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(MUNNEERIZHIVU)

Thesis Submitted to

The Tamil

Nadu

Dr. M.G.R. Medical University

In partial

fulfilment

for the award of the degree of

Doctor of Philosophy -July 2018

Dr. M. K

annan

M.D(s).,

Ph. D Scholar (part-time) Siddha Central Research Institute (CCRS, Ministry of AYUSH, Govt. of India)

Chennai - 600106

Under the supervision of

Dr. S. Justus Antony M.D(s)., Lecturer - Grade II Dept. of PG Pothu Maruthuvam

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“The pleasure, consummation and ecstasy that come along with the successful

accomplishment of any work would be incomplete unless we name the people who

made it possible, whose constant guidance and encouragement served as a beam of

light and crowned our efforts.”

In the first Place, I would like to thank the Almighty for his tremendous grace

for giving me the strength and resilience in traversing the times of distress during the

Project work and enabled me to complete arduous task satisfactorily.

I feel privileged to express my heartfelt gratitude to my Respectable Project

guide Dr. S. Justus Antony,Associate professor, Department of PG Pothu Maruthuvam,

Government Siddha Medical College, Palayamkottai, for his sincere guidance,

constructive criticism, constant encouragement and providing all necessary inputs to

initiate my research work.

I extend my sincere thanks to members of the Doctoral Advisory Committee,

Dr. S. Mohan, Former Director, NIS and Dr. P. Parthiban, Joint-Director, Directorate

of Indian Medicine & Homeopathy.

I thank with immense gratitude from the bottom of my heart to DG, CCRS,

Director in-charge, SCRI, Head of the Department, DCR, SCRI, for providing me

with all the facilities and support during academic period of the study.

I take this opportunity to express my sincere & warm thanks to Dr. G.

Dayanand Reddy, Assistant Director (Pharmacology), Siddha Central Research Institute,

Chennai and Co-guide/ Doctoral advisory committee member of my study.

I express my sincere thanks to Dr. N. Kabilan, Dr. E. M. Manikgantan and

Dr. Rajalakshmi Sivaraman, Department of Siddha, the TN Dr. MGR Medical

University, for their moral support and appropriate help from the university side during

difficult times.

I express my sincere and heartfelt thanks to the Core members of the Institute,

Dr. P. Sathiyarajeswaran, Dr. S. Natarajan, Dr. M. S. Shreedevi, Dr. V. Aarthi,

Dr. G. Kiruthiga, Dr. M. Muthuthamizh, Dr. R. Meena, Mr. N. Kirubakaran and

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I thank the Phytochemistry and analysis team Dr. K. Parthasarathy

Dr. S. Suginlal Jabaris and Dr. V. L. Reena for their support for the physiochemical and

analytical studies.

I thank Dr. K. N. Sunil Kumar and S. Brindha from Pharmacognosy

Department for their timely help in botanical authentication.

I express my deep sense of gratitude to Mr. G. Rajesh Kumar in providing

support in data analysis during entire course of the study.

I thank Dr. P. Thenmozhi, Dr. A. Lavanya for their support during thesis

preparation.

I extend my whole hearted thanks to Mr. V. Baskar, Mr. M. Vijayakumar,

Ms. R. Nandhini, Ms. A. Surya, Ms. Gayathri, for their co-operation and support during

the DTP works.

I thank Biochemistry HOD, Dr. R. Ganesan and Pathology HOD,

Dr. V. Ananthalakshmi for their moral support.

I extend my sincere thanks to Mr. D. Radhakrishna Reddy for his help in

review of literatures.

It gives me great pride and immense pleasure to extend my sincere regards and

deepest sense of gratitude to Mrs. V. Indra Johncy, Mr. Harish, Mr. Mohan,

Department of Pharmacy, Siddha Central Research Institute, who have helped in various

levels of the study.

I am thankful to Dr. C. Anbarasi, Dr. L. Juliet, Dr. K. Samraj, Dr. A. Usha,

Dr. R. Susila, Dr. S. Bhavani, Dr. Shyamala Rajkumar, Dr. Eugene Wilson, Dr. S.

Selvarajan for their referral to IGT OPD, in recruiting the IGT patients and constant

follow-up. I am thankful to all laboratory staff of SCRI who had supported me in my

study. I would like to thank all Admin and research staff for their support and constant

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final phases of this study and my mother Mrs. M. Gomathi without whom I would not

have come into being, who graced every important moments of my life and stood as Pillars

of Support in all my endeavours with her love and compassion. They are the source of

inspiration always wishing me the best from the core of their heart. I would like to give

special thanks to my Brothers-- M. Paramasivan, M. Subbiah, M. Thangarajan, and

M. Venkateshan, Sisters- In-laws of my Family for their support and Prayers for my

upliftment.

I express my hearty thanks to my better half Dr. S. Sonitha, Siddha Physician

for her invaluable help and Sacrifice, and My lovable Children K. S. Akshayaa and K.S.

Sai Adhithyaa, whose love and care were the source of energy during my difficult times.

I express my sincere thanks to Penguin Press and Citizen Printers for their

support in Printing and Binding of the thesis.

Last but not the least, I express my sincere thanks to one and all and to those

whom I might have missed to mention, who gave constant encouragement and help

throughout my academic career.

Thank you one and all.

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Chapter Title Page Number

1. Introduction 1

2. Aim & Objective 6

3. Review of Literature 7

4. Scope andPlan of Work 49

5. Materials & Methods 50

6. Result 85

7. Discussion 179

8. Summary and Conclusion 191

9. Recommendations 193

10. Bibliography

11. Annexure

Assessment Forms Table

Certificates -IHEC, CTRI,

Authentication certificates

Plagiarism report

(8)

TABLE

NO TITLE

1. Ingredients of Thiripala Chooranam Tablet

2. Preparation of Standard Solutions of concentrations

3. ICP-OES analysis

4. List of Equipments used for evaluation of Tablets

5. Compressibility index& Hausner’s ratio

6. Angle of repose:

7. Weight variation limits

8. Organisms used for Anti-Bacterial Activity

9. Ingredients of Thiripala chooranam tablet

10. Physiochemical Analysis of Individual Herbal Drugs and Thiripala Choornam

Tablets

11. Macro and Micro nutrient

12. Heavy metal Analysis of Thiripala choornam Tablet

13. Microbial analysis of Thiripala chooranam tablet formulation

14. Microbial analysis of Specific Pathogens of Thiripala chooranam tablet

formulation

15. Pesticide residue analysis of Thiripala chooranam tablet formulation

16. Aflatoxin analysis of Thiripala chooranam tablet

17. Organoleptic characters of Thiripala chooranam tablet

18. Bulk characterization of lubricated blends of optimized formulation (F3)

19. FTIR Spectrum of THIRIPALA CHOORANAM

20. FTIR Spectrum THIRIPALA CHOORNAM TABLET

21. Post compression parameters of optimized formulation (F2)

22. Post compression parameters of optimized formulation (F3)

23. Percentage inhibition of test drug Thiripala chooranam tablet on Alpha Amylase

Enzyme Inhibition Assay

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25. Percentage inhibition of Thiripala chooranam tablet on α-Glucosidase Enzyme Inhibition Assay

