“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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JAYAPRAKASH
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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
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 & 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,
KARNATAKA, BANGALORE
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)
DEPARTMENT OF RASASHASTRA & BHAISHAJYA KALPANA
SHRI DHARMASTHALA MANJUNATHESHWARA COLLEGE OF
AYURVEDA & HOSPITAL, HASSAN
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
Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
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:
DEPARTMENT OF RASASHASTRA & BHAISHAJYA KALPANA
SHRI DHARMASTHALA MANJUNATHESHWARA COLLEGE OF
AYURVEDA & HOSPITAL, HASSAN
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
Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
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)
T2 Test sample 2 (RKP 1:8:32)
T3 Test sample 3 (RKP 1:15:15)
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
12 HPTLC Densitometric scan at 366 nm 54
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
9 HPTLC Densitometric scan at 254 nm 46
10 HPTLC Densitometric scan at 366 nm 49
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…
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methods in Albino Rats” Page | 2
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 3
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 4
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…
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methods in Albino Rats” Page | 5
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…
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methods in Albino Rats” Page | 6
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…
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methods in Albino Rats” Page | 7
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
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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
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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
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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
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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
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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
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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."
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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.
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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
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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:
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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
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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.
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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.
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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
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Shodana chikitsa64
1] Antahaha Parimarjana
Virechana,
Raktamokshana
2] Bahya Parimarjana
Swedana
Churna
Pradeha (Udvartana)
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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
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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)
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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
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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)]
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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
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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
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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
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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
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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.
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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
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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
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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)
-
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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)
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Figure 9. HPTLC photo documentation at 254nm
Fig 3.a Plain Milk
Fig 3.b Kashaya method
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Fig 3.c 1:4:4
Fig 3.d 1:8:32
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Fig 3.e 1:15:15
Fig 3.f Rasona
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Figure 10. HPTLC photo documentation at 366nm
Fig 4.a Plain Milk
Fig 4.b Kashaya method
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Fig 4.c 1:4:4
Fig 4.d 1:8:32
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Fig 4.e 1:15:15
Fig 4.f Rasona
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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...
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“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...
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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
T2- Rasona Ksheerapaka (1:8:32) ratio
T3- Rasona Ksheerapaka (1:15:15) ratio
T4- Rasona Ksheerapaka by Kashaya method
EXPERIMENTAL STUDY
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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
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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%.
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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
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(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)
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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.
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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.
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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%
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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
Reagent 2 vol 125µl
Reaction temperature 37
Reaction direction Increasing
Linearity low (U/L) 0
Linearity high (U/L) 180mg/dl
Units mg/dl
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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.
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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
MES buffer (pH 6.5) 50mmol
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
MES buffer (pH 6.5) 50mmol
Detergent
TODB N, N –Bis (4sulfobutyl)-3-methylaniline 3mmol/l
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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
Reagent 1 vol 375µl
Reagent 2 vol 125µl
Reaction temperature 37
Reaction direction increasing
Linearity low (U/L) 0
Linearity high (U/L) 250mg/dl
Units mg/dl
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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)
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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
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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
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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
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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 80% - 20 minutes
Alcohol 90% - 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.
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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
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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.
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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
method)+ cholesterol
8.64ml/kg 46.33 ± 6.03* 39.16↓#
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 42.16 ± 2.33 12.766↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 31.83 ± 5.46* 34.140↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 18.33 ± 3.93** 62.073↓#
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 HDL cholesterol has been
shown in table No. 30. Administration of hyperlipidemic diet lead to significant
elevation in HDL cholesterol level in comparison to the normal control (tap water)
group.
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 21.83 ± 3.04 30.698↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 17.5 ± 2.51 44.444↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 18.33 ± 3.93 41.809↓#
Data in Mean ± SEM, *P<0.05
@- Compared with normal control, #- compared with cholesterol control,
Data related to the effect of test formulations on LDL cholesterol have been
shown in table No. 31. Administration of hyperlipidemic diet lead to significant
elevation in LDL cholesterol level in comparison to the normal control (tap water)
group. This hyperlipidemic diet induced elevation in LDL-Cholesterol level was
Experimental Study...
