TITLE OF THE STUDY -

Descriptive Title: “AN EXPLORATORY LONGITUDINAL OBSERVATIONAL AND MOLECULAR STUDY ON VARIATIONS OF CLUSTER DIFFERENTIATION MARKERS IN HEALTHY INDIVIDUALS OF EXTREME DOSHA PRAKRITI (EK DOSHAJA PRAKRITI)  IN SIX SEASONS.”

Short Title: “IMMUNO-PHENOTYPING (CD MARKERS) IN HEALTHY INDIVIDUALS OF EXTREME DOSHA PRAKRITI  (EK DOSHAJA PRAKRITI) IN SIX SEASONS”

RESEARCH QUESTION –

Is there any variation in cluster differentiation (CD) markers in healthy individuals of extreme Dosha Prakriti (Ek doshaja Prakriti)  in six seasons?

HYPOTHESIS

NULL HYPOTHESIS

The cluster differentiation (CD) markers of healthy individuals of extreme Dosha Prakriti (Ek doshaja Prakriti)  in six seasons are similar.

ALTERNATE HYPOTHESIS

The cluster differentiation (CD) markers of healthy individuals of extreme Dosha Prakriti (Ek doshaja Prakriti)  in six seasons varies.

INTRODUCTION –

               Ayurveda is based on Tridosha theory which identifies principles of Vata (motion), Pitta (metabolism), and Kapha (structure)¹. The classification of the individual constitution of Prakriti is based on differences in physical, physiological, psychological, behavioral, social, and immunological characteristics.

The Prakriti of an individual is determined at the time of conception and remains unaltered throughout life. Seven types of Prakriti are mentioned in Ayurveda based on the Predominance of which single dosha is Ekadoshaj prakriti, two doshas are Dwandvaja prakriti and the equilibrium of all three doshas is Samadosha Prakriti².  Prakriti-specific treatment including medicine, diet, and lifestyle is a distinctive feature of Ayurveda. According to Acharya Charaka, Epigenetic factors like Kalanupatini, Vayonupatini, Deshanupatini, etc³ are mentioned for the adaptations in the fixed set of Doshas in a particular range, which is again specific for each of the Prakriti. Ashtanga Samgraha has rightly depicted these variations or adaptations as Vaikrita Doshas⁴.

Among those factors, that bring about the variations/adaptations in Vaikrita Dosha, Kala is one such factor, which brings about the variations in Doshas – fortnightly as well as seasonally. As per Ayurveda kala is a most important factor. Kala can be defined as a season. There are six seasons mentioned in Charaka, Sushruta, and Vagbhata Samhita. i.e. Bruhatatrayi. Based on the position of the sun and moon in the physical environment. These seasons are divided into Adankala and Visargakala. Adankala includes three ritus Shishir, Vasant, and Grishma. Visargakala includes three ritus Varsha, Sharad, and Hemant⁵.  As seasons change, changes also occur in human’s internal body functions which has a great impact on the equilibrium of Tridosha and tends to increase the disease prevalence rates.  Kalanupatini prakriti explained in Charak Samhita³, gives a relation between seasons and bala (Immunity).   In this Acharya, Charak mentions that seasonal variation happens in Bala. which manifests that Bala is Maximum in Shishir and Hemant ritu, Moderate Bala in Sharad and Vasant ritu whereas Bala is Minimum in Varsha and Grishma ritu⁶.

Further, several reports are available on the modulation of immunity through elevating the expression profile of cluster differentiation (CD) markers. Cluster differentiation is a monoclonal antibody collection that recognizes an antigen on a particular differentiated cell type. each of the antigens recognized by such a collection of antibodies is known as a cluster differentiation marker.

The prakriti remains consistent but seasonal dosha fluctuations happen in the body.  According to Ashtang samgraha, seasonal variation, age, time of the day, and Time of meal have a great impact on the doshas we call Vaikrita Dosha⁴. thus, it can be assumed that cluster differentiation in Prakriti varies in six seasons.  

NEED OF THE STUDY –

Ayurveda proposes Prakriti influences individual variations in health and disease susceptibility. CD markers might reflect these underlying differences in immune function. Also, seasonal doshas fluctuations are Chaya, Prakop, and Prasam^7,8. These Avasthas are Vaikrit doshas changes happen in Kalanupatini prakriti. Through CD markers we associate the immune function of an individual with Prakriti.  Both Prakriti and the immune system are complex concepts with various contributing factors. 

