In-vivo Study of the Efficacy of Sanjeevani Vati in Snake Venom Poisoining

Background: Visha (Poison) is a substance, which after entering the body, disturbs natural and physiological functions of body (i.e. Dosha, Dhatu, Mala). Due to its potency, it may potentially cause death in a relatively short period. A significant proportion of Indians live in villages distant from the city and work in agriculture with their lower extremities exposed. Snake-rat habitat is more prevalent in rice and sugarcane fields. Aims and Objective: To study the efficacy of Sanjeevani Vati in common cobra venom poisoning and Russell’s viper venom as a first aid measure. Materials and Methods: The preparation of Sanjeevani Vatiis carried out in Department of Rasashastra, Govt. Ayurved College, Nagpur and venom was collected from snake farm’, Haffkine Institute for Training Research and Testing, Mumbai. Animal Experiment for efficacy of Sanjeevani Vati as a first aid measure on Common cobra venom and Russell’s viper venom was carried out in National Toxicology Center (NTC) Pune. Results: The results of survival period in Russell’s viper venom group were proved to be Original Research Article Mohurle and Lambat; JPRI, 33(50A): 47-54, 2021; Article no.JPRI.76120 48 statistically significant. P value is 0.0055(Unpaired t-test; Two tail).But in results of Common Cobra venom, it was observed that there is no delay in appearance of paralysis, convulsion & survival period. In fact all these symptoms appear near about at same time, when Sanjeevani Vati was given orally after ingestion of Cobra Venom. Conclusion: Sanjeevani Vati property is an ophidian. If you have Russell's viper venom, it is helpful as a first aid measure since it extends its life time. Poly Valent Anti snake venom serum does not interact with it (PVASVS).


INTRODUCTION
Visha (Poison) is a substance, which after entering the body, disturbs natural and physiological functions of body (i.e. Dosha, Dhatu, Mala) [1]. It also deteriorates the health of human being resulting into the death in very small time as it is more potent in small dosage.In India large number of people stay in villages far from city and engaged in manual agricultural work often with exposed lower extremities. In paddy and sugarcane farms there is more snakerat habitat [2]. Hence snake-bite cases are more common in villages but due to lack of emergency medical facilities mortality rate is very high there. Since reporting is not mandatory in many regions of the world, snakebites often go unreported [3]. However, some estimates put the number at 5.4 million snakebites, 2.5 million envenoming, resulting in perhaps 125,000 deaths. Others estimate 1.2 to 5.5 million snakebites, 421,000 to 1.8 million envenoming, and 20,000 to 94,000 deaths. Most snake envenoming and fatalities occur in South Asia, Southeast Asia, and sub-Saharan Africa [4], with India reporting the most snakebite deaths of any country. In India almost all of these deaths are caused by the Big Four, consisting of the Russell's viper, Indian cobra, saw-scaled viper, and the common krait. Maharashtra, one of the states of India with the highest incidence, reported 70 bites per 100,000 population and mortality of 2.4 per 100,000 per year. The other states with a large number of snakebite cases include West Bengal, Tamil Nadu, Uttar Pradesh, Assam & Kerala.
In snake-bite people take primary treatment at Rural Hospital and Primary Health Centers which are also away from periphery [5]. Sometimes due to lack of awareness people even go for local or traditional treatment. Currently the only scientifically valid treatment for snake venom envenomation is serotherapy i.e. Polyvalent Anti-Snake Venom Serum (PVASVS) [6]. It is made available at Rural Hospital, Primary Health Centre and Government Hospital [7].There is limited effectiveness of serotherapy against venom components and rapid local tissue damage is not reversed. Hence, we require the primary substitution or first aid measure before serotherapy, which will increase the survival period and decrease the mortality and morbidity. The most important factor in first aid measure is that should not interact with PVASVS.
In Ayurveda identification of poison and symptoms, antidotes of poisons and principle of treatment with 96 anti-poisonous kalpas are mentioned. But experimentally and on modern science parameters are not proved. So it is very need of science of Ayurveda to introduce Ayurveda medicines all over the world with the help of research and experimental study. Most of the experimental studies were done by in-vitro or pre-incubation assay method. Neutralizing actions of extractives of plants, upon shake venom are more notable in vitro but not confirmed in vivo study [8]. Poisonous snakes are classified into neurotoxic, vasculotoxic and myotoxic. Out of neurotoxic and vasculotoxic snakes, Common Cobra & Russell's viper poisoning are more common [9]. So the efficacy of drug on common cobra venom and Russell's viper venom poisoning needs to be investigated [10].
'Sanjeevani Vati' as remedy for snake venom poisoning mentioned in Sharangadhara Samhita Madhyam Khanda Cha. 7, which is subject to the experimental study.
As compared to PVASVS the Sanjeevani Vati is cheap, easy to carry, easily available and does not require trained persons for administration. Route of administration of 'Sanjeevani Vati' is oral as mentioned in Sharangadhara Samhita. It will reduce the fatality and morbidity as it will not interact with PVASVS. If this formulation will prove effective in-vivo study, then we can develop it further for clinical trials. I want to study the effect of 'Sanjeevani Vati' as a first aid measure in snake venom poisoning. If the efficacy of this drug will be proved then it will be a precious gift to the world of medical science and ultimately a blessing for humanity [11].

