EXPERIMENTAL STUDIES ON HARAGOURIRASA W.S.R TO “MRITANI LOHANI RASI BHAVANTI ”

1. P.G. Scholar, Department of Rasashastra and Bhaishajya Kalpana, NIA Jaipur. 2. P.G. Scholar, Department of Dravyaguna, NIA Jaipur. 3. P.G. Scholar, Department of Panchakarma, Parul Institute of Ayurveda Vadodara. Gujarat. 4. Professor and HOD, Department of Rasashastra and Bhaishajya Kalpana, NIA Jaipur. 5. Pharmacologist Mahatma Gandhi Ayurveda College Hospital & Reasearch Centre, Salod (H) Wardha. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 25 June 2020 Final Accepted: 30 July 2020 Published: August 2020

Ayurveda, one of the ancient systems of medicine, holds a vast domain of traditional knowledge within himself. It is considered as the science of life and has been practiced in India since times immemorial. A very systematic and elaborate step-wise procedure known as 'Bhasmikarana' converts the metal from its zero valent state to a form with higher oxidation state, which is crucial from the point of view that during this process the toxic nature of the metal and its oxide is fully destroyed while rendering the metal oxide with high medicinal value. The mentioned Verse is in the text like Rasendra mangal, Rasarnava, Ras Ratna Samuchchaya, Ras Ratnakar, The verse signifies that the absorption &assimilation of different types of Bhasma of minerals / metals / sub metals & Sindoora kalpana for internal administration in human body. Reduction in the particle size is the motto of using in the form of Bhasma or Nano medicine. The finer the particle size of bhasma allows better rate of assimilation and absorption. This is a critical attempt to understand and apply the basis of Ayurvedic concept of Bhasma and the concept of Pharmacokinetics. Pharmacokinetics supports the studies of preclinical toxicology in animals (toxicokinetics) because the drug levels in plasma or tissues are often more predictive than the dose to extrapolate the toxicity data to man.

…………………………………………………………………………………………………….... Introduction:-
Metal-based drugs prepared according to Rasa Shastra guidelines are considered to be safe and efficacious at minute doses without any adverse effects. In traditional methods of processing employed in Ayurveda, the metals are repeatedly subjected to shodana (purification with naturally available ingredients), bhavana (trituration with herbal juice) and bhasmikarana (calcination) to obtain the final product. These processes reduce the final product to nanometer size, which is believed to enhance its bioavailability and reduce its toxicity i . However, the past decade has witnessed concerns in Western countries over the use of metals in Ayurvedic formulations and several reports have emerged linking their presence in formulations to toxic effects. ii,iii 884 In Rasa shastra the metals and minerals are also termed as Dhatus and Updhatus because of their specific role in biological system that this can sustain body tissue by supplementing some of the essential elements to the tissues, whose deficiency causes many disease in the body . The mentioned Verse is in the text like Rasendra mangal, Rasarnava, Ras Ratna Samuchchaya, Ras Ratnakar, The verse signifies that the absorption and assimilation of different types of Bhasma of minerals / metals / sub metals & Sindoora kalpana for internal administration in human body. There is an urgent need of standardization and pharmacokinetics of the Ayurvedic formulations in order to achieve the uphold position in the global market. Pharmacokinetics which deals with the absorption, distribution, metabolism and excretion of the biomarkers or the new drug entity is the one of the regulatory requirement for an investigational new drug approval. Bioactive guided pharmacokinetic approach method is needed for Ayurvedic system of medicine to determine the pharmacokinetics of relevant markers in the formulation having number of markers. Also non compartmental analysis method should be applied for the analysis of pharmacokinetics of biomarkers from Ayurvedic formulations for successful pharmacokinetic evaluation.
As far as Ayurvedic formulations are concerned the concept of drug action and absorption is designed on the basis of the Panchamahabhuta theory, Agni, rasa, guna, virya, vipaka etc of the drug. Pharmacokinetics studies supports the studies of preclinical toxicology in animals (toxicokinetics) because the drug levels in plasma or tissues are often more predictive than the dose to extrapolate the toxicity data to man. To go for the bio availability of the Ayurvedic formulations especially Herbo minerals is a daunting task but they provide strong evidence base to the safety and efficacy of herbo-mineral formulations. The present pharmacokinetic study was undertaken with a view to identify a few pharmacokinetic parameters for haragourirasa.

Test Drug
The sample of Haragourirasa was prepared in the Dept. of Rasashastra and Bhaishajya Kalpana, N.I.A. Jaipur.

Experimental design and animal management
The experiment was carried out in DATTA MEGHE INSTITUTE OF MEDICAL SCIENCES SAWANGI WARDHA (Maharashtra). Ethical clearance was obtained from Institutional Animal Ethics Committee, before conducting the experiment (IAEC APPROVAL NO:DMIMS(DU)/IAEC/2018-19/08 ). The study was conducted on mature Albino rabbits, weighing 1.50 -2.00kg. Animals were acclimatized for a period of seven days in laboratory conditions prior to the experiments. Rabbits are kept at an ambient temperature of 25 ± 2°C with 12 h light: 12 h dark cycle in the animal house of DATTA MEGHE INSTITUTE OF MEDICAL SCIENCES SAWANGI WARDHA (Maharashtra). The animals were provided with standard pellet diet and water ad libitum. The Principles of Laboratory Animal Care were followed throughout the duration of the experiment.

