Preclinical Evaluation of Antidotal Property of Mritasanjeevana agada in Poisoning- A Study Protocol

Background: As the poisoning is becoming a threat to rural India, it is necessary to increase the survival time to avail the primary treatment. For the treatment of poisoning, Agada is described in Ayurveda as an antidote. Agada is a polyherbal or herbomineral formulation constituted with combination antitoxic drugs along with some antioxidant, immunomodulator or hepatoprotective drugs. But they need to be revalidated for their efficacy and safety on the basis of contemporary assessment parameters Aim: Evaluation of antidotal property of Mritasanjivana Agada in poisoning. Objectives: 1) To increase the survival time after the administration of Mritasanjivana Agada in snake venom and aluminium phosphide poisoning in albino mice. 2) To compare the efficacy of Mritasanjivana Agada and Anti-snake venom as an antidote. 3) To standardize the Mritasanjivana Agada. Methodology: Mritasanjeevana Agada will be prepared and standardized. Cobra venom poisoning and aluminium phosphide poisoning have been selected as the representative for the animate poison and artificial/ synthetic poison. After inducing poisoning in mice, one group will receive its Study Protocol Wadnerwar; JPRI, 33(38B): 271-280, 2021; Article no.JPRI.71214 272 standard antidote and other will receive standard antidote with Mritasanjivana Agada. The third group will receive only Mritasanjivana Agada without its standard antidote. All the groups will be assessed on the basis of hematology, biochemistry, Superoxide dismutase (SOD) level, Malondialdehyde (MDA) level and histopathology in case of death of the animals. Results: Mritasanjeevana agada is expected to increase the survival time in the treatment of snake venom and aluminium phosphide poisoning in albino mice. Conclusion: Mritasanjeevana agada may be as efficacious as Anti-snake venom as an antidote.


INTRODUCTION
Poisoning is very common global public health problem. It possesses 45 th rank in the total death in the whole world. Near about one million people die due to poisoning each year. Approximately 3,70,000 deaths per year are due to pesticide poisoning. According to WHO data in 2012, it is estimated that about 1,93,460 people died worldwide due to unintentional poisoning [1]. The incidence of poisoning in India is among the highest in the world. It is estimated that more than 50,000 people die every year due to poisoning in India [2]. Poisoning is the fourth most common cause of mortality especially in rural India where the mortality rate varies from 15-30% [3][4].
According to the National Poisons Information Centre, New Delhi, analysis of poisoning calls showed that the highest incidence of poisoning was due to household agents (44.1%) followed by drugs (18.8%), agricultural pesticides (12.8%), industrial chemicals (8.9%), animals bites and stings (4.7%), plants (1.7%), unknown (2.9%) and miscellaneous groups (5.6%) [2]. Treatment protocols and effective antidotes are available even though the rate of mortality due to poisoning is high. The causes of death due to poisoning depends on various factors like dose of poison, late diagnosis in case of snake bite, delayed reporting of cases especially in rural areas etc. This may lead to worsened condition of the patient.
In Ayurveda, various antidotes are described for different types of poisoning for internal as well as local use in emergency condition. Mritasanjeevana agada [5] which is a herbomineral antidote, is indicated in all types of poisoning to save the person who is likely to die or apparently dead due to poisoning. [6]: These are the twenty four treatment modalities which help to cure patient afflicted with poison. It is one of the ancient classical therapy through which the life of individual can be regained. Nowadays this therapy has disappeared. This therapy was considered as universal antidote which can be used to nullify the toxic effect of all types of poison. In this treatment Mritsanjeevana Agada is used to revive the poisoned patient who is apparently dead. It is described that this Agada was produced by Lord Brahma before the emergence of Amruta (Nectar).

Mritasanjeevana Agada [5]:
It is described that all the drugs should be collected in pushya nakshatra. All the drugs are taken in equal quantity and are triturated well to form paste. Then pills should be prepared from all the paste.
In the present study, Mritasanjeevana Agada will be prepared according to the classical reference and it will be standardized on the basis of physicochemical parameters as well as to estimate the heavy metal content with the help of inductively coupled plasma atomic emission spectroscopy (ICP-AES). Cobra venom poisoning and aluminium phosphide poisoning have been selected as the representative for the animate poison and artificial/ synthetic poison with the aim to evaluate the antidotal property of Mritasanjeevana Agada in these two poisoning conditions to increase the survival time after the administration of Mritasanjeevana Agada in snake venom and aluminium phosphide poisoning in albino mice. To find out whether Mritasanjeevana Agada is as efficacious as Antisnake venom in snake venom and aluminium phosphide poisoning respectively, the study groups will be compared with Mritasanjeevana Agada.

Methodology
Collection, Identification and Authentication of drugs: All the drugs will be collected from authentic sources.

Preparation of
Mritasanjeevan Agada: Physical impurities will be removed from all the drugs. Powder of all drugs will be prepared separately. Then powder of all drugs will be taken in equal quantity in a kharala and will triturated well. Tablets will be prepared from it.

ICP -AES (Qualitative and quantitative):
Inductively coupled plasma atomic emission spectroscopy (ICP-AES) for qualitative and quantitative analysis of Arsenic at IIT, Powai, Mumbai.

II.
Experimental Animal Study:
 Aluminium phosphide Tablets will be purchased from market.  Distilled water will be purchased from GMP certified company.