26. IC50 Values for α-Glucosidase Enzyme inhibition by Thiripala chooranam tablet

27. Zone of Inhibition data of Anti-bacterial activity

28. Age Distribution

29. Gender Distribution

30. Educational status

31. Occupation

32. Socio Economic status

33. Family History of Diabetes

34. Distribution of Body Constitution

35. BMI distribution

36. Diet Distribution

37. Duration of treatment in each patient of LTPC – Arm

38. Duration of treatment in each patient of OLSM - Arm

39. Laboratory investigation results of patients in Thiripala Chooranam tablet with

Life style modifications Arm: Arm 1 (LTPC ARM): Before Treatment

40. Laboratory investigation results of patients in Thiripala Chooranam tablet with

Life style modifications Arm: Arm 1 (LTPC ARM): After Treatment:

41. Laboratory investigation results of patients in Only Life style modifications Arm:

Arm 2 (OLSM ARM): Before Treatment

42. Laboratory investigation results of patients in Only Life style modifications Arm:

Arm 2 (OLSM ARM): After Treatment

43. Follow-up Outcome of the Patients Taking Thiripala Chooranam

44. Physical Activity Clause Interval

45. HbA1c analysis

46. The Least Square Means of HbA1c

(10)

48. Fasting Blood Sugar (FBS) Before and After between the LTPC arm and OLSM arm

49. OGTT 2 hours

50. Mean reduction of OGTT

51. Mean difference percentage of Reduction of Lipid Profile

52. Baseline Lab Results

53. Post-treatment Lab Results

54. Post-treatment Lab Results

55. Urine Sugar Analysis

56. Responder Analysis of HbA1c

57. Odds Ratio Estimates

58. Table of Treatment

59.

Calculation of Absolute Risk Reduction, Relative Risk Reduction, Relative Risk (ARR,

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FIGURE

NO TITLE

1. Thiripala chooranam Tablet

2. Ingredients of Thiripala chooranam tablet: Kadukkai

3. Ingredients of Thiripala chooranam tablet: Nellikkai

4. Ingredients of Thiripala chooranam tablet: Thandrikkai

5. Terminalia chebula

6. Emblica officinalis

7. Terminalia bellerica

8.

Maceration microscopy of Kadukkai – Terminalia chebula

fruit rind

9. Maceration microscopy of Nellikkai – Emblica officinalis fruit rind

10.

Maceration microscopy of Thandrikai – Terminalia bellerica fruit

rind

11.

Powder microscopic analysis of Kadukkai – Terminalia chebula fruit

rind

12.

Powder microscopic analysis of f Nellikkai – Emblica

officinalis-dried fruit

13.

Powder microscopic analysis of Thaandrikkai- Terminalia

bellerica pericarp

14. Thiripala chooranam tablet

15. Lignified elements in Thiripala chooranam Tablet

16. Non-lignified elements in Thiripala chooranam Tablet

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18.

HPTLC Profiling of Terminalia chebula (KADUKKAI) @ 254

nm

19.

HPTLC Profiling of Terminalia chebula (KADUKKAI) @ 366

nm

20.

HPTLC Profiling of Terminalia chebula (KADUKKAI) @ 520

nm

21. HPTLC Profiling of Emblica officinalis (Nellikkai) @ 254 nm

22. HPTLC Profiling Emblica officinalis (Nellikkai) @ 366 nm

23. HPTLC Profiling Emblica officinalis (Nellikkai) @ 520 nm

24.

HPTLC Profiling Terminalia bellerica (THANRIKKAI) @ 254

nm

25.

HPTLC Profiling Terminalia bellerica (THANRIKKAI) @ 366

nm

26.

HPTLC Profiling Terminalia bellerica (THANRIKKAI) @ 520

nm

27. HPTLC Profiling of Thiripala Choornam Tablet @ 254 nm

28. HPTLC Profiling of Thiripala Choornam Tablet @ 366 nm

29. HPTLC Profiling of Thiripala Choornam Tablet @ 520 nm

30.

UV-VISIBLE ABSORPTION PROFILE- Thiripala

Choornam[TC]

31.

UV-VISIBLE ABSORPTION PROFILE- Thiripala Tablet

[TT]

32. UV-VISIBLE ABSORPTION PROFILE- Thiripala Tablet

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33.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Arsenic

34.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Calcium

35.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Cadmium

36.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Copper

37.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Iron

38.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Magnesium

39.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Manganese

40.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Sodium

41.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Lead

42.

Inductively Coupled Plasma – Optical Emission Spectroscopy

studies: Potassium

43. Mass Spectral Studies: Gallic acid

44. Mass Spectral Studies: Quercetin

45. Mass Spectral Studies: Corilagin

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47. FTIR Spectrum of Thiripala Chooranam

48. Alpha Amylase Inhibitory Activity of Thiripala Choornam

Tablet:

49.

ALPHA - GLUCOSIDASE ENZYME INHIBITORY

ACTIVITY OF THIRIPALA CHOORANAM TABLET

50. ANTI - BACTERIAL ACTIVITY: Escherichia coli

51. ANTI - BACTERIAL ACTIVITY: Staphylococcus aureus

52. ANTI - BACTERIAL ACTIVITY: Klebsiella pneumonia

53. ANTI - BACTERIAL ACTIVITY: Enterococcus faecalis

54. Age Distribution

55. Gender Distribution

56. Educational Status

57. Occupation Status

58. Socio Economic status

59. Family History of Diabetes

60. Distribution of Body Constitution

61. BMI distribution - ARM 1 (LTPC)

62. BMI distribution - ARM 2 (OLSM)

63. Diet Distribution

64. Physical Activity Chart

65. HbA1c analysis Chart

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67. Fasting Blood Sugar (FBS) Before and After between the LTPC arm and OLSM arm

68. Fasting Blood Sugar (FBS) Mean Reduction between the LTPC arm and OLSM arm

69.

OGTT Before and After between the LTPC arm and OLSM arm

70. OGTT Mean Reduction

71. Mean difference percentage of Reduction of Lipid Profile

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➢ % - Percentage ➢ & - And

➢ ≥ - Greater than

➢ ≤ - Lesser than

➢ °C - Degree of Celsius

➢ α - Alpha

➢ β – Beta

➢ μL – Microliter

➢ AAS - Atomic Absorption Spectrophotometer

➢ AChE - Acetylcholinesterase

➢ ACP - Acid phosphatase

➢ AGEs - Advanced glycation end products

➢ ALP - Alkaline Phosphatase

➢ AYUSH - Ayurveda Yoga Unani Siddha Homoeopathy

➢ B.Wt - Body Weight

➢ BDL - Below Detection Limit

➢ CCRAS - Central Council for Research in Ayurvedic Sciences

➢ CCRS - Central council of Research in Siddha

➢ CDC - the Centres for Disease Control and Prevention

➢ CFUs - Clinical Forming Units

➢ CRF - Case Report Form

➢ CTRI - Clinical Trial Registry –India

➢ DA - Dopaminergic

➢ DC - Differential Count

➢ DPPH –2, 2- Diphenyl-1-picrylhydrazyl

➢ DMSO – Dimethyl Sulfoxide

➢ DNA - Deoxy Ribonucleic Acid

➢ EDTA - Ethylene Diamine Tetra Acetic Acid

➢ ESR - Erythrocyte Sedimentation Rate

➢ FFA - Free Fatty Acids

(17)