Dose Calculation
The dose was calculated by extrapolating the human dose to animal based on the body surface area ratio by referring to the table of Paget and Barnes (1969).

Dose conversion
Animal dose= Human Dose x 0.018x5/kg body weight. Considering the human dose of haragourirasa as 250 mg, the dose to be administered in rabbits was calculated as 22.5mg/kg body weight.

Mode of administration
Animals in each group were dosed as per the study design presented in table. Animals were kept fasting for 24 hours prior administration of drugs All the animals were weighed before dose administration and volume required for each animal was calculated according to weight. Dose formulations were prepared on the day of dosing. 10% gum acaia solution was used as vehicle for drug administration. To prepare it 90 ml RO water was taken in a clean 250 ml borosil beaker then 10 g gum acacia powder was added in it after that it was put on a magnetic stirrer till it completely dissolved in water. It took about 10 minutes. 885 1. Then a calculated amount of drug according to body weight of rabbits was dissolved in this gum acacia solution. 2. It is convenient for Rasaushadhi, because it remains in suspended form in it. 3. Then calculated dose of this suspension were given orally to rabbits by 18 No. gavanging needle and 10 ml syringe.

Procedure:
For the study 10 healthy young adult albino rabbits weighing 1.5-2kg were employed for the study and grouped in the following manner: The animals were randomly selected and marked in a specific manner to permit individual identification i.e.: Head and body Same pattern of marking was followed in the both group. The animals were kept in their cages for 7 days prior to dosing to allow for acclimatization to the laboratory conditions The Test drug (Haragourirasa) was administrated to a group of experimental animals, by its oral route at one defined dose. A solution of 10% Gum acacia will be prepared and used as vehicle to administrate the drug.

Blood Collection Technique: Marginal Ear Vein
1. Rabbit was placed in a rabbit restrainer. 2. Using clippers, hair was removed from the ear. 3. Finger was placed, loosely fitting paper clip, or other form of light tourniquet at the base of the ear. 4. A 25-gauge, 5/8-inch, 1-inch needle was used.

Precautions:
1. Small quantities can be collected from the hub of a 25-gauge needle directly into a microhematocrit tube. 2. The larger needle is attached to a syringe and blood is collected slowly into the syringe to avoid collapsing the vein. 3. If blood does not flow readily, there may be a clot formation in the needle or too much negative pressure applied to the syringe. Release the negative pressure and slowly rotate the needle. 4. Repeat these steps as needed until the desired quantity of blood has been collected.

Blood Sampling
Blood samples were collected post dosage 0 hr, 1 hr, 2 hr, 4 hr, 8hr, 10 hr and 12 hr in microfuge tubes containing K2EDTA (20 μL/mL of blood, 200 mM) as anticoagulant. , after each sampling, equal volume of heparinized saline (10 IU/mL) was injected. Plasma was harvested from blood by centrifugation of samples at 2500 g for 10 min at 4°C and stored below -20°C until bio analysis.

Observations And Results:-
After administration of vehicle to the control group A the serum the blood samples were collected at 0,1,2,4,8,10,12 hours respectively. However the serum mercury concentration was at non detectable level in all rabbits during the whole period.  As far as Ayurvedic formulations are concerned the concept of drug action and absorption is designed on the basis of the Panchamahabhuta theory, Agni, rasa, guna,virya, vipaka etc of the drug. Pharmacokinetics studies supports the studies of preclinical toxicology in animals (toxicokinetics) because the drug levels in plasma or tissues are often more predictive than the dose to extrapolate the toxicity data to man. To go for the bio availability of the Ayurvedic 887 formulations especially Herbo minerals is a daunting task but they provide strong evidence base to the safety and efficacy of herbo mineral formulations iv .

886
In the present study Albino Rabbits were selected. Typically, in Pharmacokinetic studies, following administration of the compound, serial blood samples (usually 9-10 time points) are preferred, as they give a more robust concentration versus time profile within the same animal. These samples are analyzed for the parent or metabolite using a suitable analytical technique. The objective is to determine the rate of appearance and/or disappearance of the compound in the blood/plasma. Following administration of Haragourirasa serum mercury concentration was non detectable assuming it to be zero at 0 hour followed by mean plasma concentration of 7.004 µg/L at 1 hour .The concentration then increased to 9.138 at 2 hour,11.404 at 4 hour and 22.11 at 8 hour. The concentration then decreased to 20.012 at 10 hour and to 10.496 at 12 hour. Post dosing as depicted in the graph.
The mean serum mercury concentration after administration of Haragourirasa for 7 days is 29.854. Results of the repeat-dose studies indicated that there was no accumulation of drug when given for 7 days and mercury is rapidly eliminated after discontinuation of dosing .Plasma drug levels at 8 th hour post dosage on 7 th day were similar to those obtained during the single-dose study.
The pharmacokinetics profiles of Haragourirasa thus provide useful information regarding the various aspect of drug activity of mercury preparations which can add to the evidence based study of Ayurveda which further help in the global acceptance of Ayurvedic system of medicine v .