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PHARMACOLOGICAL STUDY
Year : 2017  |  Volume : 38  |  Issue : 2  |  Page : 171-178  

Ninety days repeated dose oral toxicity study of Makaradhwaja in Wistar rats


1 Department of Pharmacology, Central Ayurveda Research Institute of Drug Development, Kolkata, West Bengal, India
2 Department of Pharmacology, Central Council for Research in Ayurvedic Sciences, Ministry of AYUSH, New Delhi, India

Date of Web Publication28-Aug-2018

Correspondence Address:
Dr. Shrirang Jamadagni
Central Ayurveda Research Institute of Drug Development, 4 CN Block, Sector 5, Bidhananagar, Kolkata - 700 091, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ayu.AYU_33_17

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   Abstract 


Context: Makaradhwaja is a Kupipakwa Rasayana. Since it contains two heavy metals, namely mercury and gold, it is essential to evaluate its safety. Hence, the present study was undertaken with an objective to evaluate toxicity and target organ of toxicity of Makaradhwaja if so. Aims: The objective was to evaluate toxicological profile, the target organ of toxicity and to find no observed effect level (NOEL) or no observed adverse effect level (NOAEL) in rats after oral administration for ninety consecutive days. Materials and Methods: Makaradhawaja preparation was administered to male and female Wistar rats for ninety consecutive days at 2.7, 13.5, and 27 mg/kg body weight. All relevant biochemical and hematological changes were observed. At termination, all the rats were sacrificed and necropsy was performed. Histopathological evaluation was also performed. Statistical Analysis Used: Dunnett's test followed by analysis of variance. Results: There was a significant increase in high-dose group kidney weight of both sexes which could not be correlated with histopathology findings and serum biochemistry. Therefore, the change was not considered as an adverse effect. Conclusions: The dose level 27 mg/kg of Makaradhwaja was found as NOAEL and dose level 13.5 mg/kg of Makaradhwaja was found as NOEL.

Keywords: Gold, Makardhwaja, mercury, rats, toxicity


How to cite this article:
Jamadagni S, Jamadagni PS, Singh RK, Upadhyay S, Gaidhani SN, Hazra J. Ninety days repeated dose oral toxicity study of Makaradhwaja in Wistar rats. AYU 2017;38:171-8

How to cite this URL:
Jamadagni S, Jamadagni PS, Singh RK, Upadhyay S, Gaidhani SN, Hazra J. Ninety days repeated dose oral toxicity study of Makaradhwaja in Wistar rats. AYU [serial online] 2017 [cited 2019 Aug 20];38:171-8. Available from: http://www.ayujournal.org/text.asp?2017/38/2/171/239953




   Introduction Top


Makaradhwaja is a classical Ayurvedic preparation categorized as Kupipakwa Rasayana (rejuvenating mercurial formulation which is prepared in glass bottle by sublimation through sand bath with specific temperature pattern). The oldest reference of its use is found in classical text of Rasendra Chintamani by Acharya Dhundhuknath in the 16th century.[1] According to Chopra,[2] its first reference can be attributed to Vaidya Bhva Mishra of 16th century. Kupipakwa Rasayana is basically powdered minerals and metals heated gradually in glass flask. They have often characteristic red (or yellow color) and their potency persists indefinitely if stored in well-stoppered bottles.[3] Gold and mercury are the heavy metal contents of the Makaradhwaja along with sulfur.[4]Makaradhwaja is aphrodisiac and nutrient to body and mind with adapto-immuno-neuro-endocrino-modulator properties. It is indicated for dyspepsia, weakness of heart, senility and fever.[4]

Users of Ayurvedic medicine may be at risk for heavy metal toxicity.[5] Repeated exposure to heavy metal containing products may lead to cumulative toxicity. Regulatory guidelines also require the product to be tested for its toxicity in rodents carrying single and repeated dose studies so as to establish the no observed effect level (NOEL) and no observed adverse effect level (NOAEL).[6] The present study was a part of multicentric toxicity studies of the Central Council for Research in Ayurvedic Sciences, hence drug was provided in a coded manner.


