jayaprakash a n - 52.172.27.147:8080

“EXPERIMENTAL STUDY ON ANTIHYPERLIPIDEMIC

ACTIVITY OF RASONA KSHEERAPAKA PREPARED BY

DIFFERENT METHODS IN ALBINO RATS”

By

JAYAPRAKASH A N

Dissertation Submitted to the

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

In partial fulfillment of the requirements for the degree of

AYURVEDA VACHASPATI

(Doctor of Medicine)

In

BHAISHAJYA KALPANA

Under the guidance of

Dr. VINAY R KADIBAGIL M.D (Ayu)

Associate Professor

Department of Rasashastra and Bhaishajya Kalpana

SDM college of Ayurveda, Hassan, Karnataka

Co- guide

Dr. RAVISHANKAR B M Sc, PhD

Director

S.D.M Research centre for Ayurveda and Allied science, Udupi

DEPARTMENT OF RASASHASTRA AND BHAISHAJYA KALPANA

SRI DHARMASTHALA MANJUNATHESHWARA

COLLEGE OF AYURVEDA & HOSPITAL

HASSAN - 573 201

2014

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2014

“EXPERIMENTAL STUDY ON ANTIHYPERLIPIDEMIC

ACTIVITY OF RASONA KSHEERAPAKA PREPARED BY

DIFFERENT METHODS IN ALBINO RATS”

By

Dr. JAYAPRAKASH A N

Dissertation submitted to the

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES


In partial fulfillment of the requirements for the degree of

AYURVEDA VACHASPATI

(DOCTOR OF MEDICINE)

In

BHAISHAJYA KALPANA

Under the guidance of

Dr. VINAY R KADIBAGIL M.D (Ayu)

Associate Professor

Department of Rasashastra and Bhaishajya Kalpana

SDM college of Ayurveda, Hassan, Karnataka

Co- guide

Dr. RAVISHANKAR B M Sc, PhD

Director

S.D.M Research centre for Ayurveda and Allied science, Udupi

DEPARTMENT OF RASASHASTRA & BHAISHAJYA KALPANA SRI DHARMASTHALA MANJUNATHESHWARA

COLLEGE OF AYURVEDA & HOSPITAL

HASSAN - 573 201

2014

DEPARTMENT OF RASASHASTRA & BHAISHAJYA KALPANA

SHRI DHARMASTHALA MANJUNATHESHWARA COLLEGE OF

AYURVEDA & HOSPITAL, HASSAN

Affiliated to

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,


DECLARATION BY THE CANDIDATE

I hereby declare that this Dissertation/thesis entitled “Experimental study on

Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different

methods in Albino Rats” is a bonafide and genuine research work carried out under

the guidance of Dr. Vinay R Kadibagil, MD (Ayu), Associate Professor,

Department of Rasashastra & Bhaishajya Kalpana, SDM College of Ayurveda,

Hassan-573201, Karnataka.

Date: Signature of Candidate

Place: Hassan (Dr. Jayaprakash A N)




Affiliated to

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

CERTIFICATE BY THE GUIDE & CO GUIDE

This is to certify that the dissertation entitled “Experimental study on


methods in Albino Rats” is a bonafide research work done by Jayaprakash A N in

partial fulfillment of the requirement for the degree of AYURVEDA

VACHASPATHI - Doctor of Medicine (AYU) in Bhaishajya Kalpana.

Guide Co Guide

Dr. Vinay R Kadibagil Dr. Ravishankar B

Associate Professor Director,

Dept. of Rasashastra & Bhaishajya Kalpana SDM Centre for Research in

SDM College of Ayurveda, Hassan Ayurveda & Allied Sciences, Udupi

Date: Date:

Place: Place:




Affiliated to

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

ENDORSEMENT BY THE HOD /HEAD OF THE INSTITUTION

This is to certify that the dissertation entitled “Experimental study on


methods in Albino Rats” is a bonafide research work done by Jayaprakash A N

under the guidance of Dr. Vinay R Kadibagil, MD (Ayu), Associate Professor,

Department of Rasashastra & Bhaishajya Kalpana. SDM College of Ayurveda,

Hassan- 573201, Karnataka.

Dr. Basavaraj Y Ganti, Dr. Prasanna N Rao,

Associate Professor & Head, Principal,

Dept. of Rasashastra & Bhaishajya Kalpana, SDM College of Ayurveda &

SDM College of Ayurveda, Hassan Hospital, Hassan

Date: Date:

Place: Hassan Place: Hassan

COPYRIGHT

Declaration by the candidate

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka shall

have the right to preserve, use and disseminate this dissertation / thesis in print or

electronic format for academic / research purpose.

Date: Signature of the Candidate

Place: (Dr. JAYAPRAKASH A N)

©RAJIV GANDHI UNIVERISTY OF HEALTH SCIENCES, KARNATAKA

ACKNOWLEDGMENT

Research is a teamwork involving participation of many individuals to bring a

fruitful outcome. This work is a product of united efforts to precede a step further in

the development of Ayurveda. Many people have contributed directly or indirectly in

the present study. This is a humble attempt to show gratitude towards the

contributors. On the ecstatic time of submitting my thesis, I bow my head down in

front of Almighty, whose omnipresence I felt on each and every step since the very

first day of my life. My faith in him has strengthened as I reached the completion of

this work.

I obeisance my head down and dedicate this Endeavour to the sacred feet of

my beloved parents SRI DR. NAGARAJACHARI and SMT. PUSHPAMMA who

taught me the lessons of life and played a key role in moulding my thoughts to be a

good human being. I would not have achieved this milestone without the love and

care they are bestowing on me in my life. It is just next to impossible to express all the

feelings about them in words. I am also grateful to my beloved brothers Chetan

kumar A N and Yogiraj A N who always remained as the source of energy and

enthusiasm to me in this world of ambiguity.

I take this opportunity to express my deep sense of gratitude towards

Poojya Padmabhushana Dr. Virendra Heggadeji, Dharmadhikari of Dharmasthala

and the founder president of SDM Educational Society Ujire for giving me an

opportunity to study in this institution.

I acknowledge gratitude to Prof. Prasanna. N. Rao, principal, S.D.M College

of Ayurveda, Hassan for his executive leadership and coordinative skill in providing

resources at my disposal and granting assistance at all levels in the organization.

I am grateful to Prof. Gurudeep Singh, Director, Dept of PG studies, for his

words of inspirations and scholarly suggestions.

I express my deep sense of gratitude towards my respected Guide, Dr. Vinay

R Kadibagil for his compassionate, constant encouragement, extensive guidance and

benevolence. I find myself highly fortunate and greatly privileged to have worked

under his guidance.

I am really thankful to Dr. B. Ravishankar, Honorable Director and Dr.

Sunil Narayanan and all the research staff of the S.D.M. Research institute, Udupi,

for providing all the facilities needed to carry out this research work.

I express my deep sense gratitude towards my respected HOD Dr. Basavaraj

Y Ganti for his concerned, extensive guidance, steady encouragement and kind-

heartedness.

I convey my deep sense of gratitude to Dr. Govind Sharma K, Dr. Gazala

Husaain, Dr. Reshma savokar, Assistant Professors, Department of Bhaishajya

Kalpana for their valuable guidance and support.

I whole heartedly express my honest gratefulness to Mr. Ravi Mundugaru

and Mr. Sudhakar Bhat, Junior research officers, S.D.M. Research institute, Udupi

for their priceless supervision, advice and help during my experimental study.

I am extremely grateful and obliged to Dr. Mallika K J, Dr. Girish K J, Dr.

Suhas Kumar Shetty and Dr. Harini, Research committee S.D.M.C.A. Hassan for

their constant support and valuable suggestions.

I am very much thankful to my dear friends Dr. Vinayak Biju, Dr. Mahesh

kumar, Dr. Nagaraj S A and special thanks to Dr. Geoshy G Kalatil who have been

predestined with me in each & every circumstances & gave me in-depth sense of

friendship.

I bestow my thanks to my batchmates Dr. Sreelal A M, Dr. Saran Babu, Dr.

Madhulika Priya, Dr. Vinyasa T.E, Dr. Priya K, for their sensible contributions,

enjoyable moments, good will and concern which always assured me of the precious

support whenever needed.

I am also thankful to my dear colleagues Dr. Manik, Dr. Arun kumar, Dr.

Rohith, Dr. Rahul, Dr. Anu , Dr. Rashmi, Dr. Bhupesh, Dr. Seetarama Kishore, Dr.

Sreekanth, Dr. Shanty and Dr. Deepthi for their support and encouragement.

I am especially grateful to Mr. Suresh and Mr. Sreedhar, Attender, practical

laboratory, Department of R.S. & B.K., S.D.M.C.A. Hassan, for their selfless help in

completing this venture.

I am very much thankful to the librarian Mr. Komal Prasad and staffs Mr.

Krishnegowda, Mr. Manjunath and Mrs. Vinoda for their timely help in completing

this project.

I beg my apology for my inability to mention by name individually everyone

associated with this work, I express my gratitude to all those, who co-operated with

me directly or indirectly in this thesis work.

Thus, I pay my thanks to one and all.

Dr. Jayaprakash A N

ABSTRACT

Background: In our classics a lot of references are available regarding Ksheerapaka

preparation but many of the references vary in their opinion about the ratio of Drug, Milk

and Water, Due to this ambiguity it is difficult to assess the better method of preparation

of Ksheerapaka which will be therapeutically more effective. Keeping this in mind, study

was done on Rasona Ksheerapaka prepared with four different ratios of milk and water

keeping the ratio of drug as constant and its analytical and experimental study was done

to know its analytical changes and its effect on Hyperlipidemia.

Methodology: Four samples of Rasona ksheerapaka were prepared. Among them one

sample was prepared exclusively as per the reference of Charaka Samhita, with the ratio

(1:4:4), other three samples were prepared according to the general method of

preparations found in Sharangadhara Samhita (1:8:32), Y T Acharyas Dravya guna

vijnana (1:15:15) and Astanga Sangraha i.e. initially kashaya was prepared out of drug

and subsequently Ksheerapaka was prepared. These samples were tested for following

analytical parameters like Organoleptic characters, Viscosity, Specific gravity, pH and

HPTLC. Samples were also administered to Albino rats to assess the Antihyperlipidemic

action.

Result: Compared to other samples Rasona Ksheerapaka by Kashaya method showed

more number of components in HPTLC densitometric scan. In reducing serum total

cholesterol level, Ksheerapaka with Kashaya method and in reducing triglycerides,

Ksheerapaka with 1:15:15 ratio were found to be statistically significant. However all

four test samples showed good lowering of serum LDL- cholesterol level.

Conclusion: Analytical parameters reveals that, Ksheerapaka prepared by kashaya

method contain more number of components which directly indicating the maximum

number of active principles in the sample. Among four samples of Ksheerapaka, that

prepared by kashaya method was found to be the best followed by Ksheerapaka with

1:15:15 ratio in producing Antihyperlipidemic effect.

Keyword: Ksheerapaka, Rasona Ksheerapaka, Hyperlipidemia,

LIST OF ABBREVATIONS

Hb Haemoglobin

HDL High density lipoprotein

HPTLC High performance thin layer liquid chromatogrphy

LDL Low density lipoprotein

MCH Mean corpuscular haemoglobin

MCHC Mean corpuscular haemoglobin concentration

MCV Mean corpuscular volume

PCV Packed cell volume

RDWCV Red cell distribution width co-efficient of variation

RDWSD Red cell distribution width standard deviation

RKP Rasona Ksheerapaka

T1 Test sample 1 (RKP 1:4:4)



T4 Test sample 4 (RKP Kashaya method)

TC Total count

TG Triglycerides

TLC Thin layer liquid chromatography

VLDL Very low density lipoprotein

TABLE OF CONTENTS

SL No TOPIC PAGE NUMBER

1 INTRODUCTION & OBJECTIVES 1-7

2 LITERARY STUDY 8-33

Drug Review 8-13

Disease review 14-33

3 METHODOLOGY 34-109

Pharmaceutical Study 34-38

Analytical study 39-54

Experimental study 55-109

4 DISCUSSION 110-128

5 CONCLUSION 129-132

6 SUMMARY 133-134

7 BIBLIOGRAPHY 135-142

8 ANNEXURE 143

List of Tables

Table

No. Table Contents Page

No.

1 Fredrickson’s classification (Phenotype) of Hyperlipidemia 20

2 ATP-III Classification for Total lipid profile 31

3 Brief treatment aspect of Medoroga according to Ayurveda 31

4 Major Drugs (Allopathic) Used For the Treatment of

Hyperlipidemia

33

5 Ratio of the Drug, Milk and the Water for Ksheerapaka

preparations

36

6 Results of Organoleptic characters of test samples with plain milk 42

7 Results of Physico- chemical parameters of test samples with

plain milk

42

8 Rf value of alcohol extract of samples at 254nm 44

9 Rf value of alcohol extract of samples at 366nm 44

10 Rf value of alcohol extract of samples after Post derivatisation 45

11 HPTLC Densitometric scan at 254 nm 53


13 Grouping of Animals with Drug and respective dose 57

14 Reagent composition for Cholesterol 60

15 Reference values of cholesterol 60

16 Reagent composition for HDL- cholesterol (R1) 61

17 Reagent composition for HDL- cholesterol (R2) 61

18 Reference values of HDL- cholesterol 62

19 General assay parameters for HDL- cholesterol 62

20 General pipetting procedure of HDL- cholesterol 63

21 Reagent composition for LDL – cholesterol (R1) 64

22 Reagent composition for LDL – cholesterol (R2) 64

23 General assay parameters of LDL- cholesterol 65

24 Reagent composition for Triglycerides 66

25 Reference values for Triglycerides 67

26 Reagent composition for Alkaline Phosphatase 67

27 Reagents composition for Creatinine 68

28 Reagent composition for urea 69

29 Effect of Test drug Rasona Ksheerapaka on serum total

Cholesterol level

73

30 Effect of Test drug Rasona Ksheerapaka on Serum HDL

cholesterol level

74

31 Effect of Test drug Rasona Ksheerapaka on serum LDL

cholesterol level

75

32 Effect of Test drug Rasona Ksheerapaka on Serum Triglycerides

level

76

33 Effect of Test drug Rasona Ksheerapaka on Haemoglobin level 78

34 Effect of Test drug Rasona Ksheerapaka on Total Count 79

35 Effect of Test drug Rasona Ksheerapaka on RBC Count 80

36 Effect of Test drug Rasona Ksheerapaka on PCV level 81

37 Effect of Test drug Rasona Ksheerapaka on MCV level 82

38 Effect of Test drug Rasona Ksheerapaka on MCH level 83

39 Effect of Test drug Rasona Ksheerapaka on MCHC level 84

40 Effect of Test drug Rasona Ksheerapaka on RDWCV level 85

41 Effect of Test drug Rasona Ksheerapaka on RDWSD level 86

42 Effect of Test drug Rasona Ksheerapaka on Platelet count 87

43 Effect of Test drug Rasona Ksheerapaka on serum Urea level 88

44 Effect of Test drug Rasona Ksheerapaka on serum Creatinine level 89

45 Effect of Test drug Rasona Ksheerapaka on serum Alkaline

phosphatase level

90

46 Effect of Test drug Rasona Ksheerapaka on changes in body Wt 91

47 Effect of Test drug Rasona Ksheerapaka on wt of Liver 92

48 Effect of Test drug Rasona Ksheerapaka on wt of Kidney 93

49 Effect of Test drug Rasona Ksheerapaka on wt of Heart 94

50 Consolidated table of the results obtained in the Experimental

study

121

LIST OF FIGURES

Fig No. Figure contents Pg No.

1 Rasona (Garlic) 38

2 Ksheera(Milk) 38

3 Separated bulbils of Garlic 38

4 Destalked Garlic 38

5 Soaked in Butter milk 38

6 Preparation of Ksheerapaka 38

7 Collection 38

8 TLC photo documentation of Rasona Ksheerapaka 43



11 3 D Display of tracks at UV 254 nm & 366 nm 52

12 Animal house 97

13 Caging of animals 97

14 Feeding cholesterol 97

15 Blood collection from Retro orbital plexus 97

16 Dissected albino Rat 97

17 Separation of organ samples 97

18 Samples- Liver, Kidney & Heart 97

19 Packing of organ samples for Histopathology 97

20 Photomicrographs of Liver 99, 101

21 Photomicrographs of Kidney 103, 105

22 Photomicrographs of Heart 107, 109

INTRODUCTION

Introduction…

“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different

methods in Albino Rats” Page | 1

INTRODUCTION

Ayurveda, the science of life has its root in antiquity, even the historians

cannot peep into the depths of that remote past. In Ayurveda, there was no separate

mention of Bhaishajya Kalpana in the early period. This branch of learning was dealt

while describing the treatment part of the diseases itself.

The entire science of Ayurveda has been framed upon Trisutras (Hetu, Linga,

Aushadha) 1

. Among them, Aushadha is most important for the alleviation of diseases

as well as for the maintenance and promotion of good health.

No branch (Astangas) of Ayurveda can exist independently without the aid of

Aushadha or Bheshaja (source may be Plant, Animal or Mineral origin). In Ayurveda,

utmost emphasis is given to the complete knowledge of drugs including identification,

procurement, processing, preparation and application under a branch of learning

called Bhaishajya kalpana.

The term Bhaishajya-Kalpana consists of two words „Bheshaja‟ and „Kalpana‟

The word Bheshaja or Aushadha is defined as “that which conquers the disease”2.

The substances which help to bring back the doshas to their normal level or that

which counteract the diseased condition and brings back the body to a healthy state is

known as Bheshaja3 also, that by which one can treat the diseases or alleviate diseases

is known as „Bheshaja‟ or „Bhaishajya‟. Kalpana refers to the method/process or a

kind of modification, transformation (Samskarana) or plan of preparation of

medicines4.

To treat any disease successfully, a physician should have good quality of

drugs at hand. Not only this, a physician become competent of curing diseases only

when he possesses the complete knowledge of all drugs including the basic principles

of Bhaishajya Kalpana and only such an ideal physician can give life to the ailing and

Introduction…



sick people5. The role of Bhaishajya kalpana in potentiating the drug action has been

explained by Charaka6 as well as by Vagbhata

7.

It is said that even a simple drug could be made into most powerful one by

simple procedures like addition or deletion of certain drugs, by keeping the drugs with

certain bhavana dravyas for specific time, by collecting the drugs during scheduled

period, by following certain adaptation procedures along with the enhancement of the

potency of the drug, it will be made fit for therapeutic administration. Charaka says,

by proper processing even fatal poison can be converted into an excellent medicine8.

Ksheerapaka

It is a special kind of preparation in the Ayurvedic pharmaceutics where Milk

is used as a media along with the water for the extraction of active principles and for

administration.

As the preparation of Ksheerapaka is concerned, a specific amount of the

Dravya (medicament) is taken. It is mixed with specific amount of Dravadravya (milk

and water) and boiled over mild flame until the volume of milk reduces to initial

volume of milk. This mixture is filtered through a cloth and used in lukewarm

condition.

Usually the teekshna (katu) and kashaya (astringent) pradhana drugs are

selected here. Main intention is to bring down the teekshnata and kashayata of the

drugs, with the help of Madhura rasa and Madhuara vipaka of milk. Since milk is also

considered as Jeevaniya, 9

it serves as a complete food for the patient.

Milk, by nature rich in protein and calcium will be a good nutrient. So along

with the medicinal support even nutritional balance is also maintained by this

preparation. So the medicines prepared using this as a media, will contain both

therapeutic as well as nutritive (dietetic) values. Digestion, absorption and

Introduction…



assimilation of the milk fat present in colloidal form will be slow, which inturn

minimizes noxious effects of teekshna dravya and facilitate in enhancing its medicinal

effects.

Though there are many references about the preparations of Ksheerapaka, in

both Brihatrayees and Laghutrayees, the acharyas might vary in their opinion in

context of ratio of Drug, Milk and Water used. Since Rasona is a known

antihyperlipidemic drug. here the active principles of Rasona are extracted through

the milk and water and used for the study.

