Evaluation of Relative Oral Bioavailability of Shankha bhasma Prepared by Two Different Methods in Healthy Volunteers: A Study Protocol

Background: Rastarangini Shankha (Conch Shell) Bhasma is one of the traditional medicines reported to be used for centuries in the treatment of calcium deficiency disorders. It proves to be a good source of calcium. But reports on its bioavailability are hardly available. This study aims to assess the methods of preparation of Shankha Bhasma and assess its relative oral bioavailability in healthy volunteers. Materials and Methods: This study will include preparation of Shankha Bhasma in Rasashastra department. The traditional method will be giving Putas. Another method includes heating Shudhha Shankha on gas flame. The products obtained with these procedures will undergo Bhasma Pariksha that includes organoleptic characteristics, physicochemical parameters, microbial contamination and other relevant tests. This will be randomized single blind control study of 90 healthy volunteers (30 in each group). The data on oral bioavailability in healthy volunteers will be analyzed by using Pair and unpaired “t” Test, One way ANOVA and descriptive statistics. Results: The relative oral bioavailability of Shankha Bhasma is expected to show all Study Protocol Dukare and Rathi; JPRI, 33(63B): 241-248, 2021; Article no.JPRI.80853 242 pharmaceutico analytical parameters within normal range and reflect significant effects on healthy volunteers. Conclusion: Significant efficacy of this product directs a new gateway for the management of bioavailability of calcium drugs.


INTRODUCTION
Ayurveda is known as age old well-documented Indian system of Medicine. Rasashastra--a popular branch of an Ayurveda pharmaceutics, flourished during the medieval period mostly deals with therapeutic uses of minerals, metals and mercurial preparations [1,2]. Rasashastra deals with the ancient pharmaceutical preparation of Ayurveda [3].The subject is related to the procedures like Shodhana, Marana, Jarana of Rasadi Dravyas like Rasa, Maharasa, Uparasa, Sadharanarasa, Dhatu and Updhatu [4].
They have additional advantages over herbal drugs i.e. Rasa-Aushadhies are effective in smaller doses, having agreeable taste, quick action and require shorter duration of administration. Almost all the substances used in Rasashastra such as metals, minerals, poisons and animal origin drugs need to be transformed to make them human friendly, pharmacotherapeutically useful and safe. Multiple pharmaceutical processing like Shodhana and Marana were developed later which render toxic substances into non-toxic, more potent for elimination of disease and promoting health. Sudhavarga is one among them, which is considered as one of the group of drugs in the field of Rasashastra. It consists of many drugs like Sudha, Shankha, Shukti, Godanti Swarasa is prepared by soaking the drug with 2 parts of water overnight and then filtering it through a piece of cloth.
Calcium bioavailability is considered as significant when an individual is losing or growing bone or when calcium intake is low. Various dietary and environmental factors such as protein, , sodium, caffeine vitamin D, fructose and phosphorous show impact on Calcium absorption in the body. in addition, individual's genetic makeup, including the vitamin D receptor genotype, may also play a key role in calcium absorption. Supplementation with various calcium preparations is now available. However it is observed that the bioavailability of many commercial calcium products is dissimilar [7]. Hence, this study is planned for the bioavailability of Shankha Bhasma compound.

Rational of Study
In today's era calcium deficiency disorders are increasing day by day. According to age dietary calcium intake is ranging from 400 to 1200 mg per day. Several modern diets failed to provide the required level of calcium. Large number of standard calcium supplements is available in market, but today's necessity is the high solubility calcium content, that could be an efficient source of calcium through dietary intakes. Bhasma contain Nano particles which is having higher absorption and distribution property. There is no information available carrying the bioavailability of Shankha Bhasma as calcium form.
There were many studies conducted regarding the preparation of Shankha Bhasma by doing shodhana in amaldravya swarasa, but Shodhana in Amlika Swasaras is not done before though this drug is freely available and economical. Shankha Bhasma is used in many formulations and indicated in many gastrointestinal diseases but oral bioavailability study of Shankha bhasma is not conducted till now. Therefore this study is planned to evaluate the oral bioavailability of Shankha Bhasma and compare with the Standard calcium supplement.

Study Design
This is the pharmaceutical, analytical and experimental study. Study will be conducted at Mahatma Gandhi Ayurved College, Hospital & Research Centre, Salod (H), Wardha. Present work will be conducted under following headings;

a)
Pharmaceutical study: This study is related to drug preparation in which two different batches of Shankha Bhasma will be prepared to establish pharmaceutical standardization. Pharmaceutical study will be done in following steps; I) Procurement of Raw materials: All raw materials required for the study will be procured from authentic reliable sources. Kumari (Aloe Vera) will be procured from Herbal Garden (MGAC), Amlika from authenticated vender. Shankha will be collected from well known raw material supplier from Nagpur.

II)
Authentication of Raw materials: Raw drugs (Herbs) will be verified and authenticated by Department of Dravyaguna of MGAC & RC. Shankha will be authenticated by Department of Rasashastra.
Raw drugs will be standardized as per A.P.I.

