Prakrithi rn

www.ayurvedicmedicinalplants.com 1

A CONCEPTUAL STUDY ON PRAKRITHI IN CORRELATION WITH MOLECULAR PROFILES

THESIS SUBMITTED TO THE UNIVERSITY OF KANNUR

IN PARTIAL FULFILLMENT FOR THE DEGREE OF

DOCTOR OF MEDICINE IN AYURVEDA

BY

K.N. AJITHKUMAR B.A.M.S

DEPARTMENT OF ROGA NIDANA

UNDER THE SUPERVISION OF

PROF. Dr. R. SREEKUMAR MD (Ay)

&

Dr. MOINAK BANERJEE PhD

DEPARTMENT OF POST GRADUATE STUDIES IN ROGA NIDANA GOVT. AYURVEDA COLLEGE, KANNUR

2006

http://www.ayurvedicmedicinalplants.com

Ayurmitra

Draft


ACKNOWLEDGEMENTS

It is a matter of great privilege for me to work under the able and highly exceptional

guidance of Dr. R. Sreekumar, former Head of the department, Department of Roga

Nidana, Govt. Ayurveda College Kannur, who has nurtured my capabilities and

always gave me ample freedom. His energetic smile, optimistic attitude, critical

judgments and above all trust in my abilities kept me going and ultimately towards

the attainment of this goal.

No less had been the incessant help and constant encouragement provided

by my co-guide Dr. Moinak Banerjee, Scientist – E1, Human Molecular Genetics lab,

Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram during the course of my

study. His scholarly guidance, experienced words, meticulous practical training,

disciplined and virtuous nature, unlimited sincerity and affection towards students

had always inspired me and I feel extremely fortunate to be his student.

Words are not enough to express my sincere and heartfelt thanks to Dr. V.N.

Parameswaran Potty and Dr. K.V. Subhadra Antherjanam and

Dr. P.K.V. Anand for their immense help in my studies. Their devotion and dedication

to work, passion for knowledge, inquisitive mind and scientific attitude have made an

everlasting impact on me and boosted my confidence. My special thanks to Dr. K.

Sankaran, Director, Ayurveda Medical Education, Dr. John K George, Head of the

department, Dr. S. Jayadevan Asst. Professor, department of social and preventive

medicine, Academy of Medical sciences, Pariyaram, Dr. V.K. Ajithkumar, Reader

and Dr. S. Gopakumar, Tutor of my department for their kind concern, critical

comments, wise counseling and timely tips which are invaluable.

I adore and respect them for their ‘Never say no’ attitude and magnanimous nature

and I owe them a lot.



Former Director Dr. R.V. Thampan, Director Dr. M. Radhakrishna Pillai

and Scientists from other departments of RGCB and especially to research scholars,

Mrs. Resmi Thomas, Miss. Neetha Vijayan, Mrs. Linda Koshi, Mr. Chandrashekhar

and technical assistants Mrs. Sudha B. Nair, Mrs. Bindu Ashokan, Mr. Antony K.P

and the summer trainee students who attended the HMG lab from various

biotechnology colleges, for their timely assistance in the lab and their invaluable help

and moral support.

I also extend my sincere thanks to my batch mates Dr. Mukesh,

Dr. Viswanath, Dr. Sudarkkodi, Dr. Surabhi Mishra, Dr. Senthil Nathan,

Dr. Surama Mishra, Dr. Anjali, Dr. Abhilash and Dr. Bindu Mary Mathew and other

P.G scholars of various Ayurveda Colleges for being there, whenever I was in need

them the most and for their constant support and encouragement.

Nevertheless, I am not missing out this opportunity to acknowledge and

appreciate some of my colleagues and very good friends like Dr. Nandalal,

Dr. Sunil Babu, Dr. Madhu, Dr. Prabha Manish, Dr. Gopikrishna, Dr. Annie Yohanan,

Dr. Vijay, Dr. Prasanth Kekuda, Dr. Brijula, Dr. Anupama,

Dr. Yogeswar Pawale, Dr. Arbind Kumar Jha, Dr. Bindu, Dr. Sudhagopal,

Dr. Eby Abraham, Dr. Vibhu Khanna, Dr. Namratha, Dr. Pradeep, Dr. Rejitha, Dr.

Vidya Menon, Dr. Sarita Mohanta, Dr. Manas Ranjan Debta, Dr. Priya,

Dr. Ananthalekshmi, Dr. Nandakumar Sahni, Dr. Shambhu Sharan, Dr. Sijin, and Dr.

Pramod whose infinite love and affection had always strengthened my determination.

Words are meaningless to express my unaccountable feelings to my father

Sri. K.V. Neelakandan Namboothiri (late) and mother Smt. C.N. Arya Devi (late);

sisters, Mrs. K.N. Anjana Sree, Mrs. K.N. Archana Sree and

Ms. V.P Aparna; brother in laws Mr. Sivanandan and Mr. Narayanan Namboothitri,



and nephew Mr. Vijay Sankar and niece Ms. Swathy for their belief in me, kind

understanding, patience and perseverance and above all in making me what I am

today. I owe them more than anything else in the world. Special thanks are also due

to my beloved wife Mrs. Shreeja Devi and daughter Ms. Devi Nanda without whose

help and support, none of my dreams would have ever come true and without whom

life would have never been the same for me.

The wealth of information provided by our college library also deserves

special thanks.

Dr. K.N.AJITHKUMAR



INTRODUCTION

IS EXCELLENCY THE PRESIDENT OF INDIA Dr. A.P.J. ABDUL KALAM in

his Address at the Inauguration of the Govt. Ayurveda College Hospital

Complex and Interaction with the Students at Govt. Ayurveda College, Trippunithura,

Ernakulam, Kerala on 17th December 2005 talked on the topic “Traditional Medicine:

Our Strength”.

He pointed out that the traditional system of medicine like Ayurveda, Siddha etc.

have advocated and practiced preventive and curative medicinal recipes specific to

individuals. The body, mind, food and environment were looked at holistically to

suggest a preventive or curative approach to health. He also stressed the need for

collaborative research of Ayurveda and biotechnology. Dr. Kalam’s focusing on

personalized medicine of Ayurvedic system of medicine has invited our attention to

the fact that in this post genomic era, the emerging concept of personalized therapy

on the basement of Pharmacogenomics is a re-definition of diseases on the

molecular level so that diagnotics and therapeutics can be targeted to specific

patient populations sub-typed on the basis of genetic make-up.

India is on a path breaking research and development activities in various areas to

combine the strength of Ayurveda and the power of Biotechnology so as to place the

nation in global leadership position in the matter of providing quality traditional health

care. The National Biotechnology development strategy addresses the utilization of

Biotechnology to add value to our traditional knowledge especially Ayurveda,

Siddha, and Unani systems as well as tribal and folk medicine. Medicinal plants are

also prime targets of bioprospecting. Besides, the tool of biotechnology can be used

for conservation and characterization of plants. Development of plant based

medicine and mechanism based screening of herbal drugs known in traditional

H



Indian system are included in the strategic action of the National Biotechnology

policy to get value added drugs quickly. For the Inaugural Address at the “DELHI

SUSTAINABLE DEVELOPMENT SUMMIT 2003” Vigyan Bhavan, New Delhi,

February 6, 2002, His Excellency the President of India Dr. A.P.J. Abdul Kalam

selected the topic ‘PROSPEROUS – PEACEFUL – SAFE HUMAN HABITAT’. While

narrating the utilization of Biotechnology for wealth generation, he also stressed on

integrative research in Ayurveda and biotechnology and stated that new

technologies as evidenced by human genome sequencing, proteomics,

chemogenomics, ultra high throughput screening is revolutionizing drug discovery1.

Medicinal plants offer enormous scope for development of drugs and he highlighted

the need to create database of traditional medicine for specific bioactivity and lead

for development of new drugs. He explained that the North Eastern States of India

have got tremendous opportunities for herbal farming and research and that there is

an integrated relationship between science, technology, environment, manufacturing

and the society.

Dr. M.S.Valiyathan has also advocated research on valuable traditional knowledge

and basic principles of Ayurveda, and not just on herbal drug development2.

Use of modern technology and Biotechnology has been identified by

Govt. of India, Department of AYUSH as a thrust area for 10th plan period for

strengthening the base for sustained propagation of Ayurveda. The objectives and

goals of Kerala Biotechnology policy is also designed to catalyze the development of

Ayurveda. The policy, among others, aim to apply biotechnology tools to “ boost the

states renowned health care practice of Ayurveda by synergising traditional

knowledge with the scientific validation and technical product profiling and clinical

data base and by evolving means to conserve and substantially use one of worlds



most valued biodiversity treasures located in the state.” It also aims at creation of an

advanced multipurpose analytical testing and standardization laboratory approved by

the National Accreditation Board for testing Calibration of Laboratories to cater the

needs of Ayurveda and pharmaceutical industry for meeting international standards.

Pharmacogenomics – the study of how an individual’s genetic make up affects the

body’s response to treatment or drug - is a rapidly growing science after the

completion of Human Genome Project was celebrated in April 2003 and it is a

developing research field still in it’s infancy. Pharmacogenomics hold the view that

drugs might one day be “tailor- made” for individuals or small populations based on

genetic make up. Modern medicine has developed based on general principles of

physiology, pathology diagnotics and treatment were as Ayurveda upholds individual

physiology, pathology, diagnostic and personalized therapy. In this post genomic

era, the emerging concept of personalized therapy on the basement of

Pharmacogenomics is infact, a re-definition of diseases on the molecular level so

that diagnotics and therapeutics can be targeted to specific patient populations sub-

typed on the basis of genetic make-up and there by offer the right treatment for right

patient population\ individual.

The emerging personalized medicine holds the view that environment, diet,

age, lifestyle and state of health all can influence a person’s response to health and

that due to genetic variations a disease may manifest itself slightly differently in

different types of patients.

Understanding the genetic make up of an individual is the key to personalized

therapy. As, at present, there is only limited knowledge of which genes are involved

in each drug response and that many genes are likely to influence a response and

obtaining a correct picture on the effect of genetic polymorphisms are complicated,



time consuming and expensive, a high speed data mining of both genotypic and

phenotypic information is essential for the development of personalized therapy. In

short, identification and analysis of millions of single gene nucleotide polymorphisms

(SNPs) to determine which drug and specific diet are more suitable to a patient is

quite impractical. Hence it has become necessary to categorize individuals on the

basis of board phenotypic clusters. Consideration of racial, ethnic and geographical

factors for classifying phenotypes to be considered collectively for genotyping is

controversial. Here, Ayurvedic concept of Prakrithi comes in to play.

According to Ayurveda, every individual is different from another and hence

should be considered as a different entity. But for diagnostic, prognostic and

therapeutic purposes Ayurveda categorizes human population in to sub-populations

such as Vatha Prakrithi, Pitha Prakrithi, Kapha Prakrithi or their combinations on the

basis of physical, physiological and psychological characteristics (phenotypes) and

completely avoid racial, ethnic and geographical considerations. In fact, the

aforesaid concept that due to genetic variations, a disease may manifest itself

slightly differently in different types of patients and the same therapy and drug will

not produce equal response in different individuals has well developed thousands of

years ago in Ayurveda. After analyzing and correlating the varying responses of

individuals to various factors like drug, food, sleep, sex, exercise, climate,

environment, and assessing various physical, physiological and psychological

characteristics (phenotypes) and sub typing the individuals showing similarities in the

above responses and characteristics, the human population is grouped in Ayurveda

in to smaller populations and designated as a particular Prakrithi such as Vatha

Prakrithi, Pitha Prakrithi, Kapha Prakrithi or combinations of either two or three of

them. Similarly, the body functions, tissues and organs are also sub-typed on the



same lines. For example, on the basis of the characteristics the pulse (nadi) is

categorized into Vatha, Pitha, Kapha or their combinations. Based on the small

variations in signs and symptoms exhibited in the above sub-typed populations,

diseases especially metabolic disorders are also sub grouped in to Vatha

predominant, Pitha predominant, Kapha predominant or combinations of either two

or three of them. Due to various factors, a disease may exhibit signs and symptoms

of Vatha/ Pitha/ Kapha/ their combination predominance in an individual irrespective

of the type of Prakrithi.

One of the basic concepts of Ayurveda is that, if a particular disease having

the symptamotology of Vatha predominant type is occurring to an

individual/population sub-typed as Vatha Prakrithi and dwelling in a place having

Vatha predominance and if the disease is occurring in an organ /tissue of Vatha

predominance at a time/season having Vatha predominance, the disease is

considered of having bad prognosis .The Prakrithi predominance of the tissue, place,

time and individual is assessed by specific criteria provided under the basic

principles of Ayurveda. An individual/population is sub-typed into a particular

Prakrithi on analyzing the phenotypic characteristics of that individual/population;

and a place by analyzing the nature of micro-climatic conditions, elevation, soil

conditions, vegetation, water, air and the phenotypic characteristics itself of the

plants and animals of that place, and that of time/season is determined by analyzing

the seasonal/timely climatic factors such as solar radiation, atmospheric pressures

(direction of wind), humidity, characteristics of clouds, etc, and the body’s response

to the above factors. Modern biotechnology upholds the view that the collective

interaction of about 30000 genes give us life and their normal function keeps the 3

trillion – cell human being healthy by producing the right proteins where as their



altered function by mutation due to exposure to harmful external and internal

environments produce wrong proteins that make disease. In short, if the etiological

factors provided by place (geopathological), time (climatic) and organism (metabolic

and genetic) are highly potential, co-existing and correlating in terms of Ayurvedic

principles for manifestation of a disease, the severity of the disease thus manifested

is the most, and the disease is most difficult to cure. The genetic make up of the

receptors or its potential and the receptor sensitivity of various factors pertaining to

tissue, time and place is an important factor that determines the severity of the

disease.

Modern western medicine has developed under the influence of

reductionism-breaking down human organism into smaller levels of such as organ,

tissue, cell and gene and focus on the diseased part and often treat to suppress the

symptom to get an immediate result rather than a long term process of correcting the

metabolic derangement. As a result, a drug therapy or gene therapy targeting on

modification of activities of a cell or group of cells produces toxicity to others and it is

estimated that even in most developed countries out of 3.5 billion annual

prescriptions 3 billion produces adverse results and the treatment adopted to one

disease give rise to another disease. With a holistic approach, Ayurveda advocates

the need for micro analysis of the tissue morbidity, the geopathological factors of the

dwelling place, the immunological status, the micro climatic elements and how they

influence the metabolic activities, the state of digestive and metabolic fire, the

genetic make-up by analyzing the phenotypic characteristics, the state of body that

depends on the course of time, the state of psyche, life style, food habits etc of a

patient for personalized diagnosis and therapy.



If the phenotype (outward appearance of an individual as a result of

genetic make up and environment, i.e. dosha Prakrithi lakshanas) and the

corresponding genotype are correlated by the tools of bio technology, it will not only

enable to highlight the ever modern principles of the oldest health science, Ayurveda

– Puranam cha Punarnavam - but also to generate data to the modern scientific

community to classify patients with same phenotypic characteristics into smaller sub

populations defined by genetic variations associated with disease, drug and food

response. Such studies will also enable the development of Pharmacogenomics and

personalized therapy from infancy to child hood more securely and safely in the

cradle of Mother Ayurveda. Many scholars and institutions in the field of medicine,

biotechnology and policy making are up in arms for research in the field of Ayurveda

in the light of modern technology and biotechnology.

Dr M S Valiyathan has pointed out the possibility of having genomic counter parts for

‘dosha Prakrithi’ mentioned in Ayurveda3.

The National Bio-technology Policy stress that the emerging discipline

of Pharmacogenomics combines both InfoTech and BioTech skills in augmenting

high speed data mining of both phenotypic and genotypic information from well

differentiated patient populations with a view to evolving new forms of diagnotics and

therapies of personalized medicine. Institute of Genomics and Integrated Biology,

one of the premier laboratories under the Council of Scientific and Industrial

Research, Govt. of India is currently working on the project: “Genotype Phenotype

Correlation based on the principles of Ayurveda with special focus on Prakrithi”.

A perusal of the observations and opinions of profound scholars,

research project initiated and proposed to initiate at various national and inter-

national institutions in the fields of medicine, bio technology and policy- making



attests that my present study which aims to analyze the role of DNA in determining

Prakrithi by conducting Polymerize Chain Reaction studies, to understand the

genetic correlation of various forms of Prakrithi and to develop standards for a

uniform guide line in analyzing Prakrithi is relevant in the Post Genomic Era and it

will add flavour to all the on going research process for the correlation of Ayur Tech,

Bio Tech and Info Tech.



PRAKRITHI - A MOLECULAR APPROACH

Microbes developed in toxins become resistant to a considerable extend to that

toxin1. Similarly, zygote developed in a particular environment also become resistant

to a considerable extends to that environment. Here the environment of the zygote is

an energic template – dosha sthithi2 – emitted and maintained unchanged through

out the span of life of an individual (Janma maranaantharala varthini nirvikarini) by

the interaction of the germ cell genotype (Sukla – Sonitha Prakrithi), climatic and

uterine environment (Kaala garbhasaya Prakrithi), the nature of mothers food and

psychophysical activities (Mathraahara vihara Prakrithi) and the nature of Pancha

bhoothas of the biosphere (Maha bhootha vikara Prakrithi) that are in operation at

the time of fertilization3. The energic template – dosha sthithi – is termed as

‘Prakrithi’ that transmits various physical, physiological and psychological phenotypic

characteristics considered as normal, when compared in relation with the abnormal

characteristics that constitute the hereditary diseases or Aadi bala pravritha roga.

The energic template – dosha sthithi – has two states – normal (Praakritha) and

abnormal (Vaikritha). Dalhana in Garbha vyakarana sareera of Susrutha samhitha

(verse 63) states that the normal energic template is the cause for Prakrithi whereas

the abnormal energic template causes genetic disorders. The effect of abnormal

energic template in causing deformity is explained on the analogy of the effect of the

force of flood. During rainy season a system of over flowing river water, the

embedded woods, rocks and surrounding land is set into action. What variations the

resultant force of flood will cause to the shape and size of a tree on the bank that fall

into the river cannot be predicted even if the general behavior of the river, the nature

of the embedded woods and rocks and the flood plain are well assessed. Similarly,

even though the nature of interacting factors such as the germ cell genotype (Sukla



– Sonitha Prakrithi), climatic and uterine environment (Kaala garbhasaya Prakrithi),

the nature of mothers food and psychophysical activities (Mathraahara vihara

Prakrithi) and the nature of Pancha bhoothas of the biosphere (Maha bhootha vikara

Prakrithi) are well assessed, what variation the resultant abnormal energic template

(Vaikritha dosha sthithi) will cause to the phenotypic characteristics of the zygote

cannot be predicted, says Charaka in Athulya Gothreeya sareera (verse 30).

Charaka considers the zygote as ‘Chatushpadi4’ because it is developed from the

first four among Pancha bhoothas – Prithwi, Ap, Thejus and Vayu derived from four

sources – 1. Sperm (sukra) 2. Ovum (sonitha) 3. Nutrient fluid (rasa) and 4.‘Athma’.

Abnormal state of any of the four factors derived from the aforesaid four different

sources may render the zygote abnormal the effect of which will be reflected in the

offspring. “Bija” (Sperm & Ovum) is a collection of functional units, which by

representing the structures and features of all parts and organs of an individual is

capable of ensuring the formation of offspring resembling the parents4a. Such

functional units are called “Bijabhagas”, which can be more or less similar to the term

“Gene” in modern genetics. Hairs on the head, mustaches, hairs on the body, bone,

nails, teeth, veins, ligaments, arteries, semen etc, which are stable, have paternal

dominance. Muscles, blood, fat, bone marrow, heart, umbilicus, liver, spleen,

intestines, anus etc that are soft have maternal dominance. Growth of the body,

strength, colour of the body, existence of the body in normalcy and its destruction

(decay) are mainly determined by factors derived from rasa dhathu (nutrients from

mothers diet). The four factors derived from Athma, are in a very subtle state so that

only the yogis can perceive it5. Vagbhata explains the transmission of such factors

associated with Athma to the zygote in the uterus on the analogy of transmission of

energy from solar radiation through a magnifying glass to the target object (cotton,



paper, firewood etc.)6. The abnormalities in the transmitted factors associated with

Athma may cause derangement mainly in sense perception and mental activities in

violation of cause and effect theory and is designated as ‘Karmaja vyadhi’. The

pattern of inheritance of the factors associated with Athma is indriyas, their

receptivity, general knowledge, special knowledge, Ayus (life span), happiness,

misery etc. Indriyas are the effect of interaction of the factors associated with Athma

with those of gametes and nutrients. Indriyas and their channels are in the form of

energy in the brain and are explained on the analogy of the radiations emitted from

the sun7. Hence a disease can be manifested as Poorvaparaadhaja (Karmaja), or

Drishtaparaadhaja (known etiology such as Sahaja/ Garbhaja/ Jathaja/ Peedakritha

etc) or by the combined effect of both (Sankara roga). Hence diseases manifested

without a traceable etiological factor of genetic, nutritional, climatic, traumatic origin

is to be considered as of Karmaja vyadhi caused by the effect of deranged state of

the four factors associated with Athma. It is noteworthy to mention that these factors

can induce heritable changes in Bija bhagas (genes). Piles can be seen as a

hereditary disorder (Sahaja Arsas)8. Here a heritable change occurs in the gene that

determines the development of rectal layers. Vagbhata says that this change can be

induced by two factors:

1. Psychophysical and nutritional defects of parents transmitted through gametes

(MathaPithraapacharatha) 2. By ‘daivam’, which means that, the invisible factors

(Susookshma bhoothas) transmitted through Athma. Charaka points out the

possibility of occurring discordance in monozygotic twins due to the effect of athmaja

bhava in the distribution of genetic material at the time of duplication and zygotic cell

division9. The both factors are simultaneously considered in Ayurveda for diagnotics,

prognotics and therapeutics.



If the 16 factors10 derived from Sperm (sukra) Ovum (sonitha) Nutrient fluid (rasa)

and ‘Athma’ are of normal state, the zygote develops into a normal child. Chart 1:

DETERMINANTS OF PRAKRITHI

Again, the characteristics which we consider as normal and transmitted by different

forms of energic templates can be considered as abnormal, if compared with the

characteristics of an ideal personality, which infact, is only conceptual as such a

personality can never be seen due to our inability to provide the superior

environmental conditions essential for the emergence of such a trait11. In this

context, Charaka considers Prakrithi (normal) as Vikrithi (abnormal) and use the term

Vathala, Pithala, Sleshmala instead of Vatha Prakrithi, Pitha Prakrithi and Kapha

Prakrithi. In short, the superior germ cell genotype (Sama Prakritihi) provides health

to the developing embryo (Garbha) and its variations such as Vathala, Pithala,

Sleshmala and combinations of either two of them cause varying degrees of

prownness to disease (sadaathura)12. Hence Vatha Prakrithi is considered as Heena

1 - Mathrja 2 - Pithrja 3 - Rasaja 4 - Athmaja

P1

A1

T1

V1

P2 P3 P4

A2

T2

V2

A3

T3

V3

A4

T4

V4

ZYGOTE



(bad), Pitha Prakrithi as Madhyama (better) and Kapha Prakrithi as Uthama (best)

and combinations of either two of them (dwantwa Prakrithis) as Nindya (worst)13. So,

such individuals are always more prown to diseases, and adverse environmental

factors can easily trigger corresponding diseases to the embryo or to the individual

depending upon the nature of factors involved. Considering the varying states of

germ cell genotype, the effect of which is reflected in the physical, physiological and

psychological characteristics of the individual (Phenotype), Ayurvedic genomics

mandate that every individual is different from another and hence should be

considered as a different entity14. But for diagnostic, prognostic and therapeutic

purposes, Ayurveda categorizes human population on the basis of relative

similarities in the aforesaid phenotypic characteristics in to 7 sub-populations such

as Vatha Prakrithi, Pitha Prakrithi, Kapha Prakrithi or their combinations of two or all.

Infact on microanalysis, Vatha (V), Pitha (P) and Kapha (K) have at least 62 states

on the basis of the percentage of degree to which they combine. Suppose 25%

increase = +1, 50% increase = +2, 100% increase = +3 and equilibrium state = 0;

whereas 25% decrease = -1, 50% decrease = -2, and 100% decrease = -3,

combinations of either two of the doshas renders 18 (9+ and 9-) states and three of

the doshas renders 26 (13+ and 13-) states and 12 +, =, and – combination states

100% increase or decrease of any one of the dosha constitute 6 {3 (+3) and 3 (-3)}

other states as detailed in table 1 below:

V+1P+2 P+1K+2 V+1P+1 V-1P-2 P-1K-2 V-1P-1

V+1K+2 K+1V+2 V+1K+1 V-1K-2 K-1V-2 V-1K-1

P+1K+2 K+1P+2 K+1P+1 P-1K-2 K-1P-2 K-1P-1

K+1V+2P+2 K+2V+1P+1 V+1K+2P+2 K-1V-2P-2 K-2V-1P-1 V-1K-2P-2

P+2K+1V+1 P+1K+2V+2 P+2K+1V+1 P-2K-1V-1 P-1K-2V-2 P-2K-1V-1



V+1P+2K+3 P+1K+2V+3 V+1K+2P+3 V-1P-2K-3 P-1K-2V-3 V-1K-2P-3

K+1P+2V+3 P+1V+2K+3 K+1V+2P+3 K-1P-2V-3 P-1V-2K-3 K-1V-2P-3

V+3 P+3 K+3 V-3 P-3 K-3

V+P0K- P+V0K- V+K0P- K+P0V- P+K0V- K+V0P-

V+K+P- V+P-K- P+K+V- P+V-K- V+P+K- K+V-P-

V+3P+3K+3 V-3P-3K-3

(Charaka samhitha Su. 17/ 40 to 44)

In Sama dosha Prakrithi, all the three doshas are in a state of balance essential to

provide superior intra zygotic environment inevitable for its healthy growth and

multiplication. A state of simultaneous provocation of all the three doshas will cause

disintegration of the zygote and hence such a Prakrithi does not occur15. Where as,

such a state in the somatic cells of the embryo or of the individual will manifest into a

disease of bad prognosis (Janma bala pravritha roga)16.

A correct perusal of the ancient and modern concepts of hereditary disease reveals

that autosomal recessive inheritance and sex linked dominant inheritance involves

both Mathrja and Pithrja bija dushti, whereas sex linked recessive inheritance is

exclusively Mathrja and all these can be included in Aadi bala pravritha roga.

