INTRODUCTION:

Kandhaga Rasayanam (KR) is a sastric Siddha medicine. The medicine is in practice for more than three decades. As of now acute toxicity study data are available for this formulation. It is a herbo mineral preparation chosen from a classical Siddha text Siddha Vaidhya Thirattu. KR is indicated for skin diseases, venereal diseases, Urinary tract infections etc¹. There are 16 ingredients in this formulation Sulphur, the sole mineral ingredient is a potent antimicrobial agent. There are 16 other herbal raw drugs used in the preparation of KR. A review on toxicity profile and chemical constituents of each ingredient was carried out and by Meena et al ². Sudhanshu Kumar Meher et al have done an extensive review on Withania somnifera, one of the major herbal ingredient of the drug Kandhaga Rasayanam³. The objective of this study is to evaluate the long term toxicity of KR in experimental rats.

MATERIALS AND METHODS:

Trial drug preparation:

All the herbal ingredients were identified by the Assistant Professor, Medicinal botany department of National Institute of Siddha.

Table 1:Ingredients of Kandhaga Rasayanam:

INGREDIENTS	QUANTITY
Purified sulphur	350 grams
*Withania somnifera*	170 grams
*Smilax china*	70 grams
*Terminaliachebula*	35 gram
*Phyllanthus emblica*	35grams
*Terminalia bellerica*	35 grams
*Root of Piper longum*	35 grams
*Piper nigrum*	35 grams
*Embelia ribes*	35 grams
*Cinnamomum zeylanicum*	35 grams
*Santalum album*	35 grams
*Cicer arietinum*	35 grams
*Semecarpus anacardium*	35 grams
*Root of Plumbago zeylanica*	35 grams
Sugar	Sufficient quantity
Honey	Sufficient quantity
Ghee	Sufficient quantity

Sulphur was authenticated by research officer (chemistry) of SCRI, Arumbakkam. All the raw drugs were purified as per the methods mentioned in the Siddha literature. The ingredients of Kandhaga Rasayanam is tabulated in table 1.

Method of preparation:

All the ingredients were powdered separately and then mixed together. Sufficient quantity of sugar, honey and ghee were then added.

Drugs and chemicals:

The chemicals for the experiments were purchased from Sigma chemical Co, St.Louis, Mo, USA.

Experimental animals:

Healthy adult male and female wistar albino rats of 8-10 weeks old were obtained from National Institute of Nutrition, Hyderabad 1.2.2013. Animals were acclimatized for 7 days in the Animal house of National Institute of Siddha prior to the experimentation. The animals were housed in polypropylene cages (55x 32.7x 19 cms). Paddy husk was the bedding material used. The animals were maintained in a controlled environment with the temperature of 23±2°c and 12 hours dark light cycle. The animals were fed with standard laboratory animal feed (Nutrilab rodents) supplied by Provimi India limited and RO water ad libitum. They were handled humanely as per CPCSEA guidelines. Randomization procedure was followed for grouping before starting the experiment. The animals were marked with picric acid on the fur and housed in cages. The body weight was recorded before the start of the experiment.

IAEC approval:

The animals and the experimental protocol were approved by the Institution Animal Ethics Committee of National Institute of Siddha, Chennai- 47.IAEC NO: NIS/IAEC/I/2011/6(A). Date of approval: 12.12.2012.

Drug preparation:

The trial drug was dissolved in 10% Carboxy Methyl Cellulose (CMC). This mixture of vehicle and drug was prepared freshly each day before administration.

Long toxicity study:

As per WHO guidelines the minimum period for long term toxicity study for a drug which is administered clinically for 45 days is 3 months. Hence 3 month duration was selected for the study.

Meena et al have reported the drug KR is nontoxic at the dose of 0.720 grams in acute toxicity study ⁴. Long term toxicity study was examined as per WHO guidelines WHO 2000. Male and female rats were randomized and grouped before the start of the experiment. 5 groups were maintained. Normal control group, vehicle control group and three experimental groups. The trial drug was administered in three concentrations daily ie, the therapeutic dose (0.072 gram), five times the therapeutic dose (0.36 gram), ten times the therapeutic dose (0.720grams) for 90 days.

