ACUTE AND SUB ACUTE TOXICITY STUDY ON SIDDHA DRUG RASA CHENDOORAM
HTML Full TextACUTE AND SUB ACUTE TOXICITY STUDY ON SIDDHA DRUG RASA CHENDOORAM
M. Jayabharathi *1, M. Mohamed Mustafa 1 andP. Sathiyarajeswaran 2
Department of Sirappu Maruthuvam, Govt. Siddha Medical College 1, Chennai, Tamil Nadu, India.
Research officer 2, SCRI, Chennai, Tamil Nadu, India.
ABSTRACT: Herbal and herbo mineral preparations are being traditionally used in Indian system of Medicine especially in Siddha Medicine. Herbo mineral preparations have longer Shelf life. Rasa Chendooram (RC) is prepared as per classical text book of Prana Rakshamirtha Sindhu, for Vadha diseases. Before conducting clinical trial, Pre clinical study should be undergone as per WHO guidelines .The clinical trial has been approved by IEC (IEC NO-GSMC-CH/1/2013/011).The present preclinical study aims to carry out safety and toxicity of RC (IAEC NO.XXXXIX/07/CLBMCP/2013dated 29.06.2013) Adult both sex of Swiss albino rats weighing 220-250 gm were used. Acute & Sub acute toxicity were carried out as per OECD guidelines 423 and 407. Hematological Parameters, biochemical parameters histopathological study were performed for all animals. The study concludes that on oral administration of 100mg/kg of bodyweight of RC to Swiss albino rats, there was no change in behaviour movements and no characteristic clinical sign of toxicity or mortality observed.
Keywords:
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Rasa chendooram, Toxicity, Vadha disease, and Oral
INTRODUCTION: Cervical spondylosis is the degenerative disease of inter vertebral discs and secondary osteo arthrosis is often asymptomatic but may be associated with neurological dysfunction, C5,C6,C7, nerve roots are most commonly affected1. In males, the prevalence was 13% in the third decade, increasing to nearly 100 % by age 70 years. In females the prevalence ranged from 5% in the fourth decade to 96% in women older than 70 years. 60% of population older than 45 years and 8% older than 65years of age account for the case of cervical spondylosis reported 2. It is more commonly reported in 25 years of age now days.
The reasons underlying are prolonged computer usage, Two-wheeler driving, poor sitting postures, and posterior neck injury 3. Even though there are treatments available in the contemporary medical system there is no complete relief. In Siddha system for cervical spondylosis, internal medicine, external therapies and exercises are advocated by Siddhars. As per Yugi Chinthamani, Cervical spondylosis correlated with features of Ceganavadham, which is one among 80 types of Vadha disease 4.
Rasa chendooram (RC), a herbo mineral drug indicated for vadha diseases is quoted in Prana Rakshamirtha Sindhu5. RC ingredients are Rasam, Gandhagam and Karunthulasi. Rasam (Mercury) - Purifies blood, strengthen nerve plexus and prevent senility and increases life span6. In animals, on administration of small doses of mercury there is diminished the amount of oxidation of tissue and an increase the number of Red blood cells ,increase in body weight and general nutrition. Gandhagam (Sulphur) is useful in skin diseases, splenomegaly, chronic fever and chronic Rheumatism7. Karunthulasi (Ocimum sanctum) has potent antioxidant anti inflammatory 8 and antistress activities 9.
The issues related to lack of scientific evidence about the efficacy and safety of herbo mineral remedies remains unresolved10. A preclinical toxicity study is mandatory in determining a safety dose for human trial 11. Prior to the initiation of human trial the safety of the drug is to be proved 12. The present pre clinical study aimed at evaluating the acute and sub acute toxicity of RC. This study provides vital information about efficacy and safety of RC.
MATERIAL AND METHODS:
Source of Drugs
Rasam (Mercury) and Gandhagam (Sulphur) were procured from Sanjeevi pharmaceuticals Pvt.Ltd. Chennai, traditional raw drugs dealer. Karunthulasi (Ocimum sanctum) leaves were collected from ABS Garden, Karipatti. Salem District. Materials were authenticated by Research Officer, (Pharmacognosy Department), SCRI, Chennai.
Purification of Rasam
Propotionate ratio of drug materials were used, Rasam –100gm, Brick powder –200gm, turmeric powder –200gm, Indian Acalypha juice (Acalypha indica) - 1.3 lt. Mercury was triturated with brick powder and turmeric power for one hour respectively and washed with water. Whereas the mercury boiled with the juice of Indian acalypha to get purified Rasam, detoxification procedure was carried out 6.