If a woman’s diet and psychophysical practices provokes tridoshas is reflected in the

whole body and also in ovum and uterus. If the dosha provocation is severe, the

zygote only disintegrates whereas the provocation is mild, and fertilization occurs,

muscles, blood, fat, bone marrow, heart, umbilicus, liver, spleen, intestines, anus etc

that are soft and are derived from the mother, become defective in the zygote as the

respective bija bhagas render defective (gene mutation), either completely or

partially. When the bija bhaga that determines the development of uterus and / or



ovum of the offspring is subjected to vitiation, it will give rise to a sterile female

(Vandhya). If the defect is in garbhasaya bija bhaga avayava, puthi praja (still birth)

or neonates with loose body tissues (klinnanga prathyanga)17. It is understood that

Dystrophin is required inside muscle cells for structural support; it is thought to

strengthen muscle cells by anchoring elements of the internal cytoskeleton to the

surface membrane. Without it, the cell membrane becomes permeable, so that

extracellular components enter the cell, increasing the internal pressure until the

muscle cell "explodes" and dies. The gene that encodes dystrophin is found on the X

chromosome. Duchenne muscular dystrophy (DMD) is one of the most prevalent

types of muscular dystrophy and is characterized by rapid progression of muscle

degeneration that occurs early in life. All are X-linked and affect mainly males — an

estimated 1 in 3500 boys worldwide18.

When the genes that determine development of uterus, ovum, and that of the

secondary sex characteristics like breast, vagina, linea alba, etc. are rendered

defective super females (sthree akrithi bhooyishtam) called Vaartha19 is born.

Vaartha and super females or Trisomy X mentioned in modern genetics shows

similarity. It affects females who have three X chromosomes, instead of the usual

two. It is the most common X-chromosome disorder in females. Triple X is a

random mutation, usually inherited from the mother. Parents who have a daughter

with Triple X usually do not have to worry about their later children having the

syndrome. The mutation occurs in one in every 1,000 to 3,000 newborn girls, but it

is often not diagnosed until later in life. Many girls and women with Triple X have no

signs or symptoms. Signs and symptoms vary a lot between individuals, but can

include: Increased space between the eyes, Vertical skin folds that may cover the

inner corners of the eyes (epicanthal folds), Tall stature (height), Small head, Speech



and language delays and learning disabilities. Delayed development of certain motor

skills, behavioral problems, seizures, delayed puberty, infertility, rarely mental

retardation20.

If a man’s diet and psychophysical practices provokes tridoshas it is reflected in the

whole body and also in genes that determines sperm and testis. If the dosha

provocation is severe, the zygote only disintegrates, whereas the provocation is mild,

and fertilization occurs, hairs on the head, mustaches, hairs on the body, bone, nails,

teeth, veins, ligaments, arteries, semen etc, which are stable, and are derived from

the father, become defective in the zygote as the respective bija bhagas render

defective (gene mutation), either completely or partially. Charaka depicts sterile

male, in relation with sperm and / or testis determining gene. If the defect is in

sukraroopa bija bhaga avayava, puthi praja (still birth) or neonates with highly firm

body tissues (sthiraanga prathyanga)21.

Fibrodysplasia Ossificans Progressiva (FOP) is an extremely rare genetic disease

that causes muscle to be turned into bone. FOP is an autosomal dominant condition,

but most cases are sporadic. FOP patients have a genetic fault, which means that

their bodies cannot switch off the mechanism that grows the skeleton in the womb.

Any small injury to connective tissue (muscles, ligaments, and tendons) can result in

the formation of hard bone around the damaged site. Children are born with a

characteristic malformation of the great toes and begin to develop heterotopic (extra)

bone formation during early childhood. Eventually, a second skeleton begins to form

that severely restricts mobility. FOP affects 1 of 2 million people. Because of the very

small numbers of patients, identifying the mutation(s) causing FOP is difficult. There

are several genes that have been implicated in the disease process. For example,

when the Noggin gene (NOG) is deleted in mice, the mice are unable to stop the



deposition of bone, causing an FOP-like disease. Another gene of interest is the

Bone Morphogenic Protein gene (BMP), which Noggin regulates. Proteins encoded

by BMP induce bone formation, and one of their roles is to stimulate the formation of

the fetal skeleton. In FOP, lymphocytes deliver BMP4 to areas of damaged muscle,

and so initiate bone growth rather than aid tissue repair. It is hoped that future

studies will pinpoint the mutation(s) occurring in FOP and lead to a better

understanding of the disease’s mechanism22.

When the gene that determine development of testis, sperm, and that of the

secondary sex characteristics like beard, penis, moustache, etc. are rendered

defective super males (purusha akrithi bhooyishtam) called Trina puthrika23 is born.

Trina puthrika and super males or XYY syndrome mentioned in modern genetics

shows similarity. An early observation that males with a second Y chromosome, XYY

were found in excess numbers in a maximum-security prison was greatly over

interpreted. Most boys and men with a second Y are normal. The origin of the XYY is

paternal non-disjunction at the second meiotic division, which produces YY sperm.

XYY syndrome is a genetic condition in males with extra Y chromosome (in 1 in

1000 male births). Symptoms: tall stature (over 6'), may including sterility,

developmental delay, learning problems24. Our acharyas have mentioned various

environmental factors that induce genetic disorders by causing bijopatapthi and

warns not to indulge such regimns or to avoid such causes for getting a meritorious

child. It is also adviced to use factors to make beneficial effects in bija bhaga to have

an offspring of desired qualities also. Psychic factors like worry, anxiety and

emotional upsets etc of the parents may produce changes in bija bhagas the effects

of which are manifested in later generations or results in reduction of phenocopies in

the next generations. Even the mental state of mother at the time of coitus and also



during the child bearing period can influence the psychogenesis and even the

morphogenesis of the fetus. Positive thoughts and mental wellbeing of the parents

are capable of endowing the gamates with factors essential for the formation of an

offspring with superior health of the body, mind and spirit. Thoughts related to

various catagories can induce changes in corresponding genes and it will produce

harmful or beneficial effects in the physical and mental functions of the offspring. If

the parents are constantly given to grief the progeny may become fearful, thin and

short lived. If they think ill of others that may make the offspring antisocial, envious,

and subjugated to women. If they indulge in the feeling of stealing that makes the

offspring exceedingly lazy, malicious, and of inactive disposition. If they constantly

resort to anger that makes the offspring fierceful, deceitful, and jelous25. Vagbhata

even dare to say that at the time of coitus if orgasm is occuring to the female first,

her mental functions may provoke Vatha and that will induce changes in the sperm

that immidiately enters vagina i.e; it will induce changes in the genes that determine

the development of testis and / or sperm, leads to disgenesis or malformation of

seminiferous tubules and vas deferens and will give birth to a child having

azoospermia or aspermia (Vathendriya)26.

Sleeping in open air and moving at night alone may result in production of insane

progeny whereas resorting to abuses and physical hazards may make the progeny

epileptic. Alcoholic addicts may make the offspring constantly thirsty, short of

memory and fickle minded. Intake of inguana fish may make the offspring suffer from

diabetes, stone in bladder and dribbling of urine. Intake of pork flesh may induce

heretable changes that produce redness in eyes, certain obstruction of respiration

and excessive roughness of hair of the offspring. Habitual intake of fish may cause

delayed closure or non closure of eyelids. Intake of sweet things may produce



offsprings suffering from diabetes, obesity, and dumpness. Habitual intake of sour

foods may render the offspring suffer from disorders of liver and spleen (Rektha

Pitha). Increase intake of salt may result in early onset of wringles, graying of hair

and baldness in the child27. It is difficult to depict all such food materials and life

styles that may produce unfavourable effects in the next generation and this does

not fall within the scope of this treatise. Most of the diseases having a genetic

component are difficult to cure and certain diseases such as cleft pallete and hairlips

were corrected by surgical measures even in ancient times. Some of the inborn

errors of metabolism can be controlled by regular replacement of the missing factors

or by preventing accumulation of toxic metabolites by appropriate manipulation of

diet and treatment. Stressing the point, prevention is better than cure to avoid

genetic abnormalities, Ayurveda priscribes several regimns if followed strictly

enables to get meritorious offsprings and thus a better and fittest species.

Dalhana coments that swabhava bala pravritha rogas are due to the effects of

Prakrithi and indicates the natural or spontaneous occurance of disease. Natural

organic phenomina such as death, ageing, hunger, thirst, and sleep are included in

this chatagory. They are subdivided into kaalakritha (timely) and akaalakritha

(untimely). Since most of the species seen to have a built in survival programme

Prakrithi is maintained unchanged through out the span of life. A change in Prakrithi

indicates deterioration of survival programme that leads to death. The synonym of

sareera – kaaya and its nirukthi, ‘cheeyathe annadibhi:’ also implies this fact.

Cheeyathe by anna means anabolism, in the same way the aadi sabda may include

the inbuilt genetic survival programme. The congenital state of dosha which interact

with genetic material (bija bhagas) for the expression of a particular Prakrithi

imposes restriction upon the span of life of an individual. Vatha Prakrithi have short



span iof life while Pitha Prakrithi have moderate span of life and Kapha Prakrithi

have long span of life.

Body of Vatha Prakrithi individuals have lean and tall shape, rough rigid, un-

proportionate, asymmetrical, marked with large number of veins and bulged calves

with dusky, cracked and lusterless hair, sole of feet and hands cold to touch.

Produce sound from joints while walking with unsteady and swift gait (movements).

Eyes are small, round, brown, lusterless, dry, and unpleasant, unattractive with thin,

partially open lid while sleeping. They are talkative and use irrelevant, obstructed,

interrupted words, in harsh voice. They are fond of music, humor and gambling.

Fond of foods – like sweet, sour, salty and hot items. They are cruel, thievish, and

ungrateful and are non-magnanimous, jealousy, unsteady in courage, memory,

thinking, movements, friendship and observation. Their dreams are fearful, and

dreams roaming on mountains, dwelling on trees, and moving in the sky.

Body of Pitha Prakrithi individual is of medium built with medium strength,

symmetrical and proportionate with very loose joints and muscles, agitated

movements, coppery red palms, soles, tongue, lips and face, hot to touch. Skin is

whitish or yellow with wrinkles, spots or pimples and scanty grey or brown hair. Eyes

are thin, small, unsteady, brown or coppery red in colour with thin, few eyelashes,

becoming red very quickly by anger, alcohol and exposure to sunlight. The voice is

harsh, irritable and high-pitched. They are fond of garlands, perfumeries, and have

moderate sexual and physical strength. Fond of foods – like sweet, astringent, bitter,

cold items. They are jealousy, highly intelligent, good behaviour, clean and

affectionate, brave, proud; with helping disposition and quick tempered, but do not



attracted by women. Their dreams are frightening, and dreams red coloured flowers

like palasa, forest fire, meteor, lightening, thunderbolt, sunrays and fire

Body of Kapha Prakrithi individual is soft, symmetrical, well-defined, good looking

with long arms, elevated chest, wide fore head, deep-seated, strong, unctuous, well-

knit joints and bones and walks with steadiness and firm stepping. The skin colours

like shining of weapons, gorochana (yellowish), padma (reddish), suvarna (golden)

with thick curly blue – black hair, slightly hot to touch. Eyes are red at angles,

unctuous, wide, and long, with well-designed white and black spheres more with

dense and dark eyelashes. Talks less, gentle, and clear with deep sounding voices

like roaring clouds, ocean, drums or lion. Speaks relevant, not harsh or abusive or

harbour enimity. Fond of foods and drinks of all tastes especially sweet, astringent,

bitter, hot items, and extra curricular activities like music, dance, drawing, sex, etc.

They are mild in nature, very sleepy, patient, magnanimous, courageous, intelligent,

truthfulness, foresight, steady memory, thinking, movements and friendship,

forgiveness, straight forwardness, obedience, and faithfulness. They dream

reservoirs of water with full of lotus, rows of birds and clouds.

Vatha Prakrithi, Pitha Prakrithi and Kapha Prakrithi are known as ‘eka doshaja

Prakrithi’ and are otherwise termed Heena (bad), Madhyama (better) and Uthama

(best) Prakrithis respectively and combinations of either two of them seen in the

same individual (dwantwa Prakrithis) is considered as Nindya (worst). Combinations

of all the characteristics of the entire first three Prakrithis seen in one individual is

termed as Sama Prakrithi and is considered as super, rare or conceptual. The

constitutional, temperamental, psychological, emotional and social characteristics of

human personality are considered in detail in Ayurveda for accurate diagnosis,



prognosis and treatment. Hence among under the ten investigation methods,

Charaka considers assessment of Prakrithi first.

Ayurveda mandates that, the study of Prakrithi should be conducted with special

reference to religion (Jati prasakta), family (Kula prasakta), and also on the basis of

the influence of geopathological factors (Desanupatini), climatic elements

(Kalanupatini), age factors (Vayonupatini), and inividual developmental traits

(Pratyatma niyata)28.

Prakrithi with reference to religion (Jati prasakta Prakrithi)

Seers, sages and visioneers from ancient to modern times have stressed the

significance of our thinking and a strong belief in what we think has an influence in

molding our character. Religion encompasses a group of individuals having an

orchestrated belief system modulated through generations having specific thinking

pattern, life style, food habits, recreation etc. It is a common belief that identical

twins have exactly the same characteristics through out their lives. Monozygotic

twins brought up under different orchestrated belief system of two different religions

will exhibit remarkable differences in genomic distribution affecting their gene

expression portrait at an older age, though they were epigenetically indistinguishable

during the early years of life. All beliefs are basically a thought. It may be our name

or an experience, belief is something we have accepted and stored in memory. If the

acceptance is unverified and blind, it is a dogma. A set of beliefs becomes a belief

system. That such a system has remarkable influence on our bodily and mental

health. Ayurveda point out that thought pattern of the parents can influence the

morphogenesis and psychogenesis of the fetus. To give birth to a child resembling

the individual of a particular race of a particular place, Charaka and Vagbhata

advises, that the parents should willfully follow up intentional activity of the mind of



that group of individuals having a specific orchestrated belief system and also follow

the life style, food habits, recreation etc of that group of individuals. Chakrapani

establishes the existence of influence of mind upon matter with examples such as

ejaculations due to sexual feelings, fetal repercussions caused by the emotions of

the mother, which centers around the non fulfilled desires during the child bearing

period, oja kshaya (low immunological status) and sukra kshaya (oligospermia) due

to grudge, fear, etc29. Dr. Benson30 writes that, “The potentially destructive stress

response typically occurs automatically when an outside stress or – such as

pressure as work, fear, or anxiety – causes the body and brain to go on full alert.

Animals and humans share this stress-response. The relaxation response (by “letting

go”) is peculiarly human, in that it tends to arise from a specific act of volition or a

conscious relaxation strategy.” Placebo effect when combined with relaxation

response magnifies the beneficial impact on health.

The effect, meditation and firm self-confidence have on our health to the extent of

even affecting the genes is being documented through collaborative research by

neuroscientists, psychiatrists and meditation practitioners. Environmental factors

including directed thinking appear to have an ability to modify the proteins that act as

gates in activating or turning off the genes, thus controlling the gene expression. It is

established that thought is a form of energy. Directed thought and meditation are

shown to have demonstrable influence in changing the neural circuits in the brain

overriding genetic disposition. Confident positive and intense thinking within a

carefully orchestrated belief system (not blind dogma) appears to have the potential

of bringing about a transformation in an individual superseding the genetic effects.

Individual’s thoughts, whether one is consciously aware or unaware, also effect the

gene expression. By creating a facilitating and enabling environment (Satsangatya),



it can be possible to alter the program in the genes to the extent that their self-

perpetuating character is curbed. Charaka, in Vimana staana 8th chapter - Jati-

soothreeya (genetic equations) - proposes performance of ‘Puthrakaameshti’ for this

purpose, where as Susrutha proposes the same in the chapter of Sukla - Sonitha

sudhi sareera.

Current brain research is establishing that brain has tremendous plasticity. This is in

contrast to the earlier view of the neuroscientists that the neural circuits, when once

formed in childhood, do not change. “Even though we are born with a set of

instructions and neuro-signatures, our brains perpetually recruit new nerve cells and

nerve-cell activation patterns to handle its daily inputs”.

Buddhist meditation technique of “Mindful Attention,” Schwartz (2003)31 could show

through brain scans the changes that came about in the activity of parts of brain

(particularly the caudate nucleus) when his four step therapy was followed by the

patients of Obsessive Compulsive Disorder. He established the plasticity of brain to

learn new things (change the neuronal connections) under the intentional activity of

the mind. Dr. Schwartz says in Chapter X:

“It seems that neuroscience has tiptoed up to a conclusion that would be right at

home in the canon of some of the eastern philosophies: introspection, willed

attention, subjective state – pick your favorite description of an internal mental state

– can redraw the contours of the mind, and in so doing can rewire the circuits of the

brain, for it is attention that makes neuro-plasticity possible. The role of attention

throws into stark relief the power of mind over brain, for it is a mental state (attention)

that has the ability to direct neuro-plasticity. Confident, positive and intense thinking

within a carefully orchestrated belief system (not blind dogma) and scientific life

styles and food habits and other instructions of any religion intended to bringing



about a transformation in an individual superseding the bad genetic effects are now

a days rarely followed by its followers and therefore the concept of assessment of

Prakrithi with special reference to religion has no relevance.

Prakrithi with reference to family (Kula prasakta)

As in the case of religion, in olden days families were also having scientific life styles,

food habits, concept of physical and mental cleanliness, etc. the effect of which are

reflected in hereditary and somatic mutations. Hence assessment of Prakrithi with

reference to familial practices was also included in the strategy of the olden times.

Individual’s born in the family of alcoholic addicts will have the ability to withstand

intoxication produced by alcohol when compared to others32. Numerous examples

are provided in Ayurvedic literature to support the above view.

Prakrithi with reference to geopathological factors (Desanupatini)

Study of geopathological factors (Desanupata) is considered as an important factor

for the pathophysiological activities of the body. Bhumidesa is classified into three

according to its particular features and ecological status – Jangala, Anupa, and

Sadharana33. The saint Parasara, in Vrikshayurveda, an ancient treatise on plant

science in Sanskrit developed parallel with Ayurveda, also depict land into the above

three classes. Instead of Sadharana desa he uses the term Misra desa.

Jangala desa is an arid land with thick evergreen forests, huge trees, dry soil, with

full of rough and hard granites and gravels; where birds such as quil, francolin

partridge, Greek partridge etc inhabits. According to Vrikshayurveda, in Jangala

desa, the tract is almost like a desert with scanty vegetation and limited water

resources. The soil is sodic (Usavantha) with abundance of gravel and sand. Kadara

(Acacia sp. with black heart wood), Khadira (Acacia catechu), Asana (Terminalia

tomentosa), Aswakarna (Shorea dalbergioides), Sallaki (Boswellia serrata), Dhava



(Anogeissus latifolia), Tinisa (Ougeinia dalbergioides), Saala (Shorea robusta),

Badara (Zizyphus jujuba), Somavalka (Ficus dalhousia), Kimsuka (butea

monosperma), Amalaka (Phylanthus embilica), Vata (Ficus bengalenesis), Aswatha

(Ficus religiosa), Sami (Leguminous sp.), Kakubha (Terminalia arjuna), Simsapa

(Dalbergia sissoo), Yava (Hordeum vulgare), Godhuma (Triticium aestivum), Bajjira

(Pennisetum americanum) and various types of legumes as well as annual grows in

Jangala desa. Due to extremely dry condition of the soil and the natural arid

environment, among the panchabhoothas, the biosphere is predominant with agni,

vayu and prithwi and hence it result in producing plants containing kashaya

(astringent), katu (pungent), and thiktha (bitter) sap. Caharaka says that in such a

place Vatha dosha will be predominant, the people are generally strong built and

hard hearted. The predominance of Vatha dosha influences the herbs, birds,

animals, and human beings and it is reflected in their Prakrithi.

Anupa desa is comparatively low place near the sea level with full of trees, coconut

gardens etc. the place is surrounded by lakes and rivers with frequent cold winds.

The atmosphere is very moist, and herbs and bushes show a very good seasonal

flowering tendency. Birds such as flamingo, ruddy goose, cranes, woodpeckers,

cuckoos, etc richly inhabit the place. Description of Anupa desa is more or less same

in Vrikshayurveda also. Here the land is green grassy and has clusters of reedy

plants (Nala), Nelumbo sp. (Kumuda), calamus rotung (vethasa) the soil is clayey.

Strong storm like wind laden with heavy moisture keeps blowing. Rows of Hindala

(Phoenix paludosa), Tamala (Garcinia morella), Kadali (Musa sp.) and Narikela

(Cocos nucifera), Venu (Bamboosa sp.), Vanira (Calamus roxburghii), bordering the

riverside present a scenic view. The forestland there appears beautiful with

assemblage of various types of trees, shrubs with blossoming young branches. Due



to the moist nature of soil and influence of Prithwi and Ap bhoothas, the herbs,

creepers and annuals flourishing in the area generally bear sap that tastes sweet or

sour. People living there are beautiful, and the place tends to produce predominance

of Kapha dosha and it is reflected in the Prakrithi of the Anupa desa inhabitants.

Sadharana desa or Misra desa is having mixed characteristics of the above two. The

people of this place are fair, strong, and most healthy. In Misra desa the soil is gray,

red or black in colour and the place is neither too dry nor too moist; neither it has an

abundance of rock particles or sand. The land is fertile sustaining all kinds of trees

and crops. Here dosha is generally maintained in a balanced state and is reflected in

the Prakrithi of the inhabitants.

Prakrithi with reference to climatic elements (Kalanupatini)

Seasonal variations of climatic factors such as solar radiation, humidity, temperature,

atmospheric pressure, cloud, etc. provides specific environment for gene expression

to constitute a particular Prakrithi. Biology tells us that we are what our genes are.

But current research on how environment can influence the gene expression has

given rise to the new science of Epigenetics. Prenatal research findings demonstrate

the influence the environment present in the mother’s womb has on the fetus and the

way the child’s mental and physical health get affected for life. ‘Kalagarbhasaya

Prakrithi’ – climatic and intra uterine environment have tremendous influence in the

expression of genetic material and have influence in the determination of Prakrithi of

an individual. Temperature effects the changes in penitrance of genes. The general

temperature at which normal phenotypes are produced is referred to as permissive

temperature and that which produces mutant phenotypes is called restrictive

temperature. Environmental factors such as temperature, light, humidity, radiation

etc. appear to have an ability to modify the proteins that act as gates in activating or



turning off the genes, thus controlling the gene expression which is reflected in the

Prakrithi of the individual.

Prakrithi with reference to age factors (Vayonupatini)

Age factor has also an important role in the physiological and psychological

development of human beings. Age, according to Ayurveda, is a state of body, which

depends on the course of time. Human beings have three state of body – age –

Baala (childhood, 1-16yrs), Madhya (adolescence, 17-60yrs), Vridha (old age, above

60yrs). The effects of predominance of Kapha, Pitha, and Vatha have most and

respective influences in the metabolic state of body during these periods34.

Charaka point out that, during childhood, the growth rate of body is comparatively

high and the tissues are not fully developed and differentiated. The individual will be

tender hearted, easily distressed by mild hurt and insult and is not at all hot

tempered with physical and mental dispositions yet to be developed. Softness,

complexion, tenderness and unctuousness of the body are in maximum in this

period.

In adolescence, strength, energy, man hood, bravery etc. reaches its peak; the

capacity to receive, preserve and recollect information are maximized and the

individual masters the art of communication by speech. The body tissues are fully

developed and differentiated in this period and are maintained undisturbed up to the

age of 60. Due to dominance of Pitha during this period, the individual is more or

less quick tempered and his actions may reflect rebellion against traditions of the

society.

During senility (old age) degenerative changes occurs in the tissues. Higher mental

activities, sensory and motoric potentialities, immunological status, libido and bravery

gradually decline. The capacity to receive, preserve and recollect information and



communication skills gradually begin to blunt and Vatha shows dominance during

the period.

Prakrithi with reference to individual developmental traits

(Pratyatma niyata)

The Prakrithi of an individual can be assessed from the physical, physiological,

psychological characteristics of an individual that are determined by germinal

mutations (Praakritha doshaja). Any variation to the above characteristics will

endanger the very existence of the individual/ organism, as it will influence the

metabolic functions of the body as a whole. Mutations occurring in a somatic cell

only produce phenotypic changes in the organ to which the mutant cell belong

(Vaikritha doshaja). Prakrithi is determined by Praakritha dosha. Hereditary diseases

due to defect in Sukla (sperm) or Arthava (ovum) of the parents such as Kushta,

Arsas, Prameha, and Kshaya etc. are called Aadi bala pravritha (dushta sukra

sonitha bala jata) as put it by Dalhana, the commentator of Susrutha samhitha35.

Though the basic body structure, color of the skin etc are defined by the genes at the

time of conception (Sukra Sonitha Prakrithi), what we are in our health and

personality depend on the environment including our biological relationship in the

womb (garbhaasaya Prakrithi) biosocial relationships in the world and how we

perceive our own position relative to the world around. Prenatal research36 shows

that it is not merely the post-birth environment that influences an individual. The

environment within the mother’s womb too has very significant effect on the health

and personality of an individual. The coded genetic information accumulated through

the millennia of years of evolution is undoubtedly stored in the DNA. What appears

to be more significant is the mechanism of gene expression rather than the presence

or absence of a specific gene. Whether a particular gene expresses itself or not or



how a protein can form through multiple pathways of instructions of several genes is

being now understood. The view that mutations and recombinations in DNA

determine the phenotypic traits is getting modified with the emerging science of

Epigenetics. Epigenetics is the study of heritable changes in gene function that occur

without a change in the DNA sequence. It provides a handle to understand the

mechanisms in phenotype transmission and development through gene activation

and inactivation without necessarily changing the genes. There are critical periods

during prenatal development when the environment in the womb is more important

than the genes on the health we enjoy throughout our life. “Chronic maternal stress

during pregnancy – both emotional and physical – can interfere with how the fetus

utilizes nutrients and can affect how well or poorly a child functions psychologically

throughout life.” The mother’s food habits and life style during pregnancy

(mathuraahara vihara Prakrithi) have a significant influence on the child’s

personality, health and disease - janmabala pravritha as put it by Susrutha, such as

Kubjata, Vamana, Jada (with Vathala food) Khalati, Pinga (with Pithala food) Switra

And Pandu (with Sleshmala food) by Vagbhata in Ashtanga Hridayam Sareera. In

short, the normal characters such as stature, facies, colour of eye, type of hair,

fertility, vigor, longevity, etc. transmitted from parent to offspring enable to assess the

Prakrithi of an individual where as the abnormal characteristics manifested help to

understand the hereditary disease (Vikrithi).

The physical, physiological, psychological characteristics of an individual that are

fixed at the time of fertilization (garbhaadi pravritha) by germinal mutations

(Praakritha doshaja) that will enable to determine the Prakrithi of an individual is

tabulated (table no 2) below:



1. General body built and other features.

In Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi

1. Ugly 1. Ugly 1. Beautiful, delightful and

lovely.

2. Emaciated and lean 2. Body parts weak and

feeble

2. Thick

3. Rough and rigid

body

3. Loosen 3. Greasy and tender

body

4. Prominent tendons 4. Loosen and delicate

musculature

4. Muscular body, smooth

musculature

5. ---- 5. Whitish or yellow

complexion

5. White and yellow

complexion

6. Net work of vessels

prominent

6. ------ 6. ------

7. ------ 7. Joint tender and

loosen

7. Joints lubricated and

smooth in working

2. Body formation.


1. Un proportionate and

asymmetrical.