Dose calculation:

Clinical dose of Kandhaga Rasayanam: 2gm b.d which means 4gm/ day. The clinical dose was converted to rat dose according to the body surface area. For a rat weighing 200gm, the dose of Kandhaga Rasayanam was calculated as 4gm x 0.018= 0.072gram (therapeutic dose). The dose for 10 times the therapeutic dose was: 0.720grams.

The animals in normal control group received normal saline 5ml/kg b.wt. The vehicle control group received 5ml/kg b.wt of 10% C.M.C. Toxic signs, mortality and body weight changes were monitored daily. At the end of the study all rats were fasted overnight and blood samples were collected from the retro orbital plexus under excessive ether anesthesia of all rats.

Haematological analysis:

The laboratory investigations of the animal samples were carried out in Siddha Central Research Institute, Ministry of AYUSH, Government of India, Arumbakkam, Chennai-106.

The blood was transferred to bottles containing EDTA sample for hematological analysis. To estimate hematological parameters, 0.08 ml blood mixed with 0.02 ml of Ethylene Diamine Tetraacetic Acid-EDTA (33.33 mg/ml) was fed to the auto analyzer (Sismes KX-21, Trans Asia).

The parameters measured were as follows: Total WBC, neutrophils count and percentage, lymphocyte count and percentage, monocyte count and percentage, hemoglobin content, packed cell volume (PCV), total RBC, platelet count, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin (MCH), and Mean Corpuscular Hemoglobin Concentration (MCHC).

Biochemical analysis:

The blood was transferred to plain sample bottles for serum generation for biochemical analysis. Serum was obtained after allowing blood to coagulate for 30 minutes and centrifugation. For estimation of biochemical parameters, requisite quantity of serum was fed to the auto analyzer (Fully automated Biochemical Random Access Analyzer, BS-200; Lilac Medicare Pvt. Ltd., Mumbai) which was automatically drawn into the instrument. Biochemical parameters measured were blood sugar, serum cholesterol, serum triglyceride, blood urea, serum creatinine, Serum Glutamic Pyruvic Transaminase, Serum Glutamic Oxaloacetic Transaminase, serum total protein, serum albumin, total bilirubin, direct bilirubin, uric acid and sodium, potassium and chlorine.

Necropsy:

All the rats were sacrificed by euthanasia after blood collection. The internal organs were grossly examined and weighed. The cut sections of the organs were preserved in 10% neutral buffered formalin solution for histopathological examination.

Histopathology:

The internal organs, the cut sections were given for histopathological examination to the Department of Veterinary Pathology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai.

Statistical analysis:

The data was analyzed by using SPSS software (version 12.0, SPSS, Chicago, IL, USA). The results were presented as Mean ± S.E.M. and statistical significance between the groups was analysed by means of an Analysis Of Variance (ANOVA)

RESULTS:

The long term toxicity was conducted for 90 days. There was no mortality throughout the treatment period in both the sexes in all the five groups. No toxic signs were observed. There was no abnormality noted in any of the observational parameters also. Significant increase in body weight was observed in drug treated groups when compared to normal control group at the end of third week. The difference in feed and water intake of the treated groups were found to be insignificant when compared to normal control group. Haematological parameters showed no significant difference when compared to normal control group. A statistical significance was observed in haemoglobin in treated group, but the values fall within the normal physiological range. The biochemical parameters showed statistical significance in creatinine, bilirubin, cholesterol, chlorine and SGPT, but the observed values were within the normal range of rats.

Effect of Kandhaga asayanam on general behaviour:

All the experimental rats showed normal general behaviour, respiratory pattern, cardio vascular signs, motor activities, reflexes and normal changes in skin and fur.

Effect of Kandhaga Rasayanam on body weight in 90 days toxicity study:

The effect of Kandhaga Rasayanam on body weight in long term toxicity is tabulated in table 2.