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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↑#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 432 ± 72.92 28.0↑#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 198.66 ± 35.23* 41.0↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 313.5 ± 74.03 7.0↓#
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 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
Experimental Study...
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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.
Experimental Study...
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 14.7 ± 0.28 4.109↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 14.53 ± 0.26 5.218↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 14.46 ± 0.55 5.675↓#
Data in Mean ± SEM
@- Compared with normal control, #- compared with cholesterol control,
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
Experimental Study...
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 10325 ± 1019.5 4.247↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 9085.11 ± 1430.5 15.738↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 10933.33 ± 1015.4 1.361↑#
Data in Mean ± SEM
@- Compared with normal control, #- compared with cholesterol control,
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.
Experimental Study...
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 7.51 ± 0.16 0.962↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 7.35 ± 0.24 3.03↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 15.41 ± 8.324 103.29↑#
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Data in Mean ± SEM,
@- Compared with normal control, #- compared with cholesterol control,
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↓#
Test-II RKP (1:8:32)+
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↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 38.9 ± 1.47 7.232↓#
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Data in Mean ± SEM
@- Compared with normal control, #- compared with cholesterol control,
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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 52.65 ± 0.53 5.02↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 52.45 ± 1.04 5.38↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 55.81 ± 1.61 0.68↑#
Data in Mean ± SEM
@- Compared with normal control, #- compared with cholesterol control,
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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
found to be statistically non-significant. In all the remaining groups including
reference standard, but except 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 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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 19.36 ± 0.21 4.07↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 19.28 ± 0.48 4.47↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 20.66 ± 0.53 2.36↑#
Data in Mean ± SEM
@- Compared with normal control, #- compared withcholesterol control,
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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
found to be statistically non-significant. In all the remaining groups including
reference standard, but except 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 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↑#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 36.78 ± 0.31 0.692↑#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 36.66 ± 0.29 0.35↑#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 37.13 ± 0.23 1.64↑#
Data in Mean ± SEM,
@- Compared with normal control, #- compared with cholesterol control,
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 14.516 ± 0.22 3.11↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 15.01 ± 0.45 0.22↑#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 15.65 ± 0.92 4.45↑#
Data in Mean ± SEM,
@- Compared with normal control, #- compared with cholesterol control,
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 26.4 ± 0.50* 9.46↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 28.55 ± 0.68 2.09↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 28.76 ± 0.67 1.37↓#
Data in Mean ± SEM, *P<0.05
@- Compared with normal control, #- compared with cholesterol control,
Experimental Study...
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 7.95 ± 0.10 11.97↑#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 8.76 ± 0.65 23.38↑#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 8.53 ± 0.55 20.14↑#
Data in Mean ± SEM
Experimental Study...
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@- 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↑#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 24.00 ± 1.77 21.02↑#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 16.50 ± 1.40 16.79↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 20.83 ± 1.86 5.04↑#
Data in Mean ± SEM,
@- Compared with normal control, #- compared with cholesterol control,
Experimental Study...
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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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 0.56 ± 0.02 15.15↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 0.35 ± 0.02* 46.96↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 0.28 ± 0.03** 57.57↓#
Data in Mean ± SEM, *P<0.05, **P<0.01
@- Compared with normal control, #- compared with cholesterol control,
Experimental Study...
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methods in Albino Rats” Page | 90
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↑#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 1030.33 ± 178.97 41.46↑#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 1082.5 ± 203.05 48.62↑#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 1666.5 ± 326.41** 128.81↑#
Data in Mean ± SEM, **P<0.01
@- Compared with normal control, #- compared with cholesterol control,
Experimental Study...
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methods in Albino Rats” Page | 91
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↑#
Test-II RKP (1:8:32)+
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
@- Compared with normal control, #- compared with cholesterol control,
Experimental Study...
