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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BENGALURU.
COMPLETED PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

EXPERIMENTAL STUDY ON REPRODUCTIVE EFFECT OF


PHALAGRITHA IN NORMAL AND D.E.S
(DIETHYLSTILBESTROL)TREATED FEMALE ALBINO RATS.
BY
Dr. KENY KRUTIKA NARENDRA
M.S. (Ayu), (SCHOLAR)
DEPT. OF PG STUDIES IN PRASOOTI TANTRA AND STREEROGA
S.D.M. COLLEGE OF AYURVEDA, UDUPI
GUIDE
Dr.V.N.K. USHA
M.D(Ayu)
HEAD OF THE DEPARTMENT
DEPT. OF PG STUDIES IN PRASOOTI TANTRA AND STREE ROGA
S.D.M. COLLEGE OF AYURVEDA, UDUPI
CO-GUIDE

RAVI MUNDUGARU
M.Sc. (Med. pharmacology)
RESEARCH OFFICER
S.D.M. RESEARCH CENTRE
UDUPI-574118.
2012-2013
DEPARTMENT OF POST GRADUATION STUDIES IN PRASOOTI TANTRA AND
STREEROGA
S.D.M COLLEGE OF AYURVEDA,KUTHPADY, UDUPI 574 118

From,
DR. KENY KRUTIKA NARENDRA
M.S.(Ayu),SCHOLAR,
DEPARTMENT OF P.G. STUDIES IN PRASOOTI TANTRA AND STREE ROGA
S.D.M COLLEGE OF AYURVEDA,UDUPI-574118

To,
THE REGISTRAR,
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,KARNATAKA,
BENGALURU.
Through,
THE PRINCIPAL
S.D.M.COLLLEGE OF AYURVEDA
KUTHPADY,UDUPI 574 118
Respected Sir,
Subject: Submission of completed proforma for registration of subject for dissertation.
I request you to kindly register the below mentioned subject against my name for
the submission of dissertation to the Rajiv Gandhi University of Health Sciences, Bengaluru for
the partial fulfillment of M.S. (Ayu) in Prasooti tantra and stree roga.
Title of Dissertation:
EXPERIMENTAL STUDY ON REPRODUCTIVE EFFECT OF
PHALAGRITHA IN NORMAL AND DES (DIETHYL STILBESTROL)
TREATED FEMALE ALBINO RATS.
Herewith I am enclosing completed Performa for registration of subject for dissertation.
Thanking you,

Place: Udupi

Yours Sincerely

Date:

1. NAME OF THE CANDIDATE :

(Dr.KENY KRUTIKA NARENDRA)

Dr.KENY KRUITKA NARENDRA


M.S(Ayu) SCHOLAR,
DEPT. OF PG. STUDIES IN PRASOOTI TANTRA
AND STREEROGA,
S.D.M.COLLEGE OF AYURVEDA,
KUTHPADY, UDUPI 574 118.

PERMANENT ADDRESS :

Dr. KENY KRUTKA NARENDRA


D/O NARENDRA KENY,
H.NO.130.JAYRAM PALACE,
P.G MARG , BHANDARWADA,
MALAD(WEST), MUMBAI-400064,
MAHARASHTA.

2. NAME OF THE INSTITUTION:

S.D.M. COLLEGE OF AYURVEDA


KUTHPADY, UDUPI 574 118

3. COURSE OF STUDY AND SUBJECT: M.S. (AYURVEDA) IN PRASOOTI TANTRA


AND STREEROGA

4. DATE OF ADMISSION OF COURSE : 16-10-2012

5. TITLE OF THE TOPIC STUDY:

EXPERIMENTAL STUDY ON REPRODUCTIVE EFFECT OF PHALAGRITHA IN


NORMAL AND DES(DIETHYLSTILBESTROL) TREATED FEMALE ALBINO RATS.