1. Symmetrical 1. Balanced and

proportionate

2. ------- 2. Strong and round body 2. Strong and round body

3. ------ 3. Soft and loose

ligaments

3. Easily movable ligaments

4. ------ 4. ------- 4. Joints, body parts and

muscles are well covered or

concealed.



3. Skin and Temperature.


1. Dry, cracked, wrinkled

(hands & feet)

1. Early wrinkled, spotted/

pimpled/ boils.

1. Smooth, oily and luster

2. Cold in touch,

temperature irregular and

low.

2. Warm in touch 2. Low and regular

temperature.

3. Intolerable to cold,

prefers hot weather.

3. Intolerable to heat and

prefers cold.

3. Tolerable to heat.

4. ------ 4. Excessive perspiration 4. -------

5. ----- 5. ------- 5. Unguent etc. are dried

lately

6. Body odourless 6. ------ 6. -----

4. Colour


1. Black and Dusty 1. White, yellowish, pale

red, saffron and pigmented

1. Golden yellow, clear

2. ------- 2. White coloured teeth 2. -------

5. Joints


1. Swift (rapid) movement

of joints

1. Movements are agitated

with a feeling of

uneasiness while moving.

1. Slow in movements

2. Loose joints 2. Relaxed joints;

ligaments loose.

2. Strong and steady joints

3. Sound produced in the 3. ------ 3. ------



movements of the joints

4. -------- 4. ------ 4. Concealed and

symmetrical joints

5. -------- 5. -------- 5. Walks steady & firm

6. Dietetic preferences.


1. Likes diet of sweet,

acidic and saltish tastes.

1. Dislikes acidic

substances.

1. Prefers sweets.

2. Prefers hot (warm)

preparations

2. Prefers cold

preparations

2. Prefers hot preparations

3. ------- 3. ------- 3. Takes light but maintains

strong structure

4. Prefers fat rich diet 4. ------- 4. Prefers dry diet and it

suits him.

7. Food habits


1. Moderate in diet 1. Excessive diet 1. Very moderate in diet

2. Eats swiftly 2. ------- 2. Eats slowly

3. Irregular in diet habits 3. Takes edibles many

times (frequently)

3. Moderate in taking food.

8. Digestion


1. Irregular digestion 1. Quick digestion 1. Weak in digestion

2. ------- 2. Intense hunger and

thirst

2. ---------



9. Bowel


1. Hard bowel 1. Soft bowel 1. Moderate bowel

2. Requires strong

purgatives for removing

constipation

2. Requires light

purgatives

2. Requires medium

purgatives

3. Constipation 3. ------ 3. -------

10. Excretion and Perspiration tendency


1. Less in quantity 1. More in quantity 1. Less in quantity

2. ----- 2. Passes urine and

excreta in more quantity.

2. --------

11. Physical strength


1. Physical strength less 1 Moderate strength 1. Strong

2. Endurance 2. Low endurance

(Incapable of bearing the

troubles)

2. Endurance

3. ----- 3. Courageous and brave 3. --------

4. Gets fatigued early. 4. ------- 4. -------

12. Smell


1. ------- 1. Unpleasant, foul and

agar like

1. Sweet



13. Hair


1. Hard and rough 1. Soft 1. --------

14. Eyes


1. Dusky 1. Coppery red color 1. Whitish

2. Spherical, small in size 2. Rounded 2. Large, broad and

elongated

3. ------- 3. Eyelashes thin 3. Lashes dense, dark

4. Thin lid 4. Thin lid 4. ------

5. Unsteady brow 5. ------ 5. -----

6. Fluctuating eyeball 6. ------- 6. ----

7. Movement of eyelids

fixed

7. ------ 7. -----

8. Rapid and much

twinkling of the eye

8. ------ 8. ------

9. Emaciated, unattractive 9. ----- 9. -----

10. ------- 10. Feels pleasant in

mist and cold

10. -----

15. Lips, Tongue and Palate


1. Unsteady and unsettled 1. Coppery 1. -------



16. Speech and Voice


1. Swift in speech 1. Talks rapidly 1. Speaks gently

2. Much talkative voice 2. Aggressive 2. Moderate in talk

17. Mental Strength


1. Coward 1. Bold 1. Courageous

2. Unstable 2. ------ 2. Steady and firm

3. Feeble minded 3. ----- 3. -----

4. Agonized with grief 4. ----- 4. -----

5. Cherishing to humble

persons

5. Haughty 5. Forgiveness

18. Reproductive Strength


1. Less children 1. Less children 1. More children

2. Un-liked by women 2. Un-liked by women 2. Liked by women

3. ------ 3. Less inclined in sex 3. More inclined in sex

4. ---- 4. Sperm quantity less 4. Profuse quantity of sperm

19. Life Span


1. Less 1. Moderate 1. Long



20. Positions and Dignity


1. Finance-poor 1. Moderate finance 1. Rich and wealthy

2. Means and luxuries-less 2. Moderate 2. ------

3. Fried circle- limited 3. ------ 3. Friendship stable

4. ------ 4. Dignified 4. Fortunate, prosperous

5. ----- 5. ----- 5. Attendants-abundant

21. Provocation of doshas


1. More susceptible to

Vatha ailments


Pitha ailments


Kapha ailments

22. Sleep


1. Sleep less 1. ------- 1. Sleepy and drowsy

2. Habit of teeth grinding

and beating of teeth in

sleep

2. ----- 2. -----

3. Eyes and mouth remain

slightly open at sleep

3. ------ 3. -----

4. Sudden sleep walking 4. ----- 4. ------

23. Dreams


1. Fearful in dreams 1. ------- 1. -------



2. Riding on camels,

walking on hells, climbing

on trees, flying in sky

2. Dreams of gold,

palasa, karnikara;

flames of fire

2. Dreams of ponds full of

lotus, Hans, rows of birds,

chakravaka, clouds.

24. Conduct and Purity


1. ------- 1. Excess and quick in

anger

1. ------

2. ----- 2. Excess in envious 2. Less envious

3. ----- 3.Pious 3. Good character

25. Similia


1. Goat, jackal, rabbit, rat,

camel, dog, vulture, crow

1. Snake, owl,

gandharva, yaksha,

monkey, tiger, bear,

mongoose

1. Brahma, rudra, varuna,

indra, lion, horse, elephant,

cow, ox, leopard, swan.

(Courtesy: Psycho- Pathology in Indian medicine)



GENES

A bird’s eye - view on genetics is essential before review of literature of the genes

included in my present study.

Gene is the fundamental physical and functional unit of heredity, which carries

information from one generation to the next. Genes make proteins. Proteins make

cells carry out the various functions of the body. Proteins are made of 20 building

blocks of life called amino acids. They combine in a thousand different ways to make

various types of proteins. Billions of proteins combine to make a cell; billions of cells

combine to make a tissue; thousands of tissues combine to make an organ, and

several organs, to make a man1.

Life begins with a single cell. By adulthood the single cell will have replicated, divided

and differentiated many times to make a three trillion-cell human being. Each cell

contains about 30000 genes and their collective interactions in our body give us life.

The normal functioning of genes keeps us healthy. When genes are exposed to

harmful environments, they alter their functions. It is called mutation. Mutations are

chemical imperfections in this process, where a base is accidentally skipped,

inserted, or incorrectly copied, or the chain is trimmed, or added to; many basic

mutations can be described as combinations of these accidental "operations".

Mutations can also occur through chemical damage (through mutagens), light (UV

damage), or through other more complicated gene swapping events. A mutated

gene makes a wrong protein and this is what makes us sick. Hence all diseases

have a genetic origin.

There are also millions of plants on earth. Many of them have developed toxins to

protect themselves against insects and environment. Those plants that produced

toxins were able to survive. These toxins act as active molecules in and have the



unique ability to bind to human genes too and are able to switch a gene on or off. If

the switching on and off of a gene is desirable, it will lead to cure of disease when

genes are switched on it produces a protein. Once produced, the protein interacts

with the many other proteins in the cell, according to the cell metabolism. This

interaction finally produces the trait. The protein floats around in our blood stream

until it comes in contact with a receptor site. If the protein is a bad one made by a

mutated gene, it can produce a disease or its simply floats around for a while and

finally is filtered out of the body.

Genes are encoded in an organism's genome, composed of DNA or RNA, and direct

the physical development and behavior of the organism. Most genes encode

proteins, which are biological macromolecules comprising linear chains of amino

acids that effect most of the chemical reactions carried out by the cell. Some genes

do not encode proteins, but produce non-coding RNA molecules that play key roles

in protein biosynthesis and gene regulation. Molecules that result from gene

expression, whether RNA or protein, are collectively known as gene products.

Most genes contain non-coding regions, that do not code for the gene products, but

often dictate gene regulation. A critical non-coding region is the promoter, a short

DNA sequence that is required for initiation of gene expression. The genes of

eukaryotic organisms often contain non-coding regions called introns which are

removed from the messenger RNA in a process known as splicing. The regions that

actually encode the gene product, which can be much smaller than the introns, are

known as exons.

The word "gene" was coined in 1909 by Danish botanist Wilhelm Johannsen for the

fundamental physical and functional unit of heredity. The word was derived from

Hugo De Vries' term pangen, itself a derivative of the word pangenesis coined by



Darwin (1868). The word pangenesis is made from the Greek words pan (a prefix

meaning "whole", "encompassing") and genesis ("birth") or genos ("origin").

Although classical genetics and evolutionary biology use the term "gene" to refer to a

conceptual entity or "unit of inheritance", modern molecular genetics typically uses

the term to refer to a physical molecule. Gene Ontology system, defines a gene as "a

locatable region of genomic sequence, corresponding to a unit of inheritance, which

is associated with regulatory regions, transcribed regions and/or other functional

sequence regions".

The word "gene" is also used in common speech to refer to the inheritance of a trait,

as in "a cancer gene" or "the gene for obesity"; however, biologists rarely use the

term in this sense because it is highly unlikely that such complex and large-scale

phenomena would be attributable to the influence of a single molecular gene.

In molecular biology, a gene is a region of DNA (or RNA, in the case of some

viruses) that determines the amino acid sequence of a protein (the coding sequence)

and the surrounding sequence that controls when and where the protein will be

produced (the regulatory sequence). The genetic code determines how the coding

sequence is converted into a protein sequence. The protein-coding regions of genes

are composed of a series of three-nucleotide sequences called codons. Each codon

specifies a particular amino acid to be added to the protein chain; thus genes

determine the protein's primary structure. Most genes are expressed in a two-stage

process: first, the DNA is transcribed by enzymes known as RNA polymerases to

produce an RNA molecule known as messenger RNA (mRNA), and second, the

mRNA is translated by specialized cellular machinery known as the ribosome into a

polypeptide chain that then folds into a functional protein. The genetic code is

essentially the same for all known life, from bacteria to humans.



Through the proteins they encode, genes govern the cells in which they reside. In

multicellular organisms, they control the development of the individual from the

fertilized egg and the day-to-day functions of the cells that make up tissues and

organs. The roles of their protein products range from mechanical support of the cell

structure to the transportation and manufacture of other molecules and to the

regulation of other proteins' activities.

Due to rare, spontaneous errors (e.g. in DNA replication), mutations in the sequence

of a gene may arise. Once propagated to the next generation, this mutation may lead

to variations within a species' population. Variants of a single gene are known as

alleles, and differences in alleles may give rise to differences in traits, for example

eye colour. A gene's most common allele is called the wild type allele, and rare

alleles are called mutants.

All the genes and intervening DNA together make up the genome of an organism,

which in many species is divided among several chromosomes and typically present

in two or more copies. The location (or locus) of a gene and the chromosome on

which it is situated is in a sense arbitrary. Genes that appear together on the

chromosomes of one species, such as humans, may appear on separate

chromosomes in another species, such as mice. Two genes positioned near one

another on a chromosome may encode proteins that figure in the same cellular

process or in completely unrelated processes. As an example of the former, many of

the genes involved in spermatogenesis reside together on the Y chromosome.

Many species carry more than one copy of their genome within each of their somatic

cells. These organisms are called diploid if they have two copies or polyploid if they

have more than two copies. In such organisms, the copies are practically never

identical. With respect to each gene, the copies that an individual possesses are



liable to be distinct alleles, which may act synergistically or antagonistically to

generate a trait or phenotype. The ways that gene copies interact are explained by

chemical dominance relationships

The existence of genes was first suggested by Gregor Mendel, who, in the 1860s,

studied inheritance in pea plants and hypothesized a factor that conveys traits from

parent to offspring. Although he did not use the term gene, he explained his results

in terms of inherited characteristics. Mendel was also the first to hypothesize

independent assortment, the distinction between dominant (An allele that determines

phenotype even when heterozygous. Also the trait controlled by that allele) and

recessive (a gene that is phenotypically manifest in the homozygous state but is

masked in the presence of a dominant allele) traits, the distinction between a

heterozygote (A diploid or polyploid with different alleles at a particular locus) and

homozygote (A diploid or polyploid with identical alleles at a particular locus), and the

difference between what would later be described as genotype and phenotype.

Mendel's concept was finally named when Wilhelm Johannsen coined the word gene

in 1909.

In the early 1900s, Mendel's work received renewed attention from scientists. In

1910, Thomas Hunt Morgan showed that genes reside on specific chromosomes. He

later showed that genes occupy specific locations on the chromosome. With this

knowledge, Morgan and his students began the first chromosomal map of the fruit fly

Drosophila. In 1928, Frederick Griffith showed that genes could be transferred. In

what is now known as Griffith's experiment, injections into a mouse of a deadly strain

of bacteria that had been heat-killed transferred genetic information to a safe strain

of the same bacteria, killing the mouse.



In 1941, George Wells Beadle and Edward Lawrie Tatum showed that mutations in

genes caused errors in certain steps in metabolic pathways. This showed that

specific genes code for specific proteins, leading to the "one gene, one enzyme"

hypothesis. Oswald Avery, Collin Macleod, and Maclyn McCarty showed in 1944 that

DNA holds the gene's information. In 1953, James D. Watson and Francis Crick

demonstrated the molecular structure of DNA. Together, these discoveries

established the central dogma of molecular biology, which states that proteins are

translated from RNA which is transcribed from DNA. This dogma has since been

shown to have exceptions, such as reverse transcription in retroviruses.

Richard Roberts and Phillip Sarp discovered in 1977 that genes can be split into

segments. This leads to the idea that one gene can make several proteins. Recently

(as of 2003-2006), biological results let the notion of gene appear more slippery. In

particular, genes do not seem to sit side by side on DNA like discrete beads. Instead,

regions of the DNA producing distinct proteins may overlap, so that the idea

emerges that "genes are one long continuum". (Pearson, 2006)

Genes under study

Molecular typing of the following genes was carried out using the polymerase chain

reaction in the present study.

1. Dopamine Receptor D2 Taq 1 D DRD2 Taq 1 D

2. Dopamine Receptor D2 Taq 1 B DRD2 Taq 1 B

3. Dopamine Receptor D2 Taq 1 A DRD2 Taq 1 A

4. Dopamine Receptor D2 Serine 311 Cysteine DRD2 S311C

5. Dopamine Receptor D2 Histidine 313 H DRD2 His 313H

6. Dopamine Receptor D3 Serine 9Glycine DRD3 S9G



7. 5-Hydroxy Tryptamine Receptor 1 B 5HTR 1B

8. Serotonin 1438 Ser 1438


10. Human Leukocyte Antigen B HLA B

11. Human Leukocyte Antigen A HLA A

Dopamine Receptor D2 Taq 1D, Taq 1B, Taq 1A

Human dopaminergic neurons are involved in the control of hormone secretion,

voluntary movement, and emotional behavior. Mediating these effects are the

dopamine D1 and D2 receptors. These macromolecules belong to a large family of

related sequences known as the G protein-coupled receptors. The D2 receptors

have been of special interest because they bind, with high affinity and specificity,

many of the commonly prescribed antipsychotic drugs.

1. Taq 1D

Kidd, Kenneth K. in a study of "A global survey of haplotype frequencies and linkage

disquilibrium at the DRD2 locus." Reported in Human Genetics Aug., 1998; 103 (2)

211-227, is the only reference that I could get about Taq 1D. He has reported that

the coding region of DRD2 gene contains four RSPs (restriction site polymorphisms),

Taq1A, Taq1B, and Taq1D. It also includes a one-dinucleotide STRP or short

tandem repeat polymorphism. DRD2, coded for by locus 11q23 in the human

chromosome, is one receptor for the neurotransmitter dopamine. This

transmembrane receptor is a member of the rhodopsin family2. As research

materials regarding the study of DRD2 Taq 1D is very rare, I have included it in my

present study.



2. Taq 1 B

A B polymorphism at the CETP (cholesteryl ester protein transfer) locus that is

detectable with the restriction enzyme TaqI is a genetic determinant of the plasma

HDL cholesterol concentration. Cholestery ester transfer protein (CETP) transfers

cholesteryl esters between lipoproteins. CETP may effect susceptibility to

atherosclerosis3.

3. Taq 1 A

The catecholamine dopamine, a precursor of noradrenaline and adrenaline, is an

endogenous neurotransmitter, which modulates a wide variety of physiologic

functions including behavior, ion transport, vascular tone, and blood pressure. There

are reports of a deficiency in renal dopamine synthesis and/or secretion in various

forms of human hypertension. As endogenous renal dopamine plays an important

role in maintaining body sodium homeostasis, renal dopaminergic deficiency may

contribute to the development and maintenance of high blood pressure, at least in a

proportion of subjects with essential hypertension Suppression of dopaminergic

activity has been observed in young normotensive subjects with a family history of

hypertension before the development of hypertension emerged. Dopamine also

plays a major role in the regulation of appetite. Dopaminergic agonist drugs, such as

dextroamphetamine, have been shown to suppress appetite and subsequently to

reduce weight, whereas a major side effect of dopamine D2 receptor (DRD2)

antagonists, such as haloperidol, is marked weight gain. The dopaminergic system

involves the interaction of dopamine with several specific dopamine receptors, which

belong to a large family of G-protein-coupled receptors. Biochemical and

pharmacological studies have shown that the physiological actions of dopamine are

mediated by interaction with two basic types of G-protein-coupled receptors, D1-like



and D2-like, which stimulate and inhibit, respectively, the enzyme adenyl cyclase.

The human DD2R gene contains eight exons, spans at least 50 kilobases (kb), and

has the unusual feature of a large (greater than 25 kb) intron (intron 1) separating

the presumed promoter region from the protein-coding region. The D2 receptor locus

has been localised to the 11q22 to 11q23 region of the human genome. . Previous

studies showed that the allelic variants of the DD2R gene play a role in the

regulation of body weight. A genome wide scan provided strong evidence on

chromosome 11q of a locus influencing susceptibility to obesity. It has been reported

that 1A polymorphism to be associated with both obesity and blood pressure in

normoglycaemic subjects, but in diabetics only the relationship with obesity was

evident4.

4. Dopamine Receptor D2 Serine 311 Cysteine

DRD2 gene has been suggested to be one missense nucleotide mutation from C to

G resulting in a substitution of serine with cystein at the codon 311 located in the

third intracellular loop of the DRD2. Variants of the DRD2 S311C play a major role in

conferring susceptibility to major psychoses, connected with disorganized and

delusional symptomatology. The schizophrenics with Cys311 tended to have a lower

age of onset and a positive family history of schizophrenia5.

5. Dopamine Receptor D2 Histidine 313 H

Dysfunction of the dopamine D2 receptor signaling has been associated with the

illness, schizophrenia. This study investigates the association of synonymous

polymorphisms (His313 and Pro319) in the dopamine D2 receptor gene may be

associated with schizophrenia. The results demonstrated that genotype distribution

for the His313 polymorphism was significantly different between schizophrenia

patients and control subjects6.



6. Dopamine Receptor D3 S9G

DRD3 S9G gene encodes the D3 subtype of the dopamine receptor. The D3 subtype

inhibits adenylyl cyclase through inhibitory G-proteins. This receptor is expressed in

phylogenetically older regions of the brain, suggesting that this receptor plays a role

in cognitive and emotional functions7.

Dopamine8 is a chemical naturally produced in the body mainly in nervous tissue

and adrenal glands. In the brain, dopamine functions as a neurotransmitter,

activating dopamine receptors. Dopamine is also a neurohormone released by the

hypothalamus. Its main function as a hormone is to inhibit the release of prolactin

from the anterior lobe of the pituitary.

Dopamine can act on the sympathetic nervous system, producing effects such as

increased heart rate and blood pressure. However, since dopamine cannot cross the

blood-brain barrier, it does not directly affect the central nervous system. To increase

the amount of dopamine in the brains of patients with diseases such as Parkinson's

disease and Dopa-Responsive Dystonia, a synthetic precursor to dopamine such as

L-DOPA can be given, since this will cross the blood-brain barrier

Functions of dopamine in the brain

Role in movement

Dopamine is critical to the way the brain controls our movements and is a crucial part

of the basal ganglia motor loop. Shortage of dopamine, particularly the death of

dopamine neurons in the nigrostriatal pathway, causes Parkinson's disease, in which

a person loses the ability to execute smooth, controlled movements.

Role in cognition and frontal cortex function



In the frontal lobes, dopamine controls the flow of information from other areas of the

brain. Dopamine disorders in this region of the brain can cause a decline in

neurocognitive functions, especially memory, attention and problem-solving.

Reduced dopamine concentrations in the prefrontal cortex are thought to contribute

to attention deficit disorder and negative schizophrenia.

Role in regulating prolactin secretion

Dopamine is the primary neuroendocrine regulator of the secretion of prolactin from

the anterior pituitary gland. Dopamine produced by neurons in the arcuate nucleus of

the hypothalamus is secreted into the hypothalamo-hypophysial blood vessels of the

median eminence, which supply the pituitary gland. The lactotrope cells that produce

prolactin, in the absence of dopamine, secrete prolactin continuously; dopamine

inhibits this secretion.

Role in pleasure and motivation

Dopamine is commonly associated with the pleasure system of the brain, providing

feelings of enjoyment and reinforcement to motivate us to do certain activities.

Dopamine is released (particularly in areas such as the nucleus accumbens and

striatum) by naturally-rewarding experiences such as food, sex, use of certain drugs

and neutral stimuli that become associated with them. This theory is often discussed

in terms of drugs (such as cocaine and amphetamines), which seem to be directly or

indirectly related to the increase of dopamine in these areas, and in relation to

neurobiological theories of chemical addiction, which argue that these dopamine

pathways are pathologically altered in addicted persons. However, cocaine and

amphetamine have different mechanisms of action. Cocaine is a dopamine

transporter blocker: it competitively inhibits dopamine uptake to increase the lifetime

of dopamine. On the other hand, amphetamines act as dopamine transporter



substrates to competitively inhibit dopamine uptake and increase dopamine efflux via

a dopamine transporter.

However, the idea that dopamine is the 'reward chemical' of the brain, a view held by

many during early stages of its research, now seems too simple. Dopamine is

released when unpleasant or aversive stimuli are encountered, so it is not

associated only with 'rewards' or pleasure. Recent research has begun to examine

whether or not the firing of dopamine neurons might function as a reward-prediction

error signal, based on evidence that, when a reward is greater than expected, there

is an increase in the firing of certain dopamine neurons (in contrast to when there is

a lesser-than-expected reward, and there is a marked decrease in the firing of the

same neurons). Some argue that dopamine may be involved in desire rather than

pleasure.

Confusion of dopamine's role in pleasure comes from studies performed on animals.

It has been shown experimentally that when the dopaminergic system of a rat is

selectively abolished it will stop eating. However when the rat is force fed food it will

still display the proper facial expressions which indicate whether they like or dislike it.

Conversely mutant hyperdopaminergic mice show higher wanting of food but not

liking. This research was taken to mean that dopamine mediated desire and

incentive salience instead of pleasure. In humans, though, drugs that reduce

dopamine activity (e.g., antipsychotics) have been shown to induce both amotivation

(lack of desire) as well as anhedonia (inability to experience pleasure). The selective

D2/D3 agonists pramipexole and ropinirole have anti-anhedonic and pro-motivational

properties as measured by the Snaith-Hamilton Pleasure Scale. Opioid and

cannabinoid transmission instead of dopamine is believed to be what modulates food

reward and palatability (liking). This explains why animals would still have the same



liking of food independent of brain dopamine concentrations. Other pleasures are

likely dependent on dopamine. Libido can be increased by drugs that enhance

dopaminergic functioning but not by ones that affect opioid peptides or other

neurotransmitters. Sociability is also closely tied to dopamine neurotransmission.

Low D2 receptor binding is found in people with social anxiety. Traits common to

negative schizophrenia (social withdrawal, apathy, anhedonia) are thought to be

related to a hypodopaminergic state in certain areas of the brain. In instances of

bipolar mania subjects can become hypersocial as well as hypersexual. This is also

believed to be due to an increase in dopamine, because it can be alleviated with

dopamine blocking antipsychotics.

7. 5-Hydroxy Tryptamine Receptor 1B, or 5-HTR 1B

5-HT1B receptors are expressed throughout the mammalian central nervous system.

These receptors are located in the axon terminals of both serotonergic and

nonserotonergic neurons, where they act as inhibitory autoreceptors or

heteroreceptors, respectively. 5-HT1B receptors inhibit the release of a range of

neurotransmitters, including serotonin, GABA, acetylcholine, and glutamate. These

receptors have been difficult to study because of the diversity of their cellular

localization and the absence of highly selective agonists and antagonists. There has

been accumulating evidence, however, that 5-HT1B receptors modulate drug

reinforcement, stress sensitivity, mood, anxiety, and aggression. The general results

of a number of studies suggest that reduced 5-HT1B heteroreceptor activities may

increase impulsive behaviors, whereas reduced 5-HT1B autoreceptor activities may

have an antidepressant-like effect. The 5-HT (1B) receptor has been implicated in

several psychopathologies, including pathological aggression, alcoholism and

suicide9.



5-hydroxytryptamine (5-HT), commonly known as Serotonin10, is released at

peripheral sites from activated enterochromaffin cells in the gastrointestinal tract

(90%), mast cells and platelets. In the body, serotonin is synthesized from the amino

acid tryptophan by a short metabolic pathway consisting of two enzymes —

TPH(1,2) and DDC. TPH1 reaction controls the flux through the pathway. Serotonin

is found in three main areas of the body: the intestinal wall; large constricted blood

vessels; and the central nervous system. The activity of serotonin arises in the

brainstem from clusters of neurons known as the raphe nucleus. The most widely

studied effects have been those on the central nervous system. From the brain,

serotonin neurons extend to virtually all parts of the central nervous system making

the branching of the serotonin network the most expansive neurochemical system in

the brain. The importance of this network becomes apparent when considering each

serotonin neuron exerts an influence over as many as 500,000 target neurons. Due

to the widespread distribution of serotonin in the nervous system, it is not surprising

that this neurotransmitter can be linked to many types of behavior. The functions of

serotonin are numerous and appear to have regulation of appetite, sleep, vomiting,

memory and learning, temperature regulation, mood, behavior (including sexual and

hallucinogenic behavior), cardiovascular function, muscle contraction, endocrine

regulation, and depression. Recent research suggests that serotonin also plays an

important role in liver regeneration and acts as a mitogen (induces cell division)

throughout the body.