➢ GOT - Glutamate Oxaloacetate

➢ GPT - Glutamate Pyruvate Transaminase

➢ g/ml – gram per milliliter

➢ H2 O2 - Hydrogen peroxide

➢ HBA1C – Glycated Hemoglobin

➢ HCl – Hydrochloric acid

➢ HDL – High Density Lipoproteins

➢ HPTLC - High Performance Thin –Layer Chromatography

➢ HPLC - High Performance Layer Chromatography

➢ Hr - Hour

➢ HRBC - Human Red Blood Cell

➢ HNO3 – Nitric Acid

➢ HUVEC - Human Umbilical Vein Endothelial Cells

➢ IHEC - Institutional Human Ethics Committee

➢ IC50 - Inhibitory Concentration At 50%

➢ ICMR - Indian council of Medical Research

➢ ICP -OES – Inductively Coupled Plasma _ Optical emission spectrometry

➢ IEC - Institution Ethics Committee

➢ IFG - Impaired Fasting Glucose

➢ IGT - Impaired Glucose Tolerance

➢ IIT - Indian Institute of Technology

➢ IPD - In Patient Department

➢ i.p - intra peritoneal.

➢ Kg - Kilogram

➢ L - Litre

➢ LDL – Low density Lipoproteins

➢ LPO - Lipid Peroxide Oxidase

➢ LTPC - Thiripala Chooranam tabletwithLife style modifications

➢ MBC - Minimum Bactericidal Concentration

➢ MCC - Microcrystalline Cellulose

➢ MCV - Mean Cell Volume

(18)

➢ Mg – Milligram

➢ Mg/ml – Milligram per millilitre

➢ Min - Minutes

➢ Ml / Kg - Millilitre Per Kilogram Body Weight

➢ Mmol/L – Millimoles Per Litre

➢ nm – nanometre

➢ OECD - Organization for Economic Co-Operation and Development

➢ OGTT – Oral glucose tolerance test

➢ OLSM - OnlyLife style modifications

➢ pH – pouvoir hydrogene, “Power of Hydrogen”

➢ PFTE – Poly tetra fluoro ethylene

➢ PLIM – Pharmacopoeial Laboratory for Indian Medicine

➢ Pre-DM - Pre-Diabetes Mellitus

➢ PPBS - Post Prandial Blood Sugar

➢ PPM – Parts per million

➢ PMN - Polymorphs nuclear leukocyte

➢ PNPG - p-nitrophenyl-α-D -glucopyranoside

➢ RDS – Raw Drug Section

➢ ROS - Reactive oxygen Species

➢ UV B - Ultra violet B rays

➢ TAC - Total Antioxidant Capacity

➢ TC – Thiripala Chooranam

➢ Tc -Total Cholesterol

➢ TCHE - Terminalia chebula hydroalcoholic Extract

➢ TLC – Thin layer chromatography

➢ TGL - Triglycerides

➢ TRF - Terminal Restriction Fragments

➢ TT – Thiripala Chooranam tablet

➢ STZ - Streptozotocin

➢ S.D - Standard Deviation

➢ S.E.M - Standard Error of Mean

(19)

➢ TNF - Tumour Necrosis Factor

➢ t-BHP - tertiary Butyl Hydro peroxide

➢ UV – Ultra violet

➢ UV B – Ultra Violet B

➢ U/ml – Units per millilitre

➢ V/V - Volume / Volume

➢ W/V - Weight / Volume

➢ W/W - Weight / Weight

➢ WBC - White Blood Cell

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Page 1

1. INTRODUCTION

Siddha system is one of the ancient systems of Indian medicine1. This system

focused on leading a healthy life style based on physical, emotional, psychological and

social wellbeing. In AYUSH systems, the promotion of preventive approach to achieve

the goal of being healthy is attained through holistic treatments. In Siddha system,

Kayakalpam plays a major role for well-being2. Kayakalpam herbs of Siddha possess

natural antioxidants which play a vital role in preventing free radical formation and thus

preventing Non-communicable diseases like Pre-diabetes, Diabetes mellitus,

Hypertension, Cancer and so on.

Non-diabetic hyperglycemia that does not satisfy the diagnostic criteria for

diabetes mellitus (DM) is generally known as prediabetes (preDM). The progression

seems to be governed by two processes: 1) a decline in sensitivity to the action of insulin

2) dysfunction and eventual exhaustion of beta-cell function and not producing as much

insulin as required.7 Beta-cell dysfunction starts 10 to 12 years prior to the presentation of

type 2 diabetes3. A preDiabetic state of dysglycemia includes Impaired Fasting Glucose

(IFG) and Impaired Glucose Tolerance (IGT) which has a strong association with insulin

resistance and increased risk of cardiovascular pathology. IGT disease progression forms

onset of Type 2 diabetes mellitus (Glucose ranges from FBS:<126 mg /dL, PPBS:

<200mg/dL and HbA1c: <6.5%) with in few years. So, it is also a risk factor for

mortality4.

The concept of IGT was first introduced by the World Health Organization

(WHO) in 1979. It is generally agreed that all forms of diabetes pass through prediabetes

state before escalating into full-blown diabetes7. Like DM, fasting plasma glucose (FPG)

and oral glucose tolerance tests are both used independently as defining criteria for

preDM5. According to World Health organization, people with IGT show abnormal

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two-Page 2 hour post-load plasma glucose values (2-h glucose 7.8–11.0 mmol/l [140–199 mg/dl]).

People with IFG only however, demonstrate an abnormal fasting plasma glucose.

According to ICMR-INDIAB study conducted in 2011 estimated 3.9 million are

in the prediabetes stage in Tamil Nadu. Projections for the whole of India would be 77.2

million preDiabetic patients6. Around 308 million people worldwide are reported to have

‘Impaired Glucose Tolerance’.

Epidemiological data from the Centers for Disease Control and Prevention (CDC)

estimate that 79 million adults aged 20 years and older in the U.S. had prediabetes in

2010, based upon fasting plasma glucose (FPG) and A1C levels14. Its prevalence is on the

rise, with an estimated 470 million people worldwide having prediabetes by 203015. It has

been proposed that 5% to 10% of individuals with prediabetes will progress to diabetes

annually, and about the same number will convert back to normoglycemia15.

Furthermore, patients with prediabetes have a 30% increased risk of developing

Type-2 diabetes mellitus over 4 years and a 70% risk over 30 years16,8. Within a decade

of the initial diagnosis ‘impaired glucose tolerance’ 25% to 75% are estimated to

progress to diabetes17.

In 2007, an estimated 308 million had Impaired Glucose Tolerance (IGT) or about

7.5% of the age group 20 to 79 years. More than 80% live in developing countries18. This

is projected to rise to 418 million, or 8.1% by 2025. Around 10% of people with

combined impaired fasting glucose (IFG) and IGT will progress to diabetes per year. The

progression rate from isolated IGT to diabetes is estimated at 3.7% per year19. The

predicted cumulative 5 to 6 years incidence of development of type 2 diabetes for people

with either IGT or IFG is 20% to 34%, those with both IGT and IFG have a progression

rate of 38% to 65%13. The pooled relative risk for development of new diabetes is 6.02 in

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Page 3 to obtain accurate epidemiological data on the prevalence of the condition due to varying

definitions used to classify individuals with elevated glycemic risk20.