6)BRIEF RESUME OF INTENDED WORK


6.1)NEED FOR THE STUDY:
Procreation is the ultimate outcome of female genital tract for which Rutu,kshetra,ambu and
beeja are responsible1. A woman provides a sourceful beeja in rutukaal which gets implanted into
healthy kshetra in the absence of vitiated vata and gets nutrition from the rasadhatu of the
mother. Vitiation at any level may interrupt the pregnancy leading to female infertility. Apart
from this yonivyapaths, artavavyapaths, manasika abhitapa, shukra dosha, ahara dosha, vihara
dosha, akaala yoga, balakshaya etc also contribute for female infertility2.
In our classics we also get special references like Revati graha3, misuse of Pancha Karma4, intake
of atiushna anapana veerya, artava,beeja become upachita5 ultimately leading to Vandhyattva.
In contemporary science also etiology of female infertility is divided into many factors.
According to FIGO manual (1990) causes for female infertility includes tubal and peritoneal
factor (25-35%), ovulatory factor (30-40%), cervical factor (5%), uterine factor or endometriosis
(1-10%)6.
As growing female infertility contributes to 50% of the total incidence of infertility it should be
checked with a better preventional and treatment protocol with efficient and safe drugs.
Hence, Phalagritha which is an age old remedy mentioned in Ayurveda widely used in clinical
practice for the treatment of Vandhyattva , is selected for the experimental study to check its
efficacy on histopathology of genital tissue and biochemical changes, to explore its mode of
action and site of action.

6.2)REVIEW OF LITERATURE:
Sushruta and Dalhana both have mentioned in Garbha sambhava samagri for
garbhotpatti.1
Sushruta described Vandhyattva under vandhya yoni vyapada.7
Harita mentioned six types of vandhya with specific etiology for each and its prognosis.8
Both Vagbhats mentioned different causes specially abeejatva as a factor responsible for
female infertility.9,10
Charaka in shareer sthana and chikitsa sthana has mentioned causes for vandhayttva.2,11,12.
Kashyapa under chapter Revati Jatharni mentioned Pushpaghni which can be co-related
with anovulatory cause of infertility with obesity and hirsutism.13

In Madhava Nidana types of Vandhyattva has been described.14


Bhela has mentioned causes for Vandhyattva and classified it under Vata vyadhis.15
Sharangadhara,Vagbhata, Yogratnakar and Bhavprakasha mentioned Phalagritha in
treatmnent of Vandhyattva.16,17,18,19,20.

EXPERIMENTAL MODEL:
Study effect of Phalagritha on internal administration in female albino rats.
PREVIOUS WORK DONE:
1. Dr.Obeja- clinical study of effect of Phala ghrita in female infertility, year 1981, Banaras

hindu university,Varanasi.
2. Mishra Deepa rani- clinical study of effect of Phalakalyanaka ghrita in female infertility,
year 1985,BHU,Varanasi.
3. Karmakar Rina- combinating female infertility with Ayurvedic herbal drugs in their
evaluation in modern prespective, year 1996, Institute of post graduate education &
research in ayurveda university of Kolkata,Kolkata.
4. Kaur Simaran pharmaceutical & chemical study on Phalakalyanaka ghrita w.s.r to
Vandhyattva, year 1998, Rishikul state ayurvedic college, Kanpur university,Kanpur.
5. Patange vinod d- an experimental study to identify the source plants of Lakshmana w.s.r
to its action on female infertility, year 2003,Dr.BK RR government ayurvedic college, AP
university, Vijayawada, Hyderabad.
6. Dr.Mahejabeen-An experimental study on Garbhapalarasa vis~a~vis teratogenicity,
year 2007,RGUHS ,S.D.M.C.A, Udupi.
In which 4 different groups were made with 8 rats in each group. 1st group was control
group where nothing was administered to the rat, 2nd group was given garbhaphala rasa,
3rd group was given known tetratogen ZnO, 4th group was given both GPR and ZnO.
Resutls obtained showed GPR depressed the CNS, depressed locomotor system and also
depressed cognitive and behavioural functions. GPR did not affect physical development
and growth in the pups.