Table 2: Effect of Kandhaga Rasayanam on body weight of rats in 90 days toxicity study

Group

Body weight in grams

1^st

week

2^nd

week

3^rd

week

4^th

week

5^th

week

6^th

week

7^th

week

8^th

week

9^th

week

10^th

week

11^th

week

12^th

week

13^th

week

Normal control

170.00

9.97

187.6

10.97

203.7

12.02

220.9

12.02

228.2

13.65

233.4

11.97

249.1

11.96

252.7

14.45

252.9

15.32

259.1

16.99

263.2

18.07

266.5

17.260

271.8

18.424

Vehicle control

169.40

6.094

198.0

5.473

226.8

6.516*

222.1

6.516

226.4

4.434

234.6

5.778

243.1

5.418

251.4

5.519

258.4

7.312

263.5

8.876

269.8

10.897

275.1

12.577

278.7

13.216

one

theraphetic

171.30

6.112

192.6

8.987

215.1

8.512

223.5

8.512

227.0

12.091

235.8

11.980

245.5

11.822

254.2

11.086

261.8

13.892

268.2

16.440

272.8

18.799*

275.0

20.637

276.1

21.099

Five

theraphetic

169.00

5.714

199.0

6.839

223.6

6.261*

231.8

6.261

232.6

10.284

240.9

11.421

252.9

10.512

260.5

10.573

267.2

11.825

270.9

13.215

275.6

14.862

278.7

15.248

280.1

15.807

Ten

therapeutic

168.90

6.21

196.0

13.06

232.1

13.26*

230.8

13.471

232.9

16.94

242.5

16.73

243.1

15.88

252.0

5.52

258.1

7.21

263.5

7.58

268.5

8.69

274.9

8.98

278.7

9.86

Values are expressed as Mean ± Standard Error of Mean. Significance with Turkey’s test followed by one way ANOVA is evaluated as * p< 0.5, **p< 0.01, ***p<0.001 versus normal control group.

Table: 3: Effect of KR on organ weight of rats

Group

Brain

Wt in grams

Heart

Wt in grams

Lung

Wt in grams

Liver

Wt in grams

Stomach

Wt in grams

Spleen

Wt in grams

Rt. Kidney

Wt in grams

Lt. Kidney

Wt in grams

Testis

Wt in grams

Ovary

Wt in grams

Normal control

1.62

0.041

0.88

0.056

1.63

0.406

8.67

0.580

1.40

0.035

0.84

0.076

1.11

0.096

1.08

0.093

2.77

0.102

0.11

0.002

Vehicle control

1.58

0.055

0.80

0.038

1.44

0.128

8.28

0.594

1.36

0.037

0.76

0.219

1.05

0.090

1.02

0.084

3.09

0. 102

0.15

0.017

1 x therapeutic dose

1.73

0.056

0.94

0.050

1.45

0.100

7.86

0.639

1.40

0.034

0.85

0.043

1.07

0.091

1.09

0.093

2.98

0.0.075

0.14

0.014

5 x therapeutic dose

1.69

0.047

0.834

0.060

1.39

0.822

8.02

0.343

1.37

0.043

0.77

0.059

0.96

0. 059

0.96

0.049

2.89

0.051

0.13

0.012

10 x therapeutic dose

1.659

0.031

0.823

0.051

1.40

0.526

8.01

0.294

1.41

0.062

0.81

0.058

1.00

0.078

0.99

0.100

3.03

0.088

0.09

0.010

Effect of Kandhaga Rasayanam on biochemical parameters:

The effect of KR on the biochemical parameters are shown in table 4.