Hence in the present study four different methods were adopted (Ratio of

Drug, Milk and Water) to prepare Rasona ksheerapaka and evaluated for their

antihyperlipidemic action in albino rats as its therapeutic indication is in Hridroga.

This is considered to mean decreasing the obesity and diseases related to heart, hence

linked to antihyperlipidemia.

Antihyperlipidemia

Today is the era of modernization and fast life. Everybody is busy and living

stressful life. Consumption of fast foods having high calories is also increasing. We

have the every comfort of living and we are not performing any kind of physical

activity and so the body fats along with cholesterol are increasing in our body, which

invites the disorders like hypertension, heart diseases and hyperlipidemia.

The industrialization, stress during the work, dietary habits, lack of exercise

and various variety of food along with the daily diet e.g. fast foods, freeze fruits,

increased amount of soft drinks and beverages, canned foods results into the

disturbance of Agni or metabolism and ultimately these food habits leads to a clinical

entity known as hyperlipidemia.

Introduction…



Hyperlipidemia is not a single condition but a range of disorders with a variety

of genetic and environmental determinants. It can be caused or modified with a range

of other disorders, and its presence can affect many different organs and systems. The

presence of hyperlipidemia is a key factor in the development of atherosclerosis and

endothelial dysfunction.

It is a condition in which the levels of lipoproteins i.e. cholesterol,

triglycerides or both are raised in plasma. Out of which cholesterol is deposited in the

arteries including the coronary arteries where it contributes to the narrowing and

blockage of the arteries that cause the symptoms of heart diseases. It is emerging as a

major health problem in the modern era. It finally leads to coronary artery diseases,

myocardial infarction and cerebro vascular accidents.

Reducing the burden of disability and death from coronary heart disease

(CHD) is one of the greatest challenges facing health professionals in the developed

world. We may be living longer than ever before, in fact, our life expectancy has

doubled in the last 150 years but too many people still die prematurely from, or are

disabled by CHD.

CVDs are the number one cause of death globally: more people die

annually from CVDs than from any other cause10

, an estimated 17.3 million

people died from CVDs in 2008, representing 30% of all global deaths10

. Of these

deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million

were due to stroke 10

.

Low and middle-income countries are disproportionally affected: over 80%

of CVD deaths take place in low and middle-income countries and occur almost

equally in men and women10

. The number of people, who die from CVDs, mainly

from heart disease and stroke, will increase to reach 23.3 million by 203010

. CVDs

Introduction…



are projected to remain the single leading cause of death10

. Most cardiovascular

diseases can be prevented by addressing risk factors such as tobacco use,

unhealthy diet and obesity, physical inactivity, high blood pressure, diabetes and

raised lipids.

9.4 million Death each year, or 16.5% of all deaths can be attributed to high

blood pressure10

. This includes 51% of deaths due to strokes and 45% of deaths due

to coronary heart disease10

. It is essential that everyone with hyperlipidemia have a

full clinical assessment, as well as appropriate treatment, so that other important

factors and co-morbidities can be identified and assessed.

Acharya Charaka has quoted Sthaulya under the eight varieties of

impediments which are designated as Ashta-Nindita Purusha11

; Ati-sthaulya

comprises one of them. He listed eight defects underlying- Sthoola Purusha,

Ayuhrasa, Javoparodha, Alpa-vyavayita, Daurbalya, Daurgandhya, Swedabadha,

Ati-trisha, Atikshudha12

.

Dalhana seems to be more explicit while commenting on Medo Roga who

specified that Agni which is involved in pathogenesis of the disease viz.

Dhatvagnimandya. An individual whose increased Meda and Mamsa Dhatu

deposition in his hips, abdomen and breasts make pendulous and whose vitality is

much less than his body size is considered Sthoola. In Sthaulya, increased Meda,

Agni and Vayu produce complications like Prameha-Pidika and Bhagandara etc.

The incidence of Diabetes mellitus, hypertension, angina pectoris, and

myocardial infarction etc. are higher among hyperlipidemic individuals.

Though the synthetic drugs in the market have played a major role in reducing

the frequency of CHD and obesity. However, side effects remain the main concern

hence there is always stress towards finding a natural based hypolipidemic agent.

Introduction…



Hence focus of the present study is Rasona Ksheerapaka (Garlic milk), which is a

well known liquid dosage for Cardiac ailments mentioned in Ayurvedic classics.

Hyperlipidaemia, Atherosclerosis these are an associated co morbid factors for CHF.

Hence this particular liquid dosage form as classically mentioned in diseases related

to heart has been chosen to explore its therapeutic value in terms of its

antihyperlipidemic activity in rats receiving hyperlipidemic diet.

AIMS & OBJECTIVES

AIM

1) To evaluate the better method for the preparation of Rasona ksheerapaka and its

relative Antihyperlipidemic activity in Albino rats

OBJECTIVES

1) Preparation of Rasona Ksheerapaka by 4 different methods

2) Physicochemical analysis of all 4 samples of Rasona Ksheerapaka

3) Analysis of Lipid profile, Hematological and Biochemical parameters of the

Animal model

PREVIOUS WORK DONE

Some of the research activities are as follows:

1] Verma R K - A clinical, Pathological study of Hrit-Shoola-Angina pectoris and its

management by Rasona Ksheerapaka. Varanasi: Banaras Hindu University; 1994

2] Shastry B S R L N - A comparative study on the effect of Arjuna Ksheerapaka and

Rasona Ksheerapaka in the management of hypertension. Hyderabad: A P University;

2003

Introduction…



PLAN OF STUDY

Present study is planned with the subsequent headings:

1. Review of Literature:

This part of study will be divided into 2 subdivisions

a. Drug review: Description of drug including chemical constituents

b. Disease review: Hyperlipidemia, its Ayurvedic and Modern perspective

2. Methodology

a. Pharmaceutical study

Preparation of 4 samples of Ksheerapaka with different ratio of Milk and Water with

the drug ratio constant. Observations during the preparation also illustrated.

b. Analytical study:

Analysis of 4 samples of Ksheerapaka using the following parameters

1. Organoleptic character: Colour, Odour, Taste

2. Viscosity

3. Specific gravity

4. pH

5. HPTLC

c. Experimental study

All 4 samples of Ksheerapaka will be subjected to the experimental study to

assess their efficacy in producing Antihyperlipidemic effect.

LITERARY STUDY

Drug Review…



DRUG REVIEW

Historical background

1) Drops of the nectar fell on the ground from severed neck of Rahu who stole

the nectar, which got transformed into Rasona. The twice born (Dvija, Brahmins) do

not eat it because it is born from the body of the Demon. Since it is actually born out

of Nectar it is the best Rasayana13

.

2) Once when Garuda tries to grab Amrutha from Indra a drop fell from it and

this drop is Rasona14

Gana

Hareetakyadi varga14

Karaveeradi gana15

Aushadhi varga16

Mulakadi varga17

Introduction18

Rasona consists of bulb of Allium sativum Linn. (Fam. Liliaceae); a perennial

bulbous plant, cultivated as an important condiment crop in the country.

Vernacular names18

Sanskrit : Rasona, Yavanesta

Assamese : Maharu

Bengali : Lasun

English : Garlic

Gujrati : Lasun, Lassun

Hindi : Lahasun

Kannada : Bellulli

Malayalam: Vellulli, Nelluthulli

Drug Review…



Marati : Lasun

Punjabi : Lasan

Tamil : Vellaipondu

Telugu : Vellulli, tellaypya, tellagadda

Urdu : Lahsan, seer

Synonyms19

Ugragandha

Yavanesta

Lashuna

Mahoushada

Mlechakanda

Properties and Action18

Rasa : Katu, Madhura

Guna : Guru, Snigdha, Tikshna, Sara, Picchila

Virya : Ushna

Vipaka: Katu

Karma: Vatahara, Kaphahara, Pittadushanakara, Raktadoshahara,

Bhagnasandhanakara, Dipana, Rasayana, Balya, Hridya, Vrshya, Varnya, Medhya,

Jantughna, Kanthya, Asthimamsasandhanakara, Chakshushya

Important formulations: Lashunadi vati, Lashunadi gritha, Vachalashunadi taila

Therapeutic uses18

Jeernajwara, Krimiroga, Gulma, Kushta, Arsa, Kasa, Shwasa, Peenasa, Sula,

Karnasula, Vatavyadhi, Hikka, Medoroga, Yoni vyapat, Visuchika, Pliha vriddhi,

Kshaya, Vishama jwara, Apasmara, Unmada, Sopha, Hrdroga, Vatasula, Trikasula,

Vrana krimi

Drug Review…



Dose

3 g

Description18

A) Macroscopic:

Drug occurs as entire bulb or isolated cloves (bulb lets); bulb sub globular, 4-6

cm in diameter, consisting of 8-20 cloves, surrounded by 3-5 whitish papery

membranous scales attached to a short, disc like woody stem having numerous, wiry

rootlets on the underside: each clove is irregularly ovoid, tapering at upper end with

dorsal convex surface, 2-3cm long, 0.5-0.8 cm wide, each surrounded by two very

thin papery whitish and brittle scales having 2-3 yellowish green folded leaves

contained within two white fleshy, modified leaf bases or scales; odour, peculiarly

pungent and disagreeable; taste, acrid gives warmth to the tongue

b) Microscopic

A clove of bulb shows triangular to tetra angular appearance in outline; outer

scale consists of an outer epidermis, followed by hypodermal crystal layer, mesophyll

made of parenchyma cells and an inner epidermis; both outer and inner epidermis

consists of sub rectangular cells; hypodermis consists of compressed, irregular,

tangentially elongated cells, each cell having large prismatic crystals of calcium

oxalate, while many cells contain small prismatic crystals also, mesophyll several

layers of parenchymatous cells having a few vascular tissues with spiral vessels; inner

epidermis similar to outer one; inner scale similar to outer scale but outer epidermis is

composed of sclerenchymatous cells; prismatic crystals in hypodermis slightly

smaller.

In surface view cells of outer epidermis elongated, narrow with thin porous

wall while those of inner epidermis similar to outer one but non porous; cells of

Drug Review…



hypodermal crystals layer ellipsoidal with thick porous walls, each cell having large

prismatic crystals of calcium oxalate, many cells also contain small prismatic crystals

in addition to bigger ones; inner scale shows markedly sclerenchymatous cells with

greatly thickened walls and very narrow lumen; cells of hypodermal crystal layer

somewhat smaller with walls more frequently pitted, size of crystals are also smaller

Chemical constituents20

Garlic contains 0.1- 0.36% of a volatile oil these volatile compounds are

generally considered to be responsible for most of the pharmacological properties of

garlic. It contains at least 33 sulphur compounds like aliin, allicin, ajoene, allylpropl,

diallyl, trisulfide, sallylcysteine, vinyldithiines, S-allylmercaptocystein, and others.

Besides sulphur compounds garlic contains 17 amino acids and their glycosides,

arginine and others.

Minerals such as selenium and enzymes like allinase, peroxidases, myrosinase,

and others. Garlic contains a higher concentration of sulphur compounds than any

other Allium species. The sulphur compounds are responsible both for garlic’s

pungent odour and many of its medicinal effects. The odour is formed by the action of

the enzyme allinase on the sulphur compound alliin. This enzyme is inactivated by

heat, which accounts for the fact that cooked garlic produces neither as strong an

odour as raw garlic nor nearly as powerful physiological effects.

However one of the most biologically active compounds, allicin (diallyl

thiousulfinate or diallyl disulfide) does not exist in garlic until it is crushed or cut;

injury to the garlic bulb activates the enzyme allinase, which metabolizes alliin to

allicin. In addition allicin is further metabolized to vinyldithiines. This breakdown

occurs within hours at room temperature and within minutes during cooking. Allilcin,

which was first chemically isolated in the 1940’s, has antimicrobial effects against

Drug Review…



many viruses, bacteria, fungi and parasites.

Like allicin, ajoene, is another chemical constituent thought to be most important to

health. Ajoene is a garlic-derived compound produced most efficiently from pure

allicin and has the advantage of a greater chemical stability than allicin. Several

clinical trials and in vitro studies of ajoene have demonstrated its best-known anti-

thrombosis, anti-microbial and cholesterol lowering activities. Recently, topical

application of ajoene has produced significant clinical response in patients with skin

basal cell carcinoma. Ajoene was shown to inhibit proliferation and induce apoptosis

of several human leukaemia CD34-negative cells including HL-60, U937, HEL and

OCIM-1.

Besides its anti-thrombosis, anti-microbial and cholesterol lowering

activities, ajoenes and dithiins are among the most active compounds formed from

fresh garlic. Ajoenes have been shown to: possess antithrombotic (anticlotting)

activity in human platelet suspensions; possess antitumor activity; display significant

antifungal activity, inhibiting the growth of Aspergillus niger , Candida albicans ,

Paracoccidioides-Brasiliensis , and Fusarium species; inactivate human gastric lipase,

a sulfhydryl enzyme involved in the digestion and adsorption of dietary fats; function

as antioxidants by inhibiting the interactions of leukocytes which mediate release of

superoxide anion.

George Barany, chemist at the University of Minnesota, reports that ajoene

is as potent as aspirin in preventing sticky red blood platelets from clumping together.

Related factors lengthen clotting time, while others actually dissolve clots. All three

effects can impact on heart attack and stroke prevention. Besides George Barany,

National Cancer Institute nutritionist Elaine Lanza states "There is still a big interest

in the area of fat and fibre, but right now, there is more emphasis on the idea of trace

Drug Review…



compounds, non-nutrients in vegetables and fruits that work against tumour

formation." "Garlic tops the list as a source of these trace elements or micronutrients."

The two major compounds in aged garlic, S-allylcysteine and S-allylmercapto-L-

cysteine, had the highest radical scavenging activity. In addition, some organosulfur

compounds derived from garlic, including S-allylcysteine, have been found to retard

the growth of chemically induced and transplantable tumours.

For thousands of years, garlic has been used for the treatment and prevention

of disease. So there has to be something there." "A huge data base exist documenting

health benefits ranging from cardiovascular effects to cancer inhibition, from the

slowing down of aging to the detoxification of heavy metals and other poisonous

chemical substances."

Disease Review…



DISEASE REVIEW

Hyperlipidemia is the term used to denote raised serum levels of one or more

of total cholesterol, low-density lipoprotein cholesterol, triglycerides, or both total

cholesterol and triglyceride (combined Hyperlipidemia). The abnormal levels of TG

and/or cholesterol in plasma are consequent to excess of substrate leading to more

production, defective transport, delayed peripheral clearance, reduced utilization of

lipoproteins or their intermediaries, or combinations of these abnormalities21

Hyperlipidemia in Ayurveda:

Hyperlipidemia does not bear a precise reference in Ayurveda though the

study of Ayurvedic literature bears some implicit allusion. This might be due to the

fact that it is a metabolic disorder and not a fully fledged disease in itself. It is

auxiliary to several other severe conditions like coronary artery disease,

cerebrovascular accidents, metabolic syndrome etc.

In Ayurveda various attempts have been made to use distinctive nomenclature

to denote the word Hyperlipidemia as follows

1. Rasagata Sneha Vriddhi

2. Rasa Raktagata Sneha Vriddhi

3. Medovriddhi

4. Medoroga or Medodosha

5. Ama Medo Dhatu.

The study of Ayurvedic literature bears certain ambiguous references

pertaining to an increase in the amount of circulating body lipids, yet the literal

meaning of hyperlipidemia is not found to be distinctly stated anywhere. The study of

hyperlipidemia can be done on the basis of studying two of the closest diseases in

Ayurveda having some amount of relation with hyperlipidemia are Atisthaulya or

Disease Review…



Medo Roga and Prameha

In the comprehensive Ayurvedic literature Medo Roga has been

synonymously ascribed to Sthaulya. Only Adhamalla while commenting on

Sharangadhara Samhita, tried to differentiate between the two types of Medo Roga22

1. Medo Roga: Adiposity including its clinical features (Sthaulya)

2. Medo Dosha: Lipid disorders where Meda acts as an etiological factor in the

genesis of other diseases. As an example of the former he has mentioned

Udaravriddhi due to fat deposition and for the latter he has quoted the morbid changes

developed due to obstruction in the channels by Meda. The obstruction of the

channels leads to vitiation of Vata in various channels due to diminished nutrition

corollary to various severe diseases having manifestations of Shwasa, Trishna,

Murcha etc.

ETIOLOGICAL FACTORS

Nidana Parivarjana or discontinuation of the etiological factors serves as the

first line of treatment in any disease23

, In hyperlipidemia the impaired Kapha Dosha

and Medo Dhatu are also found to play key role in the pathogenesis of Atisthaulya

and Prameha. Hence the causative Nidanas of the later diseases can be also

understood as the causative factors of hyperlipidemia. Acharya Dalhana has quoted

three main etiological factors of Sthaulya which encompass all the causes leading to

an increase in the Asthayi Medo Dhatu thus leading to a state of hyperlipidemia24

1. Vishistaaharavashat

It alludes to the use of specific dietary, lifestyle and psychological factors

which lead to Medo Roga. These factors either due to a similarity in the attributes or

similarity in the action result in a direct increase in the Medo Dhatu by following

Samanya Siddhanta25

. The causes of Medovaha Srotodushti can also be considered as

Disease Review…



etiological factors for hyperlipidemia as any derangement in the Srotas leads to the

Dhatu Dushti. Factors like Avyayama, Divaswapa, excessive consumption of Medya

Ahara and Varuni lead to Medovaha Srotodushti causing a state of Khavaigunya in

the Meda Dhatu26

. The following factors leading to over nutritional disorders stated

by Acharya Charaka can also be considered as etiological factors of hyperlipidemia.

These etiological factors can be classified broadly classified as Aharaja and Viharaja

Nidana27

Aharaja nidana:

They can be further classified as

Food Qualities: Guru, Madhura, Sheeta, Snigdha, Shleshmala, Atipicchil,

Abhishyandi.

The above qualities are present in Dravyas having a dominance of Prithvi and

Ap Mahabhuta due to similar Bhautika composition the above substances are seen to

cause a direct increase in the Kapha Dosha and Medo Dhatu

Food items: Navanna, Navamadya, Gramya Rasa, Audaka Rasa, Mamsa

Sevana, Paya Vikara, Dadhi, Sarpi, Ikshu vikara, Guda vikara, Shali, Godhuma,

Masha, Varuni.

The above stated foods like Navanna, Ikshu, Guda Vikara, Shali, Godhuma,

Masha are mainly seen to be rich sources of carbohydrates. Excess of ingested

carbohydrates present in the foods may be easily converted to fats. Fats formed from

carbohydrates are more saturated and have a high melting point. Excess consumption

of protein rich foods like milk and milk products may also lead to an increase in the

body lipids. If the intake of dietary protein exceeds its tissue usage it is converted into

triglycerides and stored in the body.

An obvious increase in the amount of body lipids occurs on intake of fats like

Disease Review…



Sarpi, Vasa etc. Hydrogenated oils and animal foods like milk, meat, eggs etc are rich

sources of dietary fats. Dietary fats are of two types’ unsaturated fats and saturated

fats. Unsaturated fats include monounsaturated fats, polyunsaturated fats, transfats

and omega fatty acids. Transfats and saturated fats are known to increase significantly

the risk of cardiovascular diseases by increasing the LDL levels. Unlike saturated fats,

transfats have the additional effect of decreasing levels of HDL. Thus the net increase

in LDL/HDL ratio with transfat is found to be approximately double than that due to

saturated fat28

Procedural defect of eating:

Atisampurana, Adhyashana, Vishamashana, Viruddhashana, Samashana,

Atyambupana, Bhojanottara Jalpana etc. Atisampurna or overeating results in excess

storage of energy exceeding its utilization. As seen previously an excess of

carbohydrates, proteins or fats are stored in the form of fat. Also excessive caloric

consumption of any source of food with an associated weight gain may lead to

hypertriglyceridaemia through VLDL increase.