   Materials and Methods Top


Test drug preparation and analysis

Makaradhwaja was provided by the Central Council for Research in Ayurvedic Sciences, New Delhi, in a coded manner that was decoded along with chemical analysis report after completion of the study. The drug was prepared as per the method described in Rasa Tarangini[7] a classical Ayurvedic text.

Details of the ingredients are given in [Table 1].
Table 1: Ingredients used for preparation of Makaradhwaja

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All the ingredients were collected and the final drug was as given below.

Purified gold was taken into mortar and purified mercury was added to it and triturated. Trituration was continued till amalgam of Swarna (gold) and Parada (mercury) was formed. The amalgam of Swarna and Parada and powder of Shodhit Gandhaka (purified sulphur) in specified quantity was taken and grinded to obtain homogenous, soft, fine (non lustrous) powder. The fresh juiceof flower of Rakta Karpasa (Gossypium arboreum Linn) was prepared and added to the Swarna Kajjali in adequate quantity and levigated. After completing the process, whole mixture was dried. The fresh juice of root bark of Ankol (Alangium salvifolium Linn.)was prepared and added to the Swarna Kajjali in adequate quantity and levigated. After completing the process, the whole mixture was dried. Pulp collected from fresh leaves of Kumari (Aloe vera Linn)was processed to obtain fresh juice. The fresh juice was added to the Swarna Kajjali in adequate quantity and levigated. After completing the process, whole mixture was dried. The dried Swarna Kajjali was taken in a glass bottle and placed in Valuka Yantra (sand bath). Then, the Valuka Yantra was subjected to heat adopting Kupipakva method. The temperature was increased gradually in a phased manner. At the end, Valuka Yantra was allowed to self cool and bottles were taken out. The bottles were observed for deposition of Makaradhwaja at the neck (i.e., Kanthastha Makaradhwaja). A thread soaked in kerosene was tied around the bottle just below the level of deposited Makaradhwaja and burnt to break the bottle. After this, Kanthastha Makaradhwaja was collected in mortar and was grinded to convert it in to fine powder, which was reddish (Sindoor Varneeya) in color. The residual of gold obtained from the bottom of the bottles was collected separately.

Physicochemical analysis, namely morphological description, estimation of moisture content, qualitative elemental testing, and Ayurvedic parameters, were carried out by following standard methods as per the Ayurvedic Pharmacopoeia of India Guidelines.

The quantitative elemental composition was performed on JY 2000 sequential inductively coupled plasma-atomic emission spectrometry (ICP-AES) spectrometer (Horiba Jobin Yvon, France). The operating parameters of ICP-AES were – RF power: 1000 w at 40.68 MHz, plasma gas flow rate: 12 L/min, nebulizer gas flow rate: 0.1 L/min, Sample uptake rate: 1.2 L/min, slit with: 20 micron/20 micron (entrance/exit), and monochromator: 0.64 m focal length, 2400 groves/mm, Czerny turner mounting.

Study protocol

The study protocol was provided by the sponsor Central Council for Research in Ayurvedic Sciences, New Delhi, which was designed by broadly following principles of Schedule Y of Drugs and Cosmetic Act, 1940.

Housing and environment

A total of 80 Wistar rats (40 male and 40 female) with body weight ranging from 150 g to 200 g were obtained from animal house. Ethical clearance was taken from the Institutional Animal Ethical Committee (IAEC) with letter number 6-17/2003-CRI/Tech/777 dated 02.07.2009. All the rats were maintained as per the guidelines of the committee for purpose of control and supervision of experiments on animals for laboratory animal facility. Rats were acclimatized for 7 days. Temperature and relative humidity were maintained at 25 ± 1°C and 40%–70% respectively and illumination was controlled to give approximately a sequence of 12 h light and 12 h dark. Rats were individually housed in polycarbonate cages (43 cm × 28 cm × 21 cm) with lids and rice husk bedding. Pelleted rodent diet obtained from National Institute of Nutrition, Hyderabad, was provided along with deionized water using plastic bottles with stainless steel nozzle ad libitum. Females selected were nulliparous and nonpregnant.

