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EVALUATION OF GALACTAGOGUE ACTIVITY OF LACTO VEDIC –A POLYHERBAL FORMULATION

SYNOPSIS FOR

M.PHARM DISSERTATION


SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

NARASIMHARAJU KALIDINDI

I M.PHARM

DEPARTMENT OF PHARMACOLOGY

VISVESWARAPURA INSTITUTE OF

PHARMACEUTICAL SCIENCES

BANGALORE-560070

(2009-2010)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.


ANNEXURE-II




PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION






1.


Name of the candidate and address(in block letters)

NARASIMHARAJU KALIDINDI


S/O K.ARJUNA RAJU

NR.PADMA INDUSTRIES, WARD NO-2,

VIDYANAGAR,GANGAVATHI,

KOPPAL(DT), KARNATAKA,

PIN CODE-583227






2.

Name of the institution

VISVESWARAPURA INSTITUTE OF PHARMACEUTICAL SCIENCES









3.



Course of study and subject

MASTER OF PHARMACY IN

PHARMACOLOGY









4.



Date of the admission



30th MAY 2008





5.

Title of the topic:



Evaluation of Galactagogue activity of Lacto Vedic –A Polyherbal Formulation







6.




BRIEF RESUME OF THE INTENDED WORK

6.1 NEED FOR THE STUDY

Lactating mothers use lactogenic herbs or foods for improvement of the milk production. Some of them will use several combinations of herbs to increase the pumping of milk to their children. Mothers milk is important for survival, proper development and growth of the neonate. Milk is the only source of water, organic nutrients and minerals, to which the neonates has access. Colostrum (the first milk taken from the mammary gland after posturition) and mature milk contain non nutrient substances (such as anti bodies and bioactive factors) that may be important for growth development and survival of the neonate.

Ayurvedic medicines, based on ancient scripts like Sushrutha Samhitha, Charaka Samhitha, etc are herbal formulations. Unfortunately, there is no documentary evidence to prove the medicinal properties/clinical efficacy of the herbal medicine. It necessary to generate data through systematic studies to support & justify medicinal uses of ayurvedic drugs/herbal formulations.

Low supply of milk is one of the most common reasons given for discontinuing breast feeding. Galactagogues are medications or substances believed to assist initiation, maintenance and augmentation of maternal milk production.1

The various herbal drugs having the galactagogue activity are Leptadenia reticulate, Asparagus racemosus, Ipomoea digitata, Glycerrhiza glabra, Centella asciatica, Bacopa monnieri, Anethum sowa, etc.

Lacto Vedic 500mg capsules, Manufactured by Vedic Bio Labs, Bangalore, available as natural milk enhancer, contains:
Extracts of:
1. Jivanti 100 mg

2. Shatavari 100 mg

3. Vidarikanda 100 mg

4. Yastimadhu 100 mg

5. Shatapushpa (powder) 100 mg
The present study is taken up to generate preclinical data to support the clinical use of the poly herbal formulation-(Lacto Vedic) as Galactagogue.






7

8





    1. REVIEW OF LITERATURE:



Some basic terminologies:


Galactagogues: Galactagogues are those herbs or supplements or drugs that can increase the secretion or production of milk.

Hypogalactia: It is the condition where galactagogue & lactation herbs are helpful. In this condition, there is inadequate secretion of breast milk.

Agalactia: This condition refers to loss of or complete lack of secretion of breast milk. One should consult a qualified herbalist or doctor for this condition.

Galactophyga: Herbs which diminish milk-flow and arrest the secretion of milk.

Galactopoiesis: The secretion and continuing production of milk by the mammary glands beyond need of the baby.

Galactorrhea: The excessive flow of milk from the breasts during lactation; spontaneous milk flow, such as when happens when a nursing mother hears another infant cry and her own breasts spurt milk.2

Role of hormones in lactation:

The role of galactopoietic hormones such as Prolactin & Growth hormone in maintenance of lactation is well established. Prolactin (PRL), considered the major galactopoietic hormone, is released at the time of milk removal in ruminants and nonruminants.3 PRL plays a key role in the growth and differentiation of the mammary gland as well as in the initiation and maintenance of milk production.4 Prolactin levels rise markedly during pregnancy, reach a maximum at term, and decline thereafter unless the mother breast-feeds the child. Suckling or breast manipulation in nursing mothers stimulates circulating prolactin levels, which can rise ten to one hundred fold within 30 minutes of stimulation5

