical evaluation. Animals were then euthanized, and the liver, kidney, and brain (hippocampal region) tissues were removed and preserved in 10% formalin for histopathological evaluation.
Behavioral analysis:
On the 28th day, a passive avoidance test was performed to evaluate memory retention16. However, to enhance the performance in the test, the animals were deprived of food 12 hours before the test. During the acquisition trial, the rats were placed in a well-lit chamber with an additional source of electric light, and a wooden door that separated the light chamber from the dark chamber was opened. Initial latency (IL) to step into the dark chamber was noted. Time spent in the dark compartment and light compartment were noted for 3 minutes. It was repeated for three trials, and in the third trial, electric foot shock (0.2 mA) was delivered to the floor grids for 3 seconds at the end of 3 minutes with the door closed. Five seconds of post-shock treatment, a rat was transferred from the dark chamber to its home cage. The process was repeated for all the animals in each group. After 24 hours, memory retention time was noted in the same way as in the acquisition trial. The foot shock was, however, not delivered.
Biochemical analysis:
On the 30th day, blood samples were collected from the animals by puncturing the retro-orbital sinus. The samples were used for further biochemical analysis.
Basophilic Stippling:
The blood smear was made immediately after collection to study the basophilic stippling by using Leishman's staining method17, and later the microscopic structure of RBCs was studied.
Liver Function Tests:
The blood samples were allowed to rest at room temperature for 1 hour—following which the samples were centrifuged for 5 minutes at 1000 rpm for serum separation. ALT (Alanine aminotransferase), AST (Aspartate aminotransferase) and ALP (Alkaline phosphatase) levels were estimated from the serum using standard liver function kits (Aspen laboratories, India).
Histopathological evaluation:
After euthanizing the animals by cervical dislocation, the brain, liver, and kidney were collected and carefully cut into the required size and immediately fixed in 10% formalin. They were then dehydrated using graded alcohol (50%, 70%, 90%, and 100%), xylene, and embedded in paraffin wax. Serial sections of 5µ thick were taken using a rotatory microtome. The de-paraffinized sections were stained with hematoxylin and eosin. Sections were then observed under the microscope for the possible changes in the cytoarchitecture. The areas of interest were noted and photographed and analyzed with the help of a pathologist.
Statistical Analysis:
Results were expressed as mean± SEM. The data was analyzed using Graphpad Prism software (Microsoft, San Diego, CA, USA). One-way ANOVA, followed by Dunnett's post-hoc test, was used to compare the experimental groups. P-value ≤0.05 was considered statistically significant.
RESULTS:
Behavioral analysis:
Animals treated with low doses of Ekangaveera Rasa (120 and 240mg/kg BW) spent less time in the dark compartment. However, the rats treated with a high dose (600 mg/kg BW) of Ekangaveera Rasa spent more time in the dark chamber compared to control; nonetheless, it was statistically insignificant (Figure 1).
Figure 1: Effect of Ekangaveera Rasa on passive avoidance test. 120 mg/kg, 240 mg/kg and 600 mg/kg are the groups treated with the respective doses of the drug.
Figure 2: Showing the peripheral blood smear of rats treated with different doses of Ekangaveera Rasa for 30 days
Table 1: Liver function test: Groups treated with ‘Ekangaveera Rasa’ versus control
|
Enzymes (IU/L) |
NC |
120 mg/kg |
240 mg/kg |
600 mg/kg |
|
ALT |
39.857±0.26 |
36.223±0.03 |
40.054±0.21 |
42.321±0.05 |
|
AST |
70.022±3.32 |
72.027±4.37 |
70.009±2.77 |
76.027±5.64 |
|
ALP |
81.465±0.55 |
80.021±0.51 |
81.402±0.04 |
80.868±0.22 |
NC- normal control; 120, 240, and 600 mg/kg body weight is the group treated with the respective doses of Ekangaveera Rasa. AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase. All values are expressed as mean ± SEM.
The tissues of the liver, kidney, and brain (hippocampal region) showed normal cytoarchitecture in both control and Ekangaveera Rasa (all the calculated doses) treated groups. No degenerative changes were observed in the treated groups. All the calculated doses, i.e. 120, 240, and 600 mg/kg body weight of Ekangaveera Rasa were efficient in maintaining the normal structural organisation of the vital organs such as liver, kidney and brain as is observed in the present histological findings (Figure 3).