Serotonin taken orally does not pass into the serotonergic pathways of the central

nervous system because it does not cross the blood-brain barrier. However, the

amino acid tryptophan and its metabolite 5-hydroxytryptophan (5-HTP), from which



serotonin is synthesized, can and do cross the blood-brain barrier. These agents are

available as dietary supplements and may be effective serotonergic agents.

One product of serotonin breakdown is 5-hydroxyindoleacetic acid (5 HIAA) which is

excreted in the urine. Serotonin and 5HIAA are sometimes produced in excess

amounts by certain cancer tumors, and levels of these substances may be measured

in the urine to test for these tumors.

Neurotransmission

The neurons of the Raphe nuclei are the principal source of 5-HT release. These

neurons are grouped into about nine pairs, distributed along the entire length of the

brainstem. 5-HT is thought to be released from serotonergic varicosities into the

extra neuronal space, in other words from swellings (varicosities) along the axon,

rather than from synaptic terminal buttons (in the manner of classical

neurotransmission). From here it is free to diffuse over a relatively large region of

space (>20µm) and activate 5-HT receptors located on the dendrites, cell bodies and

presynaptic terminals of adjacent neurons.

Serotonergic action is terminated primarily via uptake of 5-HT from the synapse. This

is through the specific monoamine transporter for 5-HT, 5-HT reuptake transporter,

on the presynaptic neuron.

5-Hydroxy Tryptamine Receptor 2A, or 5-HTR 2A

8. SER A-1438-G

9. SER T-102-C

Central serotonin-2A (5-HT(2A)) receptor dysfunction is regarded as an important

factor in the etiology of affective disorders. The relations between some personality

traits and the vulnerability of affective disorders are also implicated. Moreover, there

are several reports which describe the association between 5-HT(2A) receptor gene



polymorphisms and mental disorders. 5-HT(2A) receptor gene have polymorphisms

(A-1438-G, T102C, T516C, C1340T, C1354T). The 5-HT (2A) receptor belongs to

the G-protein super family. It plays an important role in vascular regulation. Reports

indicate an association of the T102C genetic polymorphism of the 5-HT (2A) receptor

with hypertension11.

Estrogen receptors and serotonin receptors coexist in cells in a wide variety of

tissues. The most active and most studied form of estrogen in mammals is 17-β

estradiol (hereafter E2). A critical review of the literature suggests that many of E2's

effects may be mediated by changes in the actions of serotonin (5HT). Serotonin is

usually considered to be a neurotransmitter, but surprisingly, only 1% of serotonin in

the human body is found in the CNS. The remaining 99% is found in other tissues,

primarily plasma, the gastro-intestinal tract, and immune tissues, where serotonin

acts as a hormone regulating various physiological functions including vasodilation,

clotting recruitment of immune cells, gastro-intestinal motility and initiation of uterine

contraction12.

HUMAN LEUCOYTE ANTIGEN GENES13

10. HLA – B

11. HLA – A

The term HLA refers to the Human Leucoyte Antigen system, which is controlled by

genes on short arm of chromosome 6. The HLA loci are part of the genetic region

known as major histocompatibility complex (MHC) (Hugh et al. 1984). The MHC

locus is pleiotropic, which is best known with its role in histocompatibility and

immune regulation. The HLA loci, by virtue of their extreme polymorphism ensure

that few individuals are identical and the population at large is well equipped to deal

with immunity threats. (Mc Devitt, 1985). Some HLA antigens are recognized on all



tissues of the body and thus the identification of HLA antigen is described as “tissue

typing” or “HLA TYPING”.

Snell first described MHC in the mouse as the H-2 system in 1948. Jean Dausset in

Paris recognized the immunological origin of the agglutination of white blood cells by

sera from transfused patients. He identified in 1953 the first leukocyte specificity

Mac, which is now called HLA-A2. Rose Payne at Stanford reported in 1958 the

appearance of similar leuko-agglutinating antibodies in multiparous women.

Independently, Jon van Rood (Leiden, The Netherlands) made similar observations

and he used computer programs for leukocyte antigen grouping from clusters of

leukoagglutinating antibodies.

The definition 4a/4b series (Jan Van Rood, 1963) and that of HLA – AI, A2, A3 (Rose

Payne and Walter Bodmer, 1964) indicated a need for international standardization

and thus was born a series of international workshops, starting in 1964 (Glen, 1991).

During the early 1960s, a growing group of investigators attempted to define

leukocyte antigen groups with serological techniques such as leukoagglutination and

complement fixation on platelets. While these assays were lacking reproducibility,

another problem was the extreme complexity of the genetics of leukocyte antigens.

A turning point in the history of leukocyte typing was the intensive international

collaboration that began as the First Workshop and Conference on Histocompatibility

organized by Bernard Amos (Durham, NC) in 1964. This was a laboratory bench

study whereby the participants compared the reactivity of their sera with various

techniques. The results were so discordant that they could not be published. The

Second Workshop held the following year in Leiden, yielded more coherent results

and several serological specificities emerged clearly. The concept was forwarded

that all of them belonged to a single, complex antigenic system analogous to the H-2



system of the mouse. Paul Terasaki and John McClelland at UCLA introduced the

complement-dependent microlymphocytotoxicity technique, which has remained the

standard serological test for HLA typing. The Third Workshop organized by Ruggero

Ceppelini (Torino, Italy) in 1967, clearly established the HLA system and two

segregant series of specificities (now called HLA-A and HLA-B) were recognized.

The success of this international collaboration has assured the continuation of the

histocompatibility workshops (Los Angeles,1970; Evian, France, 1972; Aarhus,

Denmark, 1975; Oxford, 1977; Los Angeles, 1980; Munich, 1984; New York, 1987;

Yokohama, 1991, and St Malo/Paris, 1996). After each workshop, a nomenclature

committee has incorporated salient findings towards the definition of HLA

polymorphisms including the identification of additional class I loci such as HLA-C

(Thorsby, 1970), and the class II loci HLA-DR (1977) and a few years later HLA-DQ

(formerly called MB) and HLA-DP (formerly SB). The latter comprise the HLA-D

region, which was first recognized as the MLC locus by Amos and Yunis in 1970 as

a genetic system responsible for T-cell activation in the mixed leukocyte culture. In

the eighties the introduction of new molecular biology techniques revolutionized

HLA-typing (Wake, 1982) (Owerbach, 1983). Today, with the use of various DNA-

typing techniques (Bidwell, 1994) (Begovich, 1995), many new polymorphic loci

within the MHC have been identified and new alleles are discovered continuously

(Bodmer, 1995).

Specialists use IMGT/HLA Sequence database to locate sequences of the human

major histocompatibility complex (HLA). It includes the official sequences for the

World Health Organization (WHO) HLA Nomenclature Committee for Factors of the

HLA System. The IMGT/HLA Database is part of the international ImMunoGeneTics

project (IMGT). There are currently over 1600 officially recognized HLA alleles and



these sequences are made available to the scientific community through the

IMGT/HLA database. In 1998 the IMGT/HLA database was publicly released. Since

this time the database has grown and is the primary source of information for the

study of sequences of the human major histocompatibilty complex. The numbers of

antigens and alleles named since the HLA Nomenclature Committee was first

formed in 1968, up to the end of December 2004 is given in figure 2.

Within the last years, by the extensive use of the molecular biology methods the

number of the HLA-polymorphism has been increased for 2 to 3 times, bypassing the

requirement for viable lymphocytes that express the proteins. The modern molecular

biology techniques allows more accurate typing of the HLA class I and class II

antigens at the DNA level, enabling determination of DNA sequence of each HLA

allele. Back in the 1980’s, HLA genotyping has been introduced in routine tissue

typing laboratories and for precise allele determination the main available approach

was the use of Restriction Fragment-Length Polymorphism (RFLP) protocols.

However, this method have many drawbacks such as: minimum of two weeks period

necessary to achieve a complete HLA class II typing and the need of radioactive

labeling of probes which have restricted these analyses to a reduced number of

laboratories. Introduction of the Polymerase Chain Reaction (PCR) technology adds

sensitivity and specificity to HLA typing. Most PCR based methods include: DNA

amplification with sequence specific primers (SSP) followed by hybridization with

sequence specific oligonucleotide probes (SSOP). Sequencing is taking more

importance as the number of HLA alleles are increasing and making more difficulties

in the specific accession of every allele. So far, among the most advanced

approaches of the future of HLA typing is DNA chip technology, which uses



thousands of probes attached to silicon chips. Hybridization is conducted on the

surface of the chips and the results are obtained after a fluorescence reading, in

seconds (Eggers et al, 1994).

The HLA system constituting the human MHC has gene cluster spans around a

region of about 4000 k.b on short arm of chromosome six in the distal portion of 6p

21.3 band (Lamm and Olaisen, 1985). Large number of genes with variable

expression are arranged in form of three regions in MHC as class I (36 genes), class

II (27 genes) and class III (39 genes) as first proposed by Jan Klein in

1977(Campbell And Trowsdale, 1993) as given in figure 1. Moving from telomere to

centromere, class I genes are placed first followed by class III and finally class II.

The Class I region which is the most telomeric part of the complex contains genes

for the classical HLA antigens: HLA-A, B and C, as well as four non-classical Class I

genes: HLA-E, F, G and H. The central region contains more than 20 genes. Genes

of this region encode structurally and functionally diverse molecules such as: serum

complement proteins (C2, C4A, C4B, BF), tumor necrosis factors (TNF), heat shock

proteins (HSP) and adrenal 21-hydroxylase enzyme (CYP 21B). The Class II region

encodes both alpha and beta chains of the three types of class II molecules: HLA-

DR, DQ and DP.

The Class-I and Class-II genes are among the most polymorphic genetic systems

known. While class I products play a role in determining viral specificity and MHC

restriction (Zinkernagel And Doherty, 1997; Koopmann et al. 1997), Class II are

involved in peptide presentation and cell to cell interaction during and immune

response (Pieter, 1997). The products of class III genes are components of humoral



immune response (Baltimore D, 2001). The function of MHC is to present antigenic

peptides to the antigen-specific T-cell receptors. The Major Histocompatibility

Complex controls potent transplantation antigens that elicit the rejection process.

FUNCTIONS OF MHC

The normal physiological function of the MHC remained largely unknown until the

seventies. The control of immune responses to polypeptide antigens had been

localized to the H-2 complex in 1969 (Mc Devitt, 1969) and the identity between

MHC genes and immune response (Ir) genes was confirmed in 1972 (Shevach,

1972). The immune reactions were shown to involve interactions between MHC

molecules and T-lymphocytes (Rosenthal, 1973) and the T-cell response was

subsequently shown to be restricted not only by the antigen, but also by the MHC

molecule (Zinkernagel, 1974). It was furthermore found that MHC molecules were

involved in the production of antibodies and that this process also was MHC

restricted (Kindred, 1972) by gene products from the class II region (Katz, 1973).

Today we know how MHC molecules function in detail and from crystallographic

studies also the structure of MHC class I (Bjorkman, 1987) and II molecules (Brown,

1993).

HLA - CLASS I GENES

The cell surface glycopeptide antigens of HLA – A, -B and -C series are called HLA

class I antigens (Roitt et al, 1998). They play a key role in the recognition and

destruction of cells carrying foreign antigens by cytotoxic t- Lymphocytes. The X–ray

crystallography study of HLA class I molecules (Browning And Mc Michael, 1996)

has demonstrated that the molecule has a cleft on its outermost surface for peptide



binding. The proteins are expressed on the surface of the antigen presenting cells as

heterodimeric molecules. Class I molecules are composed of heavy α - chain and a

light β2 microglobulin chain. The β2 chain encoded by genes on chromosome 15 is

needed for transporting α chain (MHC encoded) to the cell surface. Immunological

studies indicate that HLA –B which is highly polymorphic is the most significant HLA

– class I locus followed by HLA-A and then HLA –C. Nucleic acid-based methods for

allele identification have revealed more than 470 polymorphic variants at the HLA-B

locus. Screening of potential bone marrow donors with sequence specific primer

polymerase chain reactions and sequence specific oligonucleotide probe

hybridization assays revealed apparent variants within the B*58, *44, *15, and *48

allele groups.(Williams et al ,2002). Majority of differences among alleles are found

in exons 2 and 3. DNA based HLA typing focuses on these polymorphic exons to

identify HLA alleles. Genes in the MHC are important determinants of host response

to microbial infections (Hart. T.C., 1996) and also has a profound effect on the

immunogenetic susceptibility to various disease that are influenced by immune

reactions (Firatli et al, 1996)

CLINICAL RELEVANCES OF HLA

The determination of HLA alles has been extensively utilized in tissue typing for

transplantations, paternity determination and disease association. The determination

of HLA alleles, or HLA typing, has been extensively utilized in tissue typing for

transplantation, paternity determination and disease association. Upon the

approval of the idea of a link between longevity and MHC in animals, it became a

field of interest for a human longevity studies. The idea of a link between longevity

and MHC was first supported in animals in the research of Smith and Walford in



1977 and lately was approved by other gerontologists. They have shown that the

whole MHC is a region associated with a significant effect on life span. The age-

specificity may be under partial control of H-2 or HLA-linked genes (Meredith et al,

1979). Major methodological problems which make confusions in this field of impact

of polymorphic HLA genes on human longevity arise from insufficient number of the

samples included to reach statistically significant conclusions; missing of the

appropriate criteria for the selection of the participants for immunogerontological

studies; non comparison of the results with well selected control group; non

considering the sex influence on the HLA polymorphism associated with longevity

etc. The loci investigated within the HLA region differ from study to study. During the

last two decades, among the studies concerning the HLA and human longevity,

many different HLA loci belonging to both antigen classes (I and II) have been

included. In the study of several HLA-Class I (A, B, C) and HLA DR antigens by

Proust et al, in 1982, no significant differences at these loci in comparing the total

young and elderly series were found, pointing out that the sex differences should be

taken into account. Also the hypothesis that certain HLA haplotypes are associated

with the survival advantage was supported. Having the same aim, many authors

have studied certain HLA antigens in different populations such as Dutch (Laggay et

al. 1991), Chinese (Ma et al. 1997), Japanese (Akisaka et al 1997,1998), Korean

(Kyuong-Ok et al, 1996), Italian (Ricci et al., 1998), Greek (Papasteriades et al,

1997) and French (Rayana Ivanova et al., 1998) population showing very different

and poorly overlapping data. In the study of Ricci et al. four antigens (HLA-A31 (19),

-B7, -Cw7 and –DQ1) showed a higher frequency in the elder group, while

Papasteriades et al., found an increased frequency of HLA-B16 and HLA-DR7 and a

decreased frequency of HLA-B15 and HLA-DR4. Recent investigation of Ivanova et



al., showed that HLA-DR13 alleles were increased in centenarians of both genders,

HLA-DR7 was elevated in longevious men and HLA-DR11 higher in women from

sibships, unchanged in women centenarians. This and several other studies

provided evidence for association of some HLA-DR alleles with human survival and

longevity. These results are in agreement with other published data and suggest that

HLA antigens are associated with longevity, either indirectly through disease

associations or directly through involvement in the aging mechanism.

HLA typing was applied to kidney transplantations very soon after the first HLA

determinants were characterized (Terasaki 1992; Opelz, 1985; Sanfilipo et al. 1984).

During the late sixties, Terasaki, and co-workers presented early data indicating the

potential beneficial effects of HLA matching on cadaveric kidney transplant survival,

although these findings were based on typing information with a limited set of rather

crude anti-HLA antisera. Furthermore, van Rood’s group, Batchelor and Joysey, and

other investigators also found that matching for HLA improves kidney and skin graft

survivals. The conflicting presentations by Terasaki’s group at the Third International

Congress of the Transplantation Society in The Hague in 1970 produced

considerable controversies about the significance of histocompatibility matching in

kidney transplantation. Many well-matched kidney transplants failed early and

conversely, badly matched kidneys did often enough function quite well. Other

investigators had noted that same experience and it was not really surprising that

many transplant surgeons chose to ignore tissue-typing results. Of course, all these

controversies arose when HLA matching was limited to an incomplete set of HLA-A

and HLA-B antigens; there was no typing for HLA-DR and the available serological

tests had a rather low level of reproducibility. Because of improved serological

procedures and especially, the application of DNA-based techniques, HLA



compatibility can now be much better defined and there remains no doubt that HLA

matching correlates with less rejection and prolonged kidney transplant survival.

Histocompatibility testing for organ transplantation requires usually a cross match

test between recipient serum and donor cells. Starzl and co-workers reported in

1965 the first case of a patient with complement-dependent anti-donor antibodies.

This patient rejected almost immediately a kidney transplant from this donor and the

tissue pathology suggested a Shwartzmann reaction-like mechanism. Kissmeyer-

Nielsen in Copenhagen reported a similar case of what he termed a hyper acute

rejection. This experience established the cross match test as a major test in

histocompatibilty testing. The Panel-Reactive Antibody (PRA) test was first reported

by Terasaki in 1971 to identify presensitized patients at higher immunological risk of

rejecting their transplant. There is evidence that the cornea may survive slightly

better if the HLA class I antigen is matched (Mayer et al, 1983). Complete HLA

matching of bone marrow donor and recipient is crucial to the success of allogenic

bone marrow transplantations (Shankarkumar And Undevia, 1999; Ghosh, 1999;

Shankarkumar, 2001). The vast polymorphism of HLA system makes it a most

valuable tool in the field of paternity testing (Bryant, 1988).

HLA – B GENETIC DIVERSITY - POPULATION STUDIES

Initial knowledge of highly polymorphic HLA complex system that was developed

over the second half of 20th century allowed us to understand its role in the immune

response and its unique contributions to anthropology and population genetics

(Charron, 1997). Distributions of HLA antigens in various ethnic groups of the world

and India have been reported. HLA –B40 is a common B locus antigen in Asians, but

the alles underlying this antigen depend upon the population (Clayton et al, 1997).



HLA – B4006 is of high frequency among north Indians and is common among other

Asian populations (Mongolians, Chinese and Japanese). HLA-B35 is a frequent

antigen in Caucasians, the prevalent Allele being HLA – B3501 (Clayton et al, 1997;

Satz et al, 1995). North Indians have a higher frequency of B35 than other Asian

population with – B3503 as the most common subtype. B3503 is also a common B

allele in south Indians (Tait et al). HLA – B61 was the most frequent allele in

Dravidian tribal population of south India. Some of the most frequent HLA – B alleles

in the non tribal Dravidian population were similar to north Indian population and

included HLA B 07 , B 61 , B 51 , B 35 AND B 52 ( Thomas et al , 2003 ) HLA-

B44 AND HLA – B57 ( a typical Caucasian haplotype ) were found in “ Iyers ”

(Brahmins ) of Madurai ( Susuki et al , 1992 ). HLA – B5 and HLA –B7 were common

among Brahmins of western India (Shankarkumar et al, 2002). Namboothiris who are

believed to be migrants from North India showed a higher frequency of HLA B*07,

B*35, B*61and B*52 which is common among the North Indian population. HLA

B*44, common among the South Indian population was of least frequency among

Namboothiris. Two alleles (HLA B*39 and HLA B*56), which were present only in

tribal population of Kerala was observed in Namboothiri population also

HLA-B AND DISEASE SUSCEPTIBILITY

There are two general explanations for HLA and disease association (Mc Devitt,

1985). Firstly, there may be a linkage disequilibrium between alleles at a particular

disease associated locus and the HLA antigen associated with that disease.

Secondly the HLA antigen itself plays a role in disease. MHC gene products can be

involved in the pathogenesis of, especially autoimmune, diseases. The pathological

functions of certain MHC polymorphisms became known at the same time as the



normal physiological functions of the MHC gene products were reveled. In 1967, it

was found that certain MHC polymorphisms were associated with a higher risk for

developing Hodgkin's disease (Amiel, 1967) and soon thereafter many diseases

were shown to be associated with MHC genes. Major histocompatibility complex

linked immune response or immune suppressive gene (Benacerraf, 1981) has

resulted in various disease association studies at population level to seek

association with HLA (Tiwari and Terasaki, 1985). Studies on genome scan have

implicated that the genes in MHC region are contributing more to diabetes

susceptibility (Davies et al., 1994). HLA – B27 shows remarkable association with

`ankylosing spondylitis` and related spondylarthropathies in most populations in the

world (Genzalez – Roces et al., 1997); Ramos and Lopez de Castro, 2002). Work

done in Hungarian population showed a close association between HLA –B8 and `

Graves disease`. B40 and B57 are seen closely associated with `SLE`, which is an

autoimmune disease (Pradhan et al., 2004). HLA –B39 allele was found significantly

more often in insulin dependent diabetes mellitus patients (Sergei Nejentsev et al.,

1997) HLA – B54 is involved in the pathogenesis to diffuse Panbrochiolitis in Korean

patients (Myoung Lee Park et al; 1999)



Gene map of the human leukocyte antigen (HLA) region

Figure 1: Gene map of the human leukocyte antigen (HLA) region. The HLA region

spans 4 × 106 nucleotides on chromosome 6p21.1 to p21.3, with class II, class III

and class I genes located from the centromeric (Cen) to the telomeric (Tel) end. HLA

class I molecules restrict CD8+ cytotoxic T lymphocyte function and mediate immune

responses against ‘endogenous’ antigens and virally infected targets, whereas HLA

class II molecules are involved in the presentation of ‘exogenous’ antigens to T

helper cells. The HLA class III region contains many genes encoding proteins that

are unrelated to cell-mediated immunity but that nevertheless modulate or regulate

immune responses in some way, including tumour necrosis factor (TNF), heat shock

proteins (Hsps) and complement proteins (C2, C4)



Figure 2: The Graph indicates the numbers of antigens and alleles named since the

HLA Nomenclature Committee (IMGT HLA database) was first formed in 1968, up to

the end of December 200414, 15.



SYNOPSIS

Full title of the thesis

A CONCEPTUAL STUDY ON PRAKRITHI IN CORRELATION WITH MOLECULAR

PROFILES

Introduction

Prakrithi - Prakrithi means “swabhava” or nature of an individual. According to

“Arunadutta” it means physical condition. According to “Vagbhata” the Prakrithi

remains inherited throughout the life, prior to death.

The state of dosha in “sukra”(sperm) and “arthava”(ovum), the dosha predominance

at the time of fertilization, doshic state of garbhasaya (uterus), the influence of

environment on tridosha with respect to season during the intra uterine development,

the dietetics of mother, mode of living & the nature of physical & mental activities of

mother rendering the doshas susceptible to vitiation during the child bearing period,

are the factors which constitute the Prakrithi of an individual. So it is highly difficult to

provide any information regarding the actual mechanism involved in the process of

segregation of Prakrithi, but Prakrithi can be assessed from ones’ physical structure,

reaction to environment, feelings, thoughts & psychological makeup. But we can see

that only hereditary factors & post conceptional factors play a deterministic role.

To understand the actual mechanism involved in the formation of a particular

Prakrithi, knowledge of “LAW OF HEREDITY” (Anuvamsi siddhantha) is highly

essential. According to Ayurveda, “Bija” (Sperm & Ovum) is a collection of functional

units, which by representing the structures and features of all parts and organs of an

individual is capable of ensuring the formation of offsprings resembling the parents.

Such functional units are called “Bijabhagas”, which can be more or less similar to



the term “Gene” in modern genetics. So DNA can be compared to “Bijabhaga

avayava”.

Molecular Profiling (MP)

Molecular Profiling (MP) has emerged as a dynamic new discipline, capable of

generating a global view of mRNA, protein patterns, and DNA alterations in various

cell types and disease processes.

MP integrates the expanding genetic databases from the Human Genome Project

with newly developed expression analysis technologies and holds great promise to

help us:

ü Understand the molecular anatomy of normal cells and cells in various stages

of disease.

ü Develop new diagnostic and therapeutic targets for clinical intervention.

ü Explain the relationship between genotype and phenotype in humans, which

is still largely unknown.

More technical and theoretical details shall be provided with methodology of the

study.

Since this technique is of high precision and need enormous complicated procedures

it will be of greater convenience to study the DNA fingerprinting as well, which may

come out with satisfactory results.

DNA fingerprinting in humans

Genetic uniqueness of an individual is brought about by two factors,

inheritance and new mutations. Genetic fingerprinting techniques are suitable for

analyzing the diversity present within or between populations. Traditionally the term



‘Fingerprint’ refers to the pattern of fingertip ridges, which are highly characteristic for

any individual. For quite a few years, this term has also been used for the

electrophoretic and chromatographic characterization of proteins and more recently

of DNA molecules.

The size of the human nuclear genome is approximately 3x109 base pairs. All

genetic differences between individuals are laid down in the primary sequence of this

genomic DNA. An efficient strategy for identifying an individual would be to compare

those regions of the genome, which are already known to differ frequently between

individuals. Such regions are termed polymorphic sites and it has been observed

that repetitive sequences present in the genomes of virtually all species are highly

polymorphic. Genetic polymorphism is defined as the simultaneous occurrence in the

same population of two or more variants. The concept of genetic polymorphism is

fundamental to all current methods for determining genetic identity and relatedness.

The first step in characterizing a biological sample at the genome level is to purify its

genetic material, DNA. The source of DNA can be any biological sample containing

nucleated cells, such as, blood, semen, hair, bones, saliva or urine. The nuclear

DNA is compacted with proteins to form chromatin, which condenses to form

chromosomes. DNA is extracted from the other components mainly by the action of

enzymes, protein denaturing agents, salts and organic solvents like phenol and

chloroform. The isolated DNA can then be subjected to enzymatic amplification in

vitro termed as Polymerase Chain Reaction (PCR). The amplification of selective

genomic regions including single copy sequences, as well as, repetitive sequences,

like micro satellites, could be employed successfully for comparison of genotypes.

The advantage of PCR technique is that it works with small amounts of DNA, and



functions even if DNA is degraded or not very pure. The banding patterns generated

can later be used to analyze the extent of diversity /relatedness between individuals.

NEED AND SIGNIFICANCE OF THE STUDY

The assessment of Prakrithi is of great importance in identifying the body

configuration and systemic status of an individual. Prakrithi is unchangeable; it

represents the part of human personality, which is mostly determined by the

hereditary factors. Till now we have many subjective & objective parameters for

assessment. The world of genetics is growing day-by-day and evolving new

dimensions in medical research. Inorder to enter into the main stream of medical

world it is necessary to have insight into the concepts derived in genetics. Hence this

is only a preliminary study to find out the correlation of innate constitution (Prakrithi)

with DNA.

AIMS AND OBJECTIVES OF THE STUDY

1. Detailed literary study of “Prakrithi” from various texts in Ayurveda.

2. To analyze the role of DNA in determining Prakrithi by conducting a fingerprint

study.