Some research shows the relation between prakriti and CD markers but no known relation between seasonal variation and extreme prakriti has been explored. The current research on Prakriti and CD markers is preliminary. This study may confirm and understand the nature of this connection between Vaikrita Dosha and CD markers, as the seasons varies. No study has been carried out till date to assess the effect of changing Ritu on CD markers in extreme prakriti of healthy individuals. This study will be useful for understanding the vaikrit dosha concept mentioned in Ayurveda with the help of CD markers. As the Vikrita and Vaikrit Doshas, both are influenced by the Prakrita Doshas, this study aims to see the variations in the symptoms of Sanchaya, Prakopa, and Prashama in extreme Deha Prakriti persons, substantiating it with the molecular bio-markers. Furthermore, this study will be the benchmark to prevent the prevalence of diseases occurring in specific Ritus. Also, the Prakriti-specific response to the seasonal variations will be noted. This will bring forth the empirical evidence for the Ayurveda concept of epigenetic factors viz. Kalaanupatini Prakriti. Such research is scanty and clear evidence to show the differences among the different prakritis is required. Hence, the present study was performed to evaluate the ability to initiate the immune response in extreme prakriti individuals by analyzing various CD markers and relate to the concepts laid down in Ayurveda.

LITERATURE REVIEW

CLASSICAL REVIEW:

According to Ashang hridayam ,

This means at the time of conception Sukra and Aartav have vaatadi doshas which convey to the newborn baby. There are seven types of Prakriti.

As per Charak Samhita ,

     means that Kalanupatini Prakriti may change as per season but the main dominant dosha prakriti does not change. This seasonal variation, age, time of the day, and Time of meal greatly impact the doshas known as vaikrit doshas mentioned in Ashtang sangraha,

 There are many changes occur in human physiological functions as changes occur in the external environment. So it is recommended that changes must be made in diet and lifestyle according to changing seasons to prevent diseases and live a healthy life.

PREVIOUS RESEARCH WORKS

From the previous studies, it is evident that there is a relationship between CD markers and Prakriti. Another study shows seasonal variation observed in the CD19 marker; where the role of Prakriti is not considered.

Thus, from the above, it is evident that no such work has been done till date on consideration of extreme Prakriti (Ekdoshaja Prakriti) with CD markers and its variations in the different 6 seasons. Hence, the present study is exploratory longitudinal work on the said concept and is planned with the following objectives.

OBJECTIVES OF THE STUDY:

Primary Objectives:

1. To observe the effect of Ritu [ Seasonal variation ] on CD markers in healthy individuals of extreme Prakriti (Ek doshaja Prakriti).

Secondary Objectives:

1.       To assess the effect of Ritu (seasonal variation) on Chaya, Prakop, and Prasaman of different Doshas in dominant Deha Prakriti individuals, as mentioned in Ayurveda classics

2.       To assess the variations in other blood investigations and Bio-chemical profile

EXPECTED OUTCOME:

Relation between the Ritu and Cluster differentiation (CD) markers will be noted and Chaya, Prakop, and Prashama avastha can be assessed.

TRANSLATIONAL VALUE OF THE RESEARCH –
This study will serve as the benchmark for diagnosing and preventing diseases in a particular Dosha Prakriti, that manifest during specific seasons (Ritus). This will bring forth the empirical evidence for the Ayurveda concept of epigenetic factors viz. Kalaanupatini Prakriti. This study may provide baseline values for different CD markers within extreme prakriti and could be used for understanding prakriti-based disease manifestation, and etiology and improving the treatment modalities by an Ayurvedic practitioner.

MATERIALS AND METHODS

Study Design- Exploratory Longitudinal Observational & Molecular study

Timing – Prospective

Study site – AIIA, New Delhi

Duration of study - 12 Months

Assessment - Every 2 months in the middle week of the month *. Hence, 6 times in a year

Inclusion Criteria-

1. Apparently healthy individuals of age group 21- 50 years of Ek Doshaja Prakriti.

Exclusion Criteria-

·         Pregnant and lactating mothers.

·         Individuals who don’t wish to give written consent for this study.

·          Known cases of Autoimmune diseases, Systemic diseases, and Malignancy conditions.

Withdrawal criteria- The participant may be withdrawn from the study if,

·         Participant wanted to withdraw himself/herself from the study due to any reasons.

·         Participants diagnosed with any metabolic disease or co-morbid condition after enrollment, for which they need to continue the medicine for a longer time or lifetime.

MATERIALS REQUIRED:

·         Swastha Assessment Scale (SAS) for healthy individual selection.

·         Prakriti pariksha using CCRAS Prakriti assessment tool

·         Trividha Kriya Kala (Ritu-Janita Kriya Kala) questionaries for Trividha Doshas will be prepared and included in the  CRF.

·         Investigations - CBC, ESR, CRP, LFT, KFT, TFT, Lipid profile, Blood sugar (Fasting and postprandial.), HbA1c.

A] Observational  Assessment –

·         All information regarding the assessment of Prakriti, test procedures, etc. will be explained in detail to participants & written consent will be taken.

·         All participating Individuals will be screened using CCRAS PAS  and enrolled after fulfilling the inclusion criteria & after routine investigations for Ekadoshaj and Dwidoshaj Prakriti.

·         Before enrolling the subject, it will be confirmed that he / She is not on any kind of medication or Regime (Ayurveda or any other) regularly.