6.3) OBJECTIVES OF STUDY:


-

Conceptual study on effect of Stree Vandhyattva with special reference to


abeejatva(anovulation)
To evaluate effect of Phalagritha on ovulation
To evaluate effect of Phalagritha on hormonal changes
To evaluate changes on cytoarchitecture of different parts of female reproductive system

7) MATERIAL AND METHODS:


7.1) source of data:
Test drug: Phalagritha taken as test drug to be given internally.
The animals :
-

Wistar strain albino female rats will be obtained from the animal house attached to the
Pharmacology Laboratory, SDMCA, Udyavara, maintained on Amrut brand animal
pellet feed of pranav agro industries and tap water would be given ad-libitum.

The temperature and humidity would be kept at optimum and animals would be exposed to
natural day and night cycles. The experiments would be carried out in conformity with guidelines
of the Institutional Animal Ethical Committee (IAEC) after obtaining its permission

Chemicals required:
-

DES(diethyl stilbestrol)
10% Formalin
Hormonal assay kit.

Animal grouping:
Group

No. of rats

Drugs

1
2
3
4

6
6
6
6

Normal water control


DES 30mg/kg dose body wt.
Phalagritha ( TED)
Positive control (DES 30mg/kg
dose ) +6Phalagritha (TED)

Statistical analysis:
-

The data obtained would be analysed by employing one way ANOVA followed by
Dunets multiple t- test as post hoc test. Graph pad Inst 3 would be used for this purpose.
Ap value of less than 0.05 would be considered to indicate statistically significant.

Inclusion criteria:

Healthy wistar albino female rats only, with average weight of 180-220 gms, will be
selected randomly for the study.
Rats exposed to DES preventing ovulation.

Exclusion criteria:

Infected
Pregnant
The healthy wistar rats which are under other experiments.

7.2) Experimental protocol:


PROCEDURE AND DESIGN OF THE STUDY:

The selected animal will be grouped into 4 groups with 6 animals each. Group 1, only water and
normal diet would be administered. Whereas group 2 will be treated with DES only. Group 3 will
receive test drug Phalagritha at therapeutic dose. While Group 4 will receive test drug at
therapeutic dose as well as DES will be administered for 21 consecutive days.
On 21st day, the rats will be sacrificed. Blood will be collected from retro-orbital puncture and
assigned for biochemical and hormonal investigation .The uterus, Ovary, fallopian tube, liver ,

kidney and heart will be excised out from sacrificed animal ,weighed and transferred to fixing
solution (10% formalin) for Histopathological examinations.
Procedure followed for preparation of Histopathological slides:
1. Fixation: The tissue would be excised out immediately after sacrificing the animals, cleaned
of extraneous tissue, cut into pieces of appropriate thickness and would be transferred into 10%
formalin solution. The tissue would be allowed to remain in it till they are taken up for
processing.

2. Tissue processing: Tissue processing involves dehydration, clearing and infiltration of the
tissue with paraffin. Tissue should be thoroughly washed by placing it under running tap water
and then passed through a series of following solvents as per schedule for dehydration, clearing
and paraffin infiltration.
Alcohol 70%
Alcohol 80%
Alcohol 90%
Alcohol 95% (2 changes)
Isopropyl alcohol
Acetone (2 changes)
Chloroform (3 changes)
Melted paraffin wax (600C) (2 changes)

- 20 minutes
- 20 minutes
- 20 minutes
- 20 minutes
- 20 minutes
- 20 minutes
- 20 minutes
- 30 minutes

Next the tissues should be embedded in paraffin wax to prepare tissue blocks. Tissue blocks will
be fixed to metal object holder after trimming them to suitable size.
3. Section cutting: The tissue sections of the 5-6 m thickness will be cut with the help of
Spencer type rotating microtone and floated in a water bath between 50-550C for 30 minutes.
Then they will be mounted on clear glass slides with a drop of Mayers egg albumin dried on hot
plate at about 500C for 30 minutes.

4. Staining : After fixing the section on slide, the sections will be stained by serially placing
them in the following reagents.