The timing of oxytocin release relative to milk removal is an important factor affecting milk ejection. Non lactating cows will release oxytocin in response to udder stimulation. But, virgin heifers do not respond substantially to udder stimulation. Apparently maximum oxytocin release in response to udder stimulation occurs only if the mammary gland is lactating or has lactated. Oxytocin concentration during milking declines as lactation progresses.6

The role of dopamine in regulating glucocorticoid and prolactin secretion was investigated in lactating Holstein cows by characterizing serum cortisol and prolactin responses to fluphenazine, a dopamine receptor antagonist. A dopamine antagonist increased cortisol, suggesting that endogenous dopamine, at least in part, regulates cortisol and prolactin secretion. These effects are regulated through dopamine receptors in anovulatory lactating dairy cows during the early postpartum period.7



Lacto Vedic 500mg capsules available as natural milk enhancer, contains:
Extracts of:
1. Jivanti 100 mg

2. Shatavari 100 mg

3. Vidarikanda 100 mg

4. Yastimadhu 100 mg



5. Shatapushpa (powder) 100 mg

The following table shows the properties of ingredients of Lacto Vedic



Name of constituent

Botanical name

Family

Part of plant used

Chemical constituents

Medicinal uses

Jivanti8

Leptadenia reticulata

Asclpiadaceae

Roots

Leptadenol, triacontane, cetyl alcohol, leptidin1, B-sitosterol, etc

Galactagogue, nutritive, aphrodisiac, stimulant, diuretic, etc

Shatavari 9, 11.

Asparagus racemosus

Liliaceae

Roots

Shatavarin I-IV, quercetin, rutin & hyperoside, etc

Galactagogue, aphrodisiac, demulsant, etc

Vidarikanda10

Ipomoea digitata

Convolvulaceae

Tuberous root

Pterocarpanone, hydroxytuberosone, pterocarpene-anhydrotuberosine, 3-0-methyltuberosin,

Galactagogue, in hepatomegaly, in splenomegaly, anti-tubercular, etc

Yastimadhu11,12

Glycerrhiza glabra

Leguminosae

Radix

Glycyrrhizin, liquiritin, stigmasterol, B-sitosterol, glycchetinic acid, anethole, eugenol, etc

Expectorant , demulcent, anti-inflammatory, diuretic, hepatoprotective, anti-spasmodic, etc

Shatapushpa13

Anethum sowa

Umbelliferae

Flower head

Anethole, estragole, fenchone, B-sitosterol, etc

Carminative, Galactagogue, aromatic, diuretic, anti-spasmodic, expectorant, anti tussive, etc




    1. OBJECTIVE OF THE STUDY




  • Evaluation of Galactagogue activity of Lacto Vedic – A Polyherbal formulation, will be done in rats by,14

  1. Estimation of Milk yield.

  2. Estimation of Pups body weight.

  3. Estimation of serum prolactin.15, 16.

  4. Estimation of Pituitary prolactin concentration.

  5. Histology of mammary glands.


MATERIAL AND METHODS


    1. Source Of Data :

Data will be generated by performing experiments on animals. The standard information is collected from various journals, standard Text books available in library of Visveswarapura Institute of Pharmaceutical Sciences, Indian Institute of Science, RGUHS digital library and from various standard websites.


Web sites: www.sciencedirect.com

www.pubmed.com

www.google.com

www.ijp-online.com

www.himalayahealthcare.com

www.rguhs.ac.in/j-gate@helinet



    1. METHOD OF COLLECTION OF DATA:

The data will be generated by performing the experiments using animal models like rats.

Evaluation of Galactagogue activity of Lacto Vedic – A Polyherbal formulation, will be done in rats.


    1. METHODOLOGY :


Animals: Wistar fertile rats (225-250g) will be procured from NIMHANS, Bangalore. All animals will be maintained under husbandry conditions with access to food and water ad libitum.

.
Effect of oral treatment with Lacto Vedic on milk production on rats.