·         10 participants of the extreme Prakriti (Ekdoshaja Prakriti) each, in all 30 participants,  will be assessed for the Chaya, Prakopa, and Prashamana (Trividha Kriya kala questionnaire) in each season, for 6 times.

·         The CRF will be prepared in a way to rule out the involvement of the Prakrit / Vikrit           Dosha basically through Nidana Sevana.

·         The magnitude of the response will be correlated with the extreme Prakriti.

·         The season will be defined according to the Bhartiya calendar and the Jyotishshatriya Panchang system of North India.

·         Participants will be asked to report immediately for any seasonal discomfort or disease if at all they suffer from.

·         Irrespective of the seasonal disease, if anyone suffers from illness will be asked to report and consult immediately.

·         A 15-ml sample of blood from each participant’s blood will be collected for hematological, and bio-chemical investigations & molecular study, in the mid of every season.

B] MOLECULAR STUDY –

A. Rationale of selecting specific CD markers:

Based on the previous study, the CD-14 had shown significant upregulation in Pitta Prakriti, followed by Vata Prakriti, followed by Kapha Prakriti. Also, the same study had shown a significant proportion of CD-25 in the subjects of Kapha Prakriti⁹. However, this study did not repeat the assessment of the markers in all six seasons. Another study depicted the seasonal variation of CD-19 in healthy individuals; wherein Prakriti-wise variation was not taken into consideration.¹⁰ Considering these two studies, the following CD markers will be evaluated seasonally in healthy individuals of extreme Dosha Prakriti( Ek Doshaja Prakriti) and if feasible in Dwandvaja Prakriti.

B. About the selected CD markers:

C.  SOP of the Molecular Study:

The details of the molecular study of the CD markers – CD 14, 19, and 25 are annexed in Annexure – 1

D. Confounding Factors Influences Biomarkers

When assessing biomarkers such as CD14, CD19, and CD25, which are cell surface markers commonly used to identify and characterize specific immune cell populations, several confounding factors should be considered:
1. Inflammatory Conditions, Disease Status

2. Age

3. Genetic Variations

4. Environmental Exposures

5. Stress

6. Hormonal Factors

7. Nutritional Status

8. Sample Handling

The study will be conducted on healthy individuals after screening the participants using CCRAS SAS and blood tests (Hematological parameters)  in which the confounding factors, like inflammatory conditions, medications, nutritional status, disease status, Hormonal factors, and stress, etc will be ruled in advance while screening and enrollment. As the study is being conducted on 21 to 50 yrs age group individuals, age as a confounding factor will be eliminated.

SAMPLE SIZE CALCULATION:

The study aims to monitor changes in CD markers CD14, CD19, and CD25 related to three extreme Prakriti. To study the trends in six seasons. It is explored that the cost of measuring CD markers for one subject inclusion in the study is quite high approximately RS 5,000/-Therefore it is decided that we would screen eligible subjects for extreme Prakriti and select 10 subjects to follow over six seasons for each extreme Prakriti i.e. Vata, Pitta, Kapha. So the study is planned on 30 subjects.

As per the previous studies, the prevalence rate of the extreme Prakriti is less than 10%. Hence, to meet out the sample size of 30, a minimum of 300 participants need to be screened.

DATA COLLECTION & STATISTICAL ANALYSIS-

•      Descriptive baseline data, would be compared using the Chi-square test. Mean, Median, SD, Percentile, etc would be computed as necessary.

•      Data would be examined for normality and repeated measure ANOVA with Bonferroni test would be done to compare between groups and the seasonal trends. In case qualitative data is being collected by seasons Friedman test will be applied.

•      The data analysis will be done using SPSS version 26.

ACCESS TO DATA AND CONFIDENTIALITY

The Research team only will have access to the participant data & final trial dataset. Data confidentiality will be maintained and Primary data will be preserved in the concerned Department for 5 years.

ETHICS COMMITTEE CLEARANCE

·         The study will be started only after getting clearance from the Institutional Ethics Committee, AIIA, New Delhi.

·         The present study will be carried out after taking the written consent from the Study Participants.

·         The CTRI Registration will be done.

FLOW CHART FOR THE STUDY

CO-OPERATION REQUIRED:

·         Pathology lab, AIIA Hospital.

·         Staff, scholars, faculty, etc of  AIIA.

·         Molecular biology lab, AIIA, New Delhi.

·         If needed help will be taken from other departments of AIIA and various advanced laboratories with due permission from the concerned authority during the study.

FINANCIAL SUPPORT:

APPROX BUDGET:

Lab investigations                                    - Rs. 2,44,000 /- (For research purpose it is free)

Molecular biology lab investigations         - Rs.  1,50,000/-

TOTAL                                                  -Rs   3,94,000 /-

This study will be completed within the prescribed financial limit for the work. However, if more finance is required, the request will be made for due permission and approval of the extra budget to the concerned authority AIIA, in due course of research work.