Experimental study design

The animals were divided into four groups of 10/sex/group. Ninety days repeated dose oral toxicity study of Makaradhwaja was conducted by daily single administration of the drug at 27 mg/kg (high dose [HD]), 13.5 mg/kg (mid-dose) and 2.7 (low dose) mg/kg body weight along with vehicle control. The test drug was administered as suspension in vehicle, that is, honey mixed with water by gavage and control group received the vehicle. To make the drug and vehicle suspension easy to gavage, it was diluted with water in 2:3 ratio. The suspension was administered @10 ml/kg body weight. The dose calculation was as follows:

Therapeutic dose for human (70 kg body weight approximately): 30 mg/day. Dose conversion factor from human to rats as per the Paget and Barnes [8] is 0.018.

Hence, the dose for rats (200 g body weight approximately) will be 30 × 0.018 = 0.54 mg. Hence, therapeutic equivalent dose for rats per kg/body weight will be 2.7 mg which was considered as low dose. The dose that was 5 times more than therapeutic, that is 2.7 × 5 = 13.5 was considered as mid-dose, and 10 times therapeutic dose, that is, 2.7 × 10 = 27 was considered as highest dose for the study.

All animals were observed for morbidity and mortality twice daily. General clinical observations were made twice a day at the same time throughout the study. The animals were observed for changes in skin, fur, eyes, mucous membrane, occurrence of secretions and excretions. For neurological examination, the animals were taken outside the cage in a standard arena and their behavior was recorded. Body weights and feed consumption of each animal were recorded at the start of the study and thereafter at weekly intervals. At the termination of the study, that is, on 90th day, serum glucose, total protein, SGOT, SGPT and creatinine along with hematological parameters which were white blood cell and red blood cell count, hemoglobin, hematocrit (%), platelet count, mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and MCH concentration were measured for all animals. Animals were sacrificed on the 91st day using CO2 euthanasia and were subjected to a detailed postmortem examination and histopathological analysis. The adrenals, heart, kidney, spleen, testis, epididymis, ovaries, uterus, liver, brain and thymus were weighed and collected along with jejunum, duodenum, colon, cecum, ileum, stomach, lungs, pancreas, esophagus and trachea in 10% neutral-buffered formalin. All collected organs from control group and HD group were processed as per the Registry of Industrial Toxicology Animal data guidelines [9] and subjected to histopathological evaluation.

Test drug physicochemical analysis

Makaradhwaja prepared was in the form of reddish powder and had no characteristic odor. Assay of element showed 82% of mercury and 13% of sulphur. Moisture content was 0.08% which was found when loss on drying was determined at 105°C.


   Results Top


No abnormality in clinical signs was detected across all the groups throughout the study. No mortality was found in any of the study groups. No abnormality was detected across the groups during neurological examination. No treatment-related as well as dose-dependent effect on body weights, feed consumption, water consumption, fecal consistency and biochemical parameters was observed as compared to the control group. MCV of HD group male was significantly increased but was within biological limits. However, significant increase in the weights of kidneys from the male and female animals of HD group was recorded. There was no toxicity induced or any other lesion observed during the histopathology evaluation which could be correlated with the increase in weight of the kidneys. Further, there was no significant change in serum biochemistry parameters of HD group females which could be attributed to increased weight of kidneys. The results are described in [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]. No treatment-related and dose-dependent adverse changes were observed on detailed histopathological evaluation [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]. Photomicrographs of the selected organs/tissues from vehicle control group and high-dose group are provided in [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6].
Table 2: Body weight (g) of male rats

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Table 3: Body weight (g) of female rats

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Table 4: Relative organ weight of male rats

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Table 5: Relative organ weight of female rats

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Table 6: Hematological analysis of male rats at 90th day

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Table 7: Hematological analysis of female rats at 90th day

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Table 8: Serum biochemical analysis of male rats at 90th day

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Table 9: Serum biochemical analysis of female rats at 90th day

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Figure 1: Heart control group: No abnormality detected (10 × 10)

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Figure 2: Heart high-dose group: No abnormality detected (10 × 10)

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Figure 3: Kidney control group: No abnormality detected (10 × 10)

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Figure 4: Kidney High Dose Group: No abnormality detected (10× 10×)

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Figure 5: Liver control group: No abnormality detected (10 × 10)

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Figure 6: Liver high-dose group: No abnormality detected (10 × 10)