Purification of Gandhagam
The kalkam of Lawsonia inermis (100 gm) was mixed with curd of cow (1 Kg) and placed inside mud pot. The mouth of the pot was covered with a cloth. Sulphur (200gm) was placed over this cloth. The pot was covered with another pot and buried into the ground. The outer pot was subjected to puda with five dung cake. The suphur which melts and settles down at bottom was collected, and this procedure was repeated for 7 times. 6.
Preparation of Rasa chendooram
Purified Rasam and Gandhagam (each 35gm) were grind with karunthulasi juice in kalvam (stone mortar) then dried this material keep into bottle (kasi Kuppi). After this procedure, Rasa chendooram prepared by vaaluga enthiram, treat under the flame of deepakkini (mild flame-24 hour), kamalakkini (moderate flame24 hour) and kadakkini (high flame-24 hour) respectively.
Adjuvant: Amukkara chooranam (1:10 ratio)
Chemicals and Reagents & Animals
All chemicals and reagents were obtained from sigma chemicals Ltd, USA. All other reagents used in the study were of analytical grade were obtained from Qualigen fine chemicals Pvt. Ltd.
Swiss albino rats of either sex weighing 220-250grams were obtained from Animal house department, King Institute, Guindy, Chennai. Rats were housed in individually in poly propylene cages and fed with standard rodent pellet obtained and water ad libitum. The animals were subjected to a 12:12 hrs light: dark cycle under standard laboratory conditions at a temperature of 24-28ºC with a relative humidity of 60%-70%. The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC/XXXIX/07/CLBMCP/2013 dated 29.6.2013) of C.L Baid Metha Collelge of Pharmacy, Thurai Pakkam, Chennai, Tamil nadu.
Acute Oral Toxicity
Three female nulliparous and non-pregnant rats were used for acute oral toxicity study according to Organization for Economic Cooperation Development (OECD) guideline 423. 13, RC was administered orally 2000 mg/kg body weight of different groups of rats and absorbed for toxicological study. The animals were observed individually after dosing the first 30 mins, periodically during the first 24h, with special attention given during the first 4h, and daily thereafter, for 14 days. Observations included changes in skin, fur, eyes, mucous membrane (nasal), autonomic (salivation, lacrimation, perspiration, piloerection, urinary incontinence, and defecation), and central nervous system (drowsiness, gait, tremors, and convulsions) changes respectively (Table1). Mortality, if any, was determined over a period of 2 weeks.
TABLE:1 DOSE FINDING EXPERIMENT AND BEHAVIORAL SIGNS OF TOXICITY
Group | Day |
Body weight | Normal |
Assessments of posture | Normal |
Sign of Convulsion,limb paralysis | Absence of sign(-) |
Body tone | Normal |
Lacrimation | Absence |
Salivation | Absence |
Change in skin colour | No significant colour change |
Piloerection | Not observed |
Raering/Urination/Defecation | Normal |
Sensitivity response | Normal |
ocomotion | Normal |
Muscle gripness | Normal |
Food
(g/day/rat) |
Body weight(g) | ||
Control |
MEAN | 21.33 | 231.2 |
SD | 1.633 | 30.52 | |
SE | 0.6667 | 12.46 | |
Low Dose | MEAN | 23.83 | 229.3 |
SD | 2.229 | 4.967 | |
SE | 0.9098 | 2.028 | |
High Dose | MEAN | 21.5 | 224.2 |
SD | 1.871 | 2.714 | |
SE | 0.7638 | 10108 |
Sub Acute Oral Toxicity
In this study, the animals were divided into three groups of each 6 animals (3 males and 3 females) and treated with low (50 mg/kg of body weight) and high dose (100 mg/kg of body weight) levels to be administered for 28 days. Group 1 received 0.025% CMC in water and served as control, Groups 2 and 3 received 50mg/kg and 100 mg/kg RC (suspended in 0.025% CMC solution) body weight orally, respectively. The drug was administered daily for 28 days at the same time and observed at least twice for morbidity and mortality. Body weights and food consumption of the animals were evaluated weekly (Table 2). Whereas this sub-chronic oral toxicity study was carried out according to OECD guideline 40714, 15.
TABLE 2: FOOD INTAKE &BODY WEIGHT OF RATS TREATMENT WITH RC FOR28 DAYS
Values are mean of 6 animals±S.E.M. (Dunnets test) ns p>0.05
Hematological and Blood Biochemical Analysis
On the 29th day, of the sub-chronic oral toxicity, over a period of fasting, the rats were anesthetized with ether and blood sample for hematological and biochemical analysis were collected by cardiac puncture method into tubes with and without Ethylene diamine tetra acetate (EDTA), respectively. Hematological parameter observed and recorded (Table 3 and Table 4) Biochemical parameter such as serum cholesterol, LDL, HDL, Total protein, SGOT and SGPT also recorded (Table 5 and Table 6).