Factors like Adhyashana, Vishamashana, Viruddhashana, Samashana

subsequently lead to Ama formation. Ama results in Srotorodha causing further

pathogenesis of the disease.

Viharaja nidana: Avyayama, Avyavaya, Divaswapa, Asyasukha (Sedentary

lifestyle), Swapnasukha, Bhojanottara Snana, Bhojanottara Nidra etc.

All the above mentioned factors lead to lesser energy expenditure than input

resulting in corpulence.

Manas Nidana: Harshanityatva, Achintana are enumerated as the causative factors

by Acharya Charaka which are conducive to Atisthaulya.

Disease Review…



2. Adrishtavashat:

This pertains to factors mainly the heredity or genetic factors which cannot be

seen or assessed directly. These are mainly due to defects in either the Shukra or

Shonita or both which are transmitted in the progeny. All the Acharyas have

unanimously included genetic defects as a cause of Sthaulya and allied disorders like

Prameha etc. Acharya Charaka has also stated Beejadosha as one of the etiological

factors of Atisthaulya29

. These genetic factors lead to development of a state of

Khavaigunya in the Medo Dhatu which making it susceptible for the action of vitiated

Doshas.

Various genetic and hereditary factors are known to be responsible for causing

Hyperlipidemia which can also be termed as Primary Hyperlipidemia that can be

studied under the broad umbrella of “Beejadosha”. These defects maybe monogenic

(single gene defects) or polygenic (multiple gene defects). Genetic defects lead to

accumulation in the plasma of excessive amounts of lipoproteins usually due to the

failure of catabolic apparatus and seldom due to accumulation of lipoproteins caused

by increased synthesis. The failure in the catabolic apparatus may be a result of:

Deficiency of enzymes like LPL, HL, LCAT etc

Deficient cell surface receptors e.g. LDL receptors

Deficiency of apolipoproteins e.g. ApoB100

However only a small proportion of patients with clinically recognized

Hyperlipidemia have single gene mutations while most are either polygenic or a

product of the interaction of genetically determined susceptibility with environmental

factors.

Fredrickson’s classification (analysis of lipids by beta-quantification-

ultracentrifugation followed by electrophoresis) 30

differentiates five categories of

Disease Review…



Hyperlipidemias based on heredity. In this classification, all but one of the

hyperlipidemias, type IIa, are characterized by elevated triglycerides: types I, IIb, III,

IV, and V. In types I, IIb, III, and V, serum cholesterol levels also are elevated

Type I Hyperlipidemia or Familial Chylomicronemia syndrome:

It is a rare disorder characterized by severe elevations in chylomicrons and extremely

elevated triglycerides, always well above 1000 mg/dl and as high as 10,000 mg/dl or

higher. It is caused by mutations of either the gene lipoprotein lipase or its cofactor,

apo C-II. Despite the exceedingly high triglyceride elevations, these mutations do not

confer an increased risk of atherosclerotic disease. Because chylomicrons also contain

a small amount of cholesterol, serum cholesterol levels are also quite high. Type I is

the only form of Hypertriglyceridemia that does not confer an increased risk for

developing coronary artery disease

Type IIa or Familial Hypercholesterolaemia:

It is characterized by elevations in the plasma levels of LDL, which are elevated since

birth and remain so throughout life. Plasma triglyceride levels are normal and HDL

levels are reduced

Type IIb or Familial Combined Hyperlipidemia:

It is the classic mixed Hyperlipidemia (high cholesterol and triglycerides) caused by

elevations in both LDL and VLDL.

Type III or Familial Dysbetalipoproteinemia:

The patients have elevated total cholesterol and triglyceride levels and are easily

confused with patients with type II b Hyperlipidemia. Patients with type III

Hyperlipidemia have elevations in intermediate-density lipoprotein (IDL), a VLDL

remnant, and a significant risk for developing coronary artery disease.

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Type IV or Familial Hypertriglyceridaemia:

It is characterized by abnormal elevations of VLDL, and triglyceride levels are almost

always less than 1000 mg/dL. Serum cholesterol levels are normal.

Type V or Familial Hypertriglyceridaemia:

It is characterized by elevations of both chylomicrons and VLDL. Triglyceride levels

are invariably greater than 1000 mg/dL, and total cholesterol levels are always

elevated; however, LDL cholesterol levels are normal

Table no: 1. Fredrickson’s classification (Phenotype) of Hyperlipidemia

Phenotype I IIa IIb III IV V

Lipoprotein

elevated

Chylomicrons LDL LDL

and

VLDL

Chylomicron

and VLDL

remnants

VLDL Chylomicrons

and VLDL

Triglycerides + + + + -- + + + + to + + + + + + + + +

Cholesterol + to + + + +

+

+ + to

+ + +

- - to + + + + + + + +

LDL - - - -

HDL - - - - - -

Plasma

appearance

Lactescent Clear Clear Turbid Turbid Lactescent

Coronary

atherosclerosis

0 +++ +++ +/- +/- /-+

Peripheral

atherosclerosis

0 + + ++ +/- +/-

Genetic

nomenclature

FCS FH,

FDB

FCHL FDBL FHTG FHTG

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FCS – Familial Chylomicronemia syndrome FH – Familial Hypercholesterolemia

FDB – Familial Defective apo B FCHL – Familial Combined Hyperlipidemia

FDBL - Familial dysbetalipoproteinemia FHTG - Familial Hypertriglyceridaemia

3. Medosavrita Margatvat:

Commentator Dalhana has quoted that Vata Vikaras or disorders of Vata occur

due to Avarana (covering) of the Marga by Medo Dhatu. This refers to secondary

conditions leading to Hyperlipidemia like diabetes mellitus, hypothyroidism etc where

a significant role is played by Vata in the pathogenesis. The normal Gati of Vata

Dosha gets obstructed due to excess of Medo Dhatu which thus gets vitiated causing

further pathogenesis. These factors causing secondary Hyperlipidemia can also be

called as Nidanarthakari Roga since one disease serves as the etiological factor of the

other31

Some secondary conditions due to Avaranajanya Samprapti are discussed below:

Secondary Causes of Hyperlipidemia:

1. Obesity:

An increase in the adipocyte mass and decrease in insulin sensitivity seen in

obesity usually results in Hyperlipidemia. The expanded adipose tissue releases

excess of fatty acids which eventually result in an increase in VLDL or LDL. Also

plasma HDL is found to be low in obese individuals. In the recent interheart study,

abdominal obesity assessed by waist-to-hip ratio showed a strong association with

myocardial infarction32

Asian Indians are seen to have a characteristic obesity phenotype with

relatively lower BMI but with central obesity. Indians also tend to have excess body

fat, abdominal and trunkal adiposity. For any given waist circumference, they have

increased body fat accumulation and for any given body fat, they have increased

Disease Review…



insulin resistance33

, these features have been referred to as the “Asian Indian

Phenotype or Paradox”34

The World Health Organization has revised the BMI cut-off for Asian Indians

and suggested a BMI of 25 kg/m2 to define obesity against the 30 kg/m2 recommended

for Europeans35

It has been suggested that fat distributed in the abdominal region, particularly

visceral fat is more metabolically important than other fat depots making a person

more prone to metabolic defects. The increased visceral obesity leads to the resistance

of the adipocytes to the lipogenic effect of insulin. The consequence of this is higher

basal rates of lipolysis with increased release of FFA into the portal venous system.

Abdominal fat may also secrete less leptin than subcutaneous fat. Leptin potently

activates cellular fuel consumption by stimulating FA oxidation, reducing lipogenesis,

enhancing glucose entry and metabolism, and dramatically shrinking fat stores in

adipose tissue as well as in muscle and liver cells36

Thus decreased levels of leptin further add on to the hyperlipidemic picture.

Abdominal fat mass expansion is also coupled with reciprocally reduced release of

Adiponectin. Adiponectin is an adipokine (hormone) that is secreted from the fat cells

in increased amounts when a person is lean and in decreased amounts when a person

is obese37

since it improves both glucose levels and fat oxidation low levels lead to

both hyperglycemia and dyslipidemia. Abdominal fat additionally expresses higher

levels of reninangiotensin system components: increased angiotensinogen and

increased angiotensin II (Ang II) AT1 receptors eventually resulting in Hypertension38

Finally, epidemiologic studies confirm the unique significance of abdominal fat mass

in predicting microalbuminuria, diabetes mellitus and overall cardiovascular risk39

Disease Review…



2. Diabetes mellitus:

Patients with type I diabetes mellitus if under good glycemic control do not

suffer from Hyperlipidemia but diabetic ketoacidosis seen in these patients is

frequently accompanied by hypertriglyceridaemia. Insulin-resistant patients are seen

to possess a combination of dysfunctional HDL particles and increased number of

small, dense atherogenic LDL particles and triglyceride-rich remnant particles, which

can be readily assessed by the clinician through determining non-HDL-C, (apo B), or

NMR-derived LDL-particle counts40,41

3. Metabolic syndrome:

Metabolic syndrome is consistently associated with specific metabolic

abnormalities like high circulating free fatty acids (FFA) 42

and an increased

intracellular lipid content of not only white adipose tissue (WAT) but also

hepatocytes, skeletal myocytes, pancreatic cells, cardiomyocytes, gastrointestinal

enterocytes, and vascular endothelial cells 43, 44

Although vast researches have been carried out to identify the relationship

between dyslipidaemia and metabolic syndrome the relationship is still incoherent.

4. Hypothyroidism:

The reduction in hepatic LDL receptor function and delayed clearance of LDL

seen in hypothyroidism leads to an increase in the plasma LDL level. Hypothyroid

patients may have an increase in the circulating IDL and some may also be mildly

hypertriglyceridaemic.

5. Menopause:

The incidence of cardiovascular disease and coronary heart disease in women

increases after both natural and surgical menopause45

; many investigators have

studied the impact of menopause on coronary risk factors, such as serum cholesterol

Disease Review…



BP and obesity46

It has been shown consistently that menopause causes an increase in LDL

cholesterol and a decrease in HDL thereby making menopausal women more prone to

the risk of heart disease. This dyslipidaemia can be attributed to the depletion of

estrogen, which has a protective effect against cardiovascular diseases. In a

prospective population study carried out by Akahoshi et al47

showed clearly that a

significant increase in serum cholesterol preceded natural menopause by 3 years.

6. Pregnancy:

Pregnancy is normally accompanied by moderate rises in cholesterol and

triglyceride, reflecting increase in VLDL, LDL, and HDL due to an increase in

estrogens. Marked rise in cholesterol are usual in FH during pregnancy which can

exacerbate the pre-existing hypertriglyceridemia, especially when there is protein

lipase deficiency.

7. Renal Diseases:

In Nephrotic syndrome if the Creatinine clearance is relatively normal then

proteinuria is associated with increased levels of LDL cholesterol (often the level of

LDL cholesterol increase is inversely proportional to the reduction in serum albumin).

Hypertriglyceridaemia is unusual in nephrotic syndrome but may be caused due to

chronic renal failure which results in reduction of the lipoprotein lipase activity. HDL

cholesterol levels may be normal or reduced in nephrotic syndrome

8. Liver diseases:

Hypertriglyceridaemia is characteristic of hepatocellular disease due to

triglyceride rich lipoproteins with density in the VLDL and LDL range - probably

secondary to hepatic lipase deficiency and damage to remnant removal mechanisms.

HDL consists mainly of small particles with decreased cholesteryl ester secondary to

Disease Review…



a deficiency of LCAT (catalyzes the esterification of free cholesterol). Primary biliary

cirrhosis and extrahepatic biliary obstruction can cause Hypercholesterolaemia and

elevated levels of plasma phospholipids associated with increased levels of an

abnormal lipoprotein and LDL.

9. Iatrogenic:

Certain drugs like beta blockers, thiazide diuretics, and oral contraceptives are

also known to elevate the plasma lipoproteins

Beta blockers:

Beta blockers tend to result in an increase in triglyceride level (via an increase

in VLDL) and a reduction in HDL. The increase in VLDL appears to be result of

reduced clearance of triglyceride-rich lipoproteins (possibly via direct effect on

lipoprotein lipase or perhaps because of diversion of blood flow from sites rich in

lipoprotein lipase e.g. muscle vascular bed). They do not appear to affect total

cholesterol or LDL cholesterol. The beta blocker may have a marked effect in a

patient having a predisposition to hypertriglyceridaemia

Thiazide diuretics:

Thiazide diuretics raise very low density lipoprotein (VLDL) and low density

lipoprotein (LDL) by certain mechanisms which are not yet identified. The effects in

general are small - However this may be more substantial in diabetes mellitus. There

is no alteration of high density lipoprotein (HDL) levels reported with Thiazide use.

Hormones:

Oral contraceptives are associated with increased total cholesterol, HDL,

triglyceride. In persons with an underlying primary Hypertriglyceridaemia and

associated obesity, estrogenic medications can also depress triglyceride removal

mechanisms.

Disease Review…



HRT therapy with estrogen alone or combined with progesterone is associated

with an increase in HDL and triglycerides and a decrease in LDL and lipoproteins.

Anabolic steroids taken orally markedly reduce levels of HDL in contrast to injectable

testosterone, which does not adversely affect the LDL: HDL ratio.

Atypical antipsychotics:

Several studies suggest that changes are concordant with weight changes and

that clozapine and olanzapine tend to be associated with adverse changes in serum

concentrations of triglyceride and cholesterol48

Lipid Hypothesis of Atherogenesis49

The lipid hypothesis was developed in the 1850s to explain the pathogenesis

of atherosclerosis. It proposed a connection between plasma cholesterol level and the

development of coronary heart disease. The German pathologist Rudolph Virchow in

1856 suggested that blood lipid accumulation in arterial walls causes

atherosclerosis50

.

In 1913, a study by Nikolai Anitschkow showed that feeding rabbit’s with cholesterol

could induce symptoms similar to atherosclerosis, suggesting a role for cholesterol in

atherogenesis51

In 1951, Duff and McMillian formulated the lipid hypothesis in its

modern form proposing its importance in atherosclerosis in a review which appeared

in the American Journal of Medicine52

Ancel Keys was one of the most well known

early modern proponents of the theory that saturated fats and cholesterol in the blood

are the cause of heart disease53

The National Institute of Health held a consensus development conference

reviewing the scientific evidence in 1984, and established that “the lowering

definitely elevated blood cholesterol levels (specifically, blood levels of low-density

lipoprotein (LDL) cholesterol) will reduce the risk of heart attacks caused by coronary

Disease Review…



heart disease”54

. As of the end of the 1980s, the evidence accumulated through studies

resulted in general acceptance of the lipid hypothesis and the rejection of the

“cholesterol controversy”55

and by 2002, the lipid hypothesis was accepted by the

scientific community as proven56

or, as one article stated, "universally recognized as a

law.”57

Thus in accordance with the lipid hypothesis the role of raised lipids can be

established and appreciated in the pathogenesis of atherosclerosis.

Atherosclerosis:

Atherosclerosis can be understood by Dhamani Pratichaya stated in the

Ayurvedic classics. It is mentioned as one amongst the Kaphaja Nanatmaja Vikara58

The localized Kapha which is the predominant Dosha vitiates with the Asthayi

Medo Dhatu. The pathogenesis occurs due to quantitative growth of both the above

factors which brings about an obstruction to the natural movement of Vata resulting in

the vitiation of the Vata Dosha. This results in the Shoshana of the Sthanika Kapha

and Medo producing Kathinya59

this result in the hardening of blood vessels thereby

causing an obstruction to the natural circulation.

The intensity of this disease can be comprehended on evaluating its capacity to

pose a threat to the three main Marmas namely Hridaya, Shiro and Basti causing

cardiovascular diseases, cerebrovascular accidents and renal diseases respectively.

Atherosclerosis is a disease of the large and intermediate sized arteries in which fatty

lesions called atheromatous plaques develop on the inside surfaces of the arterial

walls. Abnormalities in lipoproteins and derangements in lipid metabolism rank as the

most firmly established and best understood risk factors for atherosclerosis60

since

cholesterol and allied lipoproteins have been demonstrated in the atheromatous

plaques. The plaque formation takes place by the following steps:

Disease Review…



Chronic endothelial injury: Chronic endothelial injury due to factors like

Hyperlipidemia, Hypertension, Smoking, Homocysteine etc results in endothelial

injury which further causes an inflammatory response. This can be perceived by

increased permeability, monocyte adhesion and emigration. The most important

determinant of endothelial alteration is stated to be the adverse effect of

Hypercholesterolemia.

Fatty streak formation: Emigration of the smooth muscle cells from the media to

intima occurs. Also the activated macrophages and smooth muscle cells engulf the

modified lipids (oxidized LDL) contributing to foam cell formation. These lipid filled

foam cells lead to formation of fatty streaks which are the earliest lesions of

atherosclerosis.

Atheromatous plaque formation: Fatty streaks are the precursors of plaques. These

plaques have three principle components

(a) Cells including smooth muscle cells, macrophages, and other leucocytes

(b) Extracellular matrix including collagen, elastic fibres, and proteoglycans

(c) Intracellular and extracellular lipid

Atheromatous plaques if leads to occlusion of the coronary arteries results in

myocardial infarction and angina pectoris. Atherosclerosis of the arteries supplying

the central nervous system frequently provokes stroke and transient cerebral ischemia.

In the peripheral circulation, atherosclerosis causes intermittent claudication and

gangrene and can jeopardize limb viability. Involvement of the splanchnic circulation

can cause mesenteric ischemia. Atherosclerosis can affect the kidneys either directly

(e.g., renal artery stenosis) or as a frequent site of atheroembolic disease.

Atherogenesis in humans typically occurs over a period of many years, usually

many decades. Growth of atherosclerotic plaques probably does not occur in a smooth

Disease Review…



linear fashion, but rather discontinuously, with periods of relative quiescence

punctuated by periods of rapid evolution. After a generally prolonged “silent” period,

atherosclerosis may become clinically manifest. The clinical expressions of

atherosclerosis may be chronic, as in the development of stable, effort-induced angina

pectoris or of predictable and reproducible intermittent claudication. Alternatively, a

much more dramatic acute clinical event such as myocardial infarction, a

cerebrovascular accident, or sudden cardiac death may first herald the presence of

atherosclerosis. Other individuals may never experience clinical manifestations of

arterial disease despite the presence of widespread atherosclerosis demonstrated post

mortem.

Ischemic Heart Disease (IHD):

Ischemic heart disease (IHD) causes more deaths and disability and incurs

greater economic costs than any other illness in the developed world. Round about 3

million deaths take place due to heart disease every year in India of which one third

can be attributed to coronary artery disease. India already has 50 million heart patients

and by 2010, 60% of the world’s heart disease patients will be Indians. Enas et al61

found that South Asians around the globe have the highest rates of Coronary Artery

Disease (CAD). A family history of premature IHD is an important indicator of

increased risk and should trigger a search for modifiable risk factors such as elevated

cholesterol, hypertension, smoking, obesity, low HDL levels, diabetes and lack of

physical activity which are risk factors for both men and women Total triglyceride

levels are an independent risk factor for IHD in women, but not in men. Triglycerides

appear to interact with HDL cholesterol in such a way that HDL levels fall as T G

levels rise. Low HDL is known to be harmful to the heart.

Disease Review…



By reducing the lumen of the coronary arteries, atherosclerosis limits

appropriate increase in perfusion when the demand for flow is augmented, as occurs

during exertion or excitement. When the luminal reduction is severe, myocardial

perfusion in the basal state is reduced. Coronary blood flow can also be limited by

spasm, arterial thrombi, and, rarely, coronary emboli. Epicardial coronary arteries are

the major site of atherosclerotic disease. The major risk factors for atherosclerosis

high LDL, low HDL, cigarette smoking, hypertension, and diabetes mellitus disturb

the normal functions of the vascular endothelium. These functions include local

control of vascular tone, maintenance of an anticoagulant surface and defence against

inflammatory cells. The loss of these defences leads to inappropriate constriction,

luminal clot formation and abnormal interactions with blood monocytes and platelets.