Table 4: Effect of Kandhaga Rasayanam on biochemical parameters

Biochemical parameter	Normal control	Vehicle control	1 x therapeutic dose	5 x therapeutic dose	10 therapeutic
Blood glucose (mg/dl)	99.5±4.895	93.50± 2.428	93.30±2.574	91.8±3.617	96.00±2.902
Blood urea (mg/dl)	31.5±1.544	27.9±1.402	29.20±1.209	32.9±1.130	35.7±1.770
Serum Creatinine (mg/dl)	0.61±0.023	0.55±0.030	0.58±0.049	0.61±0.028	0.7±0.026 *
Total cholesterol(mg/dl)	93.50±2.845	88.50±3.854	105.70±8.018	116.00±8.408**	110.60±2.911***
Triglyceride(mg/dl)	91.5±12.635	93.00±4.909	116.8±9.233	116.7±9.664	110.9±10.864
Total protein(g/dl)	7.54±0.268	8.12±0.270	8.72±0.429	8.350±0.1267*	8.370±0.2261
Albumin (g/dl)	3.31±0.166	3.6±0.147	3.44±0.173	3.61±0.146	3.460±0.079
Serum bilirubin (mg/dl)	0.39±0.031	0.54±0.057	0.48±0.049	0.54±0.0476*	0.500±0.0258*
SGOT(U/L)	134±5.526	127±3.587	134.50±1.344	137.60±1.416	135.60±1.647
SGPT(U/L)	51.40±1.500	48.2±2.311	52.2±3.784	50.4±1.984	57.2±1.794*
Sodium (mEq/L)	132.4±0.933	132.1±0.567	132.6±0.600	132.2±0.573	134.4±0.581
Potassium (mEq/L)	6.54±0.158	6.74±0.083	6.88±0.085	6.73±0.092	6.97±0.167
Chlorine (mEq/L)	106.3±0.651	104.60±0.510	104.70±0.367	104.30±0.367 *	105.8±0.416

Values are expressed as Mean ± Standard Error of Mean. Significance with Turkey’s test followed by one way ANOVA is evaluated as * p< 0.5, **p< 0.01, ***p<0.001 versus normal control. A statistical significance was noted in 10 therapeutic dose group of serum creatinine (* p< 0.5). In 5 and 10 therapeutic dose group, serum total cholesterol values (**p< 0.01) and bilirubin values (* p< 0.5) showed statistical significance. The total protein and chlorine in 5 therapeutic dose group showed statistical significance (* p< 0.5). SGPT of 10 therapeutic dose showed statistical significance (* p< 0.5) when compared to normal control group.

Effect of Kandhaga Rasayanam on haematological parameters:

The results of haematological parameters are tabulated in table 5.

Table 5: Effect of Kandhaga Rasayanam on haematological parameters

Haematological parameter	Normal control	Vehicle control	1x therapeutic	5 x therapeutic	10 x therapeutic
WBC (10³/ µL)	7.03 ± 0.756	6.60± 0.171	6.39 ± 0.247	6.25 ± 0.524	8.39 ± 0.471
LYM (10³/ µL)	4 ± 0.345	3.72 ± 0.115	3.8 ± 0.448	3.06 ± 0.258	3.07 ± 0.250
MON (10³/ µL)	0.24 ± 0.043	0.24 ± 0.027	0.26 ± 0.022	0.24 ± 0.022	0.30 ± 0.033
GRAN(10³/ µL)	2.81 ± 0.380	2.57 ± 0.291	2.87 ± 0.190	2.82 ± 0.282	3.36 ± 0.238
Lymphocytes %	57.37 ±1.430	55.08 ± 1.522	55.52 ± 1.572	56.27 ± 1.490	57.50 ± 1.090
Monocytes%	3.66 ± 0.140	3.48 ± 0.193	3.99 ± 0.207	3.95 ± 0.149	3.61 ± 0.231
Granulocytes%	38.97 ± 1.344	41.34 ± 1.462	40.49 ± 1.529	39.69 ± 1.525	38.90 ± 1.090
RBC (10⁶/ µL)	5.63 ± 0.257	6.09 ± 0. 226	6.41±0.180**	5.64 ± 0.291	6.79 ± 0.379*
Haemoglobin (g/dl)	9.01 ± 0.435	9.72 ± 0.300	9.89 ± 0.431	9.19 ± 0.477	10.42 ± 0.716
PLT (10³/µL)	207.50 ± 15.447	196.40± 19.515	214.10 ± 1.841	191.50 ± 14.119	212.00 ± 1.581

Values are expressed as Mean ± Standard Error of Mean. Significance with Turkey’s test followed by one way ANOVA is evaluated as * p< 0.5, **p< 0.01, ***p<0.001 versus normal control group. A statistical significance was noted in Total RBC count in 1(**p< 0.01) and 10 therapeutic group (* p< 0.5) when compared to normal control group.