Xylol (2 changes)
Acetone
Alcohol 95%
Running water
Haematoxylin stain
Running water wash
Eosin working solution
Alcohol 95% (3 changes)
Acetone (2 changes)
Xylol (2 changes)

3 min
3 min
3 min
3 min
20 min
20 min
2 min
3 min
3 min
3 min

After passing through all the above reagents and stains, the slides would be covered with D.P.X.
(Diphenyl phthalein Xylene) and cover slip will be placed. Care would be taken to avoid air
bubble formation during mounting the slide.
The slides would be viewed under binocular research Carl-Zeisss microscope (Germany) at
various magnifications to note down the changes in the microscopic features of the tissues
studied.

OBSERVATION PARAMETER:
(i)
(ii)
(iii)
(iv)

Ponderal changes: Body weight, weight of Uterus, liver, heart and kidney.
In vivo investigations: oestrous cycle monitoring, sperm plug test, gestation
period, quality of pups (morphological examinations),number of pups.
Biochemical Parameters: Serum total cholesterol ,Serum triglyceride
Hormonal assay: Serum Estradiol, Progesterone, FSH, LH,GnRH
Histopathology of uterus, Ovary, fallopian tube, liver, kidney and heart

7.3) Does the study require any investigations or interventions to be conducted on patients
or other humans or animals? If so please describe briefly:
Yes, the study will be conducted on experimental animals.

7.4) Has ethical clearance been obtained from your institution in case of 7.3?
Yes, ethical clearance is obtained through ethical committee formulated in this institution.

7) List of references:
1. Susruta ,Sushruta Samhita with Nibandhasangraha commentary of Dalhanacharya and
Nyayachandrika panjika commentary of Gayadasacharya on Nidanasthana, edited by
Vaidya Yadavji Trikamji, Chaukhambha Orientalia Publications,Varanasi,2009 edition,Pp
824,pg no.348.
2. Agnivesa,Charak Samhita,vol.2, with Vaidyamanorama hindi commentary, edited by
Acharya Vidyadhar Shukla, Chaukhambha Sanskrit Pratishthan Publication, Delhi, 2009
edition, Pp916, pg no.704.
3. Vrddhajivaka ,Kasyapa samhita, (Vrddhajivakiya Tantra), edited by Vatsya with Vidyotini
hindi commentary, Chaukhambha Sanskrit Sanstha Publication, Varanasi, 2012 edition,
Pp364, pg no. 189.
4. Vrddhajivaka ,Kasyapa samhita, (Vrddhajivakiya Tantra), edited by Vatsya with Vidyotini
hindi commentary, Chaukhambha Sanskrit Sanstha Publication, Varanasi, 2012 edition,
Pp364, pg no. 157.
5. Vrddhajivaka ,Kasyapa samhita, (Vrddhajivakiya Tantra), edited by Vatsya with Vidyotini
hindi commentary, Chaukhambha Sanskrit Sanstha Publication, Varanasi, 2012 edition,
Pp364, pg no. 205.
6. D.C Dutta,Textbook of gyneacology including contraception, edited by Hiralal
Konar,new central book agency,kolkatta,5th edition 2009,Pp 627, pg no.222.
7. Susruta ,Sushruta Samhita with Nibandhasangraha commentary of Dalhanacharya and
Nyayachandrika Panjika Commentary of Gayadasacharya on Nidanasthana, edited by
Vaidya Yadavji Trikamji, Chaukhambha Orientalia Publications,Varanasi,2009 edition,
Pp824, pg no.669.