The latter results in the subintimal collections of fat, smooth-muscle cells, fibroblasts

and intercellular matrix resulting in the formation of atherosclerotic plaques which

develop at irregular rates in different segments of the epicardial coronary tree and lead

eventually to segmental reductions in cross-sectional area. Segmental atherosclerotic

narrowing of epicardial coronary arteries is caused most commonly by the formation

of a plaque, which is subject to fissuring, erosion, haemorrhage and thrombosis. Any

of these events can temporarily worsen the obstruction, reduce coronary blood flow

and cause clinical manifestations of myocardial ischemia.

Disease Review…



Table no: 2. ATP-III Classifications of Total, LDL and HDL Cholesterol and

Triglycerides (mg/dL) 62

Total cholesterol LDL cholesterol HDL cholesterol Triglycerides

< 100 optimal

< 200 desirable 100-129 near or

above optimal

< 40 low < 150 normal

200-239 borderline

high

130-159 borderline

high

≥ 60 high 150-199 borderline

high

≥ 240 high 160-189-high 200-499 high

160-189-high ≥ 500 very high

≥ 190 very high

Table no: 3. Brief Treatment Aspect According to Ayurveda Shamana chikitsa63

AHARA AHARA VIHARA AUSHADHA

Rukshanna Sevana

Kulattha

Yava

Mudga

Aadhaki

Kshaudra

Patola

Jangala Mamsa

Kodrava

Madhu

Shyamaka

Uddalaka

Vyayama

Upavasa

Vyavaya

Prajagara

Chintana

Guduchi

Bhadra Musta

Triphala

Shilajit

Agnimantha

Arishta

Sakshaudra Abhayaprasha

Guggulu

Gomutra

Loharaja

Rasanjana

Disease Review…



Shodana chikitsa64

1] Antahaha Parimarjana

Virechana,

Raktamokshana

2] Bahya Parimarjana

Swedana

Churna

Pradeha (Udvartana)

Disease Review…



Treatment of Hyperlipidemia According To Modern Science:

Table no: 4. Major Drugs Used For the Treatment of Hyperlipidemia65

Drug Major

indications

Mechanism Common side

effects

HMG-CoA

reductase

inhibitors

Lovastatin

Pravastatin

Simvastatin

Fluvastatin

Atorvastatin

Elevated LDL Decrease

cholesterol

synthesis, decrease

hepatic LDL

receptors, decrease

VLDL production

Myalgias

Arthralgias

Elevated

transaminases

Dyspepsia

Bile acid

sequestrants

Cholestyramine

Colestipol

Colesevelam

Elevated LDL Increase bile

excretion and

increase

LDL receptors

Bloating

Constipation

Elevated TG

Nicotinic acid

Immediate release

Sustained release

Extended release

Elevated LDL,

low HDL,

elevated TG

Decrease hepatic

VLDL synthesis

Cutaneous

flushing

GI upset

Elevated glucose,

uric acid and other

liver function tests

Fibric acid

derivatives

Gemfibrizol

Fenofibrate

Elevated TG,

elevated

remnants

Increase LPL,

decrease hepatic

VLDL synthesis

Dyspepsia

Myalgia

Gallstones

Elevated

transaminases

Fish oils Severely

elevated TG

Decrease

chylomicron and

VLDL production

Dyspepsia

Diarrhoea

Fishy odour to

breath

Cholesterol

absorption

inhibitors

Ezetimibe

Elevated LDL Decrease intestinal

Cholesterol

absorption

Elevated

transaminases

METHODOLOGY

PHARMACEUTICAL STUDY

Pharmaceutical Study…



PHARMACEUTICAL STUDY

Materials and methods:

Source of the Data:

The drug Rasona required for the preparation of Ksheerapaka was collected

from Udupi, Karnataka, India

The Milk used was Pasteurized toned Nandini Milk, KMF Mangalore

Preparation of Rasona Ksheerapaka was done every day throughout the course of

study at the Dept. of Bhaishajya Kalpana, S.D.M. College of Ayurveda, Hassan.

Method of Data Collection:

Four samples of Rasona Ksheerapaka was prepared according to the

references found in the following classical Ayurvedic texts

1) Charaka Samhita66

2) Sharangadhara Samhita67

3) Dravya guna vijnana68

and

4) Astanga Sangraha69

The reference of Rasona Ksheerapaka is available in Charaka Samhita.

However the same reference is also available in Astanga Hridaya70

, Chakradatta71

and

Bhaishajya Ratnavali72

. Since there was no reference of Rasona Ksheerapaka found in

Sharangadhra Samhita, Dravya guna vijnana and Astanga Sangraha, general method

of preparation mentioned in the respective classical texts was followed.

The changes happened in the samples after the preparation was assessed by

different Analytical and Experimental study.

Ingredients:

Rasona ( Allium sativum)

Go Ksheera ( Cow’s milk)




Apparatus used

Mortar and pestle

Weighing machine

Stainless steel vessels

Spoons

Filter

Gas stove

Steps followed for Rasona shodana73

As per the protocol of Rasona shodana mentioned in Sharangadhara Samhita.

The bulbs of Rasona were separated into bulbils (bulblets). These were dehusked

(external covering was removed) and separated into two halves and mid-stalk was

removed. These destalked bulbils were soaked overnight in butter milk, next day

washed with water and used for the preparation of Ksheerapaka

General method of preparation

Specific amount of Rasona (Garlic) was crushed and added with specific

amount of Milk and water and heated on mild fire till the volume reduces to initial

volume of the Milk. Later it is filtered and used

Method of preparation of each sample

1] Sample 1(according to Charaka)

As per this reference the drug, milk and the water are taken in 1:4:4 ratio,

boiled on mild fire till the volume of the mixture reduces to the initial volume of the

milk

2] Sample 2 (according to Sharangadhara)

As per this reference the drug, milk and the water are taken in 1:8:32 ratio, boiled on

mild fire till the volume of the mixture reduces to the initial volume of the milk




3] Sample 3 (according to Yadavji Trikamji Acharya)

As per this reference the drug, milk and the water are taken in 1:15:15 ratio,

boiled on mild fire till the volume of the mixture reduces to the initial volume of the

milk

4] Sample 4 (according to Vagbhata)

As per this reference it is advised to prepare decoction of drug and water and

to that decoction equal quantity of milk is to be added and boiled till the volume of

milk reduces to the initial volume of the milk

The Ratio of the drug, milk and the water followed have been provided in the

form of consolidated statement in Table

Table no: 5. Shows Ratio of the Drug, Milk and the Water used for Ksheerapaka

preparations

Sl no Author Ratios Reduction

Drug Milk Water

1 Charaka 1

(10g)

4

(40ml)

4

(40ml)

(40ml)

2 Sharangadhara 1

(10g)

8

(80ml)

32

(320ml)

(80ml)

3 Yadavji Trikamji

Acharya

1

(10g)

15

(150ml)

15

(150ml)

(150ml)

4 Vriddha Vagbhata advices initially Kashaya (1:16=1/4) should be prepared, after

that equal quantity of Milk is to be added to that Kashaya, reheated until the

volume reduces to initial volume of the Milk [10g drug:160ml water = 40ml

kashaya] [40ml kashaya +40ml milk =40 ml (ksheeravashesha)]




Observations

During the preparation creaming on the surface was observed which was

subsided with frequent stirring

Emission of Alliaceous (Garlic) odour was observed

The pungent taste of the drug was reduced after the preparation

Precautions

The drug material used was in paste form.

It was cooked on mild fire throughout the process

Frequent stirring was done during the preparation

After the preparation it was filtered, on self cooling collected in the plastic

containers and they were named according to the ratio of drugs used for that

particular preparation




Pharmaceutical process of Rasona Ksheerapaka

Fig 7-Collection

Fig 2- Milk Fig 1-Garlic

Fig 3-Separated

bulbils

Fig 4-Destalked Garlic

Fig 5-Soaked in Butter

milk

Fig 6-Preparation

ANALYTICAL STUDY

Analytical Study……



ANALYTICAL STUDY

Introduction

Analysis is the process of breaking a complex substance into smaller parts to

gain a better understanding of it, which reveals the minor, but has important aspects

regarding the standardization of the drug. Without analytical study, research of a drug

is incomplete. It provides some standards to judge its quality and also helpful to

interpret the pharmacokinetics and pharmacodynamics of the drug.

The samples of Rasona Ksheerapaka along with the Drug Rasona and Milk

were analyzed at SDM Research Center, Udupi; using following parameters as per the

references available in protocol for testing published by CCRAS.

1. Organoleptic characters

2. Viscosity

3. Specific gravity

4. pH & h

5. HPTLC

Methodology of Analysis

1. Organoleptic characters

Organoleptic characters of the test sample were documented by means of examination

using sensory organs.

2. Viscosity74

Materials: 4 samples of Rasona Ksheerapaka, Plain milk, Viscometer and stop watch

Method: The given sample is filled in a U tube viscometer in accordance with the

expected viscosity of the liquid so that the fluid level stands within 0.2 mm of the

filling mark of the viscometer when the capillary is vertical and the specified

temperature is attained by the test liquid. The liquid is sucked or blown to the




specified height of the viscometer and the time taken for the sample to pass the two

marks is measured. Viscosity is measured using the formula

η1= ρ1t1 X η2

ρ2t2

η1 - Viscosity of sample

η2 - Viscosity of water

t1 and t 2- Time taken for the sample and water to pass the meniscus

ρ1 and ρ2 - Density of sample and water.

3. Specific gravity75

Materials: 4 samples of Rasona Ksheerapaka, plain milk, specific gravity bottle,

stopper, acetone, ether, distilled water

Method: Clean a specific gravity bottle by shaking with acetone and then with ether.

Dried the bottle and noted the weight. Cooled the sample solution to room

temperature. Carefully filled the specific gravity bottle with the test liquid, inserted

the stopper and removed the surplus liquid. Noted the weight. Repeated the procedure

using distilled water in place of sample solution.

4. Determination of pH76

Materials: 4 samples of Rasona Ksheerapaka, plain milk, pH meter, beaker, Buffer

tablets

Methods

Preparation of buffer solutions: Standard buffer solution: Dissolved one tablet of

pH 4.7 and 9.2 in 100 ml of distilled water.

Determination of pH: 1 ml of sample was taken and make up to 10 ml with distilled

water, stirred well and filtered. The filtrate was used for the experiment. Instrument

was switched on 30 minutes time was given for warming pH meter. The pH 4 solution




was first introduced and the pH adjusted by using the knob to 4.02 for room

temperature 300

C. The pH 7 solution was introduced and the pH meter adjusted to 7

by using the knob. Introduced the pH 9.2 solution and checked the pH reading without

adjusting the knob. Then the sample solution was introduced and reading was noted.

Repeated the test four times and the average reading were taken as result.

5. HPTLC77

Materials: Weighing balance, distilled ethyl acetate, water bath, HPTLC applicator, 4

samples of Rasona Ksheerapaka along with the Drug Rasona and Milk

Method: 10 µl of the sample was applied on a precoated silica gel F254 on aluminum

plates to a band width of 8 mm using Linomat 5 TLC applicator. The plate was

developed in Butanol: Water: Acetic acid: Formic acid (2.8: 0.8: 0.9: 0.2) and the

developed plates were visualized under UV 254 and 366 nm, and after derivatisation

in vanillin-sulphuric acid spray reagent and scanned under UV 254 and 366 nm. Rf,

colour of the spots and densitometric scan were recorded.




RESULTS

Table no: 6. Organoleptic characters

Plain milk Kashaya

method

1:4:4 1:8:32 1:15:15

Colour Yellowish

white

Yellowish

white

Yellowish

white

Yellowish

white

Yellowish

white

Odour Characteristic Alleacious Alleacious Alleacious Alleacious

Taste Sweet

Sweet,

Alleacious

Sweet,

Alleacious

Sweet,

Alleacious

Sweet,

Alleacious

Table no: 7. Physico-chemical parameters

Parameters Results n=3 %w/w

Plain milk Kashaya

method

1:4:4 1:8:32 1:15:15

Viscosity 2.0667 1.9687 2.1763 1.6972 1.8370

Specific gravity 1.0378 1.0332 1.0476 1.0213 1.0258

pH 6.56 6.48 6.49 6.54 6.50




Figure 8. TLC photodocumentation of alcohol extract of Ksheerapaka compared

with Rasona

At 254nm At 366nm Pot derivatisation

Track 1 - Alcohol extract of Plain milk 10 µl

Track 2 - Alcohol extract of Kashaya Method 10 µl

Track 3- Alcohol extract of 1:4:4 10 µl

Track 4 - Alcohol extract of 1:8:32 10 µl

Track 5 - Alcohol extract of 1:15:15 10 µl

Track 6- Alcohol extract of Rasona 10 µl

Solvent system – Butanol: Water: Acetic acid: Formic acid (2.8:0.8:0.9:0.2)

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6




Table no: 8. Rf value of alcohol extract of samples at 254nm - 10 µl

Plain milk Kashaya

Method

1:4:4 1:8:32 1:15:15 Rasona

- 0.27(Light

Green)

0.27(Light

Green)

0.27(Light

Green)

0.27(Light

Green)

-

- 0.55(Light

Green)

0.55(Light

Green)

0.55(Light

Green)

0.55(Light

Green)

-

- 0.65(Light

Green)

0.65(Light

Green)

0.65(Light

Green)

0.65(Light

Green)

-

- 0.73(Light

Green)

0.73(Light

Green)

0.73(Light

Green)

0.73(Light

Green)

-

Table no: 9. Rf value of alcohol extract of samples at 366nm - 10 µl

Plain milk Kashaya

Method

1:4:4 1:8:32 1:15:15 Rasona

- 0.27(Light

Violet)

0.27(Light

Violet)

0.27(Light

Violet)

0.27(Light

Violet)

0.27(Light

Violet)

- 0.51(Light

Violet)

0.51(Light

Violet)

0.51(Light

Violet)

0.51(Light

Violet)

0.51(Light

Violet)

- 0.60(Light

Violet)

0.60(Light

Violet)

0.60(Light

Violet)

0.60(Light

Violet)

-

- 0.73(Light

Violet)

0.73(Light

Violet)

0.73(Light

Violet)

0.73(Light

Violet)

-




Table no: 10. Rf value of alcohol extract of samples after Post derivatisation-10µl

Plain milk Kashaya

Method

1:4:4 1:8:32 1:15:15 Rasona

- 0.09(Light

Brown)

0.09(Light

Brown)

0.09(Light

Brown)

0.09(Light

Brown)

-

- 0.14(Light

Brown)

0.14(Light

Brown)

0.14(Light

Brown)

0.14(Light

Brown)

-

- 0.20(Light

Brown)

0.20(Light

Brown)

0.20(Light

Brown)

0.20(Light

Brown)

-

- 0.34(Dark

Brown)

0.34(Dark

Brown)

0.34(Dark

Brown)

0.34(Dark

Brown)

-

- 0.44(Brown)

0.44(Brown) 0.44(Brown) 0.44(Brown) 0.44

(Brown)

- 0.51(Light

Brown)

0.51(Light

Brown)

0.51(Light

Brown)

0.51(Light

Brown)

-

- - - - - 0.53(Light

Violet)

- - - - - 0.71(Light

Violet)

- - - - - 0.75(Light

Violet)




Figure 9. HPTLC photo documentation at 254nm

Fig 3.a Plain Milk

Fig 3.b Kashaya method




Fig 3.c 1:4:4

Fig 3.d 1:8:32




Fig 3.e 1:15:15

Fig 3.f Rasona




Figure 10. HPTLC photo documentation at 366nm

Fig 4.a Plain Milk

Fig 4.b Kashaya method




Fig 4.c 1:4:4

Fig 4.d 1:8:32




Fig 4.e 1:15:15

Fig 4.f Rasona




Figure 11. 3D Display of All the Tracks

At 254nm

At 366nm

Remarks

The organoleptic characters, pH, specific gravity and viscosity of the given sample

have been documented in the report. And also TLC photo documentation, HPTLC

densitometric scan, Rf values and 3 D display of all the tracks have been reported.

Analytical study...

Page | 53

“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different methods in Albino Rats”

Table no: 11 HPTLC Densitometric scan at 254 nm

Milk T4 T1 T2 T3 Rasona Pk Rf A % Pk Rf A % Pk Rf A % Pk Rf A % Pk Rf A % Pk Rf A %

Ab Ab 1 0.02 1.46 Ab Ab Ab

Ab Ab Ab 1 0.06 4.01 Ab 1 0.07 30.34

Ab Ab Ab Ab 1 0.09 2.79 Ab

Ab 1 0.32 4.27 2 0.32 7.74 Ab 2 0.33 10.18 Ab

Ab 2 0.35 4.33 Ab Ab Ab Ab

Ab 3 0.43 2.19 3 0.43 6.95 2 0.44 7.38 Ab Ab

Ab 4 0.63 11.06 4 0.62 8.17 3 0.62 8.73 3 0.63 11.06 Ab

1 0.74 10.48 5 0.73 28.27 5 0.74 38.46 4 0.73 41.10 4 0.74 32.44 Ab

Ab Ab Ab Ab Ab 2 0.78 69.66

2 0.82 14.96 6 0.81 19.38 6 0.81 29.18 5 0.81 24.87 5 0.82 27.71 Ab

3 0.92 2.92 7 0.91 0.81 Ab Ab Ab Ab

4 0.96 71.64 8 0.96 36.96 Ab Ab 6 0.95 3.74 Ab

Ab Ab 7 0.99 8.04 6 0.98 13.91 7 0.98 12.08 Ab

Analytical study...

Page | 54 “Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different methods in Albino Rats”

Table no: 12 HPTLC Densitometric scan at 366 nm

Milk T4 T1 T2 T3 Rasona Pk Rf A % Pk Rf A % Pk Rf A % Pk Rf A % Pk Rf A % Pk Rf A %

Ab 1 0.03 2.29 1 0.03 14.55 Ab Ab Ab

Ab Ab Ab 1 0.08 25.93 Ab Ab

1 0.30 35.85 2 0.31 8.90 Ab Ab Ab 1 0.32 100 Ab 3 0.43 5.90 2 0.44 16.24 2 0.45 10.25 Ab Ab

Ab Ab 3 0.66 17.40 3 0.67 12.06 Ab Ab

Ab 4 0.71 17.86 4 0.71 31.22 Ab 1 0.72 50.39 Ab

Ab 5 0.84 7.73 5 0.84 20.59 4 0.84 20.33 2 0.85 28.83 Ab

2 0.97 64.15 6 0.96 57.32 Ab Ab Ab Ab

Ab Ab Ab 5 0.99 31.43 3 0.99 20.78 Ab

Note

T1- Rasona Ksheerapaka (1:4:4) ratio



T4- Rasona Ksheerapaka by Kashaya method

EXPERIMENTAL STUDY

Experimental Study...



EXPERIMENTAL STUDY

INTRODUCTION

Hyperlipidemia is an elevation of lipids (fats) in the bloodstream. These

lipids include cholesterol, cholesterol esters (compounds), phospholipids and

triglycerides. It is of utmost significance because it leads to atherosclerosis of

vessels (arterial walls) leading to vascular diseases.

DIAGNOSIS

Diagnosis of hyperlipidemia is done via blood measurement of Cholesterol,

triglycerides, LDL, VLDL and HDL. LDL and VLDL can be measured indirectly

by applying the formula of FRIEDEWALD et al for calculation as

LDL = Total Cholesterol – HDL- VLDL

VLDL=Triglycerides/5 Where all the values are measured in milligrams per decilitre.