Preparation of Amlika Swarasa
Take one part fruit pulp of Amlika Soaked overnight in double quantity of portable water Next morning macerate the mixture And filter through a white clean cloth to obtain Swarasa

Flow diagram of Shodhana (Purification) of Shankha
In Khalvayantra Shankha will be made in to smaller pieces.
Then Shankha will be tied in a white clean cloth and make a Pottali.
It will be hanged in Dolayantra containing AmlikaSwarasa (It is a Shodana Dravya) As liquidmedia is subjected for Swedana of one Yama (3 hours) Later the drug in the cloth will be washed with warm water and dried and stored as Shuddha Shankha.

ShuddhaShankhawill be triturated it in Khalvayantra
Kumari(Aloe Vera) Swarasa will be added in KhalvaYantraand triturate till it becomes a fine paste SmallChakrikas will be prepared and dried The Chakrikas will be enclosed in SaravaSamput The Samputa is sealed appropriately and dried under sun Later it will be subjected for one Gajaputa with dry cow dung cakes When cool on its own, the white colored Bhasma inside the Samputa will be collected It will be Triturated and stored in airtight container as Shankha Bhasma

V) Flow diagram of Marana (Incineration) procedure of Shankha (Method 2)
The Shudhha Shankha will be heated on gas flame with using Charcoal Heat till it becomes brittle

Collect brittle Shankha and powdered in KhalvaYantra
Stored in airtight container as Shankha Bhasma.

b) Analytical study
For analytical study organoleptic characters and physicochemical parameters, microbial contamination, and other sophisticated tests like Particle size distribution analysis, SEM -EDX, AAS, FTIR, XRD, and GCMS will be done.

c) Bioavailability study
This will be randomized single blind control study of 90 healthy volunteers (30 in each group).The subject will be selected from Mahatma Gandhi Ayurveda College Hospital & Research Centre, Salod(H),Wardha.

Eligibility Criteria
Volunteers between age group 20 -40 years of male sex after physical examination and Complete blood count (CBC), Blood sugar, Liver function test, Kidney function test, Lipid profile, Blood pressure with normal values will be selected.

Interventions
Total 90 healthy volunteers divided in 3 groups (30 in each group) one standard group and other two will be study groups. For standard control group, Calcium standard supplements will be given 500mg once a day before meal and for

Investigation During Treatment
Complete blood count (CBC), Liver Function Test (LFT), , Kidney Function Test (KFT), Lipid profile, Blood sugar, Urine routine and microscopic, Urine calcium, Blood Sr. Calcium Level.

Criteria for discontinuing or modifying allocated interventions
Subjects will be withdrawn from the study if any untoward incidence, features, feature of drug sensitivity or any other disease or problem arises the subjects will be offered from free treatment till problem subsides.

Follow up period after treatment
After 24 hours, 3 rd day, 7 th day,15 th day of drug administration.

Primary outcomes
The relative oral bioavailability of Shankha Bhasma will be observed.

Implementation
Principle invigilator will allocate and enroll the patients.

Statistical Analysis
Statistical analysis will be done by applying pair and unpaired "t"test, One way ANOVA and descriptive statistics.

OBSERVATION AND RESULTS
Observations will be noted and presented in the form of tables, charts, photographs etc. The expected result of the study is that study group B and C with intervention Standard calcium supplement will be potentially more effective as a good calcium source. Patient who will take all fallow up by following Pathya and Apathya during treatment will have less chances of calcium deficiency disorders as compare to group A.

DISCUSSION
Conch is a common name that is applied to a number of different medium to large sized "sea snail "or their "shells". The term generally applies to large sea snails that have a high spire and a siphonal canal (comes to a point at both ends of the shell).True conch are marine Gastropoda Molluscs in the family Strombidae, specially in the genus Strombus and other closely related genera such as Eustrombus. Chemically it is Calcium Carbonate (CaCO 3 ). It contains mainly carbonate of calcium, iron, magnesium, sulphate, phosphate and chloride.
Medicinal plants are the major source as a medicine for the majority of the population throughout the globe. Increase in population, cost of allopathic treatment for general ailments, side effects of modern drugs and development of resistance have led to increase emphasis on the use of herbs and minerals as source of medicines and health supplements [11].
However according to the modern science, each drug needs to be scientifically, pre-clinically and clinically evaluated for the global acceptance [12]. In medical field, the analytical studies of Ayurvedic formulations help to form the base for standardization. It is the need of the hour to generate evidence for existing literature and for reproducibility [13][14][15].
Standardization of Ayurvedic medicines on pharmaceutical and analytical level is the most important aspect before beginning research on pre-clinical and clinical ground. In this protocol two different methods of preparation will be used to make Shankha Bhasma. There intervention will be with standard calcium supplement. One group will receive Shankha Bhasma prepared by method-1 and other will receive Shankha Bhsama prepared by method-2 and third group will receive Standard Calcium Supplement. The assessment will be done based on subjective and objective parameters. After that data will be analyzed by using statistical test and present in the form of table and charts [16][17][18][19][20][21][22][23][24][25][26][27][28].

CONCLUSION
This research work on Shankha Bhasma will show all pharmaceutico analytical parameters within range and significant effects on healthy volunteers to evaluate its relative oral bioavailability. As the ingredients are easily available and method of preparation does not require much time and man power therefore if this preparation show significant efficacy then this will open a new gateway for the management of bioavailability of calcium drugs.

TRANSLATORY COMPONENT
If the study is successful then this will be used in another clinical studies establishment on various calcium deficiency diseases.

NOTE
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.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.