TABLE 3: HEMATOLOGICAL PARAMETERS AFTER 28DAYS TREATMENT WITH RC IN RAT
Total Red cells count
( ×106 µl) |
Total Wight cells count
( ×103 µl) |
Platelet count
( ×103 µl) |
Packed Cell volume (%) | MCV
(fl) |
MCH
(pg) |
MCHC
(g/dl) |
||
Control |
MEAN | 7.917 | 8.517 | 562.3 | 45.5 | 55.83 | 22.17 | 34 |
SD | 0.3545 | 0.2317 | 3.933 | 1.871 | 1.472 | 2.137 | 2.28 | |
SE | 0.1447 | 0.09458 | 1.606 | 0.7638 | 0.6009 | 0.8724 | 0.9309 | |
Low Dose | MEAN | 8.35 | 9.45 | 570.2 | 46.17 | 56.17 | 22.67 | 44.83 |
SD | 0.2665 | 0.2881 | 2.639 | 1.329 | 1.722 | 2.338 | 3.545 | |
SE | 0.1088 | 0.1176 | 1.078 | 0.5426 | 0.7032 | 0.9545 | 1.447 | |
High Dose | MEAN | 8.467 | 8.3 | 566.2 | 43.33 | 56.17 | 21.5 | 44.5 |
SD | 0.2503 | 0.1789 | 3.488 | 1.966 | 1.169 | 1.517 | 1.517 | |
SE | 0.1022 | 0.07303 | 1.424 | 0.8028 | 0.4773 | 0.6191 | 0.6191 |
Values are mean of 6 animals±S.E.M. (Dunnets test) ns p>0.05
TABLE 4: HEMATOLOGICA PARAMETERS AFTER 28DAYS TREATMENT WITH RC IN RATS
HB
(g/dl) |
Neutrophils
(%) |
Lymphocytes
(%) |
Eosinophils
(%) |
Moncytes
(%) |
Basophils
(%) |
Blood sugar®(mg/dl) | ||
Control |
MEAN | 15.27 | 68.17 | 35.5 | 1.35 | 0.6833 | 0 | 75 |
SD | 0.3882 | 3.488 | 1.517 | 0.1871 | 0.1169 | 0 | 2.28 | |
SE | 0.1585 | 1.424 | 0.6191 | 0.07638 | 0.04773 | 0 | 0.9309 | |
Low Dose | MEAN | 16.33 | 64.5 | 37.17 | 1.233 | 0.7167 | 0.1667 | 76 |
SD | 1.633 | 2.51 | 2.787 | 0.1506 | 0.1329 | 0.4082 | 1.414 | |
SE | 0.6667 | 1.025 | 1.138 | 0.06146 | 0.05426 | 0.1667 | 0.5774 | |
High Dose | MEAN | 15.33 | 64.5 | 41.67 | 1.167 | 0.5333 | 0 | 76.17 |
SD | 1.033 | 1.643 | 2.944 | 0.1033 | 0.1366 | 0 | 1.835 | |
SE | 0.4216 | 0.6708 | 1.202 | 0.04216 | 0.05578 | 0 | 0.7491 |
Values are mean of 6 animals±S.E.M.(Dunnets test) ns p>0.05
TABLE 5: BIOCHEMICAL PARAMETERS AFTER 28DAYS TREATMENT WITH RC IN RATS
Serum Total
Cholesterol (mg/dl) |
Serum Triglycerides level (mg/dl) | Serum HDL Cholesterol
(mg/dl) |
Serum LDL Cholesterol
(mg/dl) |
Serum VLDL Cholesterol
(mg/dl) |
Serum Total Protein
(g/dl) |
||
Control |
MEAN | 101.2 | 45.5 | 27.83 | 44.33 | 35.67 | 5.367 |
SD | 2.317 | 1.049 | 0.7528 | 2.066 | 2.066 | 0.3724 | |
SE | 0.9458 | 0.4282 | 0.3073 | 0.8433 | 0.8433 | 0.152 | |
Low Dose | MEAN | 103.7 | 48 | 30.73 | 46.5 | 36.17 | 4.533 |
SD | 1.211 | 0.8944 | 4.355 | 2.074 | 1.835 | 0.1862/0.0760 | |
SE | 0.4944 | 0.3651 | 1.778 | 0.8466 | 0.7491 | 1 | |
High Dose | MEAN | 103.8 | 44.33 | 33.83 | 41.17 | 33.5 | 4.367/0.0816 |
SD | 2.229 | 1.366 | 1.722 | 0.9832 | 1.378 | 5/0.0333 | |
SE | 0.9098 | 0.5578 | 0.7032 | 0.4014 | 0.5627 | 3 |
Values are mean of 6 animals±S.E.M. (Dunnets test) ns p>0.05
TABLE 6: BIOCHEMICAL PARAMETERS AFTER 28DAYS TREATMENT WITH RC IN RAT
Serum
Albumin (g/dl) |
Alkaline phosphatase
(U) |
SGOT
(AST) (IU/L) |
SGPT
(ALT) (IU/L) |
Serum creatinine
(mg/dl) |
BUN
(mg/dl) |
||
Control |
MEAN | 2.65 | 255.7 | 239.5 | 62.5 | 0.8167 | 14 |
SD | 0.1049
0.0428 |
4.033 | 2.429 | 1.975 | 0.09832 | 1.414 | |
SE | 2 | 1.647 | 0.9916 | 0.8062 | 0.04014 | 0.5774 | |
Low Dose | MEAN | 2.833 | 256.8 | 215.7 | 66.5 | 0.8 | 14.25 |
SD | 0.1862
0.0760 |
1.602 | 1.211 | 3.271 | 0.08944 | 1.084 | |
SE | 1 | 0.654 | 0.4944 | 1.335 | 0.03651 | 0.4425 | |