PARAMETER - Most commonly used parameters in Hyperlipidaemia are -

1) Body Mass Index

2) Waist circumference

3) Waist / Hip ratio

4) Relative Weight (RW)

5) Ponderal Index

6) Skin fold thickness

7) Age specific weight for height table

MATERIALS AND METHODS

Collection of Plant Material

The bulbs of Rasona were collected from Local market Udupi, Karnataka,

India during September of 2013. It was authenticated by Dr. Prakash L Hegde,

Professor & Head, Dept of Dravyaguna, SDM college of Ayurveda, Hassan. The




Milk used was pasteurized toned Nandini Milk. The formulation Rasona

Ksheerapaka was prepared in the teaching pharmacy attached to SDM College

Ayurveda, Udupi, from authenticated plant material. Everyday fresh Rasona

ksheerapaka was prepared and administered throughout the course of experimental

study. The drug samples along with Rasona and Milk were subjected to chemical

profiling as per the protocol of CCRAS parameters.

Preparation of Rasona Ksheerapaka:

The Bulbs of Rasona (Allium sativum Linn), Family Liliaceae, separated into

bulbils and dehusked (external covering was removed). These dehusked bulbils

were cut into two halves and mid stalk was removed. These destalked bulbils were

soaked overnight in buttermilk, washed and used in the next morning for the

preparation of Ksheerapaka. For this preparation specified amount of previously

soaked Rasona (Garlic) was taken and added with specified amount of milk and

water, heated on mild fire till the volume reduces to initial volume of the milk. Later

it was filtered and used for the experimentation.

Experimental Animals:

Wistar strains, Albino rats of either sex between 170 to 270 g were obtained

from animal house attached to department of Pharmacology, SDM Centre for

Research for Ayurveda and Allied Sciences, Udupi. The experimental protocol was

approved by the institutional ethical committee under the reference no.

SDMCAVIAEC2012-13/HSN -02. The animals were fed with normal rat diet and

water ad libitum, cholesterol solution throughout the study. They were acclimatized

in the laboratory condition for one week prior to the experimentation. The housing

provided has the following conditions: controlled lighting of 12:12h light and dark

cycle, temperature of 25ºC and relative humidity of approximately 50%.




ANIMAL GROUPING

Table no: 13. Showing the grouping of Animals with Drug and respective dose

Sl.no Group Drug Dose No. of Animals

1 Normal

control

Normal tap water 5ml/kg 8

2 Cholesterol

control

Cholesterol+ Vanaspathi 0.5ml/100g

(40%)

8

3 Reference

standard

Atorvastatin 10mg/kg 8

4 Test –I Test-I RKP(1:4:4) + cholesterol 8.64ml/kg 8

5 Test –II Test-II RKP (1:8:32)+

cholesterol

8.64ml/kg 8

6 Test –III Test-III RKP (1:15:15)+

cholesterol

8.64ml/kg 8

7 Test –IV Test-IV RKP (kashaya

method)+ cholesterol

8.64ml/kg 8

Experimental protocol:

Wistar albino rats (56) of either sex weighing 170 g to 270g were divided into

seven different groups, eight in each group. Control group rats were administered

with normal tap water at a dose of 5 ml /kg with normal diet and water ad libitum,

the second group rats were administered with 40 % cholesterol suspended in

hydrogenated vegetable oil (Vanaspathi Ghee). The standard group was

administered with Atorvastatin 10 mg/kg along with hyperlipidemia diet, the fourth,

fifth, sixth and seventh groups were administered with Rasona ksheerapaka




(prepared with different ratios) at a dose of 8.64ml/kg with hyperlipidaemia diet.

Test drug administered at morning hour and hyperlipidaemia diet was administered

at evening hour for 28 consecutive days. The test drug was administered directly as

it was in liquid form with the aid of gastric catheter.

The hyperlipidaemia diet contained hydrogenated vegetable oil (Vanaspathi

Ghee brand Ruchirraa, manufactured by Saraiwwalaa Agrr Refineries Limited, Andra

Pradesh, batch No.022992 (08)) and cholesterol extra pure powder (Cholesterol,

puriss for biochemistry by Spectrochem pvt ltd, Mumbai, batch no 4275891) made in

to 40% suspension in Vanaspathi Ghee. The suspension was administered at a dose of

0.5ml/100g rat. On 28th

day following overnight fasting the animal were weighed

again and sacrificed with ether overdose after collecting the blood from orbital plexus.

Liver, kidney and heart were excised out, cleaned, weighed and transferred to 10%

formalin solution to tissue fixation prior to Histopathological examination.

Collection of blood sample and biochemical analysis from serum:

The blood was collected from the orbital plexuses at the end of the experiment

on 28th

day; blood was collected 4h after the last drug administration using light ether

anesthesia. Blood samples were collected separately from retro orbital sinus puncture

into sterilized dry centrifugation tubes containing sodium citrate 0.01%. Samples were

allowed to stand for 30 min at 37º C. The clear serum was separated at 2500 rpm for

10 min using micro centrifuge. The biochemical investigation was carried out to

assess total cholesterol McGowan et al., 1983), high density lipoprotein (Burstein et

al,. 1970), low density lipoprotein (Friedewald et al., 1972) and triglycerides (Buccolo

and David, 1973; Fossati and Lorenzo, 1982)




STATISTICAL ANALYSIS

The data’s were analyzed by one way ANOVA followed by Dunnet’s t- test as the

post hoc test (Graph Pad 3 was used for this purpose). A p <0.05 was considered as

statistically significant. The data were presented as Mean ± SEM.

The percentage decrease in the Lipid lowering was calculated using the following

formula:

HFD- fed animal Lipid value – Drug+ HFD- fed animal

Lipid value

Lipid lowering (%) =

HFD- fed animal Lipid value

Procedure for serum biochemical analysis:

The lipid profile and enzyme activities were estimated by using a fully

automated clinical analyzer (ERBA-EM-200). The serum processed as above was fed

to the auto sampler inlet of the apparatus. The estimation was done using commercial

kits (Erba). The brief outlines of the test details are provided below:

CHOLESTEROL

Principle

Cholesterol ester + H2OCHE Cholesterol+ Fatty acids

Cholesterol +O2

CHO Cholest-4-en-3-one +H2O2

2H2O2 + 4AAP +Phenol POD Quinoneimine dye +4H2O

CHE – Cholesterol esterase

CHO – Cholesterol Oxidase

4AAP – 4-Aminoantipyrine

POD – Peroxide

Absorbance of Quinoneimine so formed is directly proportional to cholesterol

concentration.




Table no: 14. Reagent composition of cholesterol

(Concentration and activity in the test)

Goods buffer (pH – 6.4) 100mmol/L

Cholesterol oxidase >100U/L

Cholesterol esterase >200U/L

Peroxidase >3000U/L

4- amino antipyrine 0.3mmol/L

Phenol 5mmol/L

Non reactive stabilizers and surfactants

Cholesterol Standard

Cholesterol standard- 200mg/dl

Calculation

Cholesterol (mg/dl) = Abs of teat

Abs of standard × concentration of standard (mg/dl)

Table no: 15. Reference values for cholesterol

Serum/ Plasma Mg/dl

Children ≤ 4week 50-170

2-12 months 60-190

≥1 year 110-230

Adult <200

HDL – cholesterol78

Methodology

The assay is based on a modified polyvinyl sulfonic acid (PVS) and polyethylene-

glycol-methyl ether (PEGME) coupled classic precipitation method with the

improvements in using optimized quantities of PVS/PEGME and selected detergents.




LDL, VLDL and chylomicron (CM) react with PVS and PEGME and the reaction

results in inaccessibility of LDL, VLDL and CM by cholesterol esterase (CHER). The

enzymes selectively react with HDL to produce H2O2 which is detected through a

trinder reaction.

HDL + LDL+ VLDL + CM PVS

PEGME> HDL+ (LDL+VLDL+CM). PVS/PEGME

HDL+CHOD+CHER Fatty acid+H2O2

2H2O2 + 4AA + TODB Peroxide Quenone +5H2O

Table no: 16. (R1) Reagent composition for HDL – cholesterol

MES buffer (pH 6.5) 6.5mmol

N, N- Bis (4- sulfobutyl)-3-methylaniline 3mmol

Polyvinyl sulfonic acid 50mg/l

Polyethylene glycol methyl ester 30ml/l

MgCl2 2mmol

EDTA

Detergent

Table no: 17. (R2) Reagent composition for HDL – cholesterol

MES buffer (pH 6.5) 50mmol

Cholesterol esterase 5kU/l

Cholesterol oxidase 20kU/l

Peroxidase kU/l

4-aminoantipyrine 0.9g/l

Detergent 0.5%




Stability and storage

Prior to use, when stored at 28 0 C both reagent R1 and R2 are stable until the

expiry stated on the bottle and kit label. Once opened both reagents R1& R2

are stable for 60 days at 280C, when protected from contamination.

Table no: 18. Reference values of HDL-cholesterol

HDL-C Expected Values

Adult male 35.3-79.5mg/dl

Adult female 42.0-88.0mg/dl

Table no: 19.General assay parameters for HDL-cholesterol

Mode 1 point End

Wavelength (primary) 600nm

Wavelength (secondary) 700nm

Sample volume 5µl

Reagent 1 vol 375µl


Reaction temperature 37

Reaction direction Increasing

Linearity low (U/L) 0

Linearity high (U/L) 180mg/dl

Units mg/dl




Table no: 20.General pipetting procedure for HDL-cholesterol

Pipette in tube Calibrator Sample

Reagent 1 375µl 375µl

Calibrator 5µl -

Sample - 5µl

Mix and incubate at 370 C for 5min

Add Reagent 2 125µl 125µl

Mix and incubate at 370 C for 5min. Read final absorbance at the specified

wavelength

The reagent volume (R1&R2) as well as sample can be altered proportionately as per

the requirement of the analyzer.

Calculation

HDL-C = (Abs .sample −Abs .of sample blank )

(Abs .of calib −Abs .of calib .Blank )×conc. of calib.

LDL – cholesterol

Principle

The assay is based on a modified polyvinyl sulfonic acid (PVS) and polyethylene-

glycol-methyl ether (PEGME) coupled classic precipitation method with the

improvements in using optimized quantities of PVS/PEGME and selected detergents.

LDL, VLDL and chylomicron (CM) react with PVS and PEGME and the reaction

results in inaccessibility of LDL, VLDL and CM by cholesterol esterase (CHER),

whereas HDL reacts with the enzymes. Addition of R2containing specific detergent

releases LDL from the PVS/PEGME complex. Released LDL reacts with the enzymes

to produce H2O2 which is quantified by the trinder reaction.




HDL + LDL+ VLDL + CM PVS

PEGME> HDL+ (LDL+VLDL+CM).

PVS/PEGME

HDL CHOD CHER Fatty acid+H2O2

(LDL+ VLDL+CM) PVS/PEGME Detergent LDL+ (VLDL+CM)

PVS/PEGME

LDL CHOD CHER Fatty acid+H2O2

2H2O2 + 4AA + TODB Peroxide Quenone +5H2O

Table no: 21. (R1) Reagent composition for LDL – cholesterol


Polyvinyl sulfonic acid 50mg/l

Polyethylene glycol methyl ester 30ml/l

4-aminoantipyrine 0.9g/l

Cholesterol esterase 5kU/l

Cholesterol oxidase 20kU/l

Peroxidase 5kU/l

Detergent

Table no: 22. (R2) Reagent composition for LDL – cholesterol


Detergent

TODB N, N –Bis (4sulfobutyl)-3-methylaniline 3mmol/l




R3 CAL

HDL/LDL calibrator concentration: see bottle level

Reagent preparation: Reagents R1 and R2 are liquid ready for use.

Calibrator reconstitute with 1ml of deionised water 20-250 C and mix gently (avoid

foaming). Allow to stand for at least 30 minutes until complete reconstitution before

use. Stores reconstituted calibrator at 2-80 C

Stability and storage

The unopened reagents are stable till the expiry date stated on the bottle and kit level

when stored at 2-80 C. Once opened both reagents R1 & R2 are stable for 60 days at

2-80

C, when protected from contamination.

Reagents are light sensitive. Do not let bottles remain open. Keep containers tightly

closed. The constituted calibrator is stable for 6 days at 2-80

C.

Calculation

HDL-C = (Abs .sample −Abs .of sample blank )

(Abs .of calib −Abs .of calib .Blank )× conc. of calibrator

Table no: 23. Assay parameters for LDL – cholesterol

Mode 1 point End

Wavelength (primary) 600nm

Wavelength (secondary) 700nm

Sample volume 3µl



Reaction temperature 37

Reaction direction increasing

Linearity low (U/L) 0

Linearity high (U/L) 250mg/dl

Units mg/dl




TRIGLYCERIDES79

Principle

Triglycerides +H2O LPL glucerol +fatty acids

Glycerol-3-phosphate+O2 GPO DAP+H2O2

H2O2 + 4AAP +TOOS Peroxidase Quinoneimine dye + 2H2O

The intensity of chomogen (quinoneimine) formed is proportional to the triglycdride

concentration.

Table no: 24. Reagent composition for Triglycerides

(Concentration and activity in the test)

Buffer pipes (pH – 7.0) 40mmol/L

4-Aminoantipyrine (4-AAP) 0.4mmol/L

ATP 2.0mmol/L

Mg+2

2.5mmol/L

TOOS 0.2mmol/L

Glycerol Kinase (GK) 1500U/L

Glycerol-3-Phosphate Oxidase

(GPO)

4000U/L

Peroxidase (POD) 2200U/L

Lipoprotein Lipase (LPL) 4000U/L

Also contains non reactive fillers and stabilizers

Triglycerides Standard

Triglycerides standard - 200mg/dl

Calculation

Triglycerides (mg/dl) = Abs of teat

Abs of standard × concentration of std (mg/dl)




Table no: 25. Reference values of Triglycerides

Serum/ Plasma 370C

Normal fasting levels 25-160mg/dl

Procedure for estimation of Biochemical parameters:

Alkaline Phosphatase80

Principle

Kinetic determination of ALP is according to the following reaction.

p-nitrophenyl Phosphate+H2O ALP p-nitrophenol+Inorgnic phosphate

ALP = Alkaline Phosphatase

Table no: 26. Reagent composition for Alkaline Phosphatase

Alkaline phosphatase R1 lx33ml

Diethanolamine Buffer 1 mmol/L

Magnesium Chloride 0.5 mmol/L

ALKALINE PHOSPHATASE R2 10x3mL

p-Nitrophenyl phosphate 10 mmol/L

Normal range

It is recommended that each laboratory establish its own reference values.

The following value may be used as guide line

Adults 100-290 U/L

Children 180-1200 U/L

Calculation

ALP activity (U/L) = (ΔOD min.) ×2750




CREATININE81

Principle

Creatinine reacts with picric acid to produce a coloured compound, Creatinine

alkaline picrate. The change in absorbance is proportional to the Creatinine

concentration.

Table no: 27. Reagents composition for Creatinine

Creatinine dye reagent 2×50mL

Picric Acid 8.73mmol/L

Surfactant

Creatinine base reagent 2×50mL

Sodium hydroxide 300mmol/L

Sodium phosphate 25mmol/L

Creatinine standard 1×4mL

Creatinine Standard Concentration 2mg/dL

Storage and stability

The sealed reagents are stable up to the expiry date stated on the label, when

stored at room temperature & standard at 2-8°C.

Normal range

The following value may be used as guide line.

Serum: Men:0.7-1.4mg/dL

Female: 0.6-1.2 mg/dL

Urine: 0.80 - 1.80 gm/24hr.

Calculation

Creatinine conc. (mg/dL) = (T2−T1) of Sample

T2−T1 of Standard ×2




UREA82

Principle

Enzymatic determination of urea is according to the following reactions.

Urea + H2OUrease 2NH3+ CO2

2NH3 +2-Ketoglutarate + 2NADH GLDH L-Glutamate + 2NAD

+ + 2H2O

GLDH - Glutamate dehydrogenase

Table no: 28. Reagent composition for urea

Urea U.V (S.L) R1 2×24mL/2×40mL/2×100mL

Tris buffer, (pH 7.60) 100mmol/L

ADP 0.7mmol/L

a- ketoglutarate 9.0mmol/L

Urease >6500 U/L

GLDH >1100 U/L

Urea U.V (S.L) R2 2×6mL/2×10mL/2×25mL

NADH 0.25mmol/L

2-Oxoglutorate 5mmol/L

Urea U.V STAN DARD 1×4mL

Mix and read the optical density (T1) 30 seconds after the sample or standard addition.

Take second reading (T2) exactly 60 seconds after the first reading

Calculation

Urea conc. (mg/dL) = (T1−T2) of Sample

T1−T2 of Standard ×50

Urea BUN conc. (mg/dL) = (T1−T2) of Sample

(T1−T2) of Standard × 23.4




The following procedure was employed for the histopathological studies:

1) Fixation: It is the process of preserving, hardening and preventing post-mortem

changes of the tissues. The tissues were excised out immediately after sacrificing,

cleaned of extraneous tissues, cut into pieces of such appropriate thickness that

the fixative readily penetrated throughout the tissue to be fixed. Tissue

transferred to the 10% formaldehyde solution (37-40% Formaldehyde) and

allowed to remain in it till they were taken up for processing.

2) Tissue Processing: Tissue processing involves dehydration, clearing and

infiltration of the tissue with paraffin. The usual dehydrating agent is ethyl

alcohol; acetone and isopropyl alcohol can also be used. Following dehydration,

the tissue was transferred to a paraffin solvent, which is miscible with the

dehydrating agent as well. These are known as clearing agents such as

chloroform and xylene.

Tissues were thoroughly washed by placing them under running tap water and

then conveyed through a series of following solvents as per schedule for

dehydration, clearing and paraffin infiltration

Alcohol 70% - 20 minutes



Alcohol 95% (2 changes) - 20 minutes each

Isopropyl alcohol - 20 minutes

Acetone (2 changes) - 20 minutes each

Chloroform (3 changes) - 20 minutes each

Melted paraffin wax (600C) (3 changes) - 30 minutes each.




Next the tissues were embedded in paraffin wax to prepare tissue blocks, which

are oriented so that sections are cut in desired plane of the tissue. Tissues are

fixed to metal object holder after trimming them to suitable size.

3) Section cutting: A smear of 5% Mayer's egg albumin was prepared and smeared

on to the slide and dried. The tissue section of the 6 m thickness was cut with

the help of Spencer type rotating microtome. The tissue sections were put on

slide and drops of water and then sections were floated in water on slide between

55-600C, water drained off and slides dried on hot plate at about 50

0C for 30

minutes. This section is ready for staining.

Staining procedure:

Reagents:

1) Mayer's haematoxylin stain: Dissolve 50 g of ammonium or potassium alum in

one litre of water without heating. Then dissolve haematoxylin. 1.0 gram in

this solution further adds 0.2 g. Sodium iodate, 1/0 g. citric acid and 50 g.

chloral hydrate. Shake until all of them are in solution form. The final colour

of stain is reddish violet.

2) Eosin stain, 2% w/v in alcohol.

Procedure: After fixing the section on slide, the section was stained by serially placing

them in the following reagents:

Xylol (2 changes) - 3 Minutes each

Acetone - 3 Minutes

Alcohol 95% - 3 Minutes

Haematoxylin stain - 20 Minutes

Running water - 20 Minutes

Eosin Stain - 5 Minutes




Alcohol 95% (3 changes) - 3 Minutes each

Acetone (2 changes) - 3 Minutes each

Xylol (2 changes) - 3 Minutes each.

After passing through all the above reagents and stains, the slides are mounted with

D.P.X. (Diphenyl Phthalein Xylene) and cover slip are placed. Care should be taken

for avoiding the air bubbles during mounting the slide.

The slides were viewed under Trinocular research Carl Zeiss's microscope (Germany)

at various magnifications to note down the changes in the microscopic features of the

tissues studied.




OBSERVATIONS AND RESULTS

CHANGES IN LIPID PROFILE AT THE END OF EXPERIMENT

Table no: 29. Effect of Test drug Rasona Ksheerapaka on serum total

Cholesterol level

Data in Mean ± SEM, *P<0.05, **P<0.01

@- Compared with normal control, #- compared with cholesterol control,

Data related to the effect of test formulations on serum total cholesterol are

shown in table No. 29. Administration of hyperlipidemic diet lead to significant

elevation in serum total cholesterol level in comparison to normal control group. An

apparent increase in the serum total cholesterol level was observed in test-I RKP

(1:4:4) and test-II RKP (1:8:32) groups when compared to the cholesterol control

group. However, the observed change was found to be statistically non significant.