3. To understand the genetic correlation of various forms of Prakrithi

4. To develop standards for a uniform guideline in analyzing Prakrithi

NULL HYPOTHESIS

Molecular profiles cannot help in determining the Prakrithi (constitution) of an

individual

ALTERNATE HYPOTHESIS

Molecular profiles can help in determining the Prakrithi (constitution) of an individual



METHODOLOGY

The Prakrithi of persons will be assessed using Ayurvedic references. The persons

will be divided into different groups according to Prakrithi. Blood sample taken from

these persons will be subjected to DNA fingerprint study (PCR technique). Final

analysis will be done to assess any similarity in the molecular profiles of persons

having similar Prakrithi.

Inclusion Criteria

1. Normal healthy individuals from different geographic area

2. Both sex

3. Informed consent

Exclusion Criteria

1. Individuals in the same family

2. Unwilling persons



Consent Letter

I, Dr. K.N.Ajithkumar, II MD Scholar, Govt. Ayurveda College, Kannur propose to

conduct an Ayurvedic research on Prakrithi in correlation with Molecular profiles; in

the guidance of Dr. Moinak Banerjee, Scientist, Rajiv Gandhi Centre for

Biotechnology, Thiruvananthapuram.

The data developed in the work will be kept confidential and will be used only

for research purpose and will not be used or disclosed for any other

applications. The data is not used for any commercial purpose.

I …………………………………………………….have gone through the letter and is

willing to co-operate with his work.

Signature of the Consent

Signature of the Investigator

RGCB

Date



DEPARTMENT OF ROGANIDANA GOVT.AYURVEDA COLLEGE

KANNUR CASE PROFORMA FOR A CONCEPTUAL STUDY ON PRAKRITHI IN CORRELATION WITH MOLECULAR PROFILES. By Dr.K.N.Ajithkumar under supervision of Dr.R.Sreekumar MD (Ay) & Dr.Moinak Banerjee Ph.D Case No.: 1. Name: Date: 2. Address : Spouse Name: 3. Age (Yrs) : 4. Sex : 1. Male 2. Female 5. Occupation : 1. Scientist 2. Office staff 3. Others 6. Religion : 1. Hindu 2. Muslim 3. Christian 4. Others 7. Domicile : 1. Urban 2. Rural 8. Income : 1. Rs.<5000 2. 5001-13000 3. 13001-25000 4. > 25001 9. Medical History Diabetic Respiratory disease Hepatic Renal disease Thyroid disease Neurological problems TB Others 10. Personal history Alcohol consumption Yes No Smoking Yes No Drug abuse Yes No 11. General examination a. Pulse ......................./mt b. Weight ........................Kg c. Height .......................Cms 12. Systemic Examination 1. CVS : 2. CNS : 3. R.S : 4. GIT : 5. Locomotor : 6. Uro-genital : 7. Ophthalmology & ENT : 8. Dermatology :



FORMAT FOR PRAKRITHI PAREEKSHA Vatha Pitha Kapha 1.BODY Lean Medium Stout Weak Fleshy Large Tall/ Short Delicate Soft Unshaped Unshaped Beautiful Prominent veins and tendons Loose Cold in touch Delicate joints Symmetrical Rigid Emits bad smell Firm Rough Proportionate Compact Un proportionate Lustrous Stable Undernourished Long developed Well developed Low body temperature 2. SKIN Dry Wrinkles Oily Rough Fair Soft Lusterless Warts Glossy Blackish Moles Fair Reddish / yellow White/pinkish Bluish patches Glorious Soft 3. EYE Blackish Reddish/Coppery White Dry Small RedlPink angles Lusterless Rounded Wide and long Small Quickly becomes red Pleasant Fluctuating Sharp eye sight Oily Eyelids open while sleeping Pleasant Calm Desires cold Gentle look Well designed White and Black spheres 4. EYEBROWS Unstable Nothing specific Plenty of hairs Thin Thick Small 5. EYE LASHES Small Thin More Dry Few Firm Oily



6. HAIRS OF BODY/HEAD & MOUSTACHE Scanty Soft Strong Rough Brown/coppery Thick Cracking Small Curly lusterless Few Oily Grayish Early baldness Black Curly Early graying Proportionate 7. HEAD Unstable Nothing specific Steady 8. LIPS Dry Red coloured Pinkish Cracking Soft Smooth Shapeless Thin Glossy Unstable Blackish 9. TEETH Coarse Medium Firm Thin With gap Strong Few Many Protuberant Healthy gums Cracking Even Irregular Glazing Straight Smooth Shining white 10. TONGUE Unstable Reddish / coppery Nothing specific 11. FACE Rough Fleshy Pleasant Worried look 12. FOREHEAD Small With folds Large 13. LOWER JAW Small Nothing specific Big 14. JOINTS Unstable Loose Firm Protuberant Moderately hidden Compact Sound producing Concealed on movements 15. PALMS Unstable Reddish Big Dry Oily Rough Firm Cracked 16. FOOT Unstable Reddish Nothing specific Rough Cracked 17. NAILS Rough Coppery/reddish Large Thin Soft Thick Small Small White Blackish Flat Firm



Cracking Convex Breaking Glossy 18. CHEST Small Nothing Specific Big Not well built Elevated 19. GAIT Unsteady Fast Slow Light Stable Quick Foot pressing Producing sound against ground Wandering Habit of moving hands /Iegs/shoulder 20. ACTIVITIES Unsteady Un beatable Slow Quick lazy Starts any work hastily 21. MUSCLES Bulged calves Loose / flaccid Well build 22. STRENGTH Less Moderate Good Tires Quickly Cannot withstand Does not feel physical or mental tired after Work/ exertion physical or mental Work/ exertion 23. SPEECH/ VOICE Talkative Clear Gentle Irrelevantspeech loud Speaks little Obstructed Talkative Clear voice Hoarse Good and impressive Slow Fast Good at arguments Steady Diffused Speech Firm Resonant voice 24. FOOD HABITS Eats much Excessive hunger Likes hot food Eats swifty Eats much Dry food Takes light food Like cold/warmfood East slowly Likes hot food Eats / drinks often less hungry / thirst Irregular diet Prefers sweet likes spicy Irreg. digestion Prefers bitter likes bitter Prefers sweet Prefers astringent likes astringent Prefers sour likes sweet Prefers salty Weak in digestion Prefers Oily 25. STOOL/BOWEL Hard bowel Expels frequently Yellowish well Tenaency of Large quantity formed constipation Yellowish loose Once in a day



26. URINE Obstructed Expels frequently Nothing specific Large quantity Yellowish 27. SWEATING Nothing particular Sweat very easily less sweating Foul smell 28. HUNGER/ THIRST Upredictable Good less Cannot tolerate Can tolerate 29. IMMUNITY less Moderate Good 30. LIFE SPAN Short Medium long 31. SEXUAL DESIRE less Medium More 32. REPRODUCTIVE STRENGTH Less semen / Less inclined to sex Much semen / irregular regular menstruation Unlike by women/ menstruate men Poor capacity Less semen/ovum Liked by women /men Un attracted by opposite sex Sexually powerful. 33. BEHAVIOUR Coward Aversion to heat Stability Stealing Afraid of discomfort Control over senses Atheis Affectionate to dependence Grateful Unstable Like being praised Humble No control over senses Proud Generous Quick attachtment Courageous Long concealed and detachment enimity Good behaviour Not greedy Noble less Adventurous Caim Sorrowful Jealousy Quiet Less intelligent Generous Obedient Ungrateful Scholar Straight forward Aversion to cold Intelligent Religious Biting nails Clean Honour the teachers Grinding teeth while slieeping Well educated Civilised 34. FRIENDSHIP Few Dependable Stable Unsteady 35. ANGER Quick Quick Rarely 36. FEAR Quick Rarely Not specific



37. DREAMS Climbing hills Fire /lightning Water / birds / / sky clouds 38. Sleep Interrupted Sound Sound < 6 hrs. 6 - 8 hrs. < 8 hrs. 39. GRASPING POWER Some times quickly / slowly Always quickly Always grasp late Very clever Genius 40. MEMORY Forgets,quickly Moderate Good 41. HOBBIES Music Fond of garlands Talented Humour Perfumes Hunting Decorative Gaming Music 42. INTOLARANCE Cold Hot Can tolerate Anger cold/heat/ angry/hunger thirst, Hunger physical urges Physical urges Thirst 43. PROBLEM FACING Worrying constantly Can take right Can take firm quick decision right firm calm decision and stable mind Cannot take stable decision Total number of Characters of Vata - 133 Total number of Characters of Pitta - 118 Total number of Characters of Kapha - 131 Percentage of characters of one Dosha = No. of Characters present x 100 Total No. of Characters Prakrithi of the individual Vata....................... % Pitta....................... % Kapha.................... %

= % of Characters present x 100 Total No. %



Assesment of Dhehaprakrithi of.........................................................................is Vata Pitta Kapha Vata Pitta Vata Kapha Pitta Kapha Samadosha



MATERIALS AND METHODS STUDY POPULATION

Blood samples (N = 40) were collected with informed consent from different

communities. Due care was taken to avoid sampling from related individuals

and siblings. DNA isolation was performed using standard organic extraction

methods. Molecular typing was carried out using the polymerase chain

reaction.

The main steps carried out in this analysis include:

• Preparation of DNA

• Amplification of genes

• Detection of amplification

PREPARATION OF DNA1

Ø SAMPLE REQUIREMENTS

v Whole blood is drawn into a vial with anticoagulants containing

EDTA.

Ø MATERIALS AND REAGENTS

v Lyses buffer I

v Lyses buffer II

v 2% SDS

v Proteinase K

v Tris – phenol (pH 8.0)

v Chloroform-isoamyl alcohol (24: 1)

v Absolute alcohol

v Sodium acetate (3M pH5.2)

v 70% Ethanol

v Tris – EDTA solution



EQUIPMENTS AND INSTRUMENTATION

v Deep freeze refrigerator

v Water bath

v Centrifuge

v Homogenizer

PREPARATION OF BUFFER SOLUTIONS

1. Tris (1M), 500ml [NH2C (CH2OH)3 M.W=121.14]

Take 60.5gm of Tris base and dissolve it first in little amount of water, add

more water slowly and stir well. Adjust the pH to 8 by adding required Hydrochloric

acid.

2. EDTA (0.5M), 250ml

Take 46.5gm of EDTA and dissolve in 200ml of water. Stir well. While mixing,

add Sodium hydroxide pellets and adjust the pH to 8. Make the final volume to

250ml.

3. NaCl (5M)

Take 58.4gm of Sodium chloride in 200ml of water and dissolve it by

stirring.

4. Lysis buffer I (TEN 30:5:50Mm)

Tris (1M) 7.5ml

EDTA (0.5M) 2.5ml

NaCl (5M) 2.5ml

Distilled water 250ml

5. Lysis buffer II (NE 75:2Mm)

NaCl (5M) 3.75ml



EDTA (0.5M) 1ml


6. T.E buffer (10:1Mm)

Tris (1M) 1ml

EDTA (0.5M) 0.2ml


Ø PROCEDURE

The extraction2 is first done using phenol: chloroform and then with chloroform. In

this case, deproteinisation is more efficient. Although phenol denatures proteins

efficiently, it does not completely inhibit RNAase activity and it is a solvent for RNA

molecules that contain long tracts of poly adenyline. Both these problems can be

avoided by using phenol: chloroform: isoamylalcohol. The subsequent use of

chloroform removes any trace of phenol from nucleic acid preparation.

1. To 10ml of peripheral venous blood, collected in EDTA vial, equal volume of

(10ml) RBC lysis buffer was added and frozen at –80oC for minimum of 3 Hrs.

(usually over night).

2. Sample was kept in 65oC water bath for rapid freeze thow. During this step

RBCs were lysed.

3. RBC lysed sample was centrifuged at 10,000 rpm for 10 min at 4oC.

Supernatant containing RBC (lysed) was discarded, WBC pellets obtained for

DNA isolation.

4. To the above pellet – equal volume (10ml) WBC lysis buffer was added, and

homogenized in a hand homogeniser, till the pellets get thoroughly dispersed.

5. SDS (Sodium Dodecyl Sulphate) 2% of volume (10ml blood – 1ml SDS) and

proteinase K (150 μg/ml working conc.) (10ml blood – 75μl from a stoke conc.



of 20mg/ml) was added to the above lysate, and incubated at 37oC overnight

in water bath. During this step WBC’s were ruptured by SDS and protein in

the cells were digested by proteinase K.

6. Equal volume (10ml) Tris Saturated Phenol (pH 7.5) was added and mixed

gently. Phenol removes protein contaminants.

7. Lysate was centrifuged at 10,000 rpm for 10 min. at 4oC. Phenol forms the

lower layer containing denatured proteins (organic layer). The supernatant

(aqueous layer) is collected in a fresh tube carefully using a micropipette,

without disturbing the organic layer.

8. To this aqueous layer half volume (5ml) of Tris Saturated Phenol is added and

mixed gently for 2 minutes.

9. To the above mixture half volume (5ml) of chloroform: iso-amyl alcohol (24:1)

also added. Mix gently till proper mixing occurred (almost 10 min.). Then

centrifuged the mixture at 10,000 rpm for 10 min. at 4oC.

10. The aqueous layer is collected in a fresh tube carefully using a micropipette.

To this, equal volume (10ml) of chloroform: iso-amyl alcohol (24:1) also

added. Mix gently till proper mixing occurred (almost 10 min.). Then again

centrifuged the mixture at 10,000 rpm for 10 min. at 4oC.

11. The aqueous layer was collected and 1/10th volume 3M Sodium Acetate (pH

5.2) and double volume chilled absolute

alcohol was added.

12. Mix gently for a while, the DNA will be

precipitated as a lump. Transfer the DNA

to an eppendorf tube containing 70%

ethanol using a micropipette.



13. Wash (centrifuge) the DNA twice with 70% alcohol and twice with absolute

alcohol (5000 rpm 5 min. at room temp.).

14. The pellet was air-dried and dried pellet was suspended in Tris - EDTA buffer.

It is a low ionic strength buffer. The DNA is first made to dissolve in small

amounts of Tris - EDTA, and then more is added. Rinse the walls of the tubes

well with the buffer

15. Store the DNA at 4oC. For prolonged storage –20oC or -70oC is used.

DNA QUANTIFICATION

One method

SPECTROPHOTOMETRY3

The quality and quantity of genomic DNA

was checked in a spectrophotometer

(Biospec-1601, DNA/Protein/Enzymes

Analyzer, Shimadzu). The ratio of

absorbance at 260 nm (normal light) and 280 nm (260/280) was used to estimate the

purity of the DNA.

1. Place two blank cuvettes in the spectrophotometer filled with distilled water

(100 ul)

2. Select the DNA – Protein protocol in the spectrophotometer

3. Standardize the value to ‘0’ (zero)

4. Remove the distilled water from one blank cuvette and add DNA (i.e distilled

water 98 ul and DNA 2 ul)

5. Place the cuvette in the spectrophotometer and the value will be displayed in

the monitor. (In Amstrong units)



6. Convert the Amstrong value to ngm/ul.

7. A ratio between 1.7-1.8 indicates good quality DNA without protein

contamination. The absorption of 1 OD (A) is equivalent to approximately 50

µg/mL of double stranded DNA. The concentration of DNA in 1 µL of the DNA

sample was calculated using the following equation.

50x OD A260 x Dilution factor

1000

Another method

AGAROSE GEL ELECTROPHORESIS4

1. 0.7 % Agarose in 0.5X TBE

Buffer (Stock 5X) (0.7 gm

Agarose + 100 ml 0.5X TBE)

2. Microwave heat until agarose is

dissolved and solution is clear.

3. Allow the solution to cool to about

55oC before pouring. Ethidium bromide is added and mixed well before

pouring.

4. Prepare the gel tray by sealing the ends with tape.

5. Place the comb in the gel tray and position the comb vertically such that the

teeth are about 1-2mm above the surface of the tray.

6. Pour the gel solution into the tray to a depth of 5mm. Allow the gel to solidify

for about 20 minutes at room temperature.

7. Gently remove the comb, place the tray in electrophoresis chamber and

submerge it with the same buffer used to prepare the gel.



8. To prepare samples for electrophoresis add 1ul of dye to the 1ul DNA and 4 ul

of sterile distilled water mix well using a centrifuge.

9. The samples are then pipetted into the sample wells.

10. 80 Volts current is applied. This can be confirmed by observing bubbles

coming off the electrodes.

11. DNA will migrate towards the anode, coloured red.

12. The distance DNA has migrated in the gel can be judged by monitoring the

migration of the dye.

13. After adequate migration, the gel can be viewed using a trans illuminator. The

DNA quantity can be detected

as a band.

14. Save the gel using a multi-

Imager.

TBE Buffer 5X (Stock)

Tris – 54 gm

Boric Acid - 27.5 gm

EDTA (0.5 M) – 20 ml

Distilled Water – 1000 ml

pH - 8.00

Loading dye

Single dye : Bromophenol blue - 0.25%

Glycerol - 30%

Double dye: Bromophenol blue - 0.25%

(for small fragments) Xylene cyanol - 0.25%

Glycerol - 30%



Gel showing DNA after electrophoresis for quantification

AMPLIFICATION OF GENES

MOLECULAR METHOD

The PCR is used to generate a large number of copies of a gene for rapid

detection and to reduce non-specific hybridization of oligonucleotide probes. Since

these methods are performed at genetic level, better resolution of the alleles can be

achieved.

Polymerised Chain Reaction (PCR)5

PCR is a procedure based on the ability of DNA polymerase to copy a strand of DNA

by elongation of complementary strands initiated from a pair of closely spaced

oligonucleotide primers. Each cycle of the reaction doubles the amount of the target

DNA, leading to million fold levels of amplification. PCR has high sequence

specificity because the two unique primers must hybridize in relatively close

proximity to each other on the target DNA sequence under stringent temperature and

reaction conditions before exponential amplification can occur.

Ø PRINCIPLES OF PCR6

There are three major steps in a PCR, which are repeated for 35 or 40 cycles in an

automated cycler, which can heat and cool the tubes with the reaction mixture in a

very short time (Figure 3).



1. DENATURATION AT 94 oC:

During denaturation, the double strand melts open to single stranded DNA, all

enzymatic reactions stops.

2. ANNEALING AT 50 oC:

The hydrogen bonds are formed and broken between the single stranded

template. The more stable bonds last longer and on that double stranded DNA made

of the template and the primer, the polymerase can attach and start copying the

template. Once few bases are built in, the hydrogen bond is so strong between the

template and the primer that it does not break anymore.

3. EXTENSION AT 72oC:

The primers, where there are a few bases built in, have a stronger attraction

to the template, created by hydrogen bonds, than the forces breaking these

attractions. Primers having no exact contact with the template do not give an

extension of the fragment. The bases are coupled to the primer on 3’ side, the

polymerase adds dNTPs from 5’ to 3’, reading the template from 3’ to 5’, and bases

are added complementary to the template.


2µl of the DNA is required for each PCR.


Deoxynucleoside triphosphate (dNTP) solution

PCR primers (forward and reverse)

Taq A or 10 X polymerase buffer

Taq enzyme with Mg2+ ions

Sterile distilled water



Ø EQUIPMENTS AND INSTRUMENTATION

Thermal cycler (PCR mechine, eppendorf)

Vortex mixer

Microcentrifuge

Ø PROCEDURE

1. Assemble the PCR mix for a single

sample: 2ul of PCR buffer, 2ul of dNTP

Solution, 0.2ul of each primer, about

0.3ul of Taq enzyme and water

required to bring the final volume to 20ul.

2. Aliquot approximately 2ul of the DNA samples into the appropriate reaction

tubes.

3. Vortex the PCR mix well, so that the components get evenly distributed.

4. Centrifuge in a micro centrifuge.

5. Place the samples in the Thermo cycler.

6. PCR amplification was carried out in eppendorf Thermal Cycler machine with

heated lid option. The cycling parameters for 20 µL reactions were as follows:

initial denaturation at 94°C for 5 minutes followed by 30 seconds at 94°C, 30

seconds at 50°C, 30 seconds at 72°C followed by, 5 minutes at 72°C followed

by 3 seconds at 40 C comprising one cycle.

7. The time and the temperatures for denaturation, primer annealing, and

extension will vary depending on the primers utilized and the Thermal cycler

chosen.

8. Store the amplified DNA at 4oC.



PCR is the most efficient method for the amplification of alleles. The key for

the success of PCR is Taq polymerase. This enzyme is extracted from Thermus

aquaticus, a bacterium that grows at elevated temperatures. This enzyme has

extreme thermal stability and has biochemical properties quite adequate for repeated

exposures to the denaturing temperatures required in PCR reactions. This enzyme

requires Mg+2 ion for optimal activity.



Gel showing DNA after amplification (in wells 1, 3, 4, 5, 9 shows good amplification.

11th one is marker)

POST AMPLIFICATION TECHNIQUE:

RESTRICTION AND DIGESTION7


8µl of the PCR product is required for each restriction


BSA (Bovine serum albumin) solution

10 X buffer

Sterile distilled water

Enzymes (Specified for each genes) which is listed below

Genes Enzymes

DRD2 Taq 1 D Taq 1 ∝ enzyme

DRD2 Taq 1 B Same as above

DRD2 Taq 1 A Same as above

DRD2 S311C Sau 961 enzyme

DRD His313 H NCO1 enzyme

DRD3 S9G MSC1 enzyme

5 HTR 1 B Hin C2 enzyme

Ser 1438 MSP1 enzyme



Ser 102 Same as above


Vortex mixer

Microcentrifuge

Water bath

Ø PROCEDURE

9. Assemble the PCR product for a single sample: 2ul of PCR buffer, 0.2ul of

BSA Solution, about 0.8ul of enzyme and water required to bring the final

volume to 20ul.

10. Aliquot approximately 8ul of the PCR product into the appropriate reaction

tubes.

11. Vortex the mix well, so that the components get evenly distributed.

12. Centrifuge in a micro centrifuge.

13. Place the samples in the PCR product over night in 370 C water bath.

AGAROSE GEL ELECTROPHORESIS

PRINCIPLE

The amplified PCR products are detected using this method. When electric

field is applied to an agarose gel in presence of a buffer solution, which conducts

electricity and maintains the pH, DNA fragments that are negatively charged moves

through the gel towards the positive electrode at a rate dependent on size and

shape. Small linear fragments move quickly than larger ones, which are retarded due

to entanglement with the network of agarose fibers forming the gel. As the voltage

applied to the gel is increased larger fragments migrate faster than smaller



fragments. Ethidium bromide is a fluorescent dye that intercalates between bases of

nucleic acids and allows detection of DNA fragments.


The amplified PCR products to be detected


Agarose powder (1.2%)

TBE buffer (0.5X)

Gel loading dye

Ethidium bromide


Electrophoresis chamber

Power supply

Gel casting trays

Combs to form sample wells

Trans illuminator

Multi-Imager

Micro centrifuge

Ø PROCEDURE

1. To prepare 60ml of 1.2% agarose solution, measure 0.72gm agarose into a

glass flask and add 60ml of 0.5X TBE.

2. Microwave heat until agarose is dissolved and solution is clear.

3. Allow the solution to cool to about 55oC before pouring. Ethidium bromide is

added and mixed well before pouring.



4. Prepare the gel tray by sealing the ends with tape.

5. Place the comb in the gel tray and position the comb vertically such that the

teeth are about 1-2mm above the surface of the tray.

6. Pour the gel solution into the tray to a depth of 5mm. Allow the gel to solidify

for about 20 minutes at room temperature.

7. Gently remove the comb, place the tray in electrophoresis chamber and

submerge it with the same buffer used to prepare the gel.

8. To prepare samples for electrophoresis add 5ul of dye to the 20ul PCR

product and mix well using a centrifuge.

9. The samples are then pipetted into the sample wells.

10. Current is applied. This can be confirmed by observing bubbles coming off the

electrodes.

11. DNA will migrate towards the anode, coloured red.

12. The distance DNA has migrated in the gel can be judged by monitoring the

migration of the dye.

13. After adequate migration the gel can be viewed using a trans illuminator. The

gene amplified can be detected as a band.

14. Save the gel using a multi-Imager.

Some examples are given below:



Positive results showing in Taq 1D reactions (wells 4 to 8 and 12)

Positive results showing in Taq 1B reactions (wells 3,4,6 to 10)

Positive results showing in Taq 1A reactions (wells 3 to 9 and 11)



Materials and Methods HLA Materials

Chemicals and Biologicals

Glassware and plastic wares

Isolation of Genomic DNA

DNA Quantification

HLA Typing

PCR amplification using sequence specific primers

HLA (PCR-SSP) Amplification Conditions

Agarose Gel Electrophoresis

Materials

Chemicals and Biologicals1

All the chemicals used were of either molecular biology grade or extra pure

analytical-reagent grade. Ethylene diamine tetra acetic acid (EDTA), sodium dodecyl

sulphate (SDS), agarose, dimethyl sulfoxide (DMSO), acrylamide was obtained from

(Invitrogen, Life technologies). Various chemicals like sodium chloride, chloroform,

isoamyl alcohol, phenol, Orange G, glacial acetic acid, sodium hydroxide, sodium

carbonate, isopropanol, dimethyl formamide (DMF) was obtained from Qualigens.

Proteinase K, Taq DNA polymerase, (Sigma); Boric acid, Tris base, Tris-Cl, RNase

A, X-gal, from (USB); GFX PCR DNA gel band purification system from (Amersham

Biosciences) were used in the study.



Glassware and plastic wares

Glassware was from either Borosil or Schott-Duran. Plastic ware including multi strip

PCR tubes and plates were from Axygen.

Isolation of Genomic DNA from whole blood

DNA was isolated from lymphocytes obtained from EDTA anti-coagulated blood. A

modified form of the standard organic extraction method was used for DNA

extraction2

Briefly, an equal volume of RBC lysis buffer was added to the blood sample collected

in the EDTA vial and was frozen at –80˚C for 3 hours. The sample was then freeze-

thawed at 65˚C resulting in RBC lysis. The RBC lysed sample was centrifuged at

4000 rpm for 10 minutes at 4˚C. The supernatant containing the lysed RBC was

discarded. The WBC pellet was resuspended in equal volume of WBC lysis buffer

and homogenized. SDS and Proteinase K were added to the lysate at final

concentrations of 2.0% and 150 ug/mL respectively and incubated at 37˚C for 8

hours. During this step, the SDS ruptures the WBCs and the proteins in the cells is

digested by the action of Proteinase K. Equal volume of tris-saturated phenol (pH

7.5) was added to the sample and mixed gently. The sample was centrifuged at

10000 rpm for 10 minutes at 4˚C. The aqueous layer was collected and the organic

layer containing phenol and denatured proteins was discarded. To the aqueous

layer, equal volumes of a mixture of tris-saturated phenol /chloroform/isoamyl alcohol

(25:24:1) was added and mixed gently. The sample was centrifuged at 10000 rpm

for 10 minutes at 4˚C. The aqueous layer was collected and the organic layer

containing carbohydrates and lipids was discarded. To the sample equal volume of

chloroform/isoamyl alcohol (24:1) was added and mixed gently. The sample was

centrifuged at 10000 rpm for 10 minutes at 4˚C. The aqueous layer was transferred



into a fresh tube and the organic layer was discarded. To the sample, 1/10th volume

sodium acetate (3 M, pH 5.2) and twice the volume chilled absolute ethanol were

added and mixed gently. The precipitated lump of DNA was spooled out into a

microfuge tube. The DNA was washed twice in 70% ethanol. The pellet was air-dried

and resuspended in TE buffer. The DNA samples were stored in –20˚C.