Results of Histopathology investigation:

The histopathology results of internal organs did not reveal any abnormality. The stomach of rats were non glandular and a polyp was seen in stomach of one animal in one therapeutic dose group.

The liver of one animal in vehicle control group showed vacuolar degeneration of hepatocytes. This is evident from the toxicity studies that Kandhaga rasayanam is non-toxic to experimental animals. The photographs of histopathology study results are given in figure 1.

DISCUSSION:

The drugs which are administered to humans are to be tested in animals. Though, Kandhaga Rasayanm is in practice since many years, no toixicity studies are carried out. The present study was aimed to bring out the long term ie, 90 days toxicity profile of Kandhaga Rasyanam so that it can be widely accepted by the scientific community. There was no loss of body weight during the experimental period. There was a gradual increase in body weight which is a positive sign. The feed intake and water intake of the animals are normal. There was also no toxic signs exhibited during the study period. The results of acute toxicity was confirmed by carrying out 90 days toxicity study. Three dose levels were administered to the experimental groups and there were no signs of toxicity or behavioural changes. The minor changes and fluctuation in body weight may be due to its feed and water intake variation which is considered physiological. Though it is a herbo mineral drug, the proper purification and preparation process has showed its implication in biochemical and haematological reports. There were no significant changes in biochemical and haematological parameters when compared with the normal control group. Further confirmation was done by doing histopathological examination of internal organs. The results showed no microscopic or macroscopic changes in the internal organs or tissues in treated rats. The compound formulation of this drug has been proven to be nontoxic in rats.

The feed intake and body weight showed gradual improvement in all the groups and this was slightly higher in treatment group. This was reflected in body weight gain. There was significant difference in body weight at the end of 3^rd week and 11^th week in treatment group statistically but the organ weight didn’t show any difference. This could be due to long term administration of test drug containing ghee. The Siddha literatures states Zingiber officinale, Piper nigrum and Piper longum together called as Trikadugu improves digestion and appetite. Elettaria cardamomum is also an appetizer. The probable improvement on feed can be attributed to the presence of above ingredients. There was no significant difference in the weight of vital organs in all the groups compared to the normal control group.

Laboratory investigations and histopathology investigations:

The haematology values did not show any significant difference except for a significant increase in RBCs in 1 and 10 therapeutic dose group. The study drug KR showed presence of iron in qualitative analysis and also Triphala present in KR is a haematinic which might be the cause for improvement in total red blood cells count.

The biochemical analysis report showed no significant changes in blood sugar, blood urea, triglyceride, albumin, SGOT, sodium and potassium values when compared to normal control group. A significant difference at p<0.05 confidence was observed in serum creatinine in 10 therapeutic dose group. But, the value was within normal range and hence cannot be taken as an alarming toxic sign. The other groups showed no significant difference when compared to normal. The total cholesterol and serum bilirubin in 5 therapeutic and 10 therapeutic dose group showed significant increase when compared to normal control. This increase in total cholesterol might be due to the addition of ghee in the Rasayanam. Sulphur, a major ingredient of KR is a cholagouge (pithaneer peruki). It should be noted that the cholesterol level also fall within the normal range. The total protein of 5 therapeutic dose showed p<0.05 confidence when compared with the normal control group. The SGPT value in 10 therapeutic dose group was significantly increased when compared to normal control group. The enzymes SGOT and SGPT are good indicators of liver function. The 5 therapeutic dose showed a significant decrease in chlorine value (p<0.05) which has no association with toxicity. Since there was an increase in SGPT at 10 therapeutic dose all the internal organs like brain, heart, lungs, liver, kidneys, spleen, testes, ovary, stomach, thymus were given for histopathological investigations. There was no abnormality detected in the hitopathological findings. The stomach was non glandular with a polyp in one animal’s stomach in one therapeutic dose group which is not related to toxicity. There was vacuolar degeneration of hepatocytes in the liver of one animal in vehicle control group. The results of histopathological examination confirm that the trial drug KR is non-toxic and safe for consumption. It was reported earlier that Semecarpus anacardium nut oil extracts exhibited nephrotoxicity^5,6. Jaila El Malti et al has reported in a study that Elettaria cardamomum produces toxicity at 0.3 mg/ g body weight of mouse⁷.

