Title: Anti-diabetic activity of traditional Indian gold containing preparation: Shadguna Balijarita Makaradhwaja on Streptozotocin induced diabetic rats.
Background: Makaradhwaja a gold containing mercurial preparation used for diabetes mellitus in indigenous system of medicine. It is a popular aphrodisiac and rejuvenator traditional medicine. It is prepared by using processed gold, mercury and sulfur in different ratios by applying intermittent heating pattern in Valuka Yantra. Objectives: The aim of study was to evaluate anti-diabetic effect of Shadguna Balijarita Makaradhwaja on Streptozotocin induced diabetic rats. Methods: Diabetes was induced to normal rats by injecting Streptozotocin in dose 40 mg/kg. Powdered Shadguna Balijarita Makaradhwaja and dried extract of Tinospora cordifolia were mixed with honey and administered orally for 20 days at dose 2.63 mg/kg and 42.34 mg/kg body weight respectively. The effects of treatment on body weight changes and blood glucose levels were quantified on Day 1, 5, 10, 15 and 21 of the experiments. On 21st day animals were sacrificed and gross histopathological changes in liver, kidney and pancreas were illustrated. Blood sugar level, Glyacated hemoglobin, blood urea, serum cholesterol, serum creatinine, serum triglyceride and serum protein were estimated with standard methods. Study was conducted in the year 2011. Results: Test drug observed significant decrease (P<0.001) in Glyacated hemoglobin level compared to diabetic control rats. Blood sugar level of test drug group shown significant decrease (279.11 ± 57.95) compared with diabetic rats. Conclusion: The present study demonstrates that Shadguna Balijarita Makaradhwaja and dried extract of Tinospora cordifolia with honey significantly reduces the blood glucose level and shows anti-diabetic effect.
Keywords: Anti-diabetic activity, Cinnabar gold, Kupipakwa Rasayana, Mercury sulfide Shadguna Balijarita Makaradhwaja.
Diabetes Mellitus is threatening for 21st century. It will be leading cause of morbidity and mortality in near future due to increasing incidence worldwide. Herbo-mineral-metallic drugs of Ayurveda having potential of decreasing blood sugar levels and found efficient on experimental animal models. Makaradhwaja  is such herbo-mineral-metallic composition.
Medieval Classical texts of Ayurveda quoted that Makaradhwaja is one of the best rejuvenator and aphrodisiac  agent. It is prepared by using processed gold, mercury and sulfur in the ratio of 1:8:24 or 1:8:48 by sublimation in the traditional system of heating device Valuka Yantra [1-2]. Now a days it is prepared in modified vertical electrical muffle furnace (modified Valuka Yantra) . It gives synergistic action with different herbs in the various disorders. It is therapeutically efficient in disorders like Madhumeha (diabetes mellitus), Jwara (remittent fever), Kushta (skin disorders), Raktaja Vikara (blood disorders) . Previous studies claimed its safe use without any untoward effect and toxicity. . Specific action on cell mediated immunity is proved by its immune-modulator action . In experimental and clinical studies, it is found effective in diabetes .
Guduchi (Tinospora cordifolia Linn.) is well known Madhumehahar (anti- hyperglycemic) herb described throughout Ayurvedic classics . Most commonly, its stem is used for medicinal purposes. It is well known anti-oxidant, anti-hyperglycemic, immune-modulatar and rejuvenator . Its different formulations are quoted in the Ayurvedic classics like juice, decoction, powder and Ghana (dried extract) as per diseases.
Madhu (honey) is mentioned as Madhumehahar(~anti-diabetic) agent within classical texts of Ayurveda . Its anti-diabetic activity is reported . It is used as a vehicle drug (Yogavahi) in the numerous herbo-mineral formulations. It is mentioned in the reference to Makaradhwaja as a vehicle drug . In the present study it was used as a vehicle drug as prescribed in texts of Rasashastra.
Present study was planned to evaluate anti-diabetic activity of Shadguna Balijarita Makaradhwaja and Tinospora cordifolia Linn with honey in Streptozotocin induced diabetic rats.