DNA Quantification

The quality and quantity of genomic DNA was checked in a spectrophotometer3

(Biospec-1601, DNA/Protein/Enzymes Analyzer, Shimadzu). The ratio of absorbance

at 260 nm and 280 nm (260/280) was used to estimate the purity of the DNA. A ratio

between 1.7-1.8 indicates good quality DNA without protein contamination. The

absorption of 1 OD (A) is equivalent to approximately 50 µg/mL of double stranded

DNA. The concentration of DNA in 1 µL of the DNA sample was calculated using the

following equation.

50x OD A260 x Dilution factor

1000

HLA Typing

PCR amplification using sequence specific primers

Molecular HLA typing8 was carried out using the polymerase chain reaction with

sequence specific primers (PCR-SSP) method for the HLA-A, & -B loci. Amplification

primers for HLA-B were synthesized based on published literature by Bunce et al

1995 while HLA-A primers were based on a revised paper by Tonks et al 1999. The

basic principle of the PCR-SSP typing utilizes the same PCR protocol and

parameters for typing HLA-A, & -B alleles. Most of the alleles are detected in group-

specific amplifications corresponding to serological specificities although some allele



specific detection is possible. Thirty-four primers were used for analyzing HLA-A

locus, 59 primers for HLA-B loc. The primers were purchased as crude

oligonucleotides from Sigma-Genosys. Amplification control primers giving rise to a

796 base pair fragment from the third intron of HLA-DRB1 (Olerup and Zetterquist

1992) were included in all PCR reactions as internal control. The control

amplification primer sequences are listed below:

Forward: CP 63 5-TgCCAAgTggAgCACCCAA

Reverse: CP 64 5-gCATCTTgCTCTgTgCAgAT

All primers were resuspended in sterile distilled water at stock concentrations of 100

pmoles/µL and stored at –20˚C. The primers were diluted to working concentrations

of 10 pmoles/µL.

HLA (PCR-SSP) Amplification Conditions

The final volume of all PCR reactions is 15µL. The PCR reaction mixture9 consisted

of 10 X PCR buffer (67 mM Tris Base pH 8.8, 16.6 mM ammonium sulphate, 0.01%

(v/v) Tween 20), 2.0 mM magnesium chloride, 200 µM of each deoxynucleotide

phosphate (dNTP), 0.5 pmole of each allele- specific primer, 0.5 pmole of each

control primer (CP 63 and CP 64) and 0.5 Units of Taq Polymerase (Bangalore

Genei, India). Around 50-100 ng of genomic DNA was used each PCR reaction. The

different primer mixes are shown in table 2.5.

PCR amplification was carried out in 8 well PCR strips (Axygen) in a MJ Research

Thermal Cycler (PTC 200) machine with heated lid option. The cycling parameters

for 15 µL reactions were as follows: initial denaturation at 96°C for 1 minute followed

by 5 cycles of 25 seconds at 96°C, 45 seconds at 70°C, 45 seconds at 72°C followed

by 21 cycles of 25 seconds at 96°C, 50 seconds at 65°C, 45 seconds at 72°C



followed by 4 cycles of 25 seconds at 96°C, 60 seconds at 55°C, 120 seconds at

72°C.

Table 2.5 Phototype primer mix composition for PCR-SSP reactions10

Num

ber

Prim

er M

ix Sense

Primer

Anti-

sense

Primer

Antigens Alleles

bp s

ize

1A 01A AL16 ALZ A01 A*0101-04N 575

2A 02A AL16 ALV A36 A*3601 564

3A 03A AL37 ALAW A*02 A*0201-0227,68032 940

4A 04A AL7 ALD A*03 A*0301-0304 628

5A 05A AL8 ALQ A23, A24 A*2301,2413 557

6A 06A AL8 ALR A24 A*2402-05, 2407, 2409N-11N, 2414 557

7A 07A AL11 ALC A25 A*2501, 2502 400

8A 08A AL34 ALC A26, A43 A*2601-02,2604,2607-09,2611N, 4301 402

9A 09A AL4 ALC A25, 26 A*2501-02,2601-09, 2611N,

3401-02, 6601-02

440

10A 10A AL6 ALC A25, 34,66 A*2502, 3402, 6601-02 419

11A 11A AL17 ALC A43 A*4301 442

12A 12A AL6 ALI A11 A*1101-2, 1104 520

13A 13A AL6 ALH A68, 69 A*6801-02, 6806, 6808, 6901 447

15A 14A AL4 ALAM A34, 68 A*3402, 6801-05, 6808 427

16A 15A AL25 ALH A68 A*6802 625

17A 16A AL6 ALY A69 A*6901 383

18A 17A AL35 ALF A29 A*2901-03 442

19A 18A AL12 ALG A30 A*3001-04, 3006 561

20A 19A AL10 ALF A31 A*31012 481

21A 20A AL11 ALF A32 A*3201-02 423

22A 21A AL4 ALF A33 A*3301, 3303 463

23A 22A AL24 ALAR A32, 74 A*3201-02, 7401-03 492

24A 23A AL32 ALF A29, 31,33,74 A*2901-3, 31012, 3301,

3303, 7401-02

412

25A 24A AL54 ALBK A80 A*8001 543



26A 25A AL30,

AL31

ALH A02, 23,24,68,69 A*0201-22, 0225-27,

2301, 2402-04

446

27A 26A AL30,

AL31

ALL A01, A02, A25,

A26, A30, A31,

A32, A33,

A*0101-04,0224, 2501, 2601-09, 2611,

3002-04, 3006, 31012, 3201-

02,3301,3303,3601,4301, 6603,7401-

03,8001,0301-04,1101-04,2502,2901-

03,3001,3401-02,6601-02

445

1B 34 193 221 B7, 8101 B*0702-0705,8101 619

2B 35 312 221 B703 B*0703 600

3B 36 195 212 B8, 4406 B*0801,0802,4406 543

4B 155 195 220 B8 B*0801-02 606

5B 37 280 225 B49, 50,4005,

2706,2704

B*4901,5001,4005,2704,2706 635

6B 38 208 215 B49, 59 B*4901,5901 385

7B 39 246 215 B45, 49,50 B*4501,4901,5001 600

8B 40 207 219 B45, 76 B*4501,1514 536

9B 160 202,

272

393,

285

B44 B*4402-06 546/

481

10B 42 192 220 B41 B*4101-02 605

11B 43 272 276 B49, 50,4005,

61,41, 44,45, 47

B*4002-4006,4008,4101-02,

4501,4901,5001,4402-5,4701

566

12B 44 192 247 B60, 61,4005,

41, 4801

B*4801,40011-4006 465

13B 45 272 218 B60, 61,47 B*40011-4004,4006-8,4701 627

15B 46 280 229 B60 B*40011,40012,4007 607

16B 47 243 215 B13 B*1301,1302,1303# 486

17B 48 197 127 B64, 65 B*1401,1402 389

18B 49 205 232 B65, 3904 B*1402,3904 187

19B 50 207 217 B39, 67 B*39011-3908,6701 507

20B 51 206 217 B67 B*67011,67012 548

21B 172 208,

435

217 B38 B*3801,3802 498/

508

22B 53 209 217 B38, 39,67 B*3801,3802,3901-8,67011,67012 612



23B 54 208 223 B5801, 77,

5104, 53

B*5801,5104,5301,1513 319

24B 56 194 213 B58 B*5801-3 374

25B 55 194 224 B57 B*5701,5702,5703 351

26B 166 243 438 B57, 63,13,1524 B*5701-03,1513,1516-17,1524 143

27B 57 187 214 B18 B*1801-02 458

28B 58 209 236 B55, 56,73,

3906

B*5501,5502,5601,5602,7301,

39061-2

422

1C 59 242 215 B54, 55,56,45,50 B*5401,5501,5502,5601,4501,5001 383

2C 60 203 238 B56 B*5601,5602 551

3C 61 395 236 B54 B*5401 421

4C 62 280 281,282 B27 B*2701,2709 149/150

5C 63 188 212 B37, 4406 B*3701,B*4406 606

6C 64 192 392 B37, 3902,3908 B*37,3902,3908 422

7C 65 192 228 B47 B*4701 414

8C 66 367 236 B73 B*7301 289

9C 67 203 220 B42 B*4201 594

10C 68 209 229 B4801, B8101 B*4801,8101 567

11C 69 194 225 B63 B*1516,1517 516

12C 70 240 241 B46 B*4601 459

13C 72 243 250 B62, 62V,75,

76,1521

B*1501-02,1504-08,1511-12,

1514-15,1519-21,1525,1526N, 1528

124

15C 73 192 214 B62, 72,76,

4802, 4003

B*1501,1503-07,1512,1514,

1519-20,1524-25,4802,4003

421

16C 156 271 223 B4802 B*4802 487

17C 74 209 214 B72, 72V,

71,4802,3907,

Cw0703

B*1503,1518,1523,1529,

4802,3907

486

18C 75 271 238 B4802,

71,70,72,72V

B*4802,1503,1509-10,1518,1523,1529 691

19C 76 189 238 B71,70 (NOT

B72), 1521,1523

B*1509-10,1518,1521,1523 562

20C 77 243 219, B76 B*1512,1514,1519 636-



244 637

21C 78 193 377 B62 Variants B*1508,1511,1515,1522,

A*68, 2501, 2601-5,3401,6601-2

553

22C 79 193 223 B35, 53,75,77,

5104,1521,4406

B*3501-04,3506-09,3511,

3512,5301,1502,

1513,5104,1521,4406

369

23C 80 188 237 B35, 18,78,1522 B*3501-13,18,78012, 1522 128

24C 81 195 213,

277

B35,53 B*3501-09,3511,5301 389/

340

25C 82 207 216 B78, 1509 B*7801-2,1509 400

26C 83 208 216 B51,52 B*5101-05,52011,52012 401

27C 84 193 216 B51, 78,1509 B*5101-05,7801-2,1509 451

28C 85 192 216 B52 B*52011,52012 440

Agarose Gel Electrophoresis

PCR products were electrophoresed in 1.2% agarose gels containing 0.5µg/ml

ethidium bromide after the addition of 5 µL of loading buffer consisting of 0.25%

Orange G, 30% v/v glycerol and 0.5X TBE buffer. Orange G is an excellent

electrophoresis marker as it migrates with the primer band on gel electrophoresis.

The gels were run for 68 minutes till the Orange G migrated 5 cm. The gels were

visualized using UV illumination and documented in a multi-imager system (Biorad).

A 100 bp ladder was run in all the gels as a standard for checking the sizes of the

different amplicons11.



Positive reactions for PM 03 (HLA A*02) in wells 2,3 and 12,13. 7th one is marker

Positive reactions for PM 25 (HLA A*02, 23,24,39,69) in wells 2,3,4,6,8,9,11,12,13. 7th is marker

Reactions for PM 12 and 21 (HLA A*11 and 33)



Reactions for PM 26 and 04 (HLA A*01, 02, 25,26, 30, 31, 32, 33) and A*03

Reactions for PM 155 (HLA B*8) 7th well is marker

Reactions shown for PM 69 (HLA B*63). 7th well is marker



OBSERVATIONS AND RESULTS

Observation of the data and analysis of the results are essential for a properly

conducted research. Totally 40 healthy individuals from different geographic area

and communities belonging to both the sex were selected for the study with informed

consent. Due care was taken to avoid sampling from related individuals and siblings

and unwilling and non-cooperative persons.

Blood samples were collected and DNA isolation was performed using standard

organic extraction methods. Molecular typing of the following genes were carried out

using the polymerase chain reaction (PCR), table no. 1

Table no.1 Genes under study

1. Dopamine Receptor D2 Taq 1 D DRD2 Taq 1 D

2. Dopamine Receptor D2 Taq 1 B DRD2 Taq 1 B

3. Dopamine Receptor D2 Taq 1 A DRD2 Taq 1 A

4. Dopamine Receptor D2 Serine 311 Cysteine DRD2 S311C

5. Dopamine Receptor D2 Histidine 313 H DRD2 His 313H

6. Dopamine Receptor D3 Serine 9 Glycine DRD3 S9G

7. 5-Hydroxy Tryptamine Receptor 1 B 5HTR 1B



10. Human Leukocyte Antigen B HLA B

11. Human Leukocyte Antigen A HLA A

Following are the observations of the analysis done. Out of the 40 individuals

selected the age varies from 25 to 45



Chart no.1 Distribution according to gender

70%

30%

MaleFemale

In this study maximum individuals were male (70%) and remaining 30% females

Graph 1 Distribution of Prakrithi according to gender

Gender and Prakrithi

6

10

2

8

23 3

0

6

00

2

4

6

8

10

12

Pitha Kapha Vatha Pitha Pitha Kapha Vatha Kapha

MaleFemale



Chart no.3 Distribution according to RELIGION

In this maximum individuals were Hindus (80%) Christians (15%), Muslims (5%)

Graph 2 Distribution of Prakrithi according to RELIGION

80%

5%

15%

Hindu

Muslim

Christian

Religion and Prakrithi

89

1

12

2

0

2

0 0 0

12

12

00

2

4

6

8

10

12

14

Pitha Kapha Vatha Pitha Pitha Kapha Vatha Kapha

HinduMuslimChristian



Table no.2, Graph 3 Distribution according to Prakrithi (Phenotype)

Frequency Percent

Vatha 0 0

Pitha 9 22.5

Kapha 13 32.5

Vatha Pitha 2 5.0

Pitha Kapha 14 35.0

Vatha Kapha 2 5.0

Sama 0 0

Total 40 100.0

Pitha Kapha Prakrithi is found more frequent 35% (14), Kapha Prakrithi 32.5% (13),

Pitha Prakrithi 22.5% (9), Vatha Pitha (2) and Vatha Kapha (2) 5% each. It was

observed that there were no Vatha Prakrithi phenotypes and Sama dosha Prakrithi

(combination of the three doshas) phenotypes.

9

13

2

14

2

0

2

4

6

8

10

12

14

16

Pitha Kapha VathaPitha

PithaKapha

VathaKapha



Table no.3, Graph 4 Genotype DRD2 Taq1D distribution in the population

Frequency Percent

D1/D1 7 17.5

D2/D1 20 50.0

D2/D2 13 32.5

Total 40 100.0

In this heterozygous genotype (D2/D1) individuals were found more (50%)

D 1 /D 1 D 2 /D 1 D 2 /D 2

7

2 0

1 3

0

2

4

6

8

1 0

1 2

1 4

1 6

1 8

2 0

T a q 1 D



Table no.4, Graph 5 Genotype DRD2 Taq1B distribution in the population

Frequency Percent

B1/B1 6 15.0

B2/B1 10 25.0

B2/B2 24 60.0

Total 40 100.0

In this dominant homozygous genotype (B2/B2) individuals were found frequent

(60%)

B1/B1 B2/B1 B2/B2

6

10

24

0

5

10

15

20

25

Taq 1 B



Table no.5, Graph 6 Genotype DRD2 Taq1A distribution in the population

Frequency Percent

A1/A1 9 22.5

A2/A1 13 32.5

A2/A2 18 45.0

Total 40 100.0

In this dominant homozygous genotype (A2/A2) individuals were found frequent

(45%)

A 1 /A 1 A 2 /A 1 A 2 /A 2

9

1 3

1 8

0

2

4

6

8

1 0

1 2

1 4

1 6

1 8

T a q 1 A



Table no.6, Chart 3 Genotype DRD2 S311C distribution in the population

Frequency Percent

G/G 32 80.0

G/C 8 20.0

Total 40 100.0

In this dominant homozygous genotype (G/G) individuals were found frequent (80%)

S311C

80%

20%

G/GG/C



Table no.7, Graph 7 Genotype DRD2 His 313H distribution in the population

Frequency Percent

T/T 15 37.5

C/T 19 47.5

C/C 6 15.0

Total 40 100.0

In this heterozygous genotype (C/T) individuals were found frequent (47.5%)

T /T C /T C /C

1 5

1 9

6

0

2

4

6

8

1 0

1 2

1 4

1 6

1 8

2 0

H is 3 1 3 H



Table no.8, Graph 8 Genotype DRD3 S9G distribution in the population

Frequency Percent

G/G 13 32.5

S/G 17 42.5

S/S 10 25.0

Total 40 100.0

In this heterozygous genotype (S/G) individuals were found frequent (42.5%)

G /G S /G S /S

1 3

1 7

1 0

0

2

4

6

8

1 0

1 2

1 4

1 6

1 8

S 9 G



Table no.9, Graph 9 Genotype 5 HTR 1B distribution in the population

Frequency Percent

G/G 19 47.5

C/G 16 40.0

C/C 5 12.5

Total 40 100.0

In this recessive homozygous genotype (G/G) individuals were found frequent

(47.5%)

5 HTR 1 B

19

16

5

0

2

4

6

8

10

12

14

16

18

20

G/G C/G C/C



Table no.10; Graph 10 Genotype Ser 1438 distribution in the population

Frequency Percent

G/G 10 25.0

A/G 23 57.5

A/A 7 17.5

Total 40 100.0

In this heterozygous genotype (A/G) individuals were found frequent (57.5%)

Ser 1438

10

23

7

0

5

10

15

20

25

G/G A/G A/A



Table no.11, Graph 11 Genotype Ser 102 distribution in the population

Frequency Percent

T/T 6 15.0

C/T 24 60.0

C/C 10 25.0

Total 40 100.0

In this heterozygous genotype (C/T) individuals were found frequent (60%)

Ser 102

6

24

10

0

5

10

15

20

25

30

T/T C/T C/C


www.ayurvedicmedicinalplants.com

124

Graph 12- % of Genotype distribution in the population

Table no.12, Graph 13 Correlation between Phenotype (Prakrithi type) and

HLA B alleles

Prakrithi HLA B alleles

7 8 13 15 35 37 38 39 40 44 51 52 55 56 57 58

Pitha 1 1 2 1 3 2 4 1 1 2

Kapha 3 1 3 2 1 7 2 2 2 3

Vatha Pitha 1 1 2

Pitha Kapha 3 1 3 1 2 6 5 1 1 2 1 2

Vatha Kapha 1 1 1 1

It is observed that there is a genetic basis for the three major constitutions

(Prakrithi) described in Ayurveda. The Prakrithi classification is based on

% of genotype with various Prakrithis

0.00

20.00

40.00

60.00

80.00

100.00

120.00D

2/D

1

D1/

D1

B2/

B1

B1/

B1

A2/

A1

A1/

A1

G/G C/C T/T

C/C

G/G S/S

G/G C/C

G/G A/A

C/C T/T

PithaKaphaPitha KaphaVatha PithaVatha Kapha



125

differences in physical, physiological, and psychological characteristics

and is independent of racial, ethnic, or geographical considerations. It

may provide an appropriate means of classifying phenotypes to be

considered collectively for genotyping. Of the evaluated 40 subjects both

for their Prakrithi and human leukocyte antigen (HLA) B types, we

observed that a reasonable correlation between HLA B type and Prakrithi

type. HLA B *07, *08, *13, *15, *35, *37, *38, *39, *40, *44, *51, *52, *55,

*56, *57, and *58 were observed with varying frequencies in different

Prakrithi populations. The complete absence of the HLA B*07 allele in

the Vatha Pitha and Vatha Kapha type, and of HLA B*08 and B*38 in all

the four Prakrithis except Kapha, HLA B*13 in Pitha, Kapha and Vatha

Pitha Prakrithis, HLA B*15 in the Kapha, Vatha Pitha and Vatha Kapha

Prakrithis. Interestingly, absence of HLA B*35 alleles were found only in

Vatha Pitha Prakrithi. HLA B*37 were completely absent in all the dual

constitutions of Vatha Pitha, Pitha Kapha and Vatha Kapha Prakrithis.

HLA B*39 was absent in Pitha, Kapha, Vatha Kapha. HLA B*40 and

B*44 were absent in Vatha Pitha and Vatha Kapha. HLA B*51 were

present in all the Prakrithis. HLA B*52 was absent only in Vatha Kapha.

HLA B*55 was absent in Kapha, Vatha Pitha, and Vatha Kapha. HLA

B*56 was present only in Pitha Kapha and B*57 was only in Vatha

Kapha. HLA B*58 was absent in Vatha Pitha and Vatha Kapha. A correct

perusal of table no 12 of correlation between Phenotype (Prakrithi type)

and Genotype of HLA B attest that, of the 16 alleles only B*39, B*51 and

B*52 were present in Vatha Pitha Prakrithi and B*13, B*35, B*51, B*57

were only present in Vatha Kapha Prakrithi. In Pitha Kapha Prakrithi all



126

the 12 alleles were present except B*08, B*37, B*38, and B*57. Among

the 16 alleles studied, the allele frequency of HLA B*40 was seen

highest as 16 and its distribution was highest in Kapha Prakrithi

population and Pitha Kapha Prakrithi population. The lowest allelic

frequency of HLA B was observed in B*08 & B*38 (in Kapha) B*56 (in

Pitha Kapha), and B*57 (in Vatha Kapha).

Table no. 13, Correlation between Phenotype (Prakrithi type) and HLA A

alleles

Prakrithi HLA A alleles

1 2 3 11 24 26 31 32 33 68

Pitha 1 3 1 1 4 2 3 3

Kapha 1 6 3 3 2 2 1 5 3

HLA B & Prakrithi

1 1

2

1

3

2

4

1 1

2

3

1

3

2

1

7

2 2 2

3

1 1

2

3

1

3

1

2

6

5

1 1

2

1

2

1 1 1 1

0

1

2

3

4

5

6

7

8

7 8 13 15 35 37 38 39 40 44 51 52 55 56 57 58

PithaKaphaVatha PithaPitha KaphaVatha Kapha



127

Vatha Pitha 1 1 1 1

Pitha Kapha 1 2 2 3 7 4 3 2 4

Vatha Kapha 1 1 1 1

Of the evaluated 40 subjects both for their Prakrithi and human leukocyte

antigen (HLA) A types, we observed that a reasonable correlation between

HLA A type and Prakrithi type. HLA A *01, *02, *03, *24, *26, *31, *32, *33,

*68 were observed with varying frequencies in different Prakrithi populations.

The presence of HLA A*01and HLA A*24 alleles were observed in all the five

Prakrithi populations. Complete absence of the HLA A*02 and HLA A*03

allele in the Vatha Pitha and Vatha Kapha type, and of A*11 in all the four

Prakrithis except Vatha Kapha, HLA A*26 in Kapha and Vatha Kapha

Prakrithis, HLA A*31 in the Vatha Pitha and Pitha Kapha Prakrithis. HLA A*32

absent in Pitha and Vatha Pitha where as A*33 was absent in Vatha Pitha and

Vatha Kapha. HLA A*68 was absent in Pitha, Vatha Pitha, and Vatha Kapha.

A correct perusal of table no….. of correlation between Phenotype (Prakrithi

type) and Genotype of HLA A attest that, of the 10 alleles only A*01, A*11 and

A*24 and A*26 were present in Vatha Pitha Prakrithi and A*01, A*24, A*31,

A*32 were present in Vatha Kapha Prakrithi. Among the 10 alleles studied,

the allele frequency of HLA A*24 was seen highest as 15 and its distribution

was highest in Pitha Kapha Prakrithi population and Pitha Prakrithi population.

The lowest allelic frequency of HLA A was observed in A*01 and its

distribution were equal in all the 5 Prakrithis under study.



128

HLA A & Prakrithi

1

3

1 1

4

2

3 3

1

6

3 3

2 2

1

5

3

1 1 1 11

2 2

3

7

4

3

2

4

1 1 1 1

0

1

2

3

4

5

6

7

8

1 2 3 11 24 26 31 32 33 68

PithaKaphaVatha PithaPitha KaphaVatha Kapha

Graph 14

Table no.14 Correlation between Phenotypes (Prakrithi types) and various alleles

Prakrithi

DRD2 Taq1 D1

DRD2 Taq 1

D2

DRD2 Taq1 B1

DRD2 Taq1 B2

DRD2 Taq1 A1

DRD2 Taq1 A2

DRD2 S311C

C

DRD2 S311C

G

DRD2 His313H

C

DRD2 His313H

T

DRD3S9G

S

DRD3 S9G

G 5HTR1B

C 5HTR1B

G Ser1438

A Ser1438

G Ser102

C Ser102

T

Kapha 13 13 5 21 10 16 3 23 8 18 10 16 11 15 12 14 14 12

Pitha 8 10 8 10 8 10 1 17 5 13 11 7 5 13 9 9 9 9

Pitha Kapha 11 17 8 20 12 16 3 25 14 14 14 14 9 19 12 16 17 11

Vatha Kapha 1 3 1 3 1 3 0 4 2 2 0 4 1 3 2 2 2 2

Vatha Pitha 1 3 0 4 0 4 1 3 2 2 2 2 0 4 2 2 2 2



129

Graph.15 Correlation between Phenotypes (Prakrithi types) and various

alleles

% of allele frequency with various Prakrithis

0.00

20.00

40.00

60.00

80.00

100.00

120.00

D1 D2 B1 B2 A1 A2 G C T C S G G C G A T C

PithaKaphaPitha KaphaVatha PithaVatha Kapha



130

Table no.15 Allele frequencies (%) in various Prakrithis

Test Allele Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha

Taq 1 D1 44.44 50.00 39.29 25.00 25.00

D2 55.56 50.00 60.71 75.00 75.00

Taq 1 B1 44.44 19.23 28.57 0.00 25.00

B2 55.56 80.77 71.43 100.00 75.00

Taq 1 A1 44.44 38.46 42.86 0.00 25.00

A2 55.56 61.54 57.14 100.00 75.00

DRD2 S311C G 94.44 88.46 89.29 75.00 100.00

C 5.56 11.54 10.71 25.00 0.00

DRD HIS313 H T 72.22 69.23 50.00 50.00 50.00

C 27.78 30.77 50.00 50.00 50.00

DRD3 S9G S 61.11 38.46 50.00 50.00 0.00

G 38.89 61.54 50.00 50.00 100.00

5HTR 1B G 72.22 57.69 67.86 100.00 75.00

C 27.78 42.31 32.14 0.00 25.00

SER 1438 G 50.00 53.85 57.14 50.00 50.00

A 50.00 46.15 42.86 50.00 50.00

SER 102 T 50.00 46.15 39.29 50.00 50.00

C 50.00 53.85 60.71 50.00 50.00



131

The response to Prakrithi is presumed to differ with the individual; this

variation might be due to genetic differences. Nine-receptor polymorphisms

were screened in four receptor genes. These receptor genes include

Dopamine receptor D2 and D3 genes and two Serotonin receptor gene e.g.

HTR1B and HTR2A.