Aim and Objective
To study the efficacy of Sanjeevani Vati in common cobra venom poisoning as a first aid measure.
To study the efficacy of Sanjeevani Vati in Russell's viper venom poisoning as a first aid measure.
To study whether there is any adverse reaction between Sanjeevani Vati and polyvalent anti snake venom serum (PVASVS).

Experimental Protocol
Animal Experiment for efficacy of Sanjeevani Vati as a first aid measure on Common cobra venom and Russell's viper venom was carried out in National Toxicology Center (NTC) Pune. Following Protocol is depicted in [ Table 1], Grouping is depicted in [ Table 2].

Procedure
First preliminary drug toxicity study for each sample of venom was done, In each group, weight of animals was taken first and noted; simultaneously dose of venom, PVASVS and Sanjeevani Vati were calculated. Accordingly route, venom dose was given by IM route, after 5 min drug dose was given by oral route and then PVASVS was given after 5 min by IV route. After dosing animals were observed for 24 hrs up to 7 day's, Comparative observations were tabulated.

RESULTS AND OBSERVATION
Lethal dose (LD 50 ) of our animal experiment, we gave 31 µgm of common cobra venom. For our animal experiment we gave 345 µgm of Russell's viper venom.
Analysis of common cobra venom are depicted in [ Fig. 1].
Analysis of Russell's viper venom are depicted in [Fig. 2].
In many Ayurvedic texts it is stated that remedy of Sanjeevani Vati is effective in all type of snake venom poisoning.
The results of survival period in Russell's viper venom group were proved to be statistically significant. P value is 0.0055 (Unpaired t-test; Two tail).
But in results of Common Cobra venom, it was observed that there is no delay in appearance of paralysis, convulsion & survival period. In fact all these symptoms appear near about at same time, when Sanjeevani Vati was given orally after ingestion of Cobra Venom.

DISCUSSION
Any drug which acts by any other mode of action i.e. other than chemical neutralization cannot be studied by these methods. This is particularly true of drugs acting on nervous system which primarily act by blocking of receptor sites or competitive inhibition. Both these methods give results which seldom stand true in clinical situations. Thus 'in vivo' study becomes of  Dosing: For the ease of observation albino mice were chosen for the experiment, while oral and IM dosing was not a problem, but IV dosing PVASVS proved a big hazard.
As the required amount of PVASVS was very small, its IV delivery is a very skillful and difficult procedure.