Materials and Methods:
Preparation of Shadguna Balijarita Makaradhwaja and Guduchi Ghana:
Test drug Shadguna Balijarita Makaradhwaja was prepared as per the classical text reference in the department of Rasashastra and Bhaishajya Kalpana, Institute of Post Graduate Teaching and Research in Ayurveda (IPGT & RA), Gujarat Ayurved University, Jamnagar in 2011 . Raw Material Swarna (gold) was purchased from local market and Hingula (cinnabar), Gandhaka (sulfur) were collected from Pharmacy of Gujarat Ayurved University, Jamnagar. Gold was subjected to Shodhana and after Shodhana, its foils were prepared. Cinnabar was processed to Shodhana (purification) and Parada (mercury) was procured from its sublimation by adopting Nada Yantra method . Gandhaka (sulfur) was subjected to Shodhana by melting it and pouring in cow milk and continuously heated in same milk for three hours . Processed gold foils, mercury and sulfur were brought in ratio of 1:8:48 in weight. Amalgamation was done by adding gold foils to mercury. Fine lusterless powder Kajjali was prepared by triturating sulfur with above prepared amalgam. Kajjali was levigated with juice of Kumari (Aloe barbadensis Mill.) and Japa (Hibiscus rosa sinensis) for three hours consecutively. Levigated Kajjali was dried and powdered. The fine powder was filled in seven-layer mud smeared cotton cloth wrapped glass bottle (Kacha Kupi) and heated for 12 hours in specially designed electrical muffle furnace. Heat was provided in controlled and gradually increasing temperature in modified electrical muffle furnace (modified Valuka Yantra). After the desired characteristic features of product preparation, mouth of glass bottle was sealed; furnace was switched off and subjected for self cooling. Highest recorded temperature during procedure was 6000C. Sublimed product was procured from neck of glass bottle, it was powdered and used for further analysis and study . In analytical studies, it was observed that Makaradhwaja is consisted of red sulfide of mercury and having an empirical formula of HgS (mercury sulfide). Inductively coupled Plasma optical emission spectrometry (ICPOES) was observed that it contains 1.2 % of gold with mercury and sulfur in finished product as a major element.
- cordifolia’s stems were collected from the herbal garden of Gujarat Ayurved University, Jamnagar. Stems of T. cordifolia were cut in to small pieces and crushed. These crushed pieces were cooked with four times of potable water ant reduced at 1/4th of the same to prepare decoction. The decoction was sieved, cooked to semisolid consistency. The semisolid mass was dried in hot air oven at 450C to prepare dry extract . Average 5.21 % yield was obtained from the stem extract. The Guduchi Ghana (dried extract) was powdered and stored .
Honey was collected from the local forest of Jamnagar.
Albino rats (160-220 g) of either sex were selected for study. Animals were procured from the Animal house of Pharmacology laboratory of IPGT & RA, Gujarat Ayurved University, Jamnagar. Permission for experiment was granted by animal ethical committee of the same institute (IAEC-06/09-11/02). The animals were fed pellet diet and water. As per the guidelines of National Institute of Health’s guide for the Care and Use of Laboratory Animals, study was conducted .
Collection of Blood Samples:
Tail veni-puncture method was applied for the collection of blood sample in the rats. For investigation Glucometer strip was used and reading was noted down. By adopting this procedure blood glucose level of animals was estimated, prior injection of Streptozotocin, 5th, 10th, 15th and 20th day during trials.
The sacrifice was carried out on 21st day after completion of study. Animals were anesthetized and stroked over tiles for sacrifice. The blood sample was collected by the dissection of jugular veins of animals. Biochemical parameters were investigated at pathology laboratory, IPGT & RA, Jamnagar.
Experimental Induction of Diabetes:
Diabetes was induced to rats by single intra peritoneal injection of Streptozotocin (STZ) (40 mg/kg). Streptozotocin was weighed individually for each animal, according to its weight it was solubilised with 0.2 mL saline(154mM NaCl) just prior to injection. After seventy-two hours of Streptozotocin injection, rats with diabetic hyperglycemia (blood glucose more or equal to 250 mg/dL) were selected for experiment. Suspension of Makaradhwaja and T. cordifolia with honey was started fed to selected diabetic rats.
Anti- diabetic activity:
Anti-diabetic activity was evaluated by the effect of test drugs on the ponderal and biochemical paramaeters. Ponderal parameters like gross body and different body organs weight was evaluated. Biochemical variables like blood sugar (B Sugar), glycated hemoglobin (HbA1C), serum total cholesterol, serum High density lipoprotein (HDL), serum triglyceride, serum creatinine, Blood urea (B urea), Serum glutamic-pyruvic transaminase (SGPT), Serum glutamic oxaloacetic transaminase (SGOT), total protein, albumin and globulin were estimated. Sacrificed animals gross and histological appearance of vital organs (liver, pancreas and kidney) were examined at the end of study.
Diabetic animals were divided into four groups. Food and water were provided to the animals.
- Group 1: Normal Control (NC)
- Group 2: Diabetic control (DC)
- Group 3: Diabetic control + Shadguna Balijarita Makaradhwaja and Guduchi Ghana with Honey Suspension (SBM)
- Group 4: Diabetic control + Glibenclamide (Reference Standard)(RS)
The dose of the drug was calculated by extrapolating the therapeutic dose to rat on the basis of body surface area ratio by referring to the table of Paget and Barnes (1964) .
Powdered Shadguna Balijarita Makaradhwaja 2.63mg/kg rat and dried extract of T. cordifolia 42.34mg/kg rat were grinded with honey to prepare suspension. This suspension was administered orally to test drug group SBM diabetic animals. Glibenclamide, in the dose 0.45mg/kg was administered to reference standard drug control group (RS).
All the values of were expressed as mean ± standard error mean (SEM). Statistical analysis was done by using student ‘t’ test. It was calculated by using Microsoft excel programmer.