Group Dose Total Cholesterol

(mg/dl) Mean ±

SEM

% Change

Normal control (Tap water) 10ml/kg 31.16 ± 6.00 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

76.16 ± 6.86** 144.4↑@

Atorvastatin + cholesterol

(Standard)

10mg/kg

63.66 ± 6.49 16.41↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 95.33 ± 6.65 25.17↑#

Test-II RKP (1:8:32)+

cholesterol

8.64ml/kg 84.83 ± 4.18 11.38↑#

Test-III RKP (1:15:15)+

cholesterol

8.64ml/kg 74.83 ± 13.43 1.74↓#

Test-IV RKP (kashaya


8.64ml/kg 46.33 ± 6.03* 39.16↓#




An apparent decrease in the serum total cholesterol level was observed in

standard (Atorvastatin) and test-III RKP (1:15:15) groups when compared to the

cholesterol control group. The data showed statistically non significant.

A statistically significant decrease in serum total cholesterol level in test-IV

RKP (kashaya method) group when compared to the cholesterol control group.

Table no: 30. Effect of Test drug Rasona Ksheerapaka on HDL cholesterol level

Group Dose HDL cholesterol

(mg/dl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 17.66 ± 1.05 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

48.33 ± 7.78** 173.66↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

27.83 ± 2.81** 42.416↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 42.16 ± 2.46 12.766↓#


cholesterol

8.64ml/kg 42.16 ± 2.33 12.766↓#


cholesterol

8.64ml/kg 31.83 ± 5.46* 34.140↓#



8.64ml/kg 18.33 ± 3.93** 62.073↓#



Data related to the effect of test formulations on HDL cholesterol has been


elevation in HDL cholesterol level in comparison to the normal control (tap water)

group.




This hyperlipidemic diet induced elevation in HDL-Cholesterol level was

found to be significantly decreased in Standard (Atorvastatin), Test-III RKP (1:15:15)

+ cholesterol and test-IV RKP (kashaya method) groups when compared to the

hyperlipidemic control group. The apparent decrease observed in test -1 RKP (1:4:4)

and test-II RKP (1:8:32) groups was found to be statistically non-significant.

Table no: 31: Effect of Test drug Rasona Ksheerapaka on LDL cholesterol level

Group Dose LDL cholesterol

(mg/dl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 11.66 ± 1.33 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

31.5 ± 9.56* 170.15↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

13.11 ± 1.32* 58.38↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 22.33 ± 2.61 29.11↓#


cholesterol

8.64ml/kg 21.83 ± 3.04 30.698↓#


cholesterol

8.64ml/kg 17.5 ± 2.51 44.444↓#



8.64ml/kg 18.33 ± 3.93 41.809↓#

Data in Mean ± SEM, *P<0.05


Data related to the effect of test formulations on LDL cholesterol have been


elevation in LDL cholesterol level in comparison to the normal control (tap water)

group. This hyperlipidemic diet induced elevation in LDL-Cholesterol level was




found to be apparently decreased in reference standard and test drug administered

group in comparison to the hyperlipidemic diet group. However, only the decrease

observed in reference standard group (Atorvastatin) was found to be statistically

significant.

Table no: 32. Effect of Test drug Rasona Ksheerapaka on Serum Triglycerides

level

Group Dose Triglyceride (mg/dl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 81± 2.63 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

337.5 ± 34.08** 316↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

146.5 ± 16.09 ** 56.5↓#

Test-I RKP (1:4:4) +

cholesterol

8.64ml/kg 559 ± 78.34* 65.6↑#


cholesterol

8.64ml/kg 432 ± 72.92 28.0↑#


cholesterol

8.64ml/kg 198.66 ± 35.23* 41.0↓#



8.64ml/kg 313.5 ± 74.03 7.0↓#



Data related to the effect of test formulations on serum Triglycerides have

been depicted in table No. 32. Administration of hyperlipidemic diet lead to

significant elevation in serum triglyceride level in comparison to the normal control

(tap water) group. This hyperlipidemic diet induced elevation in serum triglyceride




level was found to be significantly decreased in standard (Atorvastatin) and Test-III

RKP (1:15:15) + cholesterol groups in comparison to the hyperlipidemic control

group. Surprisingly significant elevation was observed in Test-I RKP (1:4:4) +

cholesterol treated group and non-significant moderate elevation was observed in

Test-II RKP (1:8:32) + cholesterol group in comparison to the hyperlipidemic diet

control group. In Test-IV RKP (kashaya method) + cholesterol treated group a

marginal and statistically non-significant decrease was observed.




HAEMATOLOGICAL PARAMETERS:

Table no: 33. Effect of Test drug Rasona Ksheerapaka on Haemoglobin level

Group Dose Hemoglobin (g/dl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 16.38 ± 0.31 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

15.33 ± 0.25 6.406↓@

Atorvastatin + cholesterol

(Standard)

10mg/kg

14.86 ± 0.26 3.065↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 14.85 ± 0.32 3.131↓#


cholesterol

8.64ml/kg 14.7 ± 0.28 4.109↓#


cholesterol

8.64ml/kg 14.53 ± 0.26 5.218↓#



8.64ml/kg 14.46 ± 0.55 5.675↓#

Data in Mean ± SEM


Data related to the effect of test formulations on Haemoglobin level have been

shown in table No. 33. There was decrease in the Haemoglobin level in cholesterol

control group when compared to the normal control (tap water) group. However, this

decrease was found to be statistically non-significant. In all the remaining groups




including reference standard a statistically non-significant decrease was observed in

comparison to the hyperlipidemic control group.

Table no: 34. Effect of Test drug Rasona Ksheerapaka on Total Count

Group Dose Total WBC count

103 µL, Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 12600 ± 1122.2 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

10783 ± 1269.8 14.420↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

10451.6 ± 2221.3 3.020↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 6687.71 ± 800.6 37.979↓#


cholesterol

8.64ml/kg 10325 ± 1019.5 4.247↓#


cholesterol

8.64ml/kg 9085.11 ± 1430.5 15.738↓#



8.64ml/kg 10933.33 ± 1015.4 1.361↑#

Data in Mean ± SEM


Data related to the effect of test formulations on Total WBC count have been

shown in table No. 34. There was an apparent decrease in the total WBC count in

cholesterol control group when compared to the normal control (tap water) group.




However, this decrease was found to be statistically non-significant. In all the

remaining groups including reference standard, but excluding Test-IV RKP (kashaya

method) + cholesterol group a statistically non-significant decrease was observed in

comparison

to the hyperlipidemic control group. In Test-IV RKP (kashaya method) + cholesterol

treated group a marginal and statistically non-significant increase was observed.

Table no: 35. Effect of Test drug Rasona Ksheerapaka on RBC count

Group Dose RBC(106/µl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 8.17 ± 0.26 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

7.58 ± 0.13 7.277↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

7.21 ± 0.20 4.85↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 7.38 ± 0.15 2.66↓#


cholesterol

8.64ml/kg 7.51 ± 0.16 0.962↓#


cholesterol

8.64ml/kg 7.35 ± 0.24 3.03↓#



8.64ml/kg 15.41 ± 8.324 103.29↑#




Data in Mean ± SEM,


Data related to the effect of test formulations on RBC count have been shown in table

No. 35. There was an apparent decrease in the RBC count in cholesterol control group

when compared to the normal control (tap water) group. However, this decrease was

found to be statistically non-significant. In all the remaining groups including

reference standard, but excluding Test-IV RKP (kashaya method)+ cholesterol group,

a statistically non-significant decrease was observed in comparison to the

hyperlipidemic control group. In Test-IV RKP (kashaya method) + cholesterol group

a non-significant moderate increase was observed.

Table no: 36. Effect of Test drug Rasona Ksheerapaka on PCV level

Group Dose PCV (%)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 44 .5 ± 0.83

-

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

41.93± 0.63 5.76↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

39.91 ± 0.73 4.810↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 39.58 ± 0.73 5.604↓#


cholesterol

8.64ml/kg 39.4 ± 0.72 6.040↓#

Test-III RKP

(1:15:15)+ cholesterol

8.64ml/kg 39.5 ± 0.83 5.80↓#



8.64ml/kg 38.9 ± 1.47 7.232↓#




Data in Mean ± SEM


Data related to the effect of test formulations on PCV have been shown in table No.

36. There was an apparent decrease in the PCV in cholesterol control group when

compared to the normal control (tap water) group. However, this decrease was found

to be statistically non-significant. In all the remaining groups including reference

standard a statistically non-significant decrease was observed in comparison to the

hyperlipidemic control group.

Table no: 37. Effect of Test drug Rasona Ksheerapaka on MCV level

Group Dose MCV (fL)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 54.61 ± 1.06 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

55.4 ± 0.48 1.49↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

55.08 ± 0.53 0.636↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 53.8 ± 1.31 2.49↓#


cholesterol

8.64ml/kg 52.65 ± 0.53 5.02↓#


cholesterol

8.64ml/kg 52.45 ± 1.04 5.38↓#



8.64ml/kg 55.81 ± 1.61 0.68↑#

Data in Mean ± SEM





Data related to the effect of test formulations on MCV have been shown in table No.

37. There was an apparent marginal increase in the MCV in cholesterol control group

when compared to the normal control (tap water) group. However, this increase was


reference standard, but except Test-IV RKP (kashaya method) + cholesterol group, a

statistically non-significant decrease was observed in comparison to the


a marginal but statistically non-significant increase was observed.

Table no: 38. Effect of Test drug Rasona Ksheerapaka on MCH level

Group Dose MCH (pg)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 20.05 ± 0.42 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

20.18 ± 0.23 0.66↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

20.16 ± 0.21 0.11↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 19.71 ± 0.42

2.34↓#


cholesterol

8.64ml/kg 19.36 ± 0.21 4.07↓#


cholesterol

8.64ml/kg 19.28 ± 0.48 4.47↓#



8.64ml/kg 20.66 ± 0.53 2.36↑#

Data in Mean ± SEM

@- Compared with normal control, #- compared withcholesterol control,




Data related to the effect of test formulations on MCH have been shown in table No.

38. There was an apparent marginal increase in the MCH in cholesterol control group

when compared to the normal control (tap water) group. However, this increase was


reference standard, but except Test-IV RKP (kashaya method) + cholesterol group, a

statistically non-significant decrease was observed in comparison to the


a marginal but statistically non-significant increase was observed.

Table no: 39. Effect of Test drug Rasona Ksheerapaka on MCHC level

Group Dose MCHC (g/dl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 36.78 ± 0.079 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

36.53 ± 0.26 3.406↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

36.76 ± 0.16 0.629↑#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 36.76 ± 0.24 0.629↑#


cholesterol

8.64ml/kg 36.78 ± 0.31 0.692↑#


cholesterol

8.64ml/kg 36.66 ± 0.29 0.35↑#



8.64ml/kg 37.13 ± 0.23 1.64↑#






Data related to the effect of test formulations on MCHC have been shown in table

No 39. Administration of hyperlipidemic diet did not produce any significant change

in MCHC. No change could be observed in MCHC in reference standard and test drug

administered groups in comparison to hyperlipidemic control group.

Table no: 40. Effect of Test drug Rasona Ksheerapaka on RDWCV level

Group Dose RDWCV (%)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 14.55 ± 0.28 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

14.98 ± 2.48 2.97↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

14.51 ± 0.34 3.11↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 14.36 ± 0.36 4.11↓#


cholesterol

8.64ml/kg 14.516 ± 0.22 3.11↓#


cholesterol

8.64ml/kg 15.01 ± 0.45 0.22↑#



8.64ml/kg 15.65 ± 0.92 4.45↑#






Data related to the effect of test formulations on RDW-CV have been shown in table

No 40. Administration of hyperlipidemic diet did not produce any significant change

in RDW-CV. No change could be observed in RDW-CV in reference standard and

test drug administered groups in comparison to hyperlipidemic control group.

Table no: 41. Effect of Test drug Rasona Ksheerapaka on RDWSD level

Group Dose RDWSD (fL)

Mean ± SEM

% change

Normal control(Tap

water)

10ml/kg 27.61 ± 0.47 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

29.16 ± 0.78 5.613↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

28.05± 0.59 3.80↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 27.68 ± 0.93 5.07↓#


cholesterol

8.64ml/kg 26.4 ± 0.50* 9.46↓#


cholesterol

8.64ml/kg 28.55 ± 0.68 2.09↓#



8.64ml/kg 28.76 ± 0.67 1.37↓#

Data in Mean ± SEM, *P<0.05





Data related to the effect of test formulations on RDW-SD have been shown in table

No 41. Administration of hyperlipidemic diet produced an apparent but statistically

non-significant increase in RDW-SD. An apparent decrease was observed in reference

standard and all the test formulations administered groups. However, only the

decrease observed in Test-II RKP (1:8:32) + cholesterol group was found to be

statistically significant.

Table no: 42. Effect of Test drug Rasona Ksheerapaka on Platelet count

Group Dose Platelet count (103/µl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 7.56 ± 0.31 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

7.90 ± 0.36 5.33↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

7.37 ± 0.42 3.80↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 7.83 ± 0.12 10.28↓#


cholesterol

8.64ml/kg 7.95 ± 0.10 11.97↑#


cholesterol

8.64ml/kg 8.76 ± 0.65 23.38↑#



8.64ml/kg 8.53 ± 0.55 20.14↑#

Data in Mean ± SEM




@- Compared with normal control, #- compared with cholesterol control

Data related to the effect of test formulations on platelet count have been shown in

table No 42. Administration of hyperlipidemic diet produced an apparent but

statistically non-significant increase in platelet count in comparison to normal control

rats. An apparent decrease was observed in reference standard and Test-I RKP (1:4:4)

+ cholesterol groups. In the rest of the groups mild to moderate apparent but

statistically non-significant increase was observed in comparison to the

hyperlipidemic control.

BIO CHEMICAL PARAMETERS

Table no: 43. Effect of Test drug Rasona Ksheerapaka on serum urea level

Group Dose Urea (mg/dl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 25.16 ± 1.07 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

19.83 ± 2.12 21.18↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

22.33 ± 3.78 12.60↑#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 28.33 ± 2.52 42.86↑#


cholesterol

8.64ml/kg 24.00 ± 1.77 21.02↑#


cholesterol

8.64ml/kg 16.50 ± 1.40 16.79↓#



8.64ml/kg 20.83 ± 1.86 5.04↑#






Data related to the effect of test formulations on serum urea level have been shown in

table No 43. Administration of hyperlipidemic diet produced an apparent but

statistically non-significant decrease in comparison to normal control rats. An

apparent reversal of this decrease was observed in reference standard, Test-I RKP

(1:4:4) + cholesterol, Test-II RKP (1:8:32)+ cholesterol and Test-IV RKP (kashaya

method)+ cholesterol groups. In Test-III RKP (1:15:15) + cholesterol group a further

moderate and statistically non-significant decrease was observed in comparison to

positive control group.

Table no: 44. Effect of Test drug Rasona Ksheerapaka on serum Creatinine level

Group Dose Creatinine (mg/dl)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 0.61 ± 0.17 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

0.66 ± 0.05 8.19↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

0.60 ± 0.02 9.09↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 0.53 ± 0.02 19.69↓#


cholesterol

8.64ml/kg 0.56 ± 0.02 15.15↓#


cholesterol

8.64ml/kg 0.35 ± 0.02* 46.96↓#



8.64ml/kg 0.28 ± 0.03** 57.57↓#






Data related to the effect of test formulations on serum creatinine level have been

shown in table No 44. Administration of hyperlipidemic diet produced an apparent

but statistically non-significant increase in comparison to normal control rats. An

apparent reversal of this decrease was observed in reference standard and the entire

test drug administered group. However, only the decrease observed in Test-III RKP

(1:15:15) + cholesterol and Test-IV RKP (kashaya method) + cholesterol groups was

found to be statistically significant (p<0.05 and 0.01 respectively).

Table no: 45. Effect of Test drug Rasona Ksheerapaka on serum alkaline

phosphatase level

Group Dose Alkaline phosphatase

activity (IU/L)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 359.5 49.93 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

728.33 ± 149.48 102.5↑@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

1154.83 ± 123.45 58.55↑#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 1077.5 ± 172.58 47.94↑#


cholesterol

8.64ml/kg 1030.33 ± 178.97 41.46↑#


cholesterol

8.64ml/kg 1082.5 ± 203.05 48.62↑#



8.64ml/kg 1666.5 ± 326.41** 128.81↑#

Data in Mean ± SEM, **P<0.01





Data related to the effect of test formulations on serum alkaline phosphatase activity

have been shown in table no 45. Administration of hyperlipidemic diet produced an

apparent but statistically non-significant increase in comparison to normal control

rats. In reference standard and the entire test drug administered groups an apparent

increase was observed in comparison to the hyperlipidemic control group. However,

only the increase observed with Test-IV RKP (kashaya method) + cholesterol group

was found to be statistically significant.

PONDERAL CHANGES

Table no: 46. Effect of Test drug Rasona Ksheerapaka on changes in body Wt

Group Dose Changes in body Wt in

%

MEAN ± SEM

% change

Normal control

(Tap water)

10ml/kg 6.93 ± 3.69 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

3.44 ± 2.29** 50.36↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

27.16 ± 2.02** 689.53↑#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 9.19 ± 1.83 109.01↑#


cholesterol

8.64ml/kg 6.20 ± 1.55 80.23↑#

Test-III RKP

(1:15:15)+ cholesterol

8.64ml/kg 4.55 ± 1.89 32.267↑#

Test-IV RKP

(kashaya method)+

cholesterol

8.64ml/kg 6.14 ± 3.01 78.48↑#

Data in Mean ± SEM, **P<0.01





Data related to the effect of test formulations on percentage change in body weight

have been shown in table No 46. In hyperlipidemic diet administered control group a

moderate but statistically significant decrease in percentage body weight gain was

observed in comparison to the control group. This hyperlipidemic diet induced

elevation was found to be reversed by reference standard and in all the test drug

formulations administered groups. However, only the increase observed in reference

standard group was found to be statistically significant in comparison to

hyperlipidemic control group.

Table no: 47. Effect of Test drug Rasona Ksheerapaka on wt of Liver

Group Dose Liver weight (g)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 8.6 ± 0.45 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

8.12 ± 0.33 5.58↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

8.57 ± 0.68 5.54↑#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 8.16 ± 0.88 0.49↑#


cholesterol

8.64ml/kg 7.34 ± 0.55 9.60↓#


cholesterol

8.64ml/kg 7.62 ± 0.46 6.15↓#



8.64ml/kg 8.17 ± 0.43 0.61↑#


@- Compared with normal control, #- compared with cholesterol control.




Data related to the effect of test formulations on liver weight have been shown in

table No 47. Administration of hyperlipidemic diet produced only a marginal and

non-significant decrease in liver weight in comparison to control group. Liver weight

was not affected to significant extent in reference standard and test formulation

administered groups.

Table no: 48. Effect of Test drug Rasona Ksheerapaka on wt of kidney

Group Dose Kidney weight (g)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 1.81 ± 0.06 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

1.16 ± 0.05** 35.91↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

1.36 ± 0.09 17.24↑#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 1.31 ± 0.08 12.93↑#


cholesterol

8.64ml/kg 1.18 ± 0.09 1.72↑#


cholesterol

8.64ml/kg 1.34 ± 0.11 15.51↑#



8.64ml/kg 1.54 ± 0.07* 32.75↑#






Data related to the effect of test formulations on kidney weight have been shown in

table No 48. In hyperlipidemic diet administered control group a moderate but

statistically significant decrease in kidney weight was found in comparison to the

control group. This hyperlipidemic diet induced decrease was found to be apparently

reversed in reference standard and test formulation administered groups in

comparison to hyperlipidemic diet control group. However, only the reversal observed

in Test-IV RKP (kashaya method) + cholesterol group was found to be statistically

significant.