Figure 3. Showing H&E stained images (10X magnification) of the tissue collected from the liver, kidney, and brain (CA1region of the hippocampus) of the rats fed with different doses of Ekangaveera Rasa.
CV- a central vein of the liver, arrow represent – normal sinusoid; * represent – glomeruli of the kidney.
DISCUSSION:
Ayurvedic medicines are herb-based traditional medicines. They carry a rich historic background and is one of the highest living traditions 18,19. In most of the Ayurvedic formulations, processed heavy metals, metals, and minerals are the key ingredients. Studies suggest that approximately 35–40% of Ayurvedic formulations contain at least one metal 18. Therefore it becomes imperative that the medicinal plant or plant parts used for different diseases must be checked for heavy metal contaminations in order to make it safe for human consumption12.
The metals in Ayurvedic formulations are frequently subjected to purification, i.e., Shodhana (purification and detoxification) and Marana (incineration and calcination)14,15. Practitioners develop these methods to expel the toxic effects of the raw material by chemical changes and thus modify the properties of materials to improve the therapeutic potential. The Ayurvedic formulations, therefore, lose their original elemental form and become nontoxic during the process of purification 18,20,21.
Kumar et al., in their study on Aarogyavardhini Vati, an Ayurvedic formulation at a dose of 50, 250, and 500 mg/kg of body weight for 28 rats found no adverse effect on cerebellum, liver, and kidneys. They reported that the drug was safe up to 500 mg/kg body weight 22. A similar experimental toxicity study on Makaradhwaja did not show the signs of toxicity up to a dosage of 480 mg/kg in mice. However, a 28 days' oral toxicity study on Siddha Makaradhwaja in rats showed neurodegenerative changes in the brain at a higher dose of 100 mg/kg weight 23. Aqueous extract of Coccinea Indica(roots) was studied for its toxic effects in Swiss albino mice. Herein no significant observations of behavioral changes, toxicity, mortality were observed. However, they suggested that conducting toxicity studies in natural plant products and drugs of the indigenous medicinal system is a must24.
In the present study, the values of the liver function test were found to be in the normal range. Significant changes were not observed in serum ALT, AST, and ALP levels in all the three doses of Ekangaveera Rasa. This suggests that ‘Ekangaveera Rasa’ is devoid of any drastic toxic effect in rats even at a dose almost five times higher than the standard limit.
The behavioral analysis showed no alteration at lower doses in the cognitive functioning in the rats. The histopathological findings in the CA1 region also showed no changes. The CA1 region of the hippocampus is responsible for short term memory. The rats treated with the higher dose of (600 mg/kg) Ekangaveera Rasa, showed impaired cognitive function in the behavioral analysis. It was, however, not reflected in the CA1 region of the treated rats as observed histologically.
BSE occurs due to the spontaneous aggregation of ribosomal RNA due to the poisoning of 5' nucleotidase. The finding was adverse in the present study. It indicates that the drug has no poisoning effect within the dose five times as higher than the normal equivalent human dose. The above results were also reaffirmed by the histopathological observations in the liver, kidney, and the brain- hippocampal tissues of the treated groups. The cytoarchitecture of all the three organs was found to be healthy at all the considered doses of Ekangaveera Rasa. The non-occurrence of the toxic effect could be a possible indication that the process of Ayurvedic detoxification, i.e., Shodhana, might have aided in significant changes in the characteristics of the metals. Further, it would have resulted in the elimination of toxicity, yet maintaining its pharmacological characteristics.
CONCLUSION:
"Ekangaveera Rasa" has been used clinically for centuries in the traditional Indian Medicinal System, i.e., Ayurveda. The findings of the present study endorse the nontoxic effects of this particular drug and provide valuable evidence supporting the same. The study further supports the idea that "Ekangaveera Rasa," when administered at recommended dose and duration to the human population (as is routinely practiced in Ayurveda), is harmless. The present study thereby glorifies the healing benefits that our traditional medicinal system has in store for the well-being of humanity without any side effects.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 10.06.2020 Modified on 07.02.2021
Accepted on 01.05.2021 © RJPT All right reserved
Research J. Pharm.and Tech 2021; 14(12):6491-6495.
DOI: 10.52711/0974-360X.2021.01122