In normal control rats during the course of 21 days, 13.90% weight was increased. Insignificant decrease in body weight was observed in SBM and RS group in comparison to diabetic control rats. (Table 1)
In diabetic control rats weight of kidney and liver was increased up to significant extent. Treatment with test drug did not affect the weight of these organs to significant extent in comparison to diabetic control group. (Table 2)
Initial blood sugar level was decreased by 44.27% and 44.04% in SBM and RS treated groups respectively (Table 3). Raised levels of Glycated haemoglobin (HbA1C) were significantly attenuated by test drug SBM and RS. (Table 4). Increased blood sugar levels were insignificantly decreased by SBM and RS test drugs.
Non -significant rise in Serum cholesterol, triglyceride and HDL were moderately decreased by administration of SBM. Blood urea levels decreased significantly in group SBM and moderately decreased in reference standard group. Elevated Serum creatinine levels non-significantly decreased by administration of SBM which was non-significantly increased in reference standard group in comparison to diabetic control group. Decreased SGOT (Serum glutamic oxaloacetic transaminase) parameter in diabetic control group was significantly attenuated by SBM and RS drugs. Increased levels of SGPT (Serum glutamic –pyruvic transaminase) were decreased up to significant extent by SBM and RS drugs. (Table 5).
The accumulation of fat in liver was observed in histo-pathological study. SBM and RS treated animals restored the histological changes. (Fig 1 a, b,c,d). Inflammation in blood vessels, increase in the thickness of bowman capsules, fat deposition and change in size of the glomerulus were found in kidney of diabetic rats. The treatment with SBM showed the normal histopathology of kidney without any inflammatory vessels and fat deposition (Fig 2 a, b,c,d). Decreased Islets of Langerhans, smashed size of β cells and extensive necrosis, fibrosis and atrophy were observed in pancreas of diabetic rats. STZ induced diabetic rat treated with SBM and glibenclamide restored the necrotic and fibrotic changes and up to moderate levels, raised the number of β cells. (Fig 3 a, b,c,d).
Excessive breakdown of tissue proteins decreases body weight in diabetes. It was observed in diabetic control rats in comparison to normal rats [19-21]. Treatment with SBM improved body weight to a certain extent, indicating that control over muscle wasting resulted from glycemic control.
Previous studies claimed that Streptozotocin destructs beta cells, which leads to cells less active that makes poor utilization by tissues [22-23]. This suggests that test drug may possess as insulin like effect on peripheral tissues by inhibiting hepatic gluconeogenesis by promoting glucose uptake or metabolism . Or it might be possible that test drug increases absorption of glucose into the muscles and adipose tissues  by stimulation of regeneration process and revitalization of remaining beta cells . Hence, the hypoglycemic activity of SBM may be due to its protective action against damaged pancreatic beta cells and possibly because of increased insulin release or secretion or regeneration of damaged beta cell. Makaradhwaja is a well known therapeutic medicine of rejuvenation in Indian system of medicine. Its immune-modulatory action was also established . The observed effect may be attributed to the rejuvenation property of Makaradhwaja. Previous study supports its action too . Moderate but insignificant decrease in blood sugar levels were also observed in test drug treated animals.
Higher levels of HbA1C were observed in the diabetic rats compared with those in normal rats, it might be due to poor glycemic control. SBM treated diabetic rats significantly decreased the level of HbA1C, may be glucose metabolism was improved. This action represents that SBM has an ability to prevent the development of diabetes associated complications.
Elevated levels of SGPT indicating impaired liver function due to hepato-cellular necrosis. Due to elevated transaminase activities leads to diabetic complications like increased ketogenesis and glucogenesis , test drug significantly restored this parameter towards normal levels.
Elevation of urea and creatinine levels results due to renal dysfunction caused by free radical generation mediated stress in diabetes, persistent hyperglycemia and haemo-dynamic changes within the kidney tissue [29-31]. Administration of SBM and glibenclamide to the diabetic rats significantly reduced the creatinine and urea levels, which represent the preventive action of SBM on kidney damages in diabetic condition perhaps due to the anti-oxidant properties. Blood urea level was significantly increase in diabetic rats compared to normal rats due to excessive breakdown of protein. This elevation was significantly decreased by test drug.
STZ induced diabetic rat’s increases the level of lipid peroxidation, as an indirect evidence of production of free radical . Hyperlipidemia commonly associated with diabetes . Test drug significantly attenuated serum lipid profiles in diabetic rats. STZ induced diabetic groups treated with glibenclamide and SBM brought back the increased level of total cholesterol and triglyceride near to the normal levels.
Histo-pathological changes in liver, kidney and pancreas were well restored by the test drug in comparison with diabetic control group. The observed results show anti-diabetic potential of Shadguna Balijarita Makaradhwaja. Observed results may be due to synergistic action of Makaradhwaja with adjuvant T. cordifolia and honey.
Conclusion: The present study demonstrates that Shadguna Balijarita Makaradhwaja and dried extract of Tinospora cordifolia with honey significantly reduces the blood glucose level and shows anti-diabetic effect. Restoration histo-pathological changes in different organs support safe and effective anti-diabetic action of test drug. Significant decrease in Glycated hemoglobin shows effect of Makaradhwaja on diabetes related complications.
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