PARUL UNIVERSITY, LIMDA, WAGHODIA,

VADODARA, GUJARAT

Completed Proforma for Registration of Subject for doctor of philosophy

“THE EVALUATION OF A POLYHERBAL FORMULA SHIVAGUTIKA FOR ITS ANTI INFLAMMATORY EFFECT- AN IN- VITRO AND AN EXTENDED CLINICAL PILOT STUDY”

Submitted by

DR . PRADNYA . CHITTAWADAGI

_{M.d.(ayu), M.H.Sc (APPLIED NUTRITION)}

Guide

Dr. NARESH KORE

                                                            M.D. (Ay.), Ph.D.(Ay.)

PROFESSOR

DEPARTMENT OF KAYA CHIKITSA

parul institute of ayurveda and research, limda,

waghodia, vadodara (Gujarat)

2017 - 2018

ABSTRACT:

In recent years, the prevalence of prediabetes has increased worldwide, and it is estimated that millions of adults in United States have prediabetes. Prediabetes is characterized by elevated blood glucose levels, which can lead to the development of type 2 diabetes and its associated complications.

 In addition, chronic low-grade inflammation has been identified as a key factor in the pathogenesis of type 2 diabetes. Inflammatory markers such as C-reactive protein, interleukin-6, and tumor necrosis factor-alpha are often elevated in individuals with prediabetes, indicating the involvement of inflammation in the development of the condition.

Therefore, it is imperative to investigate the relationship between prediabetes and inflammatory markers to identify potential therapeutic targets for the prevention and treatment of type 2 diabetes and its complications.

Previous studies have shown that anti-inflammatory agents can be used to inhibit the enzymes involved in the synthesis of prostaglandins and leukotrienes,adipokines which are key mediators of inflammation followed by Diabetic pathogenesis. Thus the use of herbal formulations for the management of prediabetes and inflammation is gaining interest.

In this context, the current study aims to investigate the in vitro antioxidant, anti-inflammatory, and antidiabetic activities of Shivagutika, a polyherbal formulation, in specific cell lines and human subjects.

The formulation, which has been used in traditional medicine to treat prameha, is believed to have therapeutic potential due to its unique combination of ingredients, including the main ingredient Shilajathu.

 By investigating the potential benefits of Shivagutika in the management of prediabetes and inflammation, this study aims to provide valuable insights into the development of new and effective treatment strategies for prevention of type 2 diabetes and its associated complications.

Introduction:

Prediabetes is a metabolic disorder that is characterized by higher than normal blood glucose levels but not high enough to be classified as diabetes. The prevalence of prediabetes has been increasing globally, with over 84 million adults in the United States estimated to have the condition. Prediabetes is a precursor to type 2 diabetes, which is a major cause of morbidity and mortality worldwide.

Chronic low-grade inflammation is associated with prediabetes, as evidenced by increased levels of inflammatory markers such as C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Inflammation is thought to play a key role in the pathogenesis of type 2 diabetes and its associated complications.

The importance of investigating the relationship between prediabetes and inflammatory markers is paramount, given the increasing burden of prediabetes globally. Identifying specific inflammatory markers associated with prediabetes may provide valuable clinical information for early detection and risk stratification of individuals who are at high risk of developing type 2 diabetes. Moreover, understanding the underlying mechanisms of inflammation in prediabetes could help identify novel therapeutic targets for the prevention and treatment of type 2 diabetes and its complications.

Polyphenols and flavonoids have been identified as effective antioxidant agents that can fight against diabetes, cancer, and neurodegenerative diseases. Shivagutika, a polyherbal formulation, is believed to have anti-inflammatory and antidiabetic activity due to the presence of its main ingredient Shilajathu.

The formulation has been used in the treatment of Prameha  by Lord Shiva to his son Lord Ganesha, according to mythological references due to his sedentary habits.

In this study, the in vitro anti-inflammatory activity of Shivagutika will be demonstrated using Human cellline. The study aims to extend the already established link between pre-diabetes and inflammatory markers by investigating the effects of Shivagutika in human cell lines and human subjects. The unique properties of Shivagutika, such as its ability to show Shamana (cure morbid factors with oral medicine), Brimhana (nourishment), and Rasayana effect (immunity enhancer), make it a promising formulation for investigation as a potential therapeutic agent for the treatment of prediabetic inflammatory markers and thus prevention of type 2 Diabetes with its associated complications.