A total of 40 healthy individuals participated in this study. The subjects

underwent Prakrithi analysis. We observe an increased incidence of Kapha

Prakrithi individuals. Among mixed characters we observe an increased

incidence of Pitha Kapha Prakrithi. In our sample 70% of the individuals were

males. When we compared the Prakrithis in males and females we observe

almost similar trend of Prakrithi distribution, however, males had higher

frequency of Kapha Prakrithi in compared their female counterpart. The entire

population was further classified into their religion. 80% of the individuals were

Hindus. We do not observe any significant shift in the trend of Prakrithis

based on religion; however, Hindus display lower tendencies of Vatha Pitha

Prakrithi.

DRD2 TaqI D, Taq 1A, Taq 1B, S311C, His313H, DRD3 S9G, 5HTR 1B,

5HTR 2A (Ser 1438 and Ser 102) restriction enzyme fragment length

polymorphism (RFLP) study examined genomic DNA extracted from blood

samples.

The proportions in Pitha, Kapha, Pitha Kapha, Vatha Pitha, and Vatha Kapha

of both the alleles were observed as shown in the table no.15

The RFLP study showed that the distribution of the DRD2*Taq 1D genotype

and allele frequencies differed significantly between the Prakrithis, and that



132

Vatha Pitha and Vatha Kapha Prakrithis had the highest and equal (75%)

DRD2*Taq 1D2 allele frequency than other Prakrithis. Equal distribution of

frequency of D2 and D1 alleles was observed in Kapha Prakrithi.

The RFLP study showed that the distribution of the DRD2*Taq 1B genotype


Vatha Pitha Prakrithi had the highest (100%) DRD2*Taq 1B2 allele frequency

than other Prakrithis.

The RFLP study showed that the distribution of the DRD2*Taq 1A genotype


Vatha Pitha Prakrithi had the highest (100%) DRD2*Taq 1A2 allele frequency

than other Prakrithis.

The RFLP study showed that the distribution of the DRD2*S311C genotype


Vatha Kapha Prakrithi had the highest (100%) DRD2*S311C G allele

frequency, in Pitha, in Pitha Kapha, and in Kapha Prakrithis it was 94.44%,

89.29%, 88.46% respectively.

The RFLP study showed that the distribution of the DRD2*His 313H genotype


Pitha Prakrithi had the highest (72.22%) DRD2*His 313H T allele frequency,

and in all the three dual - Pitha Kapha, Vatha Pitha and Vatha Kapha -

Prakrithis it was equal for both the alleles (T and C) and were observed as

50%.

The RFLP study showed that the distribution of the DRD3*S9G genotype and

allele frequencies differed significantly between the Prakrithis, and that Vatha

Kapha Prakrithi had the highest (100%) DRD3*S9G G allele frequency, and in



133

the two dual - Pitha Kapha, Vatha Pitha - Prakrithis it was equal for both the

alleles (S and G) and were observed as 50%.

The RFLP study showed that the distribution of the 5HTR 1B genotype and

allele frequencies differed significantly between the Prakrithis, and that Vatha

Pitha Prakrithi had the highest (100%) 5HTR 1B G allele frequency than other

Prakrithis.

The RFLP study showed that the distribution of the 5HTR 2A (Ser 1348)

genotype and allele frequencies differed significantly between the Prakrithis,

and that Pitha Kapha Prakrithi had the highest (57.14%) 5HTR 2A G allele

frequency than other Prakrithis and in the two dual - Vatha Pitha and Vatha

Kapha – Prakrithis and in Pitha Prakrithi, it was equal for both the alleles (G

and A) and were observed as 50%.

The RFLP study showed that the distribution of the 5HTR 2A (Ser 102)

genotype and allele frequencies differed significantly between the Prakrithis,

and that Pitha Kapha Prakrithi had the highest (60.71%) 5HTR 2A C allele

frequency than other Prakrithis and in the two dual - Vatha Pitha and Vatha

Kapha – Prakrithis and in Pitha Prakrithi, it was equal for both the alleles (C

and T) and were observed as 50%.

Out of nine SNPs selected we observed increased frequency of one allele in 5

polymorphisms screened in all the Prakrithis while in remaining four

polymorphisms more or less similar pattern of distribution of alleles in all

Prakrithis was observed. When individual Prakrithis and individuals

polymorphism were assessed we observe a different pattern of distribution,

however, HTR2A did not display any significant differences in frequency of

distribution of alleles among different Prakrithis.



134

When individuals polymorphism were compared in different Prakrithis we

observe increased DRD2 Taq1 D2 allele in all mixed Prakrithis, while DRD2

Taq1 B2, DRD2 Taq1 A2, DRD2 311 G and HTR1B G alleles were observed

to be increased in all Prakrithi in compared to their alternate allele.

In Pitha Prakrithi significant differences were observed in DRD2 S311C à G

allele, DRD2 313 Cà T allele and HTR1B CàG allele in compared to its

counterpart allele.

In Kapha Prakrithi significant differences were observed in DRD2 Taq1 B1à

B2, DRD2 Taq A2 à A1, DRD2 311 CàG, DRD2 313 Cà T allele.

ANALYSIS

On analysis of the results of the study of the various dopaminergic receptor

genes with respect to various Prakrithi populations a significant increase of

the allele frequencies in the Prakrithis having an association of Vatha is found.

The allele frequency of Taq 1D2 in Vatha Pitha and Vatha Kapha Prakrithis

had the highest and equal (75%). Similarly, the allele frequency of other

dopaminergic receptor genes Taq 1B2, Taq 1A2, S311C G, and His 313H (T

and C) and DRD3 S9G also shows an increasing tendency in all the Prakrithi

populations having the association of Vatha, when compared to other

Prakrithis that are having no influence of Vatha. Hence it is understood that

association of Vatha increases the allele frequency in dopaminergic receptor

genes under my study.

Similarly, serotoninergic receptor gene 5HTR 1B with respect to various

Prakrithi populations shows a significant increase of the allele frequencies in

the Prakrithis having an association of Vatha. But in the case of serotoninergic



135

receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows a significant increase

of the allele frequency in the Prakrithi having an association of Kapha.

DRD2*B2 allele and DRD2*A2 and 5HTR 1B G allele are influenced by Vatha.

DRD2 S311C and DRD3 S9G are influenced by Kapha.

Dopamine is a chemical naturally produced in the body mainly in nervous

tissue and adrenal glands. In the brain, dopamine functions as a

neurotransmitter, activating dopamine receptors. Dopamine is also a

neurohormone released by the hypothalamus. Dopamine is critical to the way

the brain controls our movements and is a crucial part of the basal ganglia

motor loop. All the functions atributed to dopamine receptors can be included

in the functions of Vatha in Ayurveda, and the allele frequency of

dopaminergic receptor genes Taq 1B2, Taq 1A2, S311C G, and His 313H (T

and C) and DRD3 S9G shows an increasing tendency in all the Prakrithi

populations having the association of Vatha.

We have also noticed that Serotoninergic 5-HT1B receptors are expressed

throughout the mammalian central nervous system and, only 1% of serotonin

in the human body is found in the CNS and also the receptor gene 5HTR 1B

shows a significant increase of the allele frequencies in the Prakrithis having

an association of Vatha. But surprisingly 99% of serotonin is found in other

tissues, primarily plasma, the gastro-intestinal tract, and immune tissues and

serotoninergic receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows a

significant increase of the allele frequency in the Prakrithis having an

association of Kapha.



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Graph 16 Allele frequency of

Graph 17 Genotypic frequency of Taq 1D in various Prakrithis

Dopamine Receptor D2 Taq 1D

44.44

53.8550.00 50.00 50.00

33.33

23.08

35.71

50.00 50.00

22.22 23.08

14.29

0.00 0.000.00

10.00

20.00

30.00

40.00

50.00

60.00

Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha

Heterozygous

D2/D2

D1/D1

D2/D1

Dopamine Receptor D2 Taq 1 D

44.4450.00

39.29

25.00 25.00

55.5650.00

60.71

75.00 75.00

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00


D1

D2



137


Graph 19 Genotypic frequency of Taq 1B in various Prakrithis

Dopamine Receptor D2 Taq 1 B

44.44

19.2328.57

0.00

25.00

55.56

80.7771.43

100.00

75.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00


B1

B2

Dopamine Recepto D2 Taq 1B

44.44

23.0814.29

0.00

50.00

33.33

69.23 64.29

100.00

50.00

22.22

7.6921.43

0.00 0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


Heterozygous

B2/B2

B2/B1B1/B1



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Graph 21 Genotypic frequency of Taq 1A in various Prakrithis

Dopamine Receptor D2 Taq 1 A

44.4438.46 42.86

0.00

25.00

55.5661.54 57.14

100.00

75.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00


A1

A2

Dopamine Receptor D2 Taq 1A

22.22

46.15

28.57

0.00

50.0044.44

38.4642.86

100.00

50.00

33.33

15.38

28.57

0.00 0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


Heterozygous

A2/A2

A2/A1

A1/A1



139


Graph 23 Genotypic frequency of S311C in various Prakrithis

Dopamine Receptor D2 Serine 311 Cysteine

94.44 88.46 89.2975.00

100.00

5.56 11.54 10.7125.00

0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


G

C

Dopamine Receptor D2 S311C

88.89

76.92 78.57

50.00

100.00

11.11

23.08 21.43

50.00

0.000.00 0.00 0.00 0.00 0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


Heterozygous

G/G

G/C

C/C



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Graph 25 Genotypic frequency of His 313H in various Prakrithis

Dopamine Receptor D2 Histidine 313 H

72.22 69.23

50.00 50.00 50.00

27.78 30.77

50.00 50.00 50.00

0.0010.0020.0030.0040.0050.0060.0070.0080.00


C

T

Dopamine Receptor D2 His 313H

66.67

46.15

14.29

0.00

50.00

11.11

46.15

71.43

100.00

0.00

22.22

7.6914.29

0.00

50.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00


HeterozygousT/C

T/T

C/C



141


Graph 27 Genotypic frequency of DRD3 S9G in various Prakrithis

Dopamine Receptor D3 S9G

61.11

38.4650.00 50.00

0.00

38.89

61.5450.00 50.00

100.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00


S

G

Dopamine Receptor D3 S9G

11.11

38.46 35.71

0.00

100.00

55.5646.15

28.57

100.00

0.00

33.33

15.38

35.71

0.00 0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


Heterozygous

S/S

G/G

S/G



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Graph 29 Genotypic frequency of 5-HTR 1B in various Prakrithis

5-Hydroxy Tryptamine Receptor 1 B

72.22

57.6967.86

100.00

75.00

27.78

42.3132.14

0.00

25.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00


G

C

5 Hydroxy Tryptamine Receptor 1B

44.44

30.77

57.14

100.00

50.0055.56 53.85

21.43

0.00

50.00

0.00

15.3821.43

0.00 0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


Heterozygous

C/G

G/G

C/C



143


Graph 31 Genotypic frequency of Ser 1438 in various Prakrithis

Serotonin 1438

50.0053.85

57.14

50.00 50.0050.0046.15

42.86

50.00 50.00

0.00

10.00

20.00

30.00

40.00

50.00

60.00


G

A

Serotonin 1438

22.2230.77 28.57

0.00 0.00

55.5646.15

57.14

100.00 100.00

22.22 23.0814.29

0.00 0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


Heterozygous

A/G

G/G

A/A



144


Graph 33 Genotypic frequency of Ser 102 in various Prakrithis

Serotonin 102

50.0046.15

39.29

50.00 50.0050.0053.85

60.71

50.00 50.00

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00


C

T

Serotonin 102

22.2230.77 28.57

0.00 0.00

55.5646.15

64.29

100.00 100.00

22.22 23.08

7.140.00 0.000.00

20.00

40.00

60.00

80.00

100.00

120.00


Heterozygous

T/C

T/T

C/C



145

DISCUSSION

Before entering to the discussion proper, I think it is proper to discuss on

certain aspects of Prakrithi, such as the importance of study of Prakrithi, the

views of various Acharyas regarding the influence of Vatha, Pitha and Kapha

on various physical, physiological and pshycological functions of human

organism such as digestion, metabolism, sense perception, thought process

and content of thinking.

Understanding how tridosha work is a big challenge. To reduce the complexity

of the problem Ayurvedic Acharyas have broken it into smaller pieces and

have discussed it in various levels and analyzed. They have discussed the

working of tridosha at molecular level by analyzing the zygote and explained

the role of Panch bhoothas and there by tridoshas in the development of

zygote to a human organism. They have also discussed how all the particles

of Pancha bhootha work together to give a cell of man, animal and plant its

special properties. How do the 16 types of bhoothas derived from four

different sources constitute the zygote, and how they function as tridoshas,

controlling its structural and functional activities are well discussed. Acharyas

have also discussed the role of tridoshas in sensing information from

perception of external world to make decisions and execute movements, in

physiological and pathological state. A review of the phenomina of visual

sense perception on the basis of Pancha bhootha / Tridosha theory will

enable to understand the effect of Prakrithi on sense perception and the

characteristics of sensory apparatus specific to the individuals of different

Prakrithis and thereby design the treatment to various diseases of the sensory

apparatus (Kaarana Dourbalya) and also to adopt personalised preventive



146

measures for better visual, olfactory , auditory, tactile and gestatory

sensations essential for a more pleasurable and healthy life.

Among the Pancha bhoothas, Thejus has a dominant role in perception of

visual sense. Theja Paramanus (Photons?) are made up of 50% Thejus and

the other 50% by the other Bhoothas in equal percentage (12.5%). The

nature of light according to Indian Metaphysics is in the form of particles,

which radiate retlinially in all directions at very high speed. When light

particles pass through a transparent medium, it penetrate through the inter-

atomic spaces with vibrations (Parispandana) of the nature of deflection

(Thiryaggamana) or reflections (Vibhaga)1. Dalhana, while explaining the

word ‘Vivarakrithi’ depicted by Susrutha to denote the nature of the outermost

layer of the eye (Cornea) explains that Cornea is highly transparent

(Athyanthaachatwa) and have inter atomic spaces (Vivarantharathwa) and

hence it does not obstruct the entry of Theja paramanus and thereby enables

to perceive the properties of objects that can be perceived by alochaka thejus

(Energy of ocular apparatus)2. The state of energy in ocular apparatus is of

apanchikritha form (100% thejus) and hence our receptors of the ocular

apparatus (or that of any electronic equipment?) is insensitive to such forms

of energy (Atheendriya) and their limitations restrict our knowledge in the field.

Acharya Nimi also considers that visual sense perception is brought about by

the interaction of two types of energies (Theja samyoga) – that emitted from

luminous objects and the Alochakathejus (energy present in the ocular

apparatus)3. The interaction of these two types of energies is designated as

Indriya-Indriyaardha Sannikarsha. By the ‘hitting’ and Samyoga, light energy

having high speed may alter the nature of energy causing visual impulse



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through Netranaadi (Optic Nerve)4. In fact, the pattern of inheritance of

Mahabhoothas derived from four different sources at the time of fertilisation

also differs and is reflected to the Prakrithi of an Individual. The percentage of

Panchabhoothas that make up various parts of the eye also varies. The

Suklamandala (Schlera and bulbar conjunctiva) have a dominance of

Jalabhootha derived from father whereas the Krishna Mandala (Cornea & Iris)

have a dominance of Vayu Mahabhootha from mother. The Mamsamandala

(mascular parts) have a dominance of Prithvi Mahabhootha derived from

mother and the Drishtimandala (Pupil, Lens and retina) have a dominance of

Agni and Jala Mahabhoothas derived equally from father and mother.

However the Nethra naadi (Optic Nerve?) and the nerve centre have a

dominance of Agni Mahabhootha derived mainly from Athmajabhaava.

Hence, any minute change in the Prakrithi may reflect in the eye of the

offspring as tabulated in Table No.2.

A Thejaparamanu (Photon) having a structure of 50% Agni and 12.5% each of

other Bhoothas produces various effects on the energy system of eye in tune

with the Prakrithi of an Individual. Or, the sensitivity of the receptors of

various Individuals have minute varations in accordance with their Prakrithi.

However, a more sharp vision is for Pitha Prakrithi Individuals and the mental

imagery of different Prakrithis at the time of dreams and hallucinations may

also vary according to the genetic make-up examples of which are provided

as itm 23 of Table No2.

While narrating the process of segregation of Prakrithi, Dalhana clearly

mentions about ‘Drishtyaarmbhaka bijabhaga’ or gene that determine the

development of lens/retina. The action of vitiated dosha on



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drishtiaarambhaka bijabhaga can produce unfavourable and potent changes

in bijabhagas (Mutations) the effect of which is manifested as Jaathyandhatha

or congenital blindness5. But if the changes occurring in the genes are not

sufficient to produce any disturbance to the general functions of the eye of the

offspring, the changes are accepted as of in normal limits but its effects will be

reflected in the development of various parts of the ocular apparatus in tune

with the nature of doshas involved. The varying characteristics of eye of

individual belonging to different Prakrithi as enumerated in the item 14 of

Table No.2. Pitha Prakrithi Individuals are more prone to Pitha predominant

eye diseases. But it does not mean that only the Pitha predominant diseases

will affect a Pitha Prakrithi Individual. Proneness to diseases of various

Individuals belonging to other Prakrithis may also be considered on the same

lines. But a correct assessment of the Prakrithi of Individuals by analysing

the Phenotypic characteristics will enable to foresight the chances for the

occurrence of eye diseases, its nature and diseases of other systems as well,

so that we can take preventive measures by adopting specific life styles, food

habits and medicines according to the Prakrithi of Individual and thereby

technologies of personalised preventive medicine of Ayurveda may be

brought in to practice.

Acharyas have also discussed the role of tridosha/ Pancha bhoothas in

producing integrated behaviors, process of dream and higher levels of human

mental activity such as self-awareness, mental imagery, and language.

Mental activity influences tridosha activity and that in turn influence the brain

activity, activities of the various systems of the body and even the gene

activity. The role of tridoshas, in the formation, association and dissociation of



149

molecules or paramanus for the formation of zygote, development of the body

and process of death are discussed in detail in our science of life. After

analyzing the functions of tridoshas at molecular, cellular, systems, behavioral

and cognitive levels, the ancient sages have developed a holistic approach in

understanding the state of human organism in health and disease and came

to a conclusion that, these two states are two sides of the same coin of

tridosha and postulated the concept that, the living and non living and life and

death are nothing but two sides of the same coin of Pancha bhootha.

Study of Prakrithi and assessment of Prakrithi of an individual is of supreme

importance in Ayurveda. It enables to understand the nature of various

categories of diseases classified on the basis of different criteria. It also helps

to predict whether a factor responsible for aggravation of Dosha is potent to

produce a disease in a person/ population assessed of having a particular

Prakrithi and to understand the state of Agni (factors or enzymes responsible

for digestion and metabolism) and maintenance of their normalcy. Without

assessing Prakrithi it is not possible to maintain health of various groups of

individuals designated as a particular Prakrithi because the preventive

measures to be adapted to various Prakrithi’s differ.

Diseases are classified into two groups, each on the basis of five different

criteria – Prognosis (curable/ incurable), Intensity (mild/ severe), Location

(mental/ physical), Nature of causative factors (endogenous/ exogenous) and

Site of origin (amasaya/ pakwasaya)6.

One substance may vitiate many Doshas, e.g. Substance having sour, saline,

and pungent taste vitiates Pitha; but those of sour taste vitiate Pitha as well as

Kapha, those of saline taste Kapha as well as Pitha and those of pungent



150

taste vitiate Pitha as well as Vatha. Similarly, spring season, which normally

aggravates Kapha, also aggravates Pitha and Vatha because of its ‘aadaana’

nature (property of absorbing water from earth). Pitha accumulated in the

rainy season gets aggravated during autumn, but there is simultaneous

aggravation of Kapha also. Similarly in summer, due to ununctuousness,

Vatha gets aggravated in view of the ununctuousness of the season and

simultaneously there is slight accumulation of Pitha due to seasonal heat. To

understand the effect of food and climate having different characteristics on

the metabolic activities of an individual, a correct knowledge of Prakrithi is of

high importance.

The metabolic fire is the most important factor that determines the state of

body. Increase or decrease of it may produce disease and its state of

equilibrium maintains health. In short, if the anabolism and catabolism are in a

balanced state the body will be in a state of health. But the state of metabolic

fire (Agni) defers according to the Prakrithi of the individual – that can be

catagorised into intense (Theekshna), mild (Manda), regular (Sama) and

irregular (Vishama). In Sama Prakrithi individuals, Agni is regular. In Vatha

Prakrithi individuals, Agni becomes irregular as the site of Agni is subdued by

Vatha. Similarly, in Pitha Prakrithi individuals, Agni is intense and in Kapha

Prakrithi individuals, it becomes mild. These varying states of Agni in

individuals having different Prakrithi denotes that digestion, absorption,

assimilation and other biological activities varies in different individuals by the

influence of tridoshas and it affects the body’s response to treatment, food or

drug. Intense metabolic fire tolerates all sorts of improper food while the mild

one got a contrary character. Improper diet easily affects the regular



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metabolic fire but otherwise remain normal, the irregular fire got the character

contrary to that of regular fire7.

Persons of eka Dosha/ DWI Dosha Prakrithi is considered as sadaathuras, as

they are more prown to disease when they come in contact with

corresponding factors that aggravate the Dosha which have dominance in

their genetic makeup. Depending upon the nature of Doshas involved, it is

necessary to adopt food habits and life styles as would be in contradiction

with the predominating Dosha, till there is normalcy of Agni. It is only after

normalcy is attained, balanced regimens should be adopted. If an individual

having a particular Prakrithi resorts to such thing as are aggravators of the

predominating Dosha in his body, the corresponding Dosha is aggravated and

afflicts the individual by manifest of diseases resulting in impairment of

strength, complexion, happiness and longevity. In short, in a Vatha Prakrithi

individual, Vatha gets immediately aggravated when Vatha aggravating things

are used. But if the same individual takes things that aggravate Pitha or

Kapha, then Pitha and Kapha do not get so vitiated to manifest a disease as it

happens in the case of Vatha.

In this connection, it is noted that certain Acharyas are of opinion, that, except

Sama Prakrithi, all other Prakrithis are to be considered as pathological, and

Charaka considers Prakrithi (normal) as Vikrithi (abnormal) and use the term

Vathala, Pithala and Sleshmala instead of Vatha Prakrithi, Pitha Prakrithi and

Kapha Prakrithi. For maintenance of positive health, adopting different

measures to the above group of individuals are essential. In the case of Sama

Prakrithi individuals who are having superior functioning of the dhathus,

measures balanced in all aspects are to be adopted, whereas in the case of



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eka Dosha Prakrithi, measures contrary to the properties of Dosha, after

considering the predominance of respective Dosha, are beneficial till Agni

becomes regular. There after balanced measure should be applied. The same

is applicable in the case of psychophysical activities and treatment regimen.

Food habits and life styles to be adopted by individuals/ population having a

particular Prakrithi can be summarized as follows:

For Vatha Prakrithi individual

1. Proper administration of oils and fomentations

2. Food having mild laxative effect prepared by addition of fat, hot

things and substances having sweet, sour and saline taste.

3. Massage, bandage, kneading, affusion, bath (hot)

4. Use of wine and asavaas (fermented drinks)

5. Fats from different sources mixed with herbs/ food materials having

digestive, stimulant, carminative, Vatha alleviating and laxative

properties.

6. Practice of Vasthi along with its regimens according to season.

For Pitha Prakrithi individuals

1. External and internal use of ghee

2. Purgatives, food and herbs having sweet, bitter and astringent taste

and cooling property.

3. Use of mild, fragrant, cooling and cordial perfumeries.

4. Use of pearls, jewels and garlands having cooling effect

5. Frequent sprinkling of cold water with cold air containing herbal

perfumeries.



153

6. Hearing of mild, sweet, agreeable songs and music, information

regarding prosperity and keeping company with friends and

agreeable ladies wearing cooling garments and garlands.

7. Residence in built in cold places in mountains and riverbanks,

which is cooled by moon rays, exposed to breezes from all sides,

with beautiful gardens and having pleasing cold and fragrant wind.

8. Use of perfumeries made of various types of lotus, rose, etc

For Kapha Prakrithi individual

1. Foods mostly ununctuous with pungent, bitter and astringent taste.

2. Running, jumping, swimming, whirling, keeping awake late at night,

wrestling, sexual intercourse, exercise, oil massage and bath.

3. Intake of strong alcohols preserved for long time, use of medicated

smoking

4. Use of warm apparels

5. Proper use of slimming therapies and use of strong and hot

elimination processes when required, and avoid lifestyles that

provokes Kapha.

Practice of drugs, foods and life styles without considering the Prakrithi of

individuals will not enable to cure or prevent disease. Such practices if

adopted to cure a disease will give rise to a new disease and may invite

adverse effect. While discussing about the importance of the study of

Prakrithi, Bhadantha Nagarjuna, in his great work, Rasa vaisheshika soothra

states ‘Praadurbhavedanyo va’ – or may develop another disease - (verse 89).

Such treatments are considered as improper. Curing of disease by treatment

without considering the Prakrithi of the individual is considered as accidental.



154

He further discusses that, focusing on the disease under treatment and

ignoring the complications associated, when treated without consideration of

Prakrithi, cannot be accepted, as complications may sometimes endanger the

very life of the patient8. Some are of opinion that, during treatment only either

of the metabolic fire, immunological status or the life style should be

considered. Bhadantha Nagarjuna is of opinion that, focus is to be given on

any factor only after considering the various stages of the disease and the

condition of the patient.

We have already seen that, any change in the Prakrithi of an individual, which

can be assessed by interpreting the changes in the phenotypic

characteristics, is fetal. Change in Prakrithi is to be understood as rishta

(symptoms of imminent death). Rasa vaisheshika soothra depicts rishta as

‘trika thraya dwandwa dikkaala vikriya’9.

Here the three triads are - Guna trika, Dosha trika and Karana trika 1. Guna

trika - Satwa, Raja, Thama 2. Dosha trika - Vatha, Pitha, Kapha 3. Karana

trika - Body, Mind and Word; Dwandwa - twin factors like heaviness &

lightness, coldness & hotness, smoothness & roughness, softness &

hardness, wetness & dryness, mobility & stiffness, bluntness & sharpness,

mild & intense along with Dik and Kaala (orientation of place and time) are the

factors to be considered to assess weather the Prakrithi of an individual is

maintained unchanged or changes have initiated indicating imminent death

(rishta)10.

A person having Satwika nature, if shows the nature of Rajasa or Thamasa or

vise versa is to be understood as initiation of changes in Prakrithi leading to

death. Satwa, Rajas And Thamas are the three forces of mind and are



155

responsible for all mental activities. They are in a state of conflict. According

to the dominance of one of them, the individual is classified into Satwika,

Rajasa And Thamasa. The characteristics of Satwika individuals are merciful,

sharing, endurant, truthful, proper in words and deeds, god fearing, self

knowledge, intelligent, retentive, recollective, resolute and those with selfless

service. Rajasa individuals are unhappy, wandering, coward, haughty, lying,

cruel, cheating, self-respectful, excited, sensual and quick tempered.