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methods in Albino Rats” Page | 92
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↑#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 7.34 ± 0.55 9.60↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 7.62 ± 0.46 6.15↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 8.17 ± 0.43 0.61↑#
Data in Mean ± SEM,
@- Compared with normal control, #- compared with cholesterol control.
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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↑#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 1.18 ± 0.09 1.72↑#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 1.34 ± 0.11 15.51↑#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 1.54 ± 0.07* 32.75↑#
Data in Mean ± SEM, *P<0.05, **P<0.01
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@- Compared with normal control, #- compared with cholesterol control,
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↓#
Test-II RKP (1:8:32)+
cholesterol
8.64ml/kg 0.64 ± 0.04 16.88↓#
Test-III RKP (1:15:15)+
cholesterol
8.64ml/kg 0.71 ± 0.03 7.79↓#
Test-IV RKP (kashaya
method)+ cholesterol
8.64ml/kg 0.72 ± 0.02 6.49↓#
Data in Mean ± SEM, *P<0.05,
@- Compared with normal control, #- compared with cholesterol control,
Experimental Study...
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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
Experimental Study...
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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.
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EXPERIMENTAL PHOTOS
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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
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Figure 20: PHOTOMICROGRAPHS OF LIVER
20.a 20.b
20.c 20.d
20.e 20.f
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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
from Test 3 (RKP 1:15:15) group (HLD con * 200)
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
Experimental study...
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Figure 20: PHOTOMICROGRAPHS OF LIVER
20.k
20.j
20.g
20.i
20.h
20.l
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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
from Test 1(RKP 1:4:4) group (HLD con * 200)
G- Glomerulus; Ct- Convoluted tubule
Note- Mild to moderate fatty changes were observed in tubular epithelium
Experimental study...
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Figure 21:PHOTOMICROGRAPHS OF KIDNEY
21.d
21.a 21.b
21.c
21.f
21.e
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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
from Test 3 (RKP 1:15:15) group (HLD con * 200)
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
Experimental study...
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Figure 21: PHOTOMICROGRAPHS OF KIDNEY
21.g
21.h
21.i
21.l
21.k
21.j
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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
from Test 1(RKP 1:4:4) group (HLD con * 200)
Note- Normal cytoarchitecture
Experimental study...
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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
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PHOTOMICROGRAPHS OF HEART
Fig – 22.a. & 22.b: Photomicrograph of representative sections of Heart of albino rat
from Test 2(RKP 1:8:32) group (HLD con * 200)
Ep- Epicardium; Mc- Myocardium
Note- Normal cytoarchitecture
Fig – 22.c. & 22.d: Photomicrograph of representative sections of Heart of albino rat
from Test 3 (RKP 1:15:15) group (HLD con * 200)
Note- Normal cytoarchitecture
Fig – 22.e. & 22.f: Photomicrograph of representative sections of Heart of albino rat
from Test 4 (RKP Kashaya method) group (HLD con * 200)
Note- Normal cytoarchitecture
Experimental study...
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Albino Rats” Page | 109
Figure 22: PHOTOMICROGRAPHS OF HEART
22.k
22.l
22.i
22.h
22.j
22.g
DISCUSSION
Discussion…
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methods in Albino Rats” Page | 110
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…
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methods in Albino Rats” Page | 111
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…
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methods in Albino Rats” Page | 112
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…
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methods in Albino Rats” Page | 113
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…
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methods in Albino Rats” Page | 114
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…
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methods in Albino Rats” Page | 115
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…
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methods in Albino Rats” Page | 116
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…
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methods in Albino Rats” Page | 117
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…
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methods in Albino Rats” Page | 118
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…
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methods in Albino Rats” Page | 119
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 120
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 121
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 122
@ - 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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 123
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 124
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 125
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 126
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 127
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 128
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 129
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 130
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 131
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 132
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 133
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…
“Experimental study on Antihyperlipidemic activity of Rasona Ksheerapaka prepared by different
methods in Albino Rats” Page | 134
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)