Table no: 49. Effect of Test drug Rasona Ksheerapaka on wt of heart

Group Dose Heart weight (g)

Mean ± SEM

% change

Normal control

(Tap water)

10ml/kg 0.88 ± 0.02 -

Cholesterol control

(Positive control)

0.5ml/100g

(40%)

0.77 ± 0.04 12.5↓@

Atorvastatin +

cholesterol

(Standard)

10mg/kg

0.61 ± 0.03* 20.77↓#

Test-I RKP(1:4:4) +

cholesterol

8.64ml/kg 0.74 ± 0.02 3.89↓#


cholesterol

8.64ml/kg 0.64 ± 0.04 16.88↓#


cholesterol

8.64ml/kg 0.71 ± 0.03 7.79↓#



8.64ml/kg 0.72 ± 0.02 6.49↓#

Data in Mean ± SEM, *P<0.05,





Data related to the effect of test formulations on heart weight have been shown in

table No 49. In hyperlipidemic diet administered control group a moderate but the

statistically non-significant decrease in heart weight was observed in comparison to

control group. An apparent decrease was also observed in reference standard and

test formulation administered groups in comparison to hyperlipidemic diet control

group. However, the observed reversal was found to be statistically non-significant.

HISTOPATHOLOGICAL EXAMINATION:

Liver: Naked eye examination of the liver from hyperlipidemic diet (HFD) showed

enlarged pale coloured liver in comparison to the red and smooth surfaced liver from

control group. Scanning of liver sections from the HFD control group rats showed

marked disturbance in the cytoarchitecture. Important among them were cell

depletion, micro and macro fatty changes in the hepatocytes and inflammatory

changes in the lobules in the form of cell infiltration and edema, in some sections

ballooning of the hepatocytes was also observed. The changes were moderate to

severe in all the rats examined in this group. In Atrovastin treated group the above

changes were found to be markedly reduced in four rats and moderately in two rats.

In test I treated group the pathological changes observed were mild to moderate

cell depletion, mild fatty change and in sections from one rat hemorrhagic streaks

were observed.

In test II treated group the pathological changes observed were mild to moderate

cell depletion, moderate micro and macro fatty changes with sinusoidal dilatation

was observed in most of the sections examined.

In test III treated group almost normal cytoarchitecture was observed in sections

from two rats, cell depletion of moderate intensity was observed in section from

one rat and mild fatty changes were observed in other sections




In test IV treated group the pathological changes observed were mild in

comparison to other groups. Almost normal profile was observed in sections from

two rats. In the rest of the sections mild fatty changes were the prominent feature

observed.

Heart: Microscopic examination of heart sections from HFD control group showed

weak to moderate myocarditis in section from two rats. Two rats exhibited almost

normal cytoarchitecture. In reference standard group the heart sections exhibited

almost normal cytoarchitecture in most of the rats. In all the four test drug treated

groups (Test 1, Test II, Test III and Test IV) the heart sections exhibited normal

cytoarchitecture.

Kidney: Microscopic examination kidney sections from HFD control group showed

mild to moderate fatty degenerative changes in four out of six rats. Mild changes were

observed in the remaining two. Cell infiltration was also observed in some rats. In

reference standard and test drug administered these changes were found to be very

much attenuated.

In test I treated group mild to moderate fatty changes were observed in tubular

epithelium otherwise the microscopic profile was normal. In test II treated group –

sections from one rat exhibited normal profile. In section from another rat mild

peri-arterial cell infiltration along with mild fatty changes were observed. In rest of

the sections mild fatty changes were observed. In test III treated group almost

normal profile was observed in sections from all the rats. In test IV treated group

section from three rats exhibited mild fatty changes in the tubular epithelium.

Intense and widespread cell infiltration was observed in section from one rat, Mild

cell infiltration was observed in another rat section.




EXPERIMENTAL PHOTOS

Experimental study...

“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different methods in

Albino Rats” Page | 98

PHOTOMICROGRAPHS OF LIVER

Fig – 20.a. & 20.b: Photomicrograph of representative sections of Liver of albino rat

from (Hyperlipidemic) Positive control group (HLD con * 200)

Kc- Kupffer cell; Nc- Normal cytoarchitecture; Hg- Haemoglobin; Bc- Ballooning of

cells; Fc- Fatty changes; Cd- Cell depletion; Hc- Hepatocytes

Note: Marked disturbance in the cytoarchitecture.

Fig – 20.c. & 20.d: Photomicrograph of representative sections of Liver of albino rat

from Standard group (HLD con * 200)

Fc – Fatty changes; Nc- Normal cytoarchitecture; Fc- Fatty changes

Note: Above changes were found to be markedly reduced but mild to moderate fatty

changes were seen.

Fig – 20.e. & 20.f: Photomicrograph of representative sections of Liver of albino rat

from Test 1(RKP 1:4:4) group (HLD con * 200)

Hc- Hepatocytes; Fc – Fatty changes

Note: Mild to moderate cell depletion, mild fatty change and haemorrhagic streaks

were observed.

*Note: RKP- Rasona Ksheerapaka




Figure 20: PHOTOMICROGRAPHS OF LIVER

20.a 20.b

20.c 20.d

20.e 20.f




PHOTOMICROGRAPHS OF LIVER

Fig – 20.g. & 20.h: Photomicrograph of representative sections of Liver of albino rat

from Test 2 (RKP 1:8:32) group (HLD con * 200)

Sn-Sinusoidal dilatation; Fc- Fatty changes; Cd- Cell depletion

Note: Mild to moderate cell depletion, moderate micro and macro fatty changes with

sinusoidal dilatation was observed

Fig – 20.i. & 20.j: Photomicrograph of representative sections of Liver of albino rat


Fc – Fatty changes; Nc- Normal cytoarchitecture; Fc- Fatty changes

Note: Almost normal cytoarchitecture with cell depletion of moderate intensity was

observed

Fig – 20.k. & 20.l: Photomicrograph of representative sections of Liver of albino rat

from Test 4 (RKP Kashaya method) group (HLD con * 200)

Hc- Hepatocytes; Fc – Fatty changes

Note: Almost normal profile with mild fatty changes were observed




Figure 20: PHOTOMICROGRAPHS OF LIVER

20.k

20.j

20.g

20.i

20.h

20.l




PHOTOMICROGRAPHS OF KIDNEY

Fig – 21.a. & 21.b: Photomicrograph of representative sections of Kidney of albino

rat from (Hyperlipidemic) Positive control group (HLD con * 200)

Fc- Fatty changes; G- Glomerulus; Ct- Convoluted tubule

Note- Mild to moderate fatty degenerative changes, Cell infiltration was also

observed

.

Fig – 21.c. & 21.d: Photomicrograph of representative sections of Kidney of albino

rat from Standard group (HLD con * 200)

G- Glomerulus; Ct- Convoluted tubule

Note- The above changes were very much attenuated.

Fig – 21.e. & 21.f: Photomicrograph of representative sections of Kidney of albino rat


G- Glomerulus; Ct- Convoluted tubule

Note- Mild to moderate fatty changes were observed in tubular epithelium




Figure 21:PHOTOMICROGRAPHS OF KIDNEY

21.d

21.a 21.b

21.c

21.f

21.e




PHOTOMICROGRAPHS OF KIDNEY

Fig – 21.g. & 21.h: Photomicrograph of representative sections of Kidney of albino

rat from Test 2 (RKP 1:8:32) group (HLD con * 200)

Note- Mild to moderate fatty changes was observed.

Fig – 21.i. & 21.j: Photomicrograph of representative sections of Kidney of albino rat


Note- Normal cytoarchitecture.

Fig – 21.k. & 21.l: Photomicrograph of representative sections of Kidney of albino

rat from Test 4 (RKP Kashaya method) group (HLD con * 200)

Note- Mild fatty changes with mild cell infiltration was observed




Figure 21: PHOTOMICROGRAPHS OF KIDNEY

21.g

21.h

21.i

21.l

21.k

21.j




PHOTOMICROGRAPHS OF HEART

Fig – 22.a. & 22.b: Photomicrograph of representative sections of Heart of albino rat

from (Hyperlipidemic) Positive control group (HLD con * 200)

Ep- Epicardium; En- Endocardium; Mc- Myocardium

Note- Weak to moderate myocarditis

Fig – 22.c. & 22.d: Photomicrograph of representative sections of Heart of albino rat

from Standard group (HLD con * 200)

Fc- Fatty changes

Note- Almost Normal cytoarchitecture

Fig – 22.e. & 22.f.: Photomicrograph of representative sections of Heart of albino rat


Note- Normal cytoarchitecture




Figure 22: PHOTOMICROGRAPHS OF HEART

22.b

22.e

8.d

8.c

8.a 8.b

22.c

22.d

22.f

22.a




PHOTOMICROGRAPHS OF HEART

Fig – 22.a. & 22.b: Photomicrograph of representative sections of Heart of albino rat


Ep- Epicardium; Mc- Myocardium


Fig – 22.c. & 22.d: Photomicrograph of representative sections of Heart of albino rat



Fig – 22.e. & 22.f: Photomicrograph of representative sections of Heart of albino rat

from Test 4 (RKP Kashaya method) group (HLD con * 200)





Figure 22: PHOTOMICROGRAPHS OF HEART

22.k

22.l

22.i

22.h

22.j

22.g

DISCUSSION

Discussion…



DISCUSSION

According to ancient research methodology, prior to establishing any

Siddhanta (theory), Upanayana (discussion) is the step preceding Nigamana

(Conclusion). Discussion is a process of re-examining and forms the base for

conclusion. Inspite of detailed classical study and experimentation by various

methods, a theory is accepted only after proper reasoning of the observations. Hence,

discussion is a crucial part of any scientific research work. Thus, the aim of discussion

should not be victory, but progress. Thereby, moving towards the future with the

guidance from past and courage from the present.

This research work has been carried out with a view to provide a scientific

basis for the claims made in various Ayurvedic texts regarding the cardio protective

nature of Rasona Ksheerapaka by noting its anti-hyperlipidemic effect. The drug was

subjected to thorough experimental study. The study comprises Pharmaceutical study

(Drug preparation), Analytical study (Analysis of Drug & formulation) &

Experimental or Pharmacological study (Animal study).

DISCUSSION (Pharmaceutical study)

Ksheerapaka is a liquid dosage form mentioned in the Ayurvedic

pharmaceutics.

Though the method of preparation resembles to that of Kwatha kalpana, specialty of

this preparation is presence of Milk media, which reduces the ushnata and teekshnata,

also mask the unpleasant taste of the drug used in the preparation.

Along with therapeutic efficacy one can attain nutritional requirement also.

Since no fixed dosage was mentioned in the classics, the dosage of Kwatha was

followed 2 pala (96ml)

Discussion…



Since different methods of preparation of Ksheerapaka are mentioned in

Ayurvedic classics. There is uncertainty as to which method of preparation is better

especially in producing therapeutic effect

So in the present study 4 samples of Rasona Ksheerapaka were prepared with

different ratios of milk and water keeping the quantity of the drug as constant and

analyzed.

Discussion of Rasona Ksheerapaka Preparations

Reference of Rasona Ksheerapaka was taken from Charaka Samhita, Gulma

chikitsa adhyaya. Where one of its indications is in Hridroga. This is considered to

decrease the obesity and diseases related to heart, hence linked to antihyperlipidimic

action. The same reference was also found in Astanga Hridaya, Chakradatta and

Bhaishajya Ratnavali.

Hence one sample of Rasona Ksheerapaka was prepared as per the ratios

mentioned in Charaka Samhita. However the other 3 samples of Rasona Ksheerapaka

were prepared as per the general method of preparation of Ksheerapaka mentioned in

Astanaga Sangraha, Sharangadhara Samhita and Dravya Guna Vijnana, so as to

conclude the better method to prepare Rasona Ksheerapaka and its efficacy in

producing antihyperlipidemic effect.

Discussion on Rasona shodana

The process of Rasona shodana was followed according to the reference

available in Sharangadhara Samhita with an intention to reduce its ushnata and

teekshna gandha so as to make the Ksheerapaka more palatable.

Discussion on Ksheerapaka prepared by Kashaya method

In the verse it is clearly stated, when the drugs are done paka with milk or

other liquids like curd etc, there will not be complete extraction of essence from the

Discussion…



drugs, so preliminarily kashaya should be prepared out of the drugs. To that obtained

kashaya equal amount of milk is to be added and heated till the volume of the mixture

reduces to the initial volume of the milk.

Discussion on Observation

Creaming on the surface of the liquid might be due to the presence of milk fat

which had a tendency to solidify on heating.

Emission of Alliaceous smell might be due to the presence of volatile oils

present in the Rasona.

Reduction in pungent taste of Rasona might be due to passage of pungent taste

into the milk media or diminished due to sweet taste of milk.

Discussion on Precautions

Rasona used was in paste form, so as to ease the passage of active principles

from drug into the liquid media.

Mild fire was maintained throughout the process- not to destroy the presence

of thermo labile phyto constituents of the drug.

Frequent stirring during the preparation was done with an intention to avoid

the charring of the drug and spilling of the milk.

Naming of the containers was done to follow the order of samples i.e. to avoid

the confusion with in the samples.

Discussion…



DISCUSSION (Analytical study)

Organoleptic characteristics

The colour of all the samples of Ksheerapaka was yellowish white in colour,

This was due to the colour of milk which was used for the preparation

The taste of all the samples of Ksheerapaka was Sweet and Alliaceous

because of the presence of Milk media and the drug Rasona used in the

preparation

The odour was also Alliaceous due to the drug Rasona used in the preparation

Viscosity

Viscosity is a property of a liquid, which is closely related to the resistance to

flow.

In the present study viscosity was 1.9687 in Ksheerapaka prepared with

Kashaya method, whereas 2.1763, 1.6972, 1.8370 in RKP (1:4:4), RKP (1:8:32) and

RKP (1:15:15) respectively. However the viscosity of the Plain milk was found to be

2.0667

The variation in the viscosity of the samples may be due to the ratio of the

milk and water used for the preparation. Here, in this study it is quite evident that

more the dilution less the viscosity of the given sample and vice versa.

Specific gravity

Specific gravity – The specific gravity of a liquid is the weight of a given

volume of the liquid at 25º (unless otherwise specified) compared with the weight of

an equal volume of water at the same temperature, all weights being taken in air.

Here the specific gravity indicates the presence of solutes (soluble or

insoluble) content in the samples of Ksheerapaka. Here solvent is milk and the solutes

may be referred to the extracted active principles from the drug Rasona and the total

Discussion…



solids but not fat (SNF) present in the plain milk.

In the present study, Specific gravity was 1.0332 in Ksheerapaka prepared

with kashaya method, whereas 1.0476, 1.0213, 1.0258 in RKP (1:4:4), RKP (1:8:32)

and RKP (1:15:15) respectively. However the specific gravity of the milk was found

to be 1.0378

pH

The pH value of an aqueous liquid may be defined as the common logarithm

of the reciprocal of the hydrogen ion concentration expressed in g, per litre.

In the present study pH was 6.48 in Ksheerapaka prepared with kashaya

method, whereas 6.49, 6.54, 6.50 in RKP (1:4:4), RKP (1:8:32) and RKP (1:15:15)

respectively. However the viscosity of the plain milk was found to be 6.56

Here it is quite evident that all the samples of Ksheerapaka were found to be

marginally acidic when compared to plain milk may be due to the presence of active

principles which were acidic in nature

TLC AND HPTLC DOCUMENTATIONS

This study reveals the chemical fingerprint profile of the test samples.

The Rf, is related to the retention of the components and their consequent separation.

It is defined as the ratio of time an analyte is retained in the stationary phase to the

time it is retained in the mobile phase. Hence, Rf = Distance travelled by the

component / distance travelled by solvent front.

Rf value is characteristic for a particular compound in a particular solvent

system and environmental conditions.

In the present study three different samples of Rasona Ksheerapaka were

compared by TLC and HPTLC at selected UV regions wavelength (at 254 nm and

366 nm). The spots/peaks due to different components were documented.

Discussion…



TLC photodocumentation of alcohol extract of Ksheerapaka compared with

Lasuna

Rf value of alcohol extract of samples at 254nm

Compound with Rf 0.27(Light green), which was present in all the test

samples, was found to be absent in the drug Rasona and plain milk

Likewise, Compounds with Rf 0.55(Light Green), 0.65(Light Green) and

0.73(Light Green) which were found to be present in all the test samples, were found

to be absent in the drug Rasona and plain milk

Rf value of alcohol extract of samples at 366nm

Compounds with Rf 0.27(Light Violet) and 0.51(Light Violet) were present in

all the test samples including the drug Rasona. However it was found to be absent in

the plain milk

Compounds with Rf 0.60(Light Violet) and 0.73(Light Violet) which were

found in all the test samples, were found to be absent in the drug Rasona and plain

milk

Rf value of alcohol extract of samples after Post derivatisation

Compounds with Rf 0.09(Light Brown), 0.14(Light Brown), 0.20(Light

Brown), 0.34(Dark Brown) and 0.51(Light Brown) which were found to be present in

all the test samples were found to be absent in the drug Rasona and plain milk

Compounds with Rf 0.44(Brown) which was present in all the test samples

including the drug Rasona was found to be absent in plain milk

Compounds with Rf 0.53(Light Violet), 0.71(Light Violet) and 0.75(Light

violet) which were present only in the drug Rasona were found to be absent in all the

test samples

Discussion…



HPTLC PHOTO DOCUMENTATION

This study reveals the chemical fingerprint profile of the test samples.

The Rf, is related to the retention of the components and their consequent separation.

It is defined as the ratio of time an analyte is retained in the stationary phase to the

time it is retained in the mobile phase. Hence, Rf = distance travelled by the

component / distance travelled by solvent front.

Rf value is characteristic for a particular compound in a particular solvent

system and environmental conditions.

In the present study three different samples of Rasona Ksheerapaka were

compared by TLC and HPTLC at selected UV regions wavelength (at 254 nm and

366 nm). The spots/peaks due to different components were documented.

HPTLC densitometric scan at 254 nm

There were 8, 7, 6, 7 peaks in sample T4, T1, T2 and T3 respectively whereas

4 and 2 peaks were found in the plain milk and the drug Rasona respectively. It was

observed that compound with Rf 0.02 (1.46%) was found only in sample T1 and

absent in all other test samples including the drug Rasona and milk.

Compound with Rf 0.09(2.79%) was found to be present only in sample T3

and absent in all the test samples including the drug Rasona and milk.

Compound with Rf 0.35(4.33%) which was present in test sample T4 was

found to be absent in all other test samples including the drug Rasona and milk.

Compound with Rf 0.78(69.66%) was present in the drug Rasona was found to

be absent in all other test samples and in the plain milk

At 366 nm

Compound with Rf 0.08(25.93%) which was present in test sample T2 was

found to be absent in all other test samples including the drug Rasona and milk

Discussion…



The HPTLC densitometry at 254 and 366 nm showed clear differences and

similarities in the samples of Rasona ksheerapaka analyzed.

Note:

T1- RKP (1:4:4)

T2- RKP (1:8:32)

T3- RKP (1:15:15)

T4- RKP (Kashaya method)

Discussion…



DISCUSSION (Experimental study)

The main objective of the present study was to determine the

antihyperlipidemic potential of Rasona Ksheerapaka on rats fed with hyperlipidaemic

diet. Rasona Ksheerapaka has been indicated as a cardiac tonic in Charaka Samhita.

Most of the Nighantus of Dravyaguna like Dhanvantari, Kaideva, Raja and

Bhavaprakasha mentioned the use of Rasona in hridroga. This is considered to mean

decreasing the obesity and diseases related to heart hence linked to antihyperlipidemic

action. Rasona Ksheerapaka has been reported to show best cardio protective action.