Sulphur has low toxicity. The risk to animal and human health is very low^8,9.

Though the compound drug contains marking nut, caramomum etc, it contains many ingredients which have hepatoprotective and nephroprotective activities. Previous research reports are available for the hepatoprotective activity of ashwagandha, harithaki, amla, Terminalia bellerica, dried ginger, pepper, long pepper, cardamom, Emeblia ribes, cinnamom, Plumbago zeylanica. Hence the drug can be considered to be safe on liver. This claim was supported by the results of histopathology.

From the results of toxicity study we can confirm the safety of KR by reverse pharmacology.

CONCLUSION:

Based on the above given results the authors conclude saying that the drug Kandhaga Rasayanam is safe to be administered in humans. Further trials in humans will add significance to the toxicity profile.

ACKNOWLEDGEMENT:

The authors would like to express their sincere thanks to Dr.V.Subha, Assistant Professor, Pharmacology Department of National Institute of Siddha, Vetenarian incharge of National institute of Siddha, Director, National Institute of Nutrition, Dr. R.Ganesan, Assistant Director (Biochemistry), Dr. Dayanand Reddy, Assistant Director (Pharmacology) of Siddha Central Research Institute, technical assistants of Siddha Central Research Institute for their help in this study. The authors also thank Dr.S.Sonitha for her help in statistical analysis. The authors would like to pay their gratitude to the Director of National Institute of Siddha for his support.

CONFLICT OF INTEREST:

Conflict of interest declared none.

REFERENCES:

1. Kuppuswamy mudhaliar, Ed.Siddha Vaithiya Thirattu.1st Edn, Department of Indian Medicine and Homoeopathy, Chennai-106, India. 1998.; 235,

2. Meena R, Ramaswamy R S. “Toxicity profile and chemical constituents of the ingredients of Siddha drug-Kandhaga Rasayanam”. International Journal of Pharma and Bio sciences, Jan 2015; 6(1): 869-886.

3. Sudhanshu Kumar Meher et al. Uses of Withania somnifera (Linn) Dunal (Ashwagandha) in Ayurveda and its Pharmacological Evidences. Res.J.Pharmacuology & P’DYNAMICS. Jan-Mar 2016; 8(1): 23-29.

4. Meena R, Ramaswamy R S. “Acute toxicity study of a Siddha formulation Kandhaga Rasayanam in rats”. International Journal of Research in Ayurveda and Pharmacy, Nov-Dec 2015; 6(6): 720-724.

5. Choudhari C V, Deshmukh PB. Acute and subchronic toxicity of Semecarpus anacardium on haemoglobin percent and R.B.C. count of male albino rat. J.Herb Med Toxicol, 2007; 1: 43-45.

6. Choudhari C V, Deshmukh PB. Effect of Semecarpus anacardium pericarp oil extract on and some enzymes of kidney in albino rat.J.Herb Med Toxicol, 2008; 2:27-32.

7. Jazila EL Malti, Driss Mountassif, Hamid Amarouch, Antimicrobial activity of Elettaria cardamomum : Toxicity, biochemical and histological studies. Food chemistry, 2007; 104 (4):1560-1568.

8. The agrochemicals handbook, The royal society of chemistry. The university Nottingham, England. 1990.

9. “Eligibility Document Facts:Sulfur.(May, 1991). Fact sheet- Environmental Protection Agency”, US Environmental Protection Agency.

Received on 17.10.2017 Modified on 17.11.2017

Research J. Pharm. and Tech 2018; 11(4): 1640-1646.

DOI: 10.5958/0974-360X.2018.00305.0