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Statistical analysis

Study observations such as body weight gain, feed consumption, blood biochemistry and hematology parameters and organ weights were recorded and analyzed statistically. One way analysis of variance and Dunnett's test were applied to compare the dose groups over the control arm.[10]


   Discussion Top


The test drug Makaradhvaja was prepared in compliance with Ayurvedic literature and contained heavy metal mercury (82%) and sulphur (13%). However, no treatment related adverse effect was observed up to 10 times therapeutic dose levels, that is, 27 mg/kg body weight in animal experiment. G. Kumar et al.[11] studied 28 days repeated dose toxicity of mercury in Ayurvedic formulation of Aarogyavardhini Vati in rats at dose of 50, 250, and 500 mg/kg of body weight with mercury content dose equivalent to 1 mg/kg to HgCl2 in HD group and reported that there was no adverse effect on cerebellum, liver and kidneys and the drug was safe up to 500 mg/kg body weight. Whereas HgCl2 at 1 mg/kg body weight alone showed pyknosis in the brain neurons and congestion of blood vessels was reported in the kidney. G. Kumar et al.[11] also attributed the nontoxic character of mercury in Arogyavardhini Vati to process of Shodhana due to which, mercury may not retain its original physiochemical form and hence toxic character. According to an experimental toxicity study,[12]Makaradhwaja when given orally did not produced mortality up to the dose of 480 mg/kg in mice. A 28 days repeated dose oral toxicity study of Siddha Makaradhwaja in rats showed neurodegenerative changes in brain at the dose of 100 mg/kg, but no histopathological changes were seen at dose level of 50 mg/kg in the kidney, liver and brain. Further no changes were seen in serum alanine aminotransferase, aspartate aminotransferase, alanine phosphatase, bilirubin, urea and creatinine implying the safety of Siddha Makaradhwaja on hepatorenal system of the rats.[13] Both Triguna and Shadaguna Makaradhwaja offered Madhumehahara property (antidiabetic) in clinical trials, but Shadguna Balijarita possessed more effect than Triguna Balijarita Makaradhwaja. It has experimentally shown antihyperglycemic, antidiabetic, renoprotective, cardiac stimulant activity and is a known immunomodulating agent in experimental studies with its use being reached in veterinary practices too.[14] A pilot clinical trial of Makaradhwaja has shown promising results for treating rheumatoid arthritis, but the study also showed mercury excretion in urine.[15] In the present study, the results are in line with earlier studies and histopathological evaluation also indicated no evidence of treatment-related or dose dependent lesions in any organ. It is apparent that Makaradhwaja is safe at dose rate of 27 mg/kg/day for ninety consecutive days. However, there was significant increase in kidney weights of females from HD group, which could not be corroborated with serum creatinine or other biochemical data and histopathological evaluation findings. Therefore, HD level, that is, 27 mg/kg body weight was decided as NOAEL and mid-dose level, that is, 13.5 mg/kg body weight was decided as NOEL was. Thus, the output of the study is in accordance with the results and conclusion by Kumar et al.[11] which underlined that the mere presence of very high proportion of mercury in Makaradhawaja does not possess any risk or toxic potential to animals or humans. The purification process adds several organic chemicals to the crude form of mercury which may lead to altered physicochemical properties of mercury and hence alters its toxic potential.[11],[16],[17],[18]


   Conclusion Top


When Makaradhwaja was administered daily for ninety consecutive days at dose levels 27, 13.5 and 2.7 mg/kg/day by oral route to Wistar rats, it was found that there was no effect on body weights, feed consumption and biochemical parameters. There was significant increase in kidney weight of male and females treated with HD which could not be correlated with histopathological and serum biochemical changes. There was no treatment-related changes observed in the histopathological evaluation at dose level 27 mg/kg. There was significant increase in MCV value of males treated with HD but was within biological limits. The dose level 27 mg/kg of Makaradhwaja was found as NOAEL and dose level 13.5 mg/kg of Makaradhwaja was found as NOEL. Ayurvedic preparation method of Makaradhwaja could be altering physicochemical properties of raw mercury rendering it to become significantly less harmful.