Experiment I:
Following groups will be made

Group 01: animals (n=6) will be administered the vehicle (0.9% Nacl) p.o. X 13 days

Group 02: animals (n=6) will be administered the Lacto Vedic (lower dose) p.o. X 13 days

Group 03: animals (n=6) will be administered the Lacto Vedic (higher dose) p.o. X 13 days

Group 04: animals (n=6) will be administered the reference standard (Domperidone) p.o.

X 13 days14



Note: Dose calculation is based on human dose.
Procedure:

Animals will be treated daily, starting on day-2 of lactation; milk production will be estimated at 18hrs after treatment. Milk production will be measured from day 3 to day 15 of lactation. Milk yield and body weight of dams and weight gain of pups will be measured each day.

Every day during the study period, after 13 hrs of treatment, the pups will be weighed (w1) and subsequently isolated from their mother for 4 hrs and then the pups will be weighed (w2), then returned to their mother and allowed to feed for 1 hr and they will be weighed (w3).

Milk yield 18 h after the gavage will be estimated as w3– w2. Daily milk yield will be corrected for weight loss due to metabolic processes in the pup (respiration, urination and defecation) during suckling. The value used will be (w2 – w1)/4. This value will be then multiplied by the number of suckling hours per day and added to the daily suckling gain.

Daily weight gain of pups will be calculated from the pup weight at w2.

Experiment II:
Following groups will be made

Group 01: animals (n=8) will be administered the vehicle (0.9% Nacl) p.o. X 13 days

Group 02: animals (n=8) will be administered the Lacto Vedic p.o. X 13 days

Group 03: animals (n=8) will be administered the reference standard (Domperidone) p.o. X

13 days14

Note: Dose calculation is based on human dose.

Procedure :

The extract will be administered orally with a gavage syringe each day. Milk production will be estimated at 18 and 23 hrs after gavage.

For the measurement of milk yield 18 hrs after gavage, the same procedure as described above for experiment I will be followed.

For measurement of milk yield 23 hrs after gavage, the pups will be subsequently isolated after measuring w3, for 4 hrs and then weighed (w4), then they will be reunited with their mother for 1 hr of feeding and, finally, they will be weighed (w5). They will be subsequently left with their mother during the night.

Milk yield 23 hrs after treatment will be estimated as w5 – w4. Daily milk yield will be corrected for weight loss due to metabolic processes in the pup (respiration, urination and defecation) during suckling.

The value used for correction for weight loss will be [(w2 – w1) + (w4 – w3)]/8. This value will be then multiplied by the number of suckling hours per day and added to the daily suckling gain.

Daily weight gain of pups will be again calculated from the pup weight at w2.14
Parameters:

Estimation of serum prolactin:15

On day 1 (at dioestrous) and day 3 (at oestrus) of the treatment, two plasma samples will be taken before and five samples after the injection, at 20-min intervals. The blood samples will be centrifuged and plasma will be stored at -20­0C until radioimmunoassay (RIA) for PRL content.14, 16.


Estimation of pituitary prolactin:

At the end of the experiment, the animals will be sacrificed under ether anaesthesia. Pituitaries will be collected for PRL extraction. Extracted with PBS pH 7·4, 0·03 M; 10 μl of the homogenate will be diluted with phosphate buffer plus 1% BSA and stored at -20°C.

Plasma and pituitary PRL levels will be measured.14
Histology of mammary tissue:

The two inguinal mammary glands will be removed, immediately fixed in alcoholic Bouin and then embedded in paraffin wax after dehydration in a graded series of ethanol and xylene. Paraffin sections (5 μm) of the mammary glands will be sliced and stained with Harris’ haematoxylin and eosin. Mammary gland structures will be identified.14




    1. STATISTICAL ANALYSIS

The results will be expressed as Mean ±SEM for each parameter. The data will be analysed by one-way ANOVA using Graph pad INSTAT followed by the Bonferroni or Scheffe test or least square difference (LSD), using the statistical package SPSS (version 7·5 for Windows). Data that will not normally distributed (plasma PRL concentrations in experiment II) will be analysed using the non-parametric Kruskal–Wallis test.


7.5 Does the study require any investigation to be conducted on patients

Or other humans or animals? If so please describe briefly

No, studies do not need any patients.

Yes, The above study requires investigation on animals. The effect of drug will be studied on various parameters using rats as animal model.

7.6 Has ethical clearance been obtained from your institution in case of 7.3?

Yes, Ethical clearance certificate is attached.