6.2. Review of Literature:

1. Detailed study on anti diabetic mechanism and its responsive cellular enzymes
2. Detailed study of Shivagutika , its composition and mode of action in prediabetic subjects.
3. Detailed study of inflammatory mechanism including- WSRT Interleukin 6 in human cell line
4. Detailed study of inflammatory mechanism including- WSRT Interleukin 6 in Pre diabetic subjects.

Previous Works done :

1)      Pharmaceutical Analysis of Shivagutika: An Ayurved Formulation 1*DR.NIKUNJ THAKKAR, 2DR. KIRTIPRAKASH UPADHYAY, 3DR KSHAMA GUPTA, 4DR PRAKASH KUMBAR, 5DR MAHESH M PARAPPAGOUDRA, 6DR. JANHAVI J. PATHARKAR- International Journal of Pharmaceutical Research | Apr - Jun 2020 | Vol 12 | Issue

2)     A CLINICAL STUDY TO EVALUATE THE ROLE OF SHIVAGUTIKA IN CYSTIC OVARIAN DISEASE

Vishwesh BN1* , Manjari Dwivedi2 1. Reader, Dept. of Prasootitantra and Stri roga, SDM College of Ayurveda, Hassan, Karnataka, India. 2. Professor, Dept. of Prasooti tantra and Stri roga, Faculty of Ayurveda, IMS, BHU, Varanasi, Uttar Pradesh, India

3)     Kishore kumar.R- A Clinical Study On Effect Of Nisha Amalaki In Madhumeha- A Controlled Study,2002,Rajiv Gandhi University of Health Sciences,Karnataka, Banglore.

3)     Dongre Raj Kumar- Clinical study on management of Diabetes gangrene(prameha janya) with the use of ayurvedic medicine,2004,Rajiv Gandhi University of Health Sciences,Karnataka, Bangalore.

4)     Vanktesh.S-Study of concept of madhumeha and its clinical evaluation wsr to   Diabetes mellitus, 2008,Rajiv Gandhi University of Health Sciences,Karnataka, Bangalore.

NEED FOR STUDY :

The relationship between prediabetes, diabetes, and inflammatory markers has already been established in previous studies. However, a new study aims to investigate the potential benefits of an herbal formulation called Shivagutika in the context of prediabetes and inflammation. This polyherbal formulation has been traditionally used in Ayurvedic medicine for the treatment of various conditions, including diabetes. By exploring the in vitro antioxidant, anti-inflammatory, and antidiabetic activities of Shivagutika, this study hopes to shed light on the potential mechanisms and clinical implications of using this herbal formulation in the management of prediabetes and its associated complications.

6.3 Scope and Statement of the problem

–  Shivagutika is one such formulation that has wide indications /phalashruti

–  Shivagutika- mode of action and terms of cellular interventions would be better established

–  1^st study to communicate in terms of biochemical influences, molecular and cellular endocrinological interventions of Shuvagutika at a cellular level using Interleukin 6 a significant anti inflammatory marker.

–  1^st study to the bridge and there by connecting phala shruti mentioned in Ayurvedic texts with biochemical and pharmacologically standardized cell-mediateded assay markers in a pathological state.

6.4 Objectives of the study

To evaluate the effect of Shivagutika on process of inflammation in vitro and in clinical pilot study  in following two sections-

–  In vitro- specific human cell line study with – IL6- Interleukin 6 markers

–  Clinical Pilot study- in pre diabetes- measuring serum- IL 6- Interleukin 6 markers.

VII.     Materials and Methods

Part 1-

–  Source of – human cell line – IL6 – National Centre for Cell Science- Savitribai  Phule Pune University(SPPU).(Dept of Biotechnology, Aided by Govt Of India)

–  Standardisation.,Extraction and application of the Shivagutika

–  Study to be done at govt licenced lab.