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8. Harita, Harita Samhita , with Nirmala hindi Commentary, edited by Vaidya Jaymini
Pandey, Chaukhambha Visvabharati publications,Varanasi,2010 edition ,Pp544 ,
pg no.463.
9. Vahat or Vrddha Vagbhata ,Astanga Sangraha with Sasilekha Sanskrit Commentary by
Indu, edited by Dr. Shivprasad Sharma, Chowkhambha Sanskrit Series
Office,Varanasi,2008, Pp 965,pg no.268.
10. Vagbhata, Astanga Hrdayam with Nirmala hindi Commentary, edited by Dr.Brahmananda
Tripathi, Chaukhambha Sanskrit Pratishthan, Delhi, 2012 edition, Pp1295, pg no. 339 and
1133.
11. Agnivesa, Charak samhita vol.2,with Vaidyamanorama Hindi Commentary, edited by
Acharya Vidyadhar Shukla, Chaukhambha Sanskrit Pratishthan Publication, Delhi, 2009
edition, Pp916, pg no.733.
12. Agnivesa, Charak samhita vol.1, with Vaidyamanorama Hindi Commentary, edited by
Acharya Vidyadhar Shukla, Chaukhambha Sanskrit Pratishthan Publication, Delhi, 2009
edition, Pp999, pg no.759.

13. Vrddhajivaka, Kasyapa samhita, (Vrddhajivakiya Tantra), edited by Vatsya with


Vidyotini Hindi Commentary, Chaukhambha Sanskrit Sanstha Publication, Varanasi,
2012 edition, Pp364, pg no.192.
14. Madhavkara, Madhava Nidanam , vol 2, edited with Vimala Madhudhara Hindi
Commentary and notes, edited by Brahmananda Tripathi,Chaukhambha Surbharati
Prakashan,Varanasi,2009 edition, Pp622 ,pg no.485
15. Bhela, Bhela Samhita with English translation, Commentary,critical notes by Dr.K.H
Krishnamurthy,edited by Prof. Priyvat Sharma, Chaukhambha
Visvabharati,Varanasi,2008 edition, Pp 660, pg no.240.

16. Sharangdhara, Sharangdhara Samhita by Pandit Sarangdharacharya with Commentary


Adhamallas dipika and Kasiramas gudhartha dipika, edited by Pandit Parasurama
Sastri,Vidysagar,Chaukhambha orientalia,Varanasi,2008 edition, Pp398, pg no.221.

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17.
Vahat or Vrddha Vagbhata ,Astanga Sangraha with Sasilekha Sanskrit
commentary by Indu, edited by Dr. Shivprasad Sharma, Chowkhambha Sanskrit Series
Office,Varanasi,2008, Pp 965,pg no.841.

18.
Vagbhata, Astanga Hrdayam with Nirmala hindi Commentary, edited by
Dr.Brahmananda Tripathi, Chaukhambha Sanskrit Pratishthan, Delhi, 2012 edition,
Pp1295, pg no. 1142.
19. Yogaratakara with Vidyotini Hindi Commentary by Vaidya Laksmipati Sastri, edited by
Bhisagratna Brahmasankar Sastri, Chaukhambha Prakashan, Varanasi, edition 2010,
Pp 504, pg no.408.
20. Bhavamisra, Bhavaprakasa,vol.2, Commentary by Dr.Bulusu Sitaram, Chaukhambha
Orientalia, Varanasi, edition 2010,Pp 770, pg no.696.

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9.

SIGNATURE OF CANDIDATE :

10.

REMARKS OF THE GUIDE:

11.

NAME & DESIGNATION OF


11.1] GUIDE :

Dr. V.N.K. USHA


M.D (Ayu)
DEPT. OF PG STUDIES,
PRASOOTI TANTRA AND STREE ROGA
S.D.M COLLEGE OF AYURVEDA,
UDUPI 574118

11.2] SIGNATURE :
11.3] CO-GUIDE :
RAVI MUNDUGARU
M.Sc. (Med. pharmacology)
RESEARCH OFFICER
S.D.M. RESEARCH CENTRE
UDUPI-574118.
11.4] SIGNATURE :
11.5] HEAD OF DEPARTMENT :
Dr. V.N.K. USHA
M.D (Ayu)
PROFESSOR AND H.O.D.
DEPT. OF PG STUDIES,
PRASOOTI TANTRA AND STREE ROGA
S.D.M COLLEGE OF AYURVEDA,
UDUPI 574118
11.6] SIGNATURE :
12] REMARKS OF THE
CHAIRMAN & THE PRINCIPAL :

12.1] SIGNATURE :

13

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