On the basis of this information it was assumed that Rasona Ksheerapaka would have

good hypolipidemic activity especially in hyperlipidemic condition. Hence the present

study was undertaken.

Before analysis of the results obtained in the present study it would be

essential to briefly summarize different steps involved in lipoprotein metabolism,

which is very important for the regulation of lipid levels in the blood. Chylomicrons

the largest of the lipoproteins are mainly involved in the transport of triglycerides of

dietary origin and are formed in the intestine. Triglycerides are removed from the

chylomicrons in different tissue through a reaction involving hydrolysis by the

enzyme lipoprotein lipase. Thus the VLDL formed in the liver acts as a vehicle for the

transport of triglycerides to different parts of the body. The VLDL triglycerides are

hydrolyzed to provide free fatty acids for storage in adipose tissue and for routing

them to β-oxidation pathway for providing energy requirement in tissues like cardiac

and skeletal muscles. After the removal of triglycerides the VLDL remnant gets

converted to LDL. Part of the remnant is converted to LDL by removing the further

amount of triglycerides. LDL is catabolised in the hepatocytes by the hydrolysis of

cholesteryl ester in its core. This cholesterol is utilized in the preparation of cell

Discussion…



membranes. HDL plays very important role in preventing the artherogenesis by taking

away the cholesterol from the arterial wall and by inhibiting the oxidation of

atherogenic lipoproteins. The protein part of this molecule is synthesized in the liver

and secreted in intestine. Surface layers of chylomicrons and VLDL acquire the lipid

part of HDL during lipolysis. It also acquires cholesterol from the tissues by an

alternate pathway. This free cholesterol is moved from the cytosol to the cell

membrane by a transporter protein termed as ABC1 from which the cholesterol is

acquired by prebeta-1-HDL. This is followed by esterification of cholesterol by the

enzyme lecithin –cholesterol acyl transferase leading to the formation of bigger HDL

molecule. From HDL cholesterol it is transferred to VLDL, IDL, LDL and

Chylomicron remnants through the activity of an enzyme known as Cholesteryl ester

transfer protein.

HDL is the only lipoprotein particle capable of receiving cholesterol from the

peripheral cells by a process known as reverse cholesterol transport. Nascent HDL

particles containing apo A-I and phospholipids are synthesized in liver. These HDLs

rapidly acquire additional un-esterified cholesterol and phospholipids from peripheral

tissues. HDL cholesterol is transported to hepatocytes by both an indirect and a direct

pathway. HDL cholesteryl esters are transferred to apo B containing lipoproteins by

cholesteryl ester transfer protein (CETP), which are then removed from liver by LDL

receptor mediated endocytosis. Modulating the activity of the important and rate

limiting enzymes and transporter proteins would lead to marked changes in the lipid

profile.

Effect on Biochemical Parameters

In the present study administration of hyperlipidemic diet (cholesterol along

with vanaspati ghee) lead to significant hyperperlipidemic condition as reflected in

Discussion…



the form of significant elevation in Total serum cholesterol, HDL-cholesterol, LDL-

cholesterol and serum triglycerides level in comparison to normal control group with

normal diet. This establishes the efficacy of the experimental protocol to induce

hyperlipidemic condition. The main objective of the study was first to ascertain the

presence of anti-hyperlipidemic activity in the test formulation - Rasona Ksheerapaka.

The second objective was to find out which of the four forms of the test formulation

provide optimum effect as anti-hyperlipidemic agent. The four formulations-

comprised three prepared from direct method of preparation using different ratios of

the ingredients and fourth prepared using kashaya method. The following is the brief

detail:

Test-I RKP (1:4:4)

Test-II RKP (1:8:32)

Test-III RKP (1:15:15)

Test-IV RKP (kashaya method)

The results obtained have been provided in the form of consolidated statement in

Discussion…



Table no: 50. Shows consolidated presentation of the result obtained in the

present study

Parameters Cholesterol

control

@

Standard

(Atorvastatin)

#

Test-I

RKP(1:4:4)

+

cholesterol

#

Test-II

RKP

(1:8:32)+

cholesterol

#

Test-III

RKP

(1:15:15)+

cholesterol

#

Test-IV

RKP

(kashaya

method)+

cholesterol

#

Lipid profile

changes

Total

cholesterol

**↑ NS↓ NS↑ NS↑ NS↓ *↓

HDL **↑ **↓ NS↓ NS↓ *↓ **↓

LDL *↑ *↓ NS↓ NS↓ NS↓ NS↓

Triglycerides **↑ **↓ *↑ NS↑ S*↓ NS↓

Hematological

changes

Hemoglobin NS↓ NS↓ NS↓ NS↓ NS↓ NS↓

Total count NS↓ NS↓ NS↓ NS↓ NS↓ NS↑

RBC NS↓ NS↓ NS↓ NS↓ NS↓ NS↑

PCV NS↓ NS↓ NS↓ NS↓ NS↓ NS↓

MCV NS↓ NS↓ NS↓ NS↓ NS↓ NS↑

MCH NS↑ NS↓ NS↓ NS↓ NS↓ NS↑

MCHC NS↓ NS↑ NS↑ NS↑ NS↑ NS↑

RDWCV NS↑ NS↓ NS↓ NS↓ NS↑ NS↑

RDWSD NS↑ NS↓ NS↓ *↓ NS↓ NS↓

Platelet count NS↓ NS↑ NS↑ NS↑ NS↑ NS↑

Biochemical

changes

Urea NS↓ NS↑ NS↑ NS↑ NS↓ NS↑

Creatinine NS↑ NS↓ NS↓ NS↓ *↓ **↓

Alkaline

phosphatase

NS↑ NS↑ NS↑ NS↑ NS↑ **↑

Ponderal

changes

Body weight **↓ **↑ NS↑ NS↑ NS↑ NS↑

Liver weight NS↓ NS↑ NS↑ NS↓ NS↓ NS↑

Kidney weight **↓ NS↑ NS↑ NS↑ NS↑ *↑

Heart weight NS↓ *↓ NS↓ NS↓ NS↓ NS↓

Discussion…



@ - Changes with reference to normal control rats

#- Changes with reference to cholesterol control

**=p<0.01, very significant

*=p<0.05, significant

NS= not significant

↑ - increased; ↓- deceased

Effect on Lipid Profile:

For a test drug to ascertain its hypolipidemic activity it is expected to reverse

the hyperlipidemic conditions. As mentioned earlier administration of hyperlipidemic

diet lead to significant elevation in all the four parameters studied. Administration of

reference standard was able to produce only a mild to moderate decrease in serum

total cholesterol level and significant decrease in serum triglyceride level. It also

produced significant decrease in LDL-Cholesterol and elevated HDL-cholesterol

levels. Among the three RKP prepared by direct method none was able to lower

serum total cholesterol level where as significant decrease was observed with RKP-

IV prepared by using kashaya. All the four produced reasonably good lowering of

LDL-cholesterol level. RKP- III and RKP- IV were found to produce best effect

among the four test formulations. RKP I - II in fact produced mild non-significant

elevation instead of lowering. Interestingly RKP III produced significant lowering

where as RKP IV produced marginal non-significant lowering in serum triglycerides

level. RKP- I produced further significant elevation where as RKP-II produced

moderate but statistically non-significant increase.

Careful analysis reveals that among the four formulations for reducing the

elevated serum total cholesterol RKP- IV- that prepared through kashaya method

should be preferred because of its remarkable cholesterol lowering effect where as for

Discussion…



lowering elevated serum triglyceride level RKP- III could be preferred. RKP- I and

RKP- II do not possess the anti-hyperlipidemic activity hence should not be preferred.

Thus the study was able to provide proof for the presence of anti-hyperlipidemic

activity in the Rasona Ksheerapaka and also the ideal type of preparation for lowering

elevated cholesterol and triglyceride level.

The results obtained indicate that methods and ratio used in the preparation of RKP-

III and RKP- IV were more effective in extracting more of the anti-hyperlipidemic

active principles.

Lowering of serum total other types of cholesterol indicates one of the

following possibilities. That is the test formulation may interfere with the absorption

of cholesterol from the GI tract by adsorption or by inhibiting lipase activity on the

dietary fats. The second modulation may be down grading cholesterol synthesis. The

third possibility is increasing the utilization of cholesterol by promoting its increased

utilization for the formation of bile salts. It seems RKP- IV has modulating effect on

all or any one or two of the above mechanisms. Lowering of triglyceride may be

indicative of modulation of Chylomicron and VLDL metabolism. RKP- III seems to

have good modulation of this probable mechanism. It may also be interfering with the

formation of triglyceride or its peripheral utilization in tissues like skeletal muscles.

HDL- cholesterol level was elevated in the present study. Some authors have

reported decrease in HDL- cholesterol in their studies where as others have indicated

elevation. It seems elevation or decrease depends up on the nature of the

hyperlipidemic diet used. The present diet most often produces significant or

moderate or near significant elevation in HDL- cholesterol level. In the present study

like reference standard RKP- III and RKP- IV produced significant lowering where as

Discussion…



RKP- I and RKP- II produced only marginal decrease. This shows that both these

formulation produce profound effect on cholesterol metabolism.

Further, it is also necessary to consider the results of histopathological

examination- as mentioned already- feeding of hyperlipidemic diet lead to moderate

to severe fatty degenerative changes in liver and moderate changes in kidney and mild

changes in heart. In reducing these degenerative changes RKP- IV was found to be

best and RKP- III almost equivalent to it. The remaining two formulations exhibited

only mild protection. Thus there is very good agreement between the effects observed

on biochemical parameters and histological examination. The degenerative changes

are linked to hyperlipidemic diet and they were found to be markedly attenuated in

reference standard confirming the link between the two. Further RKP- IV reversed

the hyperlipidemic diet induced decrease in kidney weight.

Effect on Hematological Parameters

In the present study effect of high cholesterol diet on different haematological

parameters was assessed with the aim to assess whether they are modulated by anti-

hyperlipidemic therapy or not. Administration of high cholesterol diet did not cause

remarkable changes in the haemogram profile. Though statistically significant

changes was observed in certain RBC related parameters i.e. RDWSD in the test drug

RKP- II. When the data from hyperlipidemic control were compared to the normal

control they were more or less within the normal range. The significant difference was

mainly due to high homogeneity of the data obtained.

Biochemical parameters:

Effect on Serum urea

Human kidney excretes nitrogenous wastes in the form of urea through urine.

Urea is produced on breakage of proteins by the liver cells. Decreased urea

Discussion…



concentration is seen in liver dysfunction and increased concentration in hypo-

function of kidney. In present study, administration of hyperlipidemic diet resulted in

moderate but statistically non-significant decrease in serum urea level. This may be

indicative of functional efficiency lowering in liver as a result of hyperlipidemic diet

induced degenerative changes. This was not affected to significant extent by RKP- III

and RKP- IV. Moderate but non-significant reversal was observed with RKP- I and

RKP- II. Thus the alteration in this parameter did not contribute substantially for

arriving at an inference about the efficacy of the test formulations.

Effect on Serum Creatinine

Creatinine is a good marker to detect the renal function. Creatinine is derived

from the metabolism of creatine in skeletal muscle and from dietary meat intake, with

about 1.6% of the creatine pool converted to Creatinine. At normal level, this

metabolite filtered at glomerulus but neither secreted nor absorbed by the tubules.

Administration of hyperlipidemic diet did not modulate serum Creatinine level

significantly- indicating that the kidney degenerative changes caused by

hyperlipidemic diet are not severe to produce change in Creatinine level. Surprisingly

significant lowering was observed in RKP- III and RKP- IV administered groups.

This may be indicative of decreased activity in the muscle or decreased formation of

Creatinine. It would be interesting to ascertain the exact mechanism.

Effect on serum alkaline phosphatase

The enzyme, which shows elevated activity in alkaline pH, is known as

alkaline phosphatase. It is present in bone osteoclasts, bile canaliculi, proximal

convoluted tubules of the kidney, intestinal mucosa and the placenta. This enzyme is

involved in the splitting of terminal phosphate group from the organic monophosphate

esters. If the elevation is due to hyperlipidemic diet then this elevation is expected to

Discussion…



be reversed by the test drug. In the present study a moderate but statistically non-

significant elevation was observed – it may be indicative of increased activity of the

oseteoclasts and it was not reversed by the test formulations and reference standard

which also produced elevation. The observation of further elevation in the activity of

this enzyme may be indicative of the fact the elevation is not linked to hyperlipidemic

state. It would be interesting to elucidate the probable underlying mechanism of the

observed elevation.

Effect on Ponderal Changes

Effect on body weight

Gain in body weight indicates normal progressive health status of an

organism. Decrease in body weight is indicative of degenerative changes in the body

or certain organs. In present study animals administered with hyperlipidemic diet have

shown decrease in body weight gain in comparison to normal rats with normal diet.

The observed increase was found to be statistically highly-significant. Lower body

weight gain may be due to interference with the absorption or utilization of the

nutrients in the diet by hyperlipidemic diet. Its reversal by all the formulations and

reference standard in normal course would have been linked to the presence of anti-

hyperlipidemic activity. However, increased body weight gain was observed in both

effective and non-effective formulations hence it is difficult to provide rational

explanation to the observation.

Effect on liver, kidney and heart:

Decrease in the organ weight is indicative of degenerative changes or loss of

tissue of that particular organ, increase in the weight may be due to hyper functioning

of that organ or oedematous changes. In the present study analysis of the data showed,

though marginal increase in test drug group I & IV and slight decrease in test drug

Discussion…



group II & III in relative liver weight, which was found to be non significant indicates

administration of hyperlipidemic diet did not influence the relative liver weight. Liver

weight unlike kidney weight was not affected to significant extent by hyperlipidemic

diet – this indicates its higher resilient effect. In the kidney, the test drug group I, II &

III showed slight increase in the relative kidney weight however the increase was

statistically non-significant. Whereas test drug group IV showed significant increase

in the relative kidney weight when compared to cholesterol control group. It is to be

noted that hyerlipidemic diet produced significant decrease in kidney weight may be

indicative of degenerative changes and its significant reversal by RKP- IV may be

considered as reversal of the observed degenerative changes. Heart weight was not

influenced significantly by hyperlipidemic diet in all test drug groups.

It can be concluded on the basis of careful analysis of the data generated

during the study that the test formulation Rasona Ksheerapaka has good anti-

hyperlipidemic activity. This provides unequivocal evidence for its therapeutic use-

the first objective of the study. The second objective of the study was to ascertain

which type of formulation has good biological activity. RKP- IV which is prepared

from Kashaya seems to be the best among all the formulation closely followed by

RKP- III. These two formulation produced reversal of different but more number of

hyperlipidemic diet induced changes in the studied parameters.

Further it can be suggested that among the four formulations evaluated in the present

study for reducing the elevated serum total cholesterol RKP- IV- that prepared

through kashaya should be preferred because of its remarkable cholesterol lowering

effect where as for lowering elevated serum triglyceride level RKP- III could be

preferred. RKP- I and RKP- II do not possess the anti-hyperlipidemic activity hence

should not be preferred. The reason for the observed efficacy in the former two

Discussion…



formulations is probably more extraction of the active principle or their better

synergy. The hyperlipidemic diet and test formulation did not affect haematological

parameters significantly indicating there inert nature on blood components. Changes

in biochemical parameters by the hyperlipidemic diet may be due functional

deficiency in the liver which was moderately modulated by RKP-III and RKP-IV.

Histological examination shows degenerative changes especially in liver and hearts

which were reversed in the above two groups, Thus providing extensive evidence for

their effectiveness.

CONCLUSION

Conclusion…



CONCLUSION

Pharmaceutical study

Ksheerapaka is a unique liquid dosage form mentioned in the Ayurvedic

classics, where both Milk and Water soluble active principles are extracted

through this method.

The presence of Milk in the preparation not only helps to reduce Ushnata and

Teekshnata of the drug but also make it more palatable and serves nutritional

requirement also.

Pharmaceutical study reveals that, in spite of using different ratios of Milk and

water, seldom any changes were observed in the organoleptic characters of the

samples of Rasona Ksheerapaka.

Analytical study

Analytical study reveals that, though the formulation was prepared from the

same batch drug, mere changes in the ratios of liquids (Milk and Water) and

duration of preparation- a lot of variation in the number of components in the

final products.

Analytical study also reveals more components in the final products

(Formulation) compared to the components of individual drugs

Experimental study

Results obtained from experimental study signify that- methods and ratio used

in the preparation of RKP-III and RKP- IV were found to more effective in

extracting more of the anti-hyperlipidemic active principles

Among the four formulations, for reducing the elevated serum total cholesterol

RKP- IV- that prepared through kashaya method should be preferred. Whereas

for lowering elevated serum triglyceride level RKP-III could be preferred.

Conclusion…



All the 4 samples of ksheerapaka have reasonably good lowering of LDL-

cholesterol level

In reducing the degenerative changes RKP- IV was found to be best and RKP-

III almost equivalent to it

Finally RKP- IV which was prepared from Kashaya method seems to be the

best among all the formulation, closely followed by RKP- III. These two

formulations produced reversal of different but more number of

hyperlipidemic diet induced changes in the studied parameters.

Conclusion…



LIMITATIONS OF THE STUDY

Constituents in HPTLC report could not be identified or specified, due to lack

of facility as this method needs advanced technique like phytochemical study,

Microscopic study, GLC etc...

Conclusion…



SCOPE FOR FURTHER STUDY

Same experiment can be done by using different drugs instead of Rasona and

evaluated for their specific action. So as to validate better method to prepare

Ksheerapaka.

SUMMARY

Summary…



SUMMARY

The present dissertation entitled “Experimental study on Antihyperlipidemic activity

of Rasona Ksheerapaka prepared by different methods in Albino rats” consists of

topics discussed under the following headings.

As a part of this a brief Introduction is given in the beginning of this dissertation,

which includes the importance of Ayurveda and Bhaishajya Kalpana and relevance of

study. Later the Aims, objectives and previous work done on the same have been

mentioned in brief

Review of literature

It is divided into two main sections via, Drug review comprises drug including

historical background, properties, Microscopic and Macroscopic characters chemical

composition, pharmacological action and the second section comprises of Disease

review with Ayurvedic and Modern perspective, classification and brief treatment

aspect.

The Methodology is the second part of the study which is further divided into

following parts:

1. Pharmaceutical study

2. Analytical study

3. Experimental study

Each study involves the Introduction, Methodology, Observation and Results.

Pharmaceutical study deals with the preparation of Rasona Ksheerapaka using four

different methods – 1. Ksheerapaka with Ratio (1:4:4), 2. RKP (1:8:32), 3. RKP

(1:15:15) and 4. Ksheerapaka by (Kashaya method). These methods have been

explained as four practicals with procedure, observation and precautions.

Summary…



Analytical study: Deals with subjecting drug with different Analytical parameters

mentioned in the study and tabulation of their results.

Experimental study: Deals with the effect of test samples on various Biochemical

parameters like Lipid profile, Haematological parameters, Serum total Urea,

Creatinine and Alkaline phosphate and Ponderal changes

Discussion: In this part, an attempt has been made to analyze the data obtained from

the study.

Conclusion: Here conclusive remarks have been made on each study i.e.

Pharmaceutical study, Analytical study and Experimental study. It also includes the

limitation and scope of the study.

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ANNEXURE

Annexure…

Page | 143

Department of Dravya Guna

SDM college of Ayurveda and

Hospital, Hassan

Drug Authenticity Certificate

This is to certify that Dr. Jayaprakash A N, student of SDM college of

Ayurveda, dept. of Bhaishajya Kalpana, SDM College of Ayurveda,

Hassan has selected research topic for her M.D. degree which is given

below

“EXPERIMENTAL STUDY ON ANTIHYPERLIPIDEMIC ACTIVITY OF

RASONA KSHEERAPAKA PREPARED BY DIFFERENT METHODS IN ALBINO

RATS.”

Under this study specimens which has been submitted to me for the

botanical standardizations and authentication was identified &

confirmed as

1. Rasona (Allium sativum)

jayaprakash a n - 52.172.27.147:8080

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