–  Results- to be interpreted statistically

Methods

–  Plant Material Collection –

–  Extract Preparation Methanol and water were taken as polar and non-polar solvents for extraction purposes.

–   For this 10g of medicine powder dissolved in 100 ml of the respective solvents and then kept for 48 hrs in a rotatory shaker. The extracts were filtered and kept for drying at 30°C. The dried extract was scrapped and refrigerated for further use at 4°C

–  Phytochemical analysis is done by both qualitative and quantitative methods.

–  Qualitative Phytochemical Analysis The leaves were analyzed for various qualitative methods of phytochemicals.

–  The presence of alkaloids was determined by the Mayer test. Borntrager’s test was performed to determine the presence of glycosides .

–   Sodium hydroxide and lead acetate tests were performed to show.

–  The presence of flavonoids showed by .

–   Molish test was used to determine the presence of carbohydrates. The analysis of terpenoids was done by the Salkowski test.

–   Froth test determines the presence of Saponin.

–   Phenols presence showed by ferric chloride test.

–  Quantitative method The quantitative phytochemical analysis was done for the total phenolic count and flavonoids content.

–  Total phenolic determination Total phenolic content of each Shivagutika extract was determined by  with some modifications and taking gallic acid as standard and the assay performed in a microtitre plate.

–  Test sample was taken in 2.5µl, 5µl, 7.5µl and 10µl concentrations and was diluted with distilled water to make a final volume.

–   Subsequently, the solution was mixed with 10µl of fresh Folin Ciocalteu reagent and after 5 min 100µl of Na2CO3 (7.5%) solution was added with 130µl of distilled water.

–  The plates were incubated for 90 minutes at 30°C.

–   The absorbance was measured at 750nm.

–  The results were expressed as Gallic acid equivalents (mg gallic acid/g dried extract).

–  Total flavonoids determination The total flavonoid content was determined by with some modifications and taking quercetin acid as standard, the assay performed in a microtitre plate.

–   The plant extracts samples were taken in different 2.5µl, 5µl, 7.5µl and 10µl concentrations and the final volume were prepared adding more distilled water.

–  To each sample 10µl of NaNO2 solution (5%) was added.

–  After 5 minutes, 10µl of an AlCl3 solution (10%) was added and allowed to stand for 6 minutes at room temperature.

–  A solution (1M) was added to the mixture and absorbance was measured at 510nm.

–  Rutin was used as a standard for the calibration curve.

–  Total flavonoids content of the extracts was expressed as mg quercetin acid equivalents per gram of sample (mg/g).

Part 2-

7.1       Source of Data

In the present study, the research scholar proposes to take the patients attending the Outpatient Wing of

a) Department of Kaya chikitsa of Khemdas Ayurveda Hospital, the teaching hospital of Parul Institute of Ayurveda and Research.

b) Department of Kaya chikitsa of Shree Mahaganapathy Ayurveda Medical College & Hospital, Dharwad (Karnataka).

7.2       Methods of collection of Data

            A clinical survey of patients attending OPD and IPD of Department of Kaya Chikitsa of

       a) Khemdas Ayurveda Hospital, teaching hospital of Parul Institute of Ayurveda and Research. the teaching hospital of Parul Institute of Ayurveda and Research.

 b)Shree Mahaganapathy Ayurveda Medical College & Hospital, Dharwad (Karnataka)will be made and patients fulfilling the criteria of diagnosis as per the proforma will be selected for the study.

A clinical evaluation of patients will be done by collection of data through information obtained by history, physical examination, and blood tests.Review of literature will be collected from Central Library, Department of Kaya chikitsa of Shree Mahaganapathy Ayurveda Medical College & Hospital, Dharwad (Karnataka) and from Authentic Research Journals, Websites, Digital Publications etc.The data, which are obtained by the clinical trial will be statistically analysed by applying Students‘t’ test.

c)     Source of the medicine- pharmacy SDM Udupi.

A)  Inclusion Criteria          

·       Cases of prediabetes, on the basis of the laboratory investigation between the age group 30 to 40 years of both sexes.

·       HbA1C  between 5.7 to 6.4 mg/dL.