It has been observed that glucose measures that define IFG and IGT are able to

identify about 10% of the adults who have prediabetes19. Unfortunately, measures based

on glycated hemoglobin (A1C) levels identify a significantly lower proportion of the

population15. There is a fair amount of evidence to demonstrate that glucose levels lower

than those meeting the current definition of prediabetes may also be associated with

similar risks, particularly in high-risk individuals21. It is well recognized that the risk

of developing type 2 diabetes is higher in men than in women38.

Recently, hemoglobin A1c (HbA1c) has also been used in the diagnosis of

preDM5, 3. It is well-established that the risks of type 2 DM (T2DM) and cardiovascular

disease are significantly increased in preDM subjects.

Pre-diabetic stage is treated in contemporary western medicine with

hypoglycemic drugs. Since lifestyle modifications and poor dietary habits are also

responsible for this disease, earlier evidences have proved that combined effect of diet,

exercise and drugs (Metformin, Acarbose) are effective at preventive progression to DM

from IGT subjects6. According to DPP study intensive lifestyle intervention reduced the

development of diabetes by 58%14. Lifestyle modification is the ideal method of delaying

or preventing diabetes as it also reduces cardiovascular risk profile5. Lifestyle

interventions remain an essential part of management of prediabetes36. But lifestyle

modifications are treated separately and not approached holistically.

Pharmacological studies of the herbal formulations for the treatment of diabetes

indicate that the mechanisms of action of these interventions might be multifactorial.

Medicinal plants enhance the micro-circulation, increase the availability of insulin and

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Page 4 Herbs containing polysaccharides restore the function of the pancreatic tissues

causing increased insulin output by the beta-cells. Polysaccharides are the most abundant

and the most diverse materials found on earth which possess potent antioxidant activity

and can protect the liver and kidneys from the injurious effects of diabetes.

Polysaccharides can have number of effects including anti-inflammatory,

immune-stimulating, complement activation, anti-thrombotic, antidiabetic and infection protectant

activity and many more 22,23,24,25,26. Most polysaccharides especially fructans and

mannans are derived from higher plants. They are relatively nontoxic and do not cause

significant side effects, which is a major problem associated with immunomodulatory

bacterial polysaccharides and synthetic compounds 27,28,29,30. This provides a window of

opportunity to prevent or delay the development of type 2 diabetes, and potentially

diminish the risk of cardiovascular and other complications37. Both IGT and IFG are

insulin-resistant states, but they differ in site of insulin resistance39.

However, it is possible to halt or reverse the progression of pre-diabetes, or at

least delay the development of diabetes using Kayakalpa herbs with Siddha lifestyle which regulate blood sugar levels.

Pre - Diabetes can be analogous with Munneerizhivu or piramya rogam in Siddha

system which is caused due to increase in Aiyakuttram. However, bio-combustion

challenge being the concern in the DM is related to Azhal, on slow deterioration of Azhal

leads to DM. Its complications however related to Aiyam. It is indirectly meant that pre-

DM stage, there will be decrease in Azhal and full-blown DM, increase in Aiyam31.

In drugs selection it becomes essential to identify a drug which will reduce the

increased Azhalkutram. Astringent taste regularizes Azhalkutram. Astringent taste

regularizes excessive fluid secreted out of the body. It also checks the disease caused due

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Page 5

Thiripala32 Chooranam (TPC) being a Kayakarpam drug in Siddha system of

medicine, having 3 major astringents, regularizes Azhal and reduces the Aiyam.

Thiripala chooranam tablet is a poly herbal Siddha formulation, comprising of three herbs Terminalia chebula, Terminalia bellerica, and Emblica officinalis which is proven as an anti-diabetic drug33. Some studies have investigated the possible

anti-diabetic properties of combination drug Thiriphala chooranam in animal models, one in a

high fructose diet induced and another in alloxan diabetic rats34. The results of these

studies shows that the administration of the extracts reduced the blood sugar level.

Gallic acid is the component present in the composition of Thiripala chooranam

having anti-diabetic activity35. More over the same studies highlighted the anti-oxidant,

and anti-obesity effects of this drug40. These effects are assumed to play a major role to

reduce the chances of transition of IGT patients to established Diabetics. These become

the rationale for the selection of “Thiripala Choornam Tablet” as an interventional drug.

In this study, prediabetics (Munneerizhivu) is treated holistically with the study

drug (Thiripala Choornam tablet) and Siddha Life style modification. So, the above said

poly-herbal formulation, Thiripala Choornam Tablet is more effective, cost effective and

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Page 6 2. AIM & OBJECTIVES

To pharmacognostically identify the selected individual plant powders, Kadukkai

(Terminalia chebula), Nellikai (Emblica officinalis), Thandrikai (Terminalia bellerica) for authentication.

To develop the Thiripala Chooranam tablet (Siddha formulation) of the selected plants.

To Standardize individual plant crude powders and Thiripala Chooranam tablet (Siddha

formulation) through analytical tests such as HPTLC, UV-Visible Absorption Profile

Studies, FTIR, ICP- OES, AAS, LC-MS.

To evaluate safety of the trial drug through microbial study, pesticide, aflatoxin and

heavy metal analysis.

To develop and analyze Thiripala Chooranam tablet by Pre - formulation and Post - compression studies as per AYUSH guidelines.

To measure the glycosidic enzyme - inhibiting activity of Thiripala Chooranam tablet

(Siddha formulation) on alpha amylase and alpha glucosidase ( In - Vitro studies).

To evaluate the clinical efficacy of the trial drug in controlling progress to full blown

diabetes with an active control in parallel to prove its efficacy of reducing and

maintaining blood glucose levels in normo - glycemic range and with respect to body

constitution (Udaliyal).

To assess the hypolipidemic efficacy of the trial drug through clinical trial.

To bring about conclusive and demonstrable scientific support for the Thiripala

Chooranam tablet- a traditional Siddha formulation in treating and reversal of Impaired

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Page 7

3. REVIEW OF LITERATURE

A detailed and thorough literature survey was carried for the individual

selected plants and Thiripala Chooranam Tablet from Libraries of well-established

institutes (IIT Chennai, CLRI Chennai, The T.N. Dr. M.G.R. Medical University

Chennai, Govt. Siddha Medical College, Chennai and Palayamkottai, National

Institute of Siddha, Chennai, SCRI, Chennai), and various literature sources such as

Medline via Pub Med, Science Direct, and Google Scholar. The collected scientific

reports were presented.

3.1 ETHNOBOTANICAL REVIEW

3.1.1 PLANT PROFILE (Terminalia chebula Retz)

Botanical Source : Terminalia chebula Retz.

Family : Combretaceae

Synonyms : Buceras chebula (Retz.) Lyons, Myrobalanus chebula (Retz.)

Gaertn.

Parts Used : Dried pericarp

Taxonomic classification

Kingdom : Plantae

Division : Tracheophyta

Class : Magnoliopsida

Order : Myrtales

Family : Combretaceae

Genus : Terminalia

Species : Terminalia chebula

Vernacular Names

Sanskrit : Kayastha

Hindi : Harra

Kannda : Halle

Malayalam : Kattuka

English : Chebulic myrobalan

Tamil : Kadukkai

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Page 8

Distribution

The plant is found throughout India chiefly in deciduous forests. It occurs

abundantly in North India. Its range extends southwards at 300 to 900 m altitude41.