High Dose | MEAN | 2.75 | 256.2 | 215.8 | 76 | 0.5667 | 14.67 |
SD | 0.251 | 1.835 | 1.329 | 1.549 | 0.1211 | 2.582 | |
SE | 0.1025 | 0.7491 | 0.5426 | 0.6325 | 0.04944 | 1.054 |
Histopathological Study
Animals in the study were also subjected to a full, detailed gross necropsy. The positions, shapes, sizes and colors of internal organs (heart, kidney, brain and liver) were also recorded. The liver heart, kidney, brain samples from each groups were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Sections of thickness of about 5 μm were cut and stained with hematoxylin and eosin for histopathological investigation. 16
Statistical Analysis
All of the data were expressed as mean ± SEM. Statistical significance between more than two groups were using one way ANOVA followed by Bonferroni multiple comparision test. Calculations were done using GraphPad prism softwere. The significance level was set at p value ≤ 0.05 for all tests.
DISCUSSION:
- All the animals both control and dose treated groups upto 100mg/kg survived throughout the period of 28 days.
- No signs of major or significant intoxication were observed in animals from lower to higher dose groups during the dosing period of 28 days.
- No sign of Behavioral changes, Heamatological and Biochemical abnormalities were observed.
- Food consumption and Body weight gain were found to be comparable throughout the dosing period of 28 days.
- Opthalmoscopic examination, conducted prior to and at the end of dosing periods on animals from control and all the treated dose groups did not reveal any abnormality.
- Gross pathological examination did not reveal any abnormality.
- Histopathological examination, Liver shows Lumen of hepatic veins appears normal. No signs of necrosis. Kidney shows normal arrangement of nephrotic bundle in all the three groups Heart shows no signs of lesion or infracts was observed in all the three groups Brain shows normal histology with regular neuronal alignment further there was no considerable observation of signs of edema or degeneration.
Histopathological analysis of Sub-Acute toxicity study:
CONCLUSIONS: Based on above findings, no toxic effects were observed upto 100 mg/kg of body weight of Rasa Chendooram treated via oral
route over a period of 28 days. So this study concluded that the Rasa Chendooram is suitable for therapeutic use in human with the dosage recommendations of upto 100mg/kg of body weight p.o.
ACKNOWLEDGEMENTS: The Grateful Author HOD. Dept. of Sirappu Maruthuvam, Govt. Siddha Medical College, Chennai, Tamil Nadu
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How to cite this article:
Jayabharathi M, Md. Mustafa MandSathiyarajeswaranP: Acute and Sub Acute Toxicity Study on Siddha Drug Rasa Chendooram. Int J Pharm Sci Res2014; 5(12): 5463-68.doi: 10.13040/IJPSR.0975-8232.5 (12).5463-68.
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IJPSR
M. Jayabharathi *, M. Mohamed Mustafa and P. Sathiyarajeswaran
Post.Graduate Scholar Sirappu Maruthuvam Dept. GSMC, Chennai. Tamil Nadu.India.
bharathivelan84@gmail.com
07 May, 2014
26 July, 2014
18 August, 2014
http://dx.doi.org/10.13040/IJPSR.0975-8232.5(12).5463-68
01 December 2014