·       FBS levels falling between- 100 to120 mg/dL

·       PPBS levels falling between- 140 to 199 mg/dL

·       Onset less than 6 months.

·       BMI Above 21.

·       A Positive family history of type 2 DM.

·       Higher than normal values of lipid profile values.

B) Exclusion Criteria                                                                      

·       Subjects other than the inclusion criteria.

·       Subjects having DM Type I [IDDM], GDM, Patient of DM Type II who is on Insulin therapy, DM with complications, FBS [>120mg/dL], PPBS [>250mg/dL] and

·       Hypertension.

·       Subjects having any serious illness, H.I.V; HbsAG, VDRL, Malignancy and other immuno compromised conditions.

Parameters of Study:

Subjective parameters                                                                    

ü  Pipasa (Polydypsia),

ü  Prabhoot mutrata(Polyurea),

ü  Avil mutrata(Turbid urine),

ü  Sharira Gaurava(Heavyness in body),

ü  Tandra (Drowsiness),

ü  Hast paad tal daah(Burning sensation in palm and sole),

ü  Kara Pada Suptata(Numbness in palms and sole)

ü  Saad(Lethargy) and Panduvarna Mutra(Yellowish white urine) were assessed before and after the trial study.

 Objective parameters

1.     Fasting plasma glucose (FPG), PPBS.

2.     HbA1C.

3.     IL6

4.     Lipid profile

5.     BMI

Study Design             :  A Single group pilot study.

Sample size                :  A Minimum of 15 cases categorized as pre diabetes will be selected incidentally and randomly.

Duration                    :  90 Days

Follow up                   : 15 Days

Investigations

•        Fasting plasma glucose (FPG), PPBS.

•        HbA1C.

•        IL6

•        Lipid profile

Interventions:

Shivagutika in a tablet form

Dose                                     : 500 mgs- 1-0-1 – twice a day.

Time                                    :  after food

Anupana                             : ushna jala

Duration                             :  90 days

Assessment criteria

Change in Subjective and objective features of Pre Diabetes before and after the treatment. The results will be recorded as;

                        Marked relief              -           Above 75% improvement

                        Moderate relief           -           50%-75% improvement

                        Mild relief                   -           25%-50% Improvement

                        No relief                      -           Below 25% improvement

List of References:

1. Professor Dattatreya Anant Kulkarni, Rasaratnasamuchaya, vijnanabodinihindi translation and commentary, Prathama bhaga, chapter 2, slogam. No. 116, page. No. 34, MerchandLachhmandas publications, reprinted in 1998

2. Journal of Ayurveda and Holistic Medicine, January 2014, Volume 2, Issue 1, A Clinical study to evaluate the role of Shivagutika in Pelvic inflammatory disease.

3. Translated by Prof. K. R. Srikanta Murthy, Ashtanga sangraha of vagbhata, Text, English translation,notes and index, Uttara Sthana, chapter 49, page. No. 506, slogam. No. 309-325, Chaukhambha Orientalia 5th Edition 2005.

4. Edited and translated by Priyavrat Sharma, Cakradatta, chapter LXVI, slogam. No. 172-193, page. No. 593 (Sanskrit text with English translation, Chaukhambha publishers, Varanasi, 2013.

5. By Vaidya Lakshmipatisastri, Yogaratnakara with Vidyotinihindi commentary, edited by BhisagratnaBrahmasankarSastri, chapter 11, slogam. No. 101-109, Chaukhambha Prakashan, Edition reprinted 2013.

6. Analysis with appendixed by Shri KavirajAmbikadatta Shastri Ayurvedacharya, Bhaishajyaratnavali of Govind das, Edited and enlaged by Bhisagratna Shri Brahma Shankar Misra, Vidyotini Hindi commentary Editor Shri Rajeswardatta Shastri, chapter 73, slogam. No. 151-175, Chaukhambha Sanskrit Sansthan Varanasi, Eighteen revised Edition, 2005.

7. International Ayurvedic Medical Journal, Volume 1, Issue 3, May-June 2013, Clinical study to evaluate the therapeutic effect of Shivagutika in patients with H.I.V.