Description of drug

Fruit is a hard, stony drupe, greenish yellow in colour, odourless, ovate,

longitudinally wrinkled, 3.5 to 4 cm in length, 1.5 to 2 cm wide and has 5 to 6 ridges

(longitudinal ribs). In some, the basal portion is narrower and somewhat elongated on

tapering42.

Habit

Moderate sized or large tree48.

Chemical Constituents

Fruit

Tannins (20-40%)43, which on hydrolysis give chebulic acid and D-galloyl

glucose, chebulagic acid, chebulinic acid, ellagic acid and gallic acid; a tannin

terchebin44, an ellagitannin terchebulin45, syringic acid46, gallic acid (1.21%)47.

Traditional Medicinal Uses

Jaundice, eye diseases, bleeding disorders, laxative, ascites48.

PHYTOCHEMICAL INVESTIGATIONS

Terminalia chebula contains the triterpenes arjun glucoside 1,

arjungenin and the chebulosides 1&2. Other constituents contain

tannins up to 30%, chebulic acid 3-5%, chebulinic acid 30%, tannic

acid 20-40%, ellagic acid, 2,4-chebulyi–β-D-glucopyranose, gallic

acid, ethyl gallate, punicalagin, terflavin A, terchebin, anthraquinone,

flavonoids like luteolin, rutins, and quercetin etc.

PHARMACOLOGICAL ACTION

➢ The water-soluble fraction of T. chebula (WFTC) was effective against

systemic and local anaphylaxis. Shin et al. showed that the injection of

WFTC with doses of 0.01–1.0 g/kg inhibited anaphylactic shock

100%. When WFTC was pretreated at concentrations ranging from

(31)

dose-Page 9

dependently. In addition, this study showed that WFTC increased

anti-dinitrophenol and IgE-induced tumor necrosis factor- (TNF-) α

production49.

T. chebula has pharmacological activities relevant to dementia therapy.

Different extracts from T.chebula have exhibited

concentration-dependent inhibitory activities on AChE and BChE. T. chebula with a

high content of phenolic constituents exhibits strong antioxidant and

neuroprotective properties in vitro and in vivo. Hence T. chebula

proves to be beneficial in the treatment of Alzheimer’s disease 50.

Anti-ulcerogenic activity: Animals pretreated with doses of 200 and

500 mg/kg, Terminalia chebula hydroalcoholic extract showed

significant reduction in lesion index, total affected area and percentage

of lesion in comparison with control group (p<0.05 and p<0.01) in the

aspirin, ethanol and cold restraint stress-induced ulcer models.

Similarly extracts increased mucus production in aspirin and

ethanol-induced ulcer models. At doses of 200 and 500 mg /kg Terminalia

chebula extract showed antisecretory activity in pylorus ligated model which lead to a reduction in the gastric juice volume, free acidity, total

acidity and significantly increased gastric pH. This activity thus lends

pharmacological credence to the suggested use of the plant as a natural

remedy in the treatment or management of ulcer 51,52.

Neuroprotective activity: The methanol and water extracts of

Terminalia chebula exhibit neuroprotective activities against H202-induced toxicity toward PCl 2 cells and are potential candidates for the

treatment of H202-induced neurodegenerative disease. The effective

neuroprotective activity of the water extract is consequence of its "OH

and HP2 scavenging activities, its greatest extraction yield and its total

phenolic and tannin content 53,54.

Anti-convulsant activity: The ethanolic extract of Terminalia chebula

significantly reduced the duration of seizures induced by maximal

electroshock. The ethanol extract in doses of 200 and 500 mg/ kg

(32)

Page 10

same doses also protected animals from pentylenetetrazole-induced

tonic seizures and significantly delayed the onset of tonic seizures

produced by picrotoxin55. The ethanolic extract of Terminalia chebula

(EETC) possess anticonvulsant activity since it reduced the duration of

seizures produced by maximal electroshock and delayed the latency

of seizures produced by pentylenetetrazole and picrotoxin55. This

provides a pharmacological justification for the traditional use of the

plants fruits in the management of epilepsy in some rural parts of

India.

Anti-oxidant activity: Terminalia chebula are new potential sources

of natural antioxidants for food and nutraceutical products. The

methanol extract of Terminalia chebula had the greatest total

triterpenoid content and exhibited good antioxidant activity in the

HRP-luminol-H202 assay56. The water extract appeared to have the

greatest total phenolic and tannin content and showed good antioxidant

activities in both CuS04-Phen-Vc-HP2 and luminol - H202 assays. The

95% ethanol extract exhibited good antioxidant activity in the

pyrogallol-luminol assay. Thus, the three extracts present various

levels of ROS scavenging efficiency due to differences between the

mechanisms of the four ROS chemiluminescence systems. Aqueous

extract of natural herb, Terminalia chebula inhibitsxanthine / xanthine

oxidase activity and is also an excellent scavenger of DPPH radicals.

It is concluded that the aqueous extract of T. chebula acts as a potent antioxidant and since it can protect cellular organelles from the

radiation-induced damage, it may be considered as a probable

radioprotector57. Protective effects of an aqueous extract of Terminalia

chebula fruit on the tert-butyl hydroperoxide (t-BHP)-induced oxidative injury was observed in cultured rat primary hepatocytes and

rat liver58,59. It has stronger antioxidant activity than alpha-tocopherol.

HPLC analysis with diode array detection indicated the presence of

hydroxybenzoic acid derivatives, hydroxycinnamic acid derivatives,

flavonol aglycones and their glycosides, as main phenolic

(33)

Page 11

➢ On comparison with the typical aqueous extraction method the

extraction efficiency was highest for microwave treatment followed by

ultrasonication61. The study revealed a 17 .6% increase in the

yield of phenolics and a 14% increase in the tannin content of the

microwave extracts. A 20.6% increase in the antioxidant activity of

tthe microwave extract was also obtained. The sonication extracts

showed an increase of 0.6, 5 and 9.69% in the yield of phenolics,

tannins and antioxidant activity, respectively.

➢ Hepatoprotective activity: Terminalia chebula extract was found to

prevent the hepatotoxicity caused by the administration of rifampicin

(RIF), isoniazid (INH) and pyrazinamide (PZA) combination in

sub-chronic model in mice (12 weeks)62.

Cardioprotective activity: Terminalia chebula extract pretreatment

was found to ameliorate the effect of isoproterenol on lipid peroxide

formation and retained the activities of the diagnostic marker enzymes

m - isoproterenol induced myocardial damage in rats63. Its pericap has

also been reported to have cardioprotective activity in isolated frog

heart model64.

Cytoprotective activity: Ethanolic extract of Terminalia chebula

fruit exhibited significant cytoprotective effect against UV B-induced

oxidative damage. These observations were attributed to the

inhibitory effect of the Terminalia chebula extract on the age dependent shortening of the telomere length as shown by the

Southern Blots of the Terminal Restriction Fragments (TRFs) of

DNA extracted from sub-culture passages. Cytoprotective effect on

oxidative stress and inhibitory effect on cellular aging of its fruits

(34)

Page 12

Antidiabetic and retino-protective activity: The anti-diabetic

property of medicinal plants and its relationship with their

antioxidant potential have long been established66. The methanolic

extract of Terminalia chebula, Terminalia bellerica, Emblica

officinalis and their combination named 'Triphala' was found to inhibit lipid peroxide formation and scavenge hydroxyl and superoxide

radicals in the diabetic rats confirming their antidiabetic potential67.