Thamasa types are gloomy, atheistic, unrighteous, unintelligent, spiritually

ignorant, evil minded, indolent and drowsy. Infact the characteristics of all the

three type are seen in one individual and on the basis of dominance the

mental makeup of the individual is designated as Satwika, Rajasa, or

Thamasa or combinations of either two or three of them.

Tridosha vaikrithi is the variation in the Prakrithi of an individual and is

assessed by alternations in the earlier tabulated phenotypic characteristics.

Karana trika vaikrithi includes deterioration of mental faculty such as

alternation of consciousness and disorientation of people/ day/ date etc is

included in mano vikrithi. Derangement in physical properties such as gaining

of weight by starvation, emaciation without any cause etc. is Sareera vikriya.

Vak vikrithi (irrelevant words) pointing disorders of thought process.

Dwandwa vikriya (change in twin factors) denotes alternations in sense

perception, such as hot as cold, soft as hard, sweet as astringent, microscopic

as macroscopic, sweet smell as foul smell etc.

Dik vikrithi means disorientation of space such as feeling of north as south

and vise versa etc.



156

Kaala vikrithi is disorientation of time including the sensing of day as night, or

otherwise. Expression of altered physical, physiological and psychological

characteristics without any cause is considered as symptoms of alternation of

Prakrithi, which is also considered as the symptoms of imminent death.

Detailed description of the phenomena is included in Ayurvedic books,

especially in the 12 chapters of Indriya sthaana of Charaka samhitha. The

studies of Prakrithi in comparison with Vikrithi (rishta) enable to predict

prognosis of the disease and span of life of the individuals.

In the introductory part, it was discussed that the traditional system of

medicine like Ayurveda, Siddha etc. have advocated and practiced preventive

and curative medicinal recipes specific to individuals. The body, mind, food

and environment were looked at holistically to suggest a preventive or

curative approach to health. His Excellency the President of India has pointed

out the need for collaborative research of Ayurveda and biotechnology. Dr.

Kalam’s focusing on personalized medicine of Ayurvedic system of medicine

has invited our attention to the fact that in this post genomic era, the emerging

concept of personalized therapy on the basement of Pharmacogenomics is a

re-definition of diseases on the molecular level so that diagnotics and

therapeutics can be targeted to specific patient populations sub-typed on the

basis of genetic make-up. Use of modern technology and Biotechnology has

been identified by AYUSH as a thrust area for 10th plan period. The Kerala

Biotechnology policy is also designed with an objective to catalyze the

development of Ayurveda by collaborative research. The National

Biotechnology development strategy addresses the utilization of

Biotechnology to add value to Ayurveda. The emerging personalized medicine



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upholds the views of Ayurveda that environment, diet, age, lifestyle and state

of health all can influence a person’s response to health and that due to

genetic variations a disease may manifest itself slightly differently in different

types of patients. Ayurveda upholds individual physiology, pathology,

diagnostic and personalized therapy whereas Modern medicine base on

general principles of physiology, pathology diagnotics and treatment.

Study of the genetic make up of an individual is essential for personalized

therapy. A high-speed data mining of both genotypic and phenotypic

information is essential for the development of personalized therapy on the

basement of Pharmacogenomics. To categorize individuals on the basis of

board phenotypic clusters, consideration of racial, ethnic and geographical

factors to be considered collectively for genotyping is controversial. Hence

study of the concept of Prakrithi is beneficial. For diagnostic, prognostic and

therapeutic purposes, Ayurveda categorizes human population in to sub-

populations such as Vatha Prakrithi, Pitha Prakrithi, Kapha Prakrithi or their

combinations on the basis of physical, physiological and psychological

characteristics (phenotypes) and completely avoid racial, ethnic and

geographical considerations.

My present study aims to analyze the role of DNA in determining Prakrithi by

conducting Polymerize Chain Reaction studies, genetic correlation of various

forms of Prakrithi and to develop standards for a uniform guide line in

analyzing Prakrithi in this study I have tried to correlate Ayur Tech, Bio Tech

and Info Tech by seeking guidance and assistance of experts from the

respective fields.



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An attempt was made to analyze the molecular basis of Prakrithi. As in the

case of microbes, zygote also become resistant to the medium in which it is

developed, whereas the medium for microbe is a toxin and that of the zygote

is an energic template – Dosha sthithi – emitted and maintained unchanged

through out the span of life by the interaction of the extra zygotic and intra

zygotic environments at the time of fertilization. The zygote or Chatushpadi is

the sum total of the first four Maha-bhoothas – Prithwi, Ap, Thejus and Vayu

derived from four different sources – 1. Sperm (sukra) 2. Ovum (sonitha) 3.

Nutrient fluid (rasa) and 4. ‘Athma’ and any abnormalities of the four factors

derived from the aforesaid sources may render the zygote abnormal, the

effect of which will be reflected in the offspring. “Bija” (Sperm & Ovum) is

considered as collection of Genes. The superior germ cell genotype (Sama

Prakritihi) provides health to the developing embryo (Garbha) and its

variations Vathala, Pithala, Sleshmala and combinations of either two of them

cause varying degrees of prownness to disease (sadaathura). Hence Vatha

Prakrithi is considered as Heena (bad), Pitha Prakrithi as Madhyama (better)

and Kapha Prakrithi as Uthama (best) and combinations of either two of them

(dwantwa Prakrithis) as Nindya (worst). Diseases are mainly manifested as

Poorvaparaadhaja (Karmaja), or Drishtaparaadhaja (known etiology such as

Sahaja/ Garbhaja/ Jathaja/ Peedakritha etc) or by the combined effect of both

(Sankara roga).

Autosomal recessive inheritance and sex linked dominant inheritance involves

both Mathrja and Pithrja bija dushti, whereas sex linked recessive inheritance

is exclusively Mathrja and all these can be included in Aadi bala pravritha

roga. Defect in the somatic cells of the embryo will manifest into disease



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called Janma bala pravritha roga. The difference between diseases of aadi

bala pravritha and janma bala pravritha is that the former is due to the defect

in the germ cell and the later in the somatic cell due to the intra uterine

environment. Super females (sthree akrithi bhooyishtam) called Vaartha is

born due to the defect in genes that determine development of uterus, ovum,

and that of the secondary sex characteristics like breast, vagina, linea alba,

etc. Vaartha and super females or Trisomy X mentioned in modern genetics

shows phenotypic similarity. A sterile female (Vandhya) is born by defective

bija bhaga (gene) that determines the development of uterus and / or ovum of

the offspring. Defect in garbhasaya bija bhaga avayava, give rise to puthi

praja (still birth) or neonates with loose body tissues (klinnanga prathyanga).

Duchenne muscular dystrophy (DMD) is one of the most prevalent types of

muscular dystrophy and shows similarity to the above condition. Similarly in

super males called trina puthrika, a sterile male (Vandhya) and puthi praja or

sthiraanga prathyanga are occurring due to defect in sperm, shows

corresponding similarity with XYY syndrome, Fibrodysplasia Ossificans

Progressiva (FOP). Charaka clearly explains the bija bhagas (genes) that

determines the development of uterus and/ or ovum, testis and/ or sperm and

also those of the male and female secondary sex characteristics. The

expression of bija bhagas that determine the development of breast and

beard by adolescence is clearly explained. The influence of mental activity

over gene activity and how the mental disturbances of parents and that of the

mother during coitus and child bearing period explained by Charaka seems to

be an important subject of research for modern geneticists. Innumerable

factors pertaining to physical, physiological, and psychological aspect of



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mother that renders the offspring susceptible to various diseases, enumerated

by Charaka under ‘garbhopaghathakara bhavas’ was also discussed in detail

in the introductory part. The phenotypic characteristics of individuals

belonging to different Prakrithis are also explained and tabulated. The study of

Prakrithi with special reference to religion (Jati prasakta), family (Kula

prasakta), and also on the basis of the influence of geopathological factors

(Desanupatini), climatic elements (Kalanupatini), age factors (Vayonupatini),

and inividual developmental traits (Pratyatma niyata) are discussed in detail.

A review of literature of genes was included in my study, before discussing

the nature and functions of the genes under my study. Genes are the

fundamental physical and functional unit of heredity. Genes make proteins,

which are made of 20 amino acids. Billions of proteins combine to make a

cell; billions of them to make a tissue; thousands of tissues combine to make

an organ, and several organs, to make a man. The normal functioning of

genes keeps us healthy. When genes are exposed to harmful environments,

they alter their functions. It is called mutation. Plants interact with environment

and produce toxins, which act as active molecules that have the unique ability

to bind to human genes too and are able to switch a gene on or off. If the

switching on and off of a gene is desirable, it will lead to cure of disease when

genes are switched on it produces a protein. Genes are encoded in an

organism's genome, composed of DNA or RNA, and direct the physical

development and behavior of the organism. DNA does not change during an

individual’s lifetime but its functional properties like RNA and protein may

change depending on the environment. Various species carry more than one

copy of their genome within each of their somatic cells. These organisms are



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called diploid if they have two copies or polyploid if they have more than two

copies. In such organisms, the copies are practically never identical. With

respect to each gene, the copies that an individual possesses are liable to be

distinct alleles, which may act synergistically or antagonistically to generate a

trait or phenotype. Sir John gregor Mendel was the first to hypothesize

independent assortment, the distinction between dominant (An allele that

determines phenotype even when heterozygous, and also the trait controlled

by that allele) and recessive (a gene that is phenotypically manifest in the

homozygous state but is masked in the presence of a dominant allele) traits,

the distinction between a heterozygote (A diploid or polyploid with different

alleles at a particular locus) and homozygote (A diploid or polyploid with

identical alleles at a particular locus), and the difference between what would

later be described as genotype and phenotype.

Molecular typing of the following genes was carried out.

1. Dopamine ReceptorD2 Taq1D

2. Dopamine Receptor D2 Taq1B

3. Dopamine Receptor D2 Taq1A

4. Dopamine Receptor D2 S311C

5. Dopamine Receptor D2 His 313H

6. Dopamine Receptor D3 S9G

7. 5 Hydroxy Tryptamine Receptor 1B

8. Serotonin 1438

9. Serotonin 102

10. Human Leukocyte Antigen B

11. Human Leukocyte Antigen A



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A detailed study about each of the above genes was reviewed to understand

their nature and function.

To analyze the role of DNA in determining Prakrithi, to understand the genetic

correlation of various forms of Prakrithi and to develop standards for a uniform

guideline in analyzing Prakrithi, the study was conducted. As per criteria fixed

earlier, the Prakrithi of 40 unrelated individuals were assessed with informed

consent. Blood samples were collected. DNA isolation was performed using

standard organic extraction methods. Quantification of DNA was done by two

methods – spectrophotometry and agarose gel electrophoresis. Amplification

of genes was carried out using the polymerase chain reaction. Molecular

typing (Detection of amplification) was done by agarose gel electrophoresis

method and documented the gels using multi imager.

Of the 40 individuals selected for the study, maximum individuals were male

70% and remaining 30% females; Hindus (80%) Christians (15%), Muslims

(5%). The distribution according to various Prakrithis was noted. Kapha

Prakrithi 32.5% (13), Pitha Prakrithi 22.5% (9), Pitha Kapha Prakrithi is found

more frequent 35% (14), Vatha Pitha (2) and Vatha Kapha (2) 5% each. It

was observed that there were no Vatha Prakrithi phenotypes and Sama

Dosha Prakrithi (combination of the three Doshas) phenotypes (graph 3).

The genotype distribution in the population was as follows:

Dopamine ReceptorD2 Taq1D heterozygous genotype (D2/D1) individuals (50%);

Dopamine Receptor D2 Taq1B dominant homozygous genotype (B2/B2) (60%);

Dopamine Receptor D2 Taq1A dominant homozygous genotype (A2/A2) (45%);

Dopamine Receptor D2 S311C dominant homozygous genotype (G/G) (80%);

Dopamine Receptor D2 His 313H heterozygous genotype (C/T) (47.5%); Dopamine

Receptor D3 S9G heterozygous genotype (S/G) (42.5%); 5 Hydroxy Tryptamine



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Receptor 1B recessive homozygous genotype (G/G) (47.5%); Serotonin 1438

heterozygous genotype (A/G) (57.5%); Serotonin 102 heterozygous genotype (C/T)

(60%); Human Leukocyte Antigen B allele B*40 was frequent (40%); Human

Leukocyte Antigen A allele A*24 was frequent (37.5%)

Analysis of the results of the study was conducted by statistical analysis using

the software SPSS version 10 for windows and Microsoft excel sheet for

preparing graphs and charts.

Of the various dopaminergic receptor genes with respect to various Prakrithi

populations a significant increase of the allele frequencies in the Prakrithis

having an association of Vatha is found. The allele frequency of dopaminergic

receptor genes Taq 1B2, Taq 1A2, S311C G, and His 313H (T and C) and

DRD3 S9G also shows an increasing tendency in all the Vatha associated

Prakrithi populations. Hence it is understood that association of Vatha

increases the allele frequency in dopaminergic receptor genes under my

study.

Similarly, serotoninergic receptor gene 5HTR 1B shows a significant increase

of the allele frequencies in the Vatha associated Prakrithis. But in the case of

serotoninergic receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows a

significant increase of the allele frequency in the Kapha associated Prakrithi.

Dopamine functions as a neurotransmitter, activating dopamine receptors.

Dopamine is also a neurohormone released by the hypothalamus. Dopamine

is critical to the way the brain controls our movements and is a crucial part of

the basal ganglia motor loop. All the functions attributed to dopamine receptor

can be included in the functions of Vatha in Ayurveda.

Serotoninergic 5-HT1B receptors are expressed throughout the mammalian

central nervous system and, only 1% of serotonin in the human body is found



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in the CNS and also the receptor gene 5HTR 1B shows a significant increase

of the allele frequencies in the Vatha associated Prakrithis. 99% of serotonin

is found in other tissues, primarily plasma, the gastro-intestinal tract, and

immune tissues and serotoninergic receptor gene 5HTR 2A (Ser 1438 and

Ser 102) shows a significant increase of the allele frequency in the Kapha

associated Prakrithis.

Among the 16 alleles studied, the allele frequency of HLA B*40 was seen

highest in Kapha Prakrithi and Pitha Kapha Prakrithi. The lowest allelic

frequency of HLA B was observed in B*08 & B*38 (in Kapha) B*56 (in Pitha

Kapha), and B*57 (in Vatha Kapha).

Among the 10 alleles studied, the allele frequency of HLA A*24 was seen

highest in Pitha Kapha Prakrithi and Pitha Prakrithi. The lowest allelic

frequency of HLA A was observed in A*01 and its distribution were equal in all

the 5 Prakrithis.

Human leukocyte antigen (MHC class I) genes are important determinants of

host response to microbial infections and also have a profound effect on the

immunogenetic susceptibility to various diseases that are influenced by

immune reactions. It has gene cluster spans around a region of about 4000

k.bps on short arm of chromosome six in the distal portion of 6p 21.3 band.

Large number of genes with variable expression is arranged in form of three

regions in MHC as class I (36 genes), class II (27 genes) and class III (39

genes). Class I products play a role in determining viral specificity and were

involved in the production of antibodies. They play a key role in the

recognition and destruction of cells carrying foreign antigens by cytotoxic

t- Lymphocytes.



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The defense mechanism in the human body is associated with Kapha in terms

with the doctrines of Ayurveda; oja otherwise called as bala is ‘somathmaka’;

‘sleshma somah:’ so it is understood that Kapha plays an important role in

maintaining the immunological status of the body. Kapha Prakrithi is

considered as best. Human leukocyte antigen (MHC class I) genes having

role in host response to infections and profound effect on the immunogenetic

susceptibility to various diseases, the frequency of which is high in Kapha

Prakrithi attest the scientific basis of tridosha theory.

Analysis of the result shows that all genes show varying frequencies in

different Prakrithis having predominance of Vatha, Pitha, Kapha or their

combinations. It is due to the influence of tridoshas, that genes show

variations in their frequency in different individuals with varying dominance of

Vatha, Pitha and Kapha. Ayurveda clearly states that, it is the due to the

influence of tridoshas on the genetic material that the corresponding

variations are occurring in phenotypic characteristics of different individuals

sub typed into different Prakrithis. When Vatha influences the innumerable

genes that determines the development of various organs and tissues of the

body, the organs and systems are developed with specific characteristics so

that, the individual can be grouped in Vatha Prakrithi. Further studies with

large samples are essential to establish the influence of tridoshas on genetic

material. Infact the aim of my study was to analyze the role of DNA in

determining Prakrithi, but it is found that tridoshas influence DNA so that an

individual is developed with the specific characteristics of a particular

Prakrithi. Prakrithi, which also means the ‘basic cause’, represents the Dosha



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sthithi – energic template – that influence the gentic material to determine the

development of the specific characteristics of an individual.



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CONCLUSIONS

$ It is observed that there is a genetic basis for the three major

constitutions (Prakrithi) described in Ayurveda, and that tridoshas

influence DNA at the time of fertilization, so that an individual is

developed with the specific characteristics of a particular Prakrithi.

$ This study enables to uphold the Ayurvedic concepts of personalized

therapy by analyzing the population on the basis of phenotypic clusters

of different genotypes.

$ Association of Vatha increases the allele frequency in dopaminergic

receptor genes and serotoninergic receptor gene 5HTR 1B under my

study. All the functions atributed to dopamine receptors are included in

the functions of Vatha in Ayurveda.

$ Serotoninergic receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows

a significant increase of the allele frequency in the Kapha associated

Prakrithi.

$ Kapha plays an important role in maintaining the immunological status

of the body. Kapha Prakrithi is considered as most healthy with high

immunological status. Human leukocyte antigen (MHC class I) genes

having role in host response to infections and profound effect on the

immunogenetic susceptibility to various diseases, the frequency of

which is high in Kapha Prakrithi attest the scientific basis of tridosha

theory.

$ Study on the association of Pitha on any receptor genes were not

conducted due to the lack of availability of enzymes at the time of my

study.



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$ Of the 40 individuals selected none was having Sama dosha Prakrithi

and it attest the Ayurvedic concept that such a Prakrithi, though super,

is rare or conceptual due to our inability to provide the superior

environmental conditions essential for the emergence of such a trait.

$ Absence of Vatha Prakrithi and increased frequency of Kapha or

Kapha associated Prakrithi indicate the high health status of the

population under study.

$ When we compared the Prakrithis in males and females we observe

almost similar trend of Prakrithi distribution, males had higher

frequency of Kapha Prakrithi in compared their female counterpart.

$ Though, 80% of the individuals were Hindus, we do not observe any

significant shift in the trend of Prakrithis based on religion.

$ More study is essential to develop standards for a uniform guideline in

analyzing Prakrithi, with large and equal sample size of all the six types

of Prakrithi.

$ A repetition of the study of the individuals included in my study at their

old age may enable to interpret the influence of tridoshas on DNA at

the time of old age in tune with the basic concepts of tridosha theory.



169

REFERENCES

Introduction

1. www.presidentofindia.nic.ni

2. Dr. M.S. Valiyathan, 70th annual meeting in Indian academy of science

News, current science, vol.88

3. Dr. M.S. Valiyathan, in the book -The legacy of Charaka - 2003

Prakrithi

1. Susrutha samhitha Sareera sthaana 4/ 79

2. Rasa vaisheshika soothra translated by Naarasimha 1/ 7

3. Charaka samhitha Vimana sthaana 8/ 95

4. Charaka samhitha Sareera sthaana 2/ 3

a. Charaka samhitha Sareera sthaana 3/ 7, Chakrapani

5. Charaka samhitha Sareera sthaana 2/ 31, Ibid Chakrapani

6. Ashtanga hridayam Sareera sthaana 1/ 3

7. Charaka samhitha Siddhi sthaana 9/ 4, Ibid Chakrapani

8. Ashtanga Hridayam Nidana sthaana 7/ 16

9. Charaka samhitha Sareera sthaana 2/ 16, 26, 35, Ibid Chakrapani

10. Charaka samhitha Sareera sthaana 2/ 26

11. Charaka samhitha Vimana sthaana 6/ 13, Ibid Chakrapani

12. Charaka samhitha Suthra sthaana 7/ 39, Ibid Chakrapani

13. Ashtanga hridayam Suthra sthaana 1/ 10

14. Dr. M.S. Valiyathan, in the book -The legacy of Charaka - 2003

15. Charaka samhitha Suthra sthaana 17/ 40 to 44

16. Susrutha samhitha Suthra sthaana 24/5, Ibid Dalhana



http://www.presidentofindia.nic.ni


170

18. http://www.mdausa.org/home.html/genes and diseases


20. http://www.med.umich.edu/ university of michigan


22. http://www.ifopa.org/ genes and diseases


24. www.yale.edu/ynhti/comments.html and Glossary of Genetic Terms,

University of Kansas Medical Center


26. Ashtanga sangraha Sareera sthaana 2/ 21, Ibid Indu


28. Charaka samhitha Indriya sthaana 1/ 5, and Ashtanga sangraha

Sareera sthaana 8/ 17

29. Charaka samhitha Sareera sthaana 2/ 22 – 27, Chakrapani

30. Benson, H. with Stark, M., (1996), “Timeless Healing: The Power

and Biology of Belief”, Scribner, NY, pp: 350. and online Journal of

Indology, Biology of Belief. mht by Dr. Ramesam

31. Schwartz, J.M. and Bagley, S., (2003), “The Mind and the Brain:

Neuroplasticity and the Power of Mental Force”, Reagan Books

32. Ashtanga Hridaya Nidana staana 6/ 11

33. Ashtanga Hridaya Suthra staana 1/ 23


35. Susrutha samhitha Suthra sthaana 24/ 5, Ibid Dalhana

36. Nathanielsz, P. with Vaughan, C., (2001), “The Prenatal

Prescription”, Harper Collins, NY


http://www.mdausa.org/home.html/genes

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http://www.ifopa.org/

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171

Genes

1. http//en.wikipedia.org/wiki/genes

2. http://www.bio.davidson.edu/courses/genomics/2001/powell/drd2

3. http://www.ncbi.nlm.nih.gov/ Gene Taq 1 B

4. The Pharmacogenomics Journal (2005), Association between the

DRD2 gene Taq1A

5. http://www.ncbi.nlm.nih.gov/entrez/PubMed/ drd2 s311c

6. http://lib.bioinfo.pl/his 313 rs6275

7. http://www.ncbi.nlm.nih.gov/entrez/PubMed/meta-analysis suggests

that the DRD3 gene Ser9Gly variation confers susceptibility to

schizophrenia

8. http//en.wikipedia.org/wiki/dopamine

9. http://www.neurotransmitter.net/index/5-ht1bReceptors and

Aggression/ Impulsivity -- Neurotransmitter_net.

10. http//en.wikipedia.org/wiki/serotonin

11. http://medgenmed.medscape.com/medgenmed/ Serotonin 5-HT(2A)

receptor gene polymorphism, 5-HT(2A) receptor function and

personality traits in healthy subjects a negative study

12. An Overlooked Connection: Serotonergic Mediation of Estrogen-

Related Physiology and Pathology, by Leszek A. Rybaczyk; Meredith

J. Bashaw; Dorothy R. Pathak; Scott M. Moody; Roger M. Gilders;

Donald L. Holzschu.

13. Thomas, R., Nair, S. B. & Banerjee, M. (2004) HLA-B and HLA-C

alleles and haplotypes in the Dravidian tribal populations of southern

India.


http://www.bio.davidson.edu/courses/genomics/2001/powell/drd2

http://www.ncbi.nlm.nih.gov/

http://www.ncbi.nlm.nih.gov/entrez/PubMed/

http://lib.bioinfo.pl/his

http://www.ncbi.nlm.nih.gov/entrez/PubMed/meta-analysis

http://www.neurotransmitter.net/index/5-ht1bReceptors

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14. James Robinson,Matthew J.Waller, Peter Parham ,Natasja de Groot,

Ronald Bontrop, Lorna J.Kennedy, Peter Stoehr and Steven

G.E.Marsh. IMGT/HLA and IMGT/MHC:sequence databases for the

study of the major histocompatibility complex. Nucleic Acids Research,

2003, Vol. 31, 311–314

15. Marsh, S.G.E., Bodmer, J.G., Albert, E.D., Bodmer, W.F., Bontrop,

R.E., Dupont, B., Erlich, H.A., Hansen, J.A., Mach, B., Mayr, W.R. et al.

(2001) Nomenclature for factors of the HLA system, 2000. Tissue

Antigens, 57, 236–283.

Materials and methods

1. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: a Laboratory

Manual. New York: Cold Spring Harbour, 1982.

2. Birnboim HC and Dolly J. A rapid alkaline extraction procedure for

screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-

23.1979.

3. http://en.wikipedia.org/w/Spectrophotometer

4. http://www.life.uiuc.edu/molbio/geldigest/electrophoresis

5. Molecular Biology Techniques Manual, Third Edition by: Vernon E

Coyne, M Diane James, Sharon J Reid and Edward P Rybicki.

Standard PCR Protocol January 1994, February 2001.


Coyne, M Diane James, Sharon J Reid and Edward P Rybicki PCR

PRIMER DESIGN AND REACTION OPTIMISATION


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173

7. Abhilash, R., Dileepkumar, P. and Suresh, B. N. Genomic Profile

Analyser. Master of Science dissertation, 2002. University of Kerala,

Trivandrum, India.

8. Comas, D., Mateu, E., Calafell, F., Perez-Lezaun, A., Bosch, E.,

Martinez-Arias, R. & Bertranpetit, J. (1998) HLA class I and class II

DNA typing and origin of Basques. Tissue Antigens 51, 30-40.


Coyne, M Diane James, Sharon J Reid and Edward P Rybicki.

Standard PCR Protocol January 1994, February 2001

10. Bunce M, O’Neill CM, Barnado MCNM, Krausa P, Browning, MJ, Morris

P.J, Welsh KI: Phototyping: comprehensive DNA typing for HLA-A, B,

C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes

utilizing sequence specific primers (PCR-SSP). Tissue Antigens 46:

355, 1995.

11. Thomas, R., Nair, S. B. & Banerjee, M. (2004) HLA-B and HLA-C

alleles and haplotypes in the Dravidian tribal populations of southern

India. Tissue Antigens 64, 58-65.

Discussion 1. Thesis titled ‘Clinical management of Aganthuja Kshata Sukla

with special reference to Murivenna’ by Dr. K.V. Subhadra

Antherjanam, University of Kerala, 1988.

2. Susrutha samhitha Uthara sthaana 7/ 2-4, Dalhana

3. Susrutha samhitha Suthra sthaana 5/ 7, Dalhana quoted Nimi

4. Tharka samgraha Pradhama Khanda Deepika & Neelakanti

tikas

5. Susrutha samhitha Sareera sthaana 4/ 63, Dalhana



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8. Rasa vaisheshika soothra, soothra 91, 92

9. Rasa vaisheshika soothra by Bhadantha Nagarjuna, soothra 97

10. Sri. Raghavan thirumulppad: Rasa vaisheshika soothra by

Bhadantha Nagarjuna, soothra 97, Malayalam translation, 1977.

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