Acknowledgment

The authors are thankful to Director General, Central Council for Reseach in Ayurvedic Sciences, New Delhi, for his support and providing necessary infrastructure.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Acharya Dhundhuknath, Hartala Prakarana. In: Mishra SN, editor. Rasendra Chintamani. Siddhiprada Teeka. 1st ed. Varanasi: Chaukhamba Orientalia Publication; 2011. p. 254-5.  Back to cited text no. 1
    
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Chopra RN. Indigenous Drugs of India. Calcutta: The Art Press; 1933. p. 449.  Back to cited text no. 2
    
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Government of India. Ayurvedic Formulary of India Part 1. 2nd Revised ed., Ch. 15. New Delhi: Controller of Publication, Under Ministry of Health and Family Welfare, Government of India; 2003. p. 562.  Back to cited text no. 3
    
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Government of India. Ayurvedic Formulary of India Part 2. 1st English ed., Ch. 16:40. Controller of Publication, New Delhi: Under Ministry of Health and Family Welfare, Government of India; 2000.  Back to cited text no. 4
    
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Saper RB, Kales SN, Paquin J, Burns MJ, Eisenberg DM, Davis RB, et al. Heavy metal content of ayurvedic herbal medicine products. JAMA 2004;292:2868-73.  Back to cited text no. 5
    
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Government of India. Schedule “Y” Drugs and Cosmetics (II Amendment) Rules. Ministry of Health and Family Welfare, Government of India; 2005.  Back to cited text no. 6
    
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Sharma HS. Rasendra Mangalam of Nagarjuna. 1st Part. 1st ed., Ch. 1-4. Varanasi: Chaukhamba Orientalia Publication; 2008.  Back to cited text no. 7
    
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Paget GE, Barnes JM. Evaluation of drug activities. In: Lawrence DR, Bacharach AL, editors. Pharmacometrics. Vol. 1. New York: Academic press; 1969. p. 161.  Back to cited text no. 8
    
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Morawietz G, Ruehl-Fehlert C, Kittel B, Bube A, Keane K, Halm S, et al. Revised guides for organ sampling and trimming in rats and mice – Part 3. A joint publication of the RITA and NACAD groups. Exp Toxicol Pathol 2004;55:433-49.  Back to cited text no. 9
    
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Kumar G, Srivastava A, Sharma SK, Gupta YK. Safety evaluation of an ayurvedic medicine, Arogyavardhini Vati on brain, liver and kidney in rats. J Ethnopharmacol 2012;140:151-60.  Back to cited text no. 10
    
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Lavekar GS, Ravishankar B, Gaidhani S, Shukla VJ, Ashok BK, Padhi MM, et al. Mahayograj guggulu: Heavy metal estimation and safety studies. Int J Ayurveda Res 2010;1:150-8.  Back to cited text no. 11
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Patgiri BJ, Prajapati PK, Ravishankar B. A toxicity study of Makaradhwaja prepared by Astasamskarita Parada. AYU Int Q J Res Ayurveda 2006;27:55.  Back to cited text no. 12
    
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Kumar G, Srivastava A, Sharma SK, Gupta YK. Safety evaluation of mercury based Ayurvedic formulation (Siddha Makardhwaja) on brain cerebrum, liver and kidney in rats. Indian J Med Res 2014;139:610-8.  Back to cited text no. 13
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Dhundi SN, Prajapati PK. Review on various experimental and clinical studies conducted on Makaradhwaja. Ann Ayurvedic Med 2013;2:99-103.  Back to cited text no. 14
    
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Kumar G, Srivastava A, Sharma SK, Rao TD, Gupta YK. Efficacy & safety evaluation of ayurvedic treatment (Ashwagandha powder & Siddha Makardhwaja) in rheumatoid arthritis patients: A pilot prospective study. Indian J Med Res 2015;141:100-6.  Back to cited text no. 15
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Gogtay NJ, Bhatt HA, Dalvi SS, Kshirsagar NA. The use and safety of non-allopathic Indian medicines. Drug Saf 2002;25:1005-19.  Back to cited text no. 16
    
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Garnier R, Poupon J. Lead poisoning from traditional Indian medicines. Presse Med 2006;35:1177-80.  Back to cited text no. 17
    
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Dash B. Alchemy and Metallic Medicines in Ayurveda. New Delhi: Concept Publishing Company; 1986.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]



 

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