Anti-diabetic potential of Terminalia chebula fruits was effective on streptozotocin (STZ) -induced experimental diabetes in rats. Oral

administration of ethanolic extract of the fruits (200 mg/kg body

weight/rat/day) for 30 days significantly reduced the levels of blood

glucose, glycosylated hemoglobin and stimulated insulin in diabetic

rats. Also, a decrease in the number of secretory granules of β-cells

was observed in the STZ induced diabetic rats and these pathological

abnormalities were normalized after treatment with T. chebula

extract68.

Terminalia chebula methanolic extract (TCE) containing 2.7%

chebulic acid showed preventive effects against the formation

of advanced glycation end products (AG Es) and endothelial cell

dysfunction69. When the effects of TCE on AGE formation and

on protein crossing linking by glycation with D-threose and lens

crystallines were examined, TCE showed inhibitory activity in a

dose-dependent manner and the concentration of 1000 µg mL_,

presented an activity similar to that of 5 mM aminoguanidine as

a positive control. The incubation of Human Umbilical Vein

Endothelial Cells (HUVEC) with 100 µg mL_, of advanced glycation

end products (AGEs) caused a considerable increase in THP-1

monocytic cell adhesion, but this adhesion was reduced by the

treatment of TCE. This showed that TCE is a potential agent

(35)

Page 13

Hypolipidaemic activity: Terminalia chebula was found to possess

significant hypolipidaemic activity. In atherogenic diet induced

hyperlipidemic model, the rats receiving treatment with Terminalia chebula showed significant reduction in total cholesterol, triglycerides, total protein and elevation of high density lipoprotein

cholesterol. The results also suggested that Terminalia chebula at 1.05

and 2.10 mg kg concentrations are an excellent lipid-lowering

agent70,71.

Anti-arthritic effect: Terminalia chebula hydroalcoholic Extract

(TCHE) has the potential to be used as a disease-modifying

agent in treatment of rheumatoid arthritis72,73. TCHE produced

a significant inhibition of joint swelling as compared with

control in both formaldehyde-induced and Complete Freund's

adjuvant CFA-induced arthritis. The TCHE treatment also reduced

serum TNF-a level and synovial expression of TNF-Rl, IL-6 and

IL-1 ~- Results of acute toxicity study showed that the oral LD50

of TCHE was >2000 mg kg ". Chronic administration also did

not produce any significant physiological changes as compared

with normal rats.

Antimutagenic/anticarcinogenic activity: The inhibitory action on

cancer cell growth by the phenolics of Terminalia chebula fruit

and found that chebulinic acid, tannic acid and ellagic acid were the

growth inhibitory phenolics74. Acetone extract of bark and fruit

powder of Terminalia chebula harbors constituents with promising

antimutagenic/anticarcinogenic activity75.

Molluscicidal activity: Terminalia chebula fruit is a potential

source of biomolluscicides against Lymnaea acuminata76. These

snails are the intermediate host of liver fluke Fasciola gigantica which

causes 94% fascioliasis in the buffalo population of northern India 77.

The active molluscicidal component of Terminalia chebula fruit is soluble in carbon tetrachloride, chloroform, ether, acetone and ethanol.

(36)

Page 14

component present is more soluble in ethanol than other organic

solvents. The tannic acid is the active component present in

Terminalia chebula fruit by High Performance Liquid

Chromatography76. Further it was evaluated that in vivo and in

vitro exposure of tannic acid significantly inhibited the

acetylcholinesterase (AChE), acid phosphatase (ACP) and alkaline

phosphatase (ALP) activity in the nervous tissue of Lymnaea

acuminata78.

Immunomodulatory effect: The immunosuppressive effects of gallic

acid and chebulagic acid79, the active phytoconstituents of Terminalia

chebula extract., on cytotoxic T - lymphocyte (CTL) mediated cytotoxicity. It has been noted that gallic acid and chebulagic acid

blocked the CTL- mediated cytotoxicity. Moreover, gallic acid and

chebulagic acid has been shown to inhibit the killing activity of CD8 +

CTL clone at IC 50 values of 30 and 50 µM respectively.

Additionally, the granular exocytosis in response to anti-CD3

stimulation was also blocked by gallic acid and chebulagic acid that

further evidenced its immunosuppressive effect.

Anaphylactic effect: Inhibitory action of water soluble fraction of

Terminalia chebula on systemic and local anaphylaxis has also been

evaluated. The effects of the water soluble fraction of Terminalia

chebula showed the reduction and frequency of anaphylactic shock that further confirmed the fact that it may possess a strong anti-

anaphylactic action80.

Anti-caries effect: The potential of the aqueous extract of Terminalia

chebula as an anticaries agent have also been evaluated. The extract strongly inhibited the growth, sucrose-induced adherence and

glucan-induced aggregation of Streptococcus mutans81.

Wound healing: The herbal paste preparation obtained from T.

(37)

Page 15

and deposition of collagen. Thus, it offers a distinct advantage to

wound healing82.

Prokinetic effect: The oral administration of Terminalia chebula

on gastric emptying has been investigated to confirm its potent

prokinetic effect. Metoclopramide significantly increased the

gastric emptying (76.33±12.37%; p<0.01) and atropine inhibited the

motility percent gastric emptying (7.26±19.76%; p<0.01 ).

Terminalia chebula extract was found to increase the percent gastric

emptying (86.57±6.65%; p<0.01) which showed that Terminalia

chebula extract may serve as a useful alternative to prokinetic drugs available benefits.

TOXICITY STUDIES

➢ To support the safety-in-use of the ethyl acetate (EtOAc)-soluble

portion of a T. chebula ethanol extract containing 29.4% chebulic acid

content, the prepared portion was tested in an in vitro mutagenicity

assay, and a single and 14-day repeated dose oral toxicity study. In the

bacterial mutation assay, up to 5000 µg/mL concentration of the

EtOAc-soluble portion, the numbers of colonies did not increase

whether with or without metabolic activation. In the oral toxicity study,

the single oral dose of the extract at 2000 mg/kg did not produce

mortality or abnormal lesions in the internal organs of rats. The results

of a 14-day orally repeated dose showed that the EtOAc-soluble

portion of T. chebula ethanol extracts gave no adverse effects at dosages of 2000 mg/kg in rats83.

ANTI MICROBIAL ACTIVITY

➢ Anti-bacterial activity: The extract of Terminalia chebula shows

broad spectrum activitiy84. The ethanol extract at a concentration of

1mg per disc showed maximum inhibition against Salmonella

epidermidis followed by Bacillus subtilis85. The methanolic and

(38)

Page 16

against gram-negative bacteria particularly to the agents causing

gastroenteritis86. Terminalia chebula exhibited antibacterial activity

against a number of bacterial species87. It is effective in inhibiting

the urease activity of Helicobactor pylori88, an ubiquitous bacterium implicated in the development of gastritis, ulcers and stomach

cancers. Gallic acid and its ethyl ester isolated from ethanolic extract

of Terminalia chebula showed antimicrobial activity against methicillin-resistant Staphylococcus aureus89. It has also growth inhibitory action against Salmonella typhi90 and intestinal bacteria91.