Administration of test drug (Mritasanjeevana Agada): Fine suspension of Mritasanjeevana
Agada will be prepared in distilled water as per dose level and it will administered 30 minutes before the administration of snake venom or aluminium phosphide to animals of the experimental group on the first day of experiment. From the second day, fresh suspension of the test drug will be prepared every day and administered between 10:00 am to 10:30 am daily for minimum seven consecutive days or till the recovery to each mouse by a single oral gavage. The animals will be dosed using a stainless steel intubation needle fitted onto a suitably graduated syringe.
The dosage volume administered to individual mice will be adjusted according to its most recent recorded body weight.

Route of administration of Drug:
 Snake venom will be administered intramuscular (IM)  Inj Polyvalent Antisnake Venom (PVASV) will be administered intravenous (IV).  Aluminium Phosphide will be administered orally.  Study drug will be administered orally.

Duration of study drug administration:
Minimum 7 Days and till the recovery of animals.

Comparison: All Groups are compared.
Dose calculations: Dose of the drug will be calculated by extrapolating the human therapeutic dose to mice on the basis of body surface area ratio.

Formula for conversion of dose Animal dose=
Human dose x 0.018 (conversion factor)

1) Cobra venom
Fatal dose -12mg of dried venom for human beings Animal dose=7.2mg/kg body weight of mice IM

2) Aluminium phosphide
Fatal dose -3 gm for human beings Animal dose=1.8 mg/kg body weight of mice orally

3) Inj Polyvalent Antisnake Venom
Therapeutic dose for human beings-bolus dose of 200 ml ASV and repeated doses of 100 ml ASV every 6 hours Animal dose=120 ml/kg body weight of mice IV

Housing and feeding conditions
As per the OECD Guidelines following conditions should be maintained,  The temperature in the experimental animal room should be 22ºC (±3ºC).  Humidity should be at least 30% and preferably not exceed 70%, other than during room cleaning; the aim should be 50 -60%.  Lighting should be conventional laboratory diet may be used with an unlimited supply of drinking water.  Animals may be housed individually or be caged in small groups of the same sex.

OBSERVATIONS
The following parameters will be observed during the course of the study.
 Body weight: Before the start of drug administration and at the end of the study.  Food Intake: Before the start of drug administration, then once daily and at the end of the study.  Survival time in all groups  Survival rate in all groups  Hematological examination -Before the start of drug administration and at the end of the study.  Renal and hepatic function tests -Before the start of drug administration and at the end of the study.  Superoxide dismutase (SOD) Level  Malondialdehyde (MDA) Level  Animals found dead during the examination will be autopsied as soon as possible. A macroscopic examination will be done of organs and tissues.  Organ measurement and Histopathological examination will be performed to identify the cause of death and the nature (severity or degree) of the toxic changes present.

Statistical Analysis
The results will be presented as Mean+ Standard Error (SE) of means in each group. Statistical comparisons will be performed by both paired, unpaired student's t test followed and One Way ANOVA test to determine the significant difference between the groups at P<0.05 (level of significance).

RESULTS
 Mritasanjeevana Agada is expected to increase the survival time in the treatment of snake venom and organophosphorus poisoning in Swiss albino mice.  It is expected to be as efficacious as Antisnake venom as an antidote.  If so, it can also be used in the cases where no antidote is available.  The drug will be standardized for future reference and use.  There will be exploration of the fundamental concept of Agada in Ayurveda.  There will be standardization and validation of safety and efficacy of at least one

DISCUSSION
Mritasanjeevana Agada cures all types of poison, makes a person victorious. It revives a person who is apparently dead because of poisoning. It also cures fever. If it is inhaled, applied externally as an ointment, carried in the body as an amulet, smoked or kept in the house, it annihilates the afflictions by evil spirits, poisons, germs, sin, mantra, thunder-bolt and enemies. It counteracts the evil effects of bad dreams and stri-dosha (poisons secretly given by women). It prevents untimely death, fear of water and fear of thieves. It endows a person with wealth, food-grains and success in undertakings. It promotes auspiciousness, nourishment and longevity. Mritasanjeevana Agada is an excellent recipe helps in the revival of a dead person. This agada is indicated in 8 th vega (Impulse) of Visha (poison) [8]. Acharya Sushruta has also mentioned similar Agada with the name Sanjeevana Agada to regain the life of dead person [9].
Agada is an antidote mentioned in Ayurvedic toxicology. It not only counteracts the action of poison but it also possesses the therapeutic efficacy. In the literature survey, some reviews are found on Agada [10] but a very few preclinical studies are found in the context of Bilwadi Agada [11] Panchashirish Agada [12], Dashang Agada [13], Paravatadi Agada [14] and Maha Agada [15]. Hence it is very necessary to standardize the Agada and to evaluate their safety and efficacy so that they can be used in the management of poisoning cases [16][17][18][19].
In the present scenario, as the death rates are increasing due to poisoning, Mritasanjeevana Agada may increase the survival time and survival rate of the poisoned patients. As the scientific research is not conducted to evaluate the efficacy of this ancient antidote, an experimental study is designed in animals with the intention to standardize the drug and to evaluate the efficacy of Mritasanjeevana Agada so that the evidences can be generated to use it in the emergency management of poisoning as an adjuvant therapy to increase the survival period of the patient which is a golden time for the treatment.

CONCLUSION
Mritasanjeevana agada may be as efficacious as Anti-snake venom as an antidote.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors CONSENT It is not applicable.