Graph showed analysis of russell's viper venom
Initially it was decided that 80% fatal dose of venom should be used in cobra group & 100 % fatal dose of venom should be used in Russell's viper group. According to previous studies done at Tilak Ayurved Mahavidyalaya, Pune, they suggest that in future periments of this kind 100% fatal dose of 120% fatal dose of Russell's viper venom should be injected.To resolve this experimentally, we conducted the pilot study using two animals per dosage group. In the first ven 31, 41 & 51 µgm gm (in Russell's viper) dose levels of venom respectively. At this level in cobra group 100% animal died in 140 minutes (average) at minimum dose i.e. 31µgm. But in Russell's viper group no fatality or bservable morbidity was seen. The reason for this discrepancy may be loss of potency of venom during storage. Therefore in Russell's viper group two more stages by increasing venom doses are carried, & at 345 µgm fatality gm of cobra venom & gm of Russell's viper venom were used.
For the ease of observation albino mice were chosen for the experiment, while oral and IM dosing was not a problem, but IV dosing PVASVS proved a big hazard.
As the required amount of PVASVS was very small, its IV delivery is a very skillful and difficult During Observations: It was very difficult to observe and distinguish the pre paralytic signs of the Common cobra venom. It was impossible to record the pre-paralytic signs. In paralytic signs tremors was not observed. Other signs i.e. paralysis, convulsion in Common cobra venom i.e. control group [I] were observed. As Russell's viper venom is haemostatic, external bleeding from mouth, nose, necrosis at the bite site are the common symptoms in humans. But these symptoms were not observed in Russell's viper venom group i.e. control group [V].
In Common cobra control group (Gr. I) appearance of paralysis was observed after 130 min (average) and that of drug group (Gr. II) it was after 111.67 min (average) i.e. time duration of appearance of paralysis was decreased by 18.33 min in Sanjeevani Vati group, which is statistically not significant. P value is 0.2237 (Unpaired t tail).
In Common cobra control group (Gr. I) appearance of convulsions was after 148.33 min (average) and in drug group (Gr. II) it was after 145 min (average) i.e. time duration of appearance of convulsions was decreased by 3.33 min in Sanjeevani Vati group, but P value is notstatistically significant. P value is 0.7878 (Unpaired t-test; Two tail).

Duration Of Survival
; Article no.JPRI.76120 It was very difficult to observe and distinguish the pre-paralytic and paralytic signs of the Common cobra venom. It paralytic signs. In paralytic signs tremors was not observed. Other signs i.e. paralysis, convulsion in Common cobra venom i.e. control group [I] were observed. As Russell's viper venom is haemostatic, external bleeding from mouth, nose, ear and necrosis at the bite site are the common symptoms in humans. But these symptoms were not observed in Russell's viper venom group i.e.
In Common cobra control group (Gr. I) appearance of paralysis was observed after 130 age) and that of drug group (Gr. II) it was after 111.67 min (average) i.e. time duration of appearance of paralysis was decreased by 18.33 min in Sanjeevani statistically not significant. P value is 0.2237 (Unpaired t-test; Two In Common cobra control group (Gr. I) appearance of convulsions was after 148.33 min (average) and in drug group (Gr. II) it was after 145 min (average) i.e. time duration of arance of convulsions was decreased by group, but P value is notstatistically significant. P value is 0.7878 In Common cobra control group (Gr. I) duration of survival was 151.33 min (average) & that of drug group (Gr. II) duration of survival was 148.66 min (average) i.e. duration of survival was decreased by ~3 min in Sanjeevani Vati group, but P value is notstatistically significant. P value is 0.8375 (Unpaired t-test; Two tail).
In Common cobra venom + PVASVS (standard group III) all mice survived completely without showing any signs.
In Common cobra venom + Sanjeevani Vati+ PVASVS group (Gr. IV) all mice survived completely without showing any signs. No adverse interaction between Sanjeevani Vati and PVASVS was seen.
In Russell's viper venom group (Gr. V) duration of survival was 2020 min (average) & in drug group (Gr. VI) duration of survival was 2470 min (average) i.e. duration of survival was delayed by 450 min in Sanjeevani Vati group, which is statistically significant. P value is 0.0055 (Unpaired t-test; Two tails).
In Russell's viper venom + PVASVS group (Gr. VII), all mice survived without showing any signs.In Russell's viper venom + Sanjeevani Vati+ PVASVS group (Gr. VIII), all mice survived without showing any signs. No adverse interaction between Sanjeevani Vati and PVASVS was seen.

CONCLUSION
The Present study partially proves the 'Vishaghna' property i.e. Ant ophidian property of the Sanjeevani Vati. Itis useful as a first aid measure in Russell's viper venom because, Itincreases the survival period in Russell's viper venom. It does not interact with Poly Valent Anti Snake Venom Serum (PVASVS).

RESEARCH SIGNIFICANCE
The study highlights the efficacy of "Ayurveda" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.

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.

ETHICAL APPROVAL
It is not applicable.