The extracts of Terminalia chebula proves to be an effective

anti-bacterial agent by forming the inhibitory zone against

Pseudomonas aeruginosa, P. fluorescens, Bacillus bronchiseptica, Staphylococcus aureus, Salmonella epidermidis, B. cereus, B. pumilis, Shigella boydii and Escherichia coli92.

Antifungal activity: An aqueous extract of Terminalia chebula

exhibits antifungal activity against a number of dermatophytes and

yeasts93,94. It is effective against the pathogenic yeast Candida

albicans and dermatophytes Epidermophyton, Floccosum,

Microsporum gypseum and Trichophyton rubrum95.

Antiviral activity: Terminalia chebula fruits afforded four

immunodeficiency virus type 1 (HIV-1) integrate inhibitors, gallic

acid and three galloy glucoses. Their galloyl moiety plays a major role

for inhibition against the 3'-processing of HIV-1 integrase of the

compounds (Ahn et al., 2002). It protects epithelial cells against

Influenza A virus, supporting its traditional use for aiding in

recovery from acute respiratory infections96. The therapeutic

activity against Herpes simplex virus (HSV) both in in vitro and in

vivo tests97. Terminalia chebula's effect on human cytomagalovirus

(CMV). They found that Terminalia chebula was effective in

inhibiting the replication of human cytomagalovirus in vitro and in

anAIDS model with immunosuppressed mice and concluded that it

(39)

Page 17

immunocompromised patients. It is also helpful in sexually transmitted

diseases and AIDS98.

CLINICAL STUDIES

➢ Oral rinsing with extract of T. chebula was found to significantly

reduce both total bacterial counts and streptococcal counts in saliva samples. The protective effect lasted for about 3 h after rinsing,

demonstrating a potential role of T. chebula in the prevention of dental

caries99.

➢ A short term clinical trials have been carried out on patients with

simple constipation. T. chebula increases the stools and has got

property of evacuating the bowel completely100.

➢ Besides, some Ayurvedic drugs, consisting of T. chebula as one of the

constituents have been subjected to clinical trials regarding their

effects on constipation, mental and physical disability, allergic rhinitis

and mental stress. In all the cases T. chebula containing drugs showed

good effects in the treated groups when compared to their normal

(40)

Page 18

3.1.2 PLANT PROFILE (Phyllanthus emblica L.)

Botanical Source : Phyllanthus emblica L.

Family : Phyllanthaceae

Synonyms : Emblica officinalis Gaertn, Cicca emblica (L.) Kurz, Diasperu

emblica (L.) Kuntze .

Parts Used : Dried fruit

Taxonomic classification

Kingdom : Plantae

Division : Tracheophyta

Class : Magnoliopsida

Order : Malphigiales

Family : Phllanthaceae

Genus : Phyllanthus L.

Species : Phllanthus emblica

Vernacular Names

Sanskrit : Amalaki

Hindi : Amla, Amlika

Kannda : Nellikayi, Amalaka

Malayalam : Nellikai, Nelli

English : Emblic myrobalan

Tamil : Nellikai, Nelli

Telugu : Amalakamu, Nelli

Distribution

The tree is found in the mixed deciduous forests of India ascending to 1280 m in

the hills. It is often cultivated in gardens and homeyards103

Description of drug

Drug consists of curled pieces of pericarp of dried fruit occurring as separated

segments; 1 to 2 cm, long or united with 3 or 4 segments; bulk colour grey to black,

pieces showing a broad, highly shriveled and wrinkled external convex surface to

somewhat concave, transversely wrinkled lateral surface, external surface shows a few

whitish specks, occasionally, some pieces show a portion of stony testa; texture rough

(41)

Page 19

Habit

Small or medium sized deciduous tree103

Chemical Constituents

Fruit

The fruits are reported to possess most stable ascorbic acid content and tannins,

and can be retained even when dried, powdered, or prepared candies or tablets105,106.

Some of the tannins which were found in all tissues except seeds in all stages of growth

were glucogallin, corilagin, gallic acid, ellagic acid. Immature fruits consisted of

chebulagic acid, chebulinic acid, 3, 6-dialloylglucose and five unidentified tannin

components107. Pericarp consisted emblicanins A and B, punigluconin and

pedunculagin108. The juice of the fruit contains tannic acid, gallic acid, phoroglucinol,

pyrogallol and catechol109. The major amino acids found in the fruits were glutamic

acid, proline, aspartic acid, alanine and lysine110. The free amino acids found in the

fruits were serine and traces of alanine and beta-alanine along with fructose, glucose

and sucrose as sugars111. The organic acids in the fruits were citric, malic, oxalic and

tartaric acid112. The macro and micro contents of the fruits showed the presence of

potassium, phosphorus, calcium, magnesium, sulphur, iron, manganese, zinc, boron,

copper, sodium, chloride, selenium and silica110.

Traditional Medicinal Uses

The fruits are used as carminative113, appetizer114, laxative, astringent115,

purgative, refrigerant116, nausea 117, vomiting115, stomach troubles118, constipation119,

indigestion120, diarrhea, dysentery, for gastritis, jaundice116, stomach ache121,

flatulence122, liver tonic123, liver disorders124, hepatitis125, cholera126, burning sensation,

emaciation, peptic ulcer, anorexia, dyspepsia115, to promote salivary secretion127, skin

diseases128, leucoderma129, scurvy130, hair tonic131, alopecia132, for dandruff133,

bronchitis134, aphrodisiac115, abortifacient135, menorrhagia136, leucorrhoea137,

spermatorrhoea138, anaemia116, high blood pressure139, urinary ailments140,

hemorrhage116, cardiac disorders141, painful micturition during pregnancy142, alexiteric,

(42)

Page 20

PHYTOCHEMICAL INVESTIGATIONS

Emblica officinalis Gaertn contains most stable ascorbic acid content

and tannins, and can be retained even when dried, powdered, or

prepared candies or tablets105,106. Some of the tannins which were

found in all tissues except seeds in all stages of growth were

glucogallin, corilagin, gallic acid, ellagic acid. Immature fruits

consisted of chebulagic acid, chebulinic acid, 3, 6-dialloylglucose and

five unidentified tannin components107. Pericarp consisted emblicanins

A and B, punigluconin and pedunculagin108. The juice of the fruit

contains tannic acid, gallic acid, phoroglucinol, pyrogallol and

catechol109. The major amino acids found in the fruits were glutamic

acid, proline, aspartic acid, alanine and lysine110. The free amino acids

found in the fruits were serine and traces of alanine and beta-alanine

along with fructose, glucose and sucrose as sugars111. The organic

acids in the fruits were citric, malic, oxalic and tartaric acid112. The

macro and micro contents of the fruits showed the presence of

potassium, phosphorus, calcium, magnesium, sulphur, iron,

manganese, zinc, boron, copper, sodium, chloride, selenium and

silica110.

PHARMACOLOGICAL ACTION

Hepatoprotective Activity: Hepatoprotective activity of Emblica

officinalis (EO) and chyawanprash (CHY) extracts was studied using

Carbon tetrachloride induced liver injury model in rats. Emblica

officinalis and CHY extracts were found to inhibit the hepatotoxicity produced by acute and chronic administration as seen from the

decreased levels of serum and liver lipid peroxides (LPO),

glutamate-pyruvate transaminase (GPT), and alkaline phosphatase (ALP). Chronic

CCI (4) administration was also found to produce liver fibrosis as seen

from the increased levels of collagen hydroxyl proline and pathological

analysis. Emblica officinalis and CHY extracts were found to reduce the

elevated levels significantly, indicating that the extract could inhibit the

(43)

Page 21

Anti-Inflammatory Activity: Leaves and fruits of Phyllanthus emblica

L. have been used for the anti-inflammatory and antipyretic treatment of

rural populations in its growing areas in subtropical and tropical parts of

China, India, Indonesia, and the Malay Peninsula. In the present study,

leaves of Phyllanthus emblica were extracted with ten different solvents

(n-hexane, diethyl ether, and methanol, tetrahydrofuran, acetic acid,

dichloromethane, 1, 4 - dioxane, toluene, chloroform and water).

The inhibitory activity of the extracts against human poly morphs

nuclear leukocyte (PMN) and platelet functions was studied. Methanol,

tetrahydrofuran, and 1, 4-dioxane extracts (50 micrograms/ml) inhibited

leukotriene B4-induced migration of human PMNs by 90% and

N-formyl-L-methionyl- L-phenylalanine (FMLP)-induced degranulation

by 25-35%. Diethyl either extract (50 micrograms/ml) inhibited calcium

ionosphere A23187-induced leukotrienes Release form human PMNs by

40% thromboxane B2 production in platelet during blood clotting by

40% and adrenaline induced platelet aggregation by 36%. Ellagic acid,

garlic acid and rutin all compounds isolated earlier from P. emblica.

Anti-inflammatory activity was found in the water fraction of methanol

extract of the plant leaves. The water fraction of the methanol extract

inhibited migration of human PMNs in human platelets during clotting;

suggesting that the mechanism of the anti-inflammatory action found in

the rat paw model does not involve inhibition of the synthesis of the

measured lipid mediators144.

Cardio-protective Activity: The effects of chronic oral administration

of fresh fruit homogenate of Amla on myocardial antioxidant system

and oxidative stress induced by ischemic-reperfusion injury (IRI) were

investigated on heart in rats. Chronic Emblica officinalis administration

produces myocardial adaptation by augmenting endogenous antioxidants

and protects rat hearts from oxidative stress associated with IRI145.

Anti-cancer activity: The important advantages claimed for therapeutic

use of medicinal plants in chemoprevention. Chemoprevention is a

rapidly growing area of oncology which focuses towards the cancer

preventive strategy of natural or synthetic interventions, nowadays

(44)

Page 22

use of synthetic agents which shows toxic and harmful side effects.

Amla is one of the foremost plants utilized from antiquity till to date.

Emblica officinalis is valued for its unique tannins and flavanoids, which

exhibit very powerful antioxidant properties. The inhibition of tumor

incidences by fruit extract of this plant has been evaluated on two-stage

process of skin carcinogenesis in Swiss albino mice. Chemopreventive

potential of Emblica officinalis fruit extract on

7,12-dimethylbenz(a)anthracene (DMBA) induced skin tumourogenesis in

Swiss albino mice have been found146.

➢ Experiment using Emblica officinalis powder (EOP) to understand the

activity in Alzheimer’s disease147, total serum cholesterol level and

brain cholinesterase activity in mice. EOP was administered orally in

three different doses for 15 days to different groups of young and aged

mice. Study was performed using different models to test the cognition

activity. Diazepam, Scopolamine and ageing induced amnesia served

as the interceptive behavioral model, different blood biochemical

parameters were also estimated. EOP showed a dose dependent growth

in learning behavior of young and elderly mice. It also reversed the

amnesia induced by scopolamine and diazepam. EOP helped to reduce

brain cholinesterase and total cholesterol levels. EOP may prove to be

a useful medication for Alzheimer’s disease.

➢ The antiaging effect148 of Embelica officinalis fruit (EO).

Mitochondrial activity on human skin fibroblasts was established using

MTT assay. Valuation of pro-collagen-1 and matrix

metalloproteinase-1 (MMP-metalloproteinase-1) release was performed. EO demonstrated inhibitory

activity of hyaluronidase. EO also banned UVB disturbed cell cycle to

normal phase. EO helps to control UVB induced photoaging in human

skin fibroblasts via its strong ROS scavenging ability.

Photoprotection efficacy149 of Amla (Emblica officinalis) fruit extract

through various in vitro models. It was found that

1-O-Galloyl-β-D-glucose (β-glucogallin) active in Amla extract contributes for photoprotection activity in cell cultures. β-glucogallin efficacy was a at range between 10 to 80μg/mL in B16F1 mouse melanoma cells which

(45)

Page 23

melanogenesis. It showed very high oxygen radical absorbance

capacity (ORAC) value of 4200μmol Trolox equivalents/g.

β-glucogallin from amla extract has potential photo protection efficacy.

➢ The efficacy of Emblica officinalis (EO) against radiation and

cadmium induced biochemical changes150. Total proteins, glycogen,

cholesterol, acid phosphatase, alkaline phosphatase activity, DNA and

RNA was examined. The value of cholesterol, glycogen, RNA, acid

phosphatase and alkaline phosphatase activity increased till day 14 in

non-drug treated groups and in day-7 (EO) treated groups and

thereafter decreased up to the last autopsy interval studied.

➢ An experiment to evaluate the antitumor efficacy151 of the ethanolic

extract of Amalakyadi churna (AC) against Ehrlich Ascites Carcinoma

(EAC) tumor model. The ethanolic extract showed significant

increased survival time, life span and increases body weight of treated

tumorous mice was almost normal and reducing the rate of tumor

incidence. This experiment informs that AC possesses significant

antitumor activity when tumor is at preliminary stage. The chemical

compounds present in the extract might synergistically exhibit the

antitumor activity and provided compounds responsible for the folk

anticancer uses of this plant.

Anti-oxidant properties152 of Emblica officinalis (Amla) fruits on

Streptozotocin induced type-I diabetes rats. Diabetic induced animals

showed significant increase in BSL, malonadialdehyde and reduced

superoxide dismutase, catalase and decreased glutathione levels in

liver tissue compared to control groups. This infers potent antioxidant

and lipid peroxidation inhibiting activity of fresh juice and

hydroalcoholic extract of fruits of E.officinalis in diabetic rats. The

study indicates link between oxidative stress and diabetes.

Anti-diabetic activity153 of fresh and hydroalcoholic extract of fruits

of E.officinalis on Streptozotocin (STZ) induced type-I diabetic rats. Treatment with freshly extracted juice and hydroalcoholic extract

significantly reduced elevated fasting glucose levels in type-I diabetic

rats. Treatment with freshly extracted juice and hydroalcoholic extract

Figure

Figure No. 33: Arsenic
Figure No. 37: Iron
Figure No. 38: Magnesium
Figure No. 39: Manganese
+7

References

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Laryngeal salivary gland carcinomas are rare dis- eases, cause the low density of minor glands in the lar- ynx (between 23 and 47 glands/cm2) accounting for less than 1% of