REFERENCES:


  1. Sjolin S, Hofvander Y, Hillervik C. Factors related to early termination of breastfeeding: A retrospective study in Sweden. Acta Paediatr Scand, 1977,(66):505-11.

  2. http://www.holistic-herbalist.com/galactagogue.htm (accessed on 16/11/2008).

  3. Tucker HA, Knobil E, Neill JD. In: Lactation and its hormonal control, In: The Physiology of Reproduction. Raven Press, New York, 1994, pp. 1065-98. (Vol. 2).

  4. Tucker HA, Physiological control of mammary growth: Lactogenesis and Lactation. J Dairy Sci, 1981,(64):1403-21.

  5. Trott JF, Vonderhaar BK, Hovey RC. Historical perspectives of Prolactin and Growth Hormone as Mammogens, Lactogens and Galactagogues—Agog for the Future. J Mammary Gland Biol Neoplasia, 2008,(13):3–11.

  6. Brunton LL, Lazo JS, Parker KL. Goodman & Gilman’s The Pharmacological Basis of Therapeutics.11th ed., McGraw-Hill, New York, 2006, pp. 1543-46.

  7. Ahmadzadeh A, Barnes MA, Gwazdauskas FC, Akers RM. Dopamine Antagonist Alters Serum Cortisol and Prolactin Secretionin Lactating Holstein Cows. J. Dairy Sci., 2006,(89):2051-55.

  8. http://www.himalayahealthcare.com/herbfinder/h-leptad.htm#c (accessed on 18/11/2008).

  9. Goyal RK, Singh J, Lal H. Asparagus Racemosus - An Update. Ind J Med Sci., 2003,9(57):407-14.

  10. http://www.holisticonline.com/Herbal-Med/_Herbs/h203.htm (accessed on 18/11/2008).

  11. Moharana D. Shatavari, Jastimadhu and Aswagandha: The Ayurvedic Therapy. Orissa Review, 2008,72-7.

  12. Kokate CK, Purohit AP, Gokhale SB. In: Drugs containing glycosides, Pharmacognosy. 39th ed., Nirali Prakashan, Pune, 2007, pp. 212-20.

  13. http://www.himalayahealthcare.com/herbfinder/h-shatapush.htm (accessed on 18/11/2008)

  14. Ouedrago ZL, Heide DV, Beek EMV, Swarts HJM, Mattheij JAM, Sawadogo L. Effect of aqueous extract of Acacia nilotica ssp adansonii on milk production and prolactin release in the rat. Journal of Endocrinology. 2004, (182):257-66.

  15. Duhau L, Grassi J, Grouselle D, Enjalbert A, Grognet JM. An enzyme immunoassay for rat prolactin, Application to the determination of plasma levels. J. Immunoassay, 1991, 12(2):233-50.

  16. Grassi J, Pradelles PH. Compounds labelled by the acetyl cholinesterase of Electrophorus Electricus: Its preparation process and its use as a tracer or marquer in enzymo-immunological determinations. United States patent. 1991,1,047,330.

  17. Ramesh PT, Mitra SK, Suryanarayna T, Sachan A. Evaluation of Galactin: A Herbal Galactagogue preparation in dairy cows. The Veterinarian, 2000, (24).1-3.

  18. Plaut K, Bauman DE, Agergaard N, Akers RM. Effect of exogenous prolactin administration on lactational performance of dairy cows. Domest. Anim. Endocrinol. 1987, (4):279-90.

  19. Sagi RC, Gorewit DB, Wilson Role of exogenous oxytocin in eliciting milk ejection in dairy cows. J Dairy Sci. 1980, 63(12):2006-11.














































































































09.

SIGNATURE OF THE CANDIDATE





10.

REMARKS OF THE GUIDE





11.




NAME AND DESIGNATION








11.1 NAME OF THE GUIDE



Dr. MEERA SUMANTH

PROFESSOR

DEPARTMENT OF PHARMACOLOGY

V.I.P.S, BANGALORE-70.



11.2 SIGNATURE






11.3 CO-GUIDE (IF ANY)






    1. SIGNATURE

.




    1. HEAD OF THE

DEPARTMENT



Dr. RAJU B KONERI

H.O.D; PROFESSOR

DEPARTMENT OF PHARMACOLOGY

V.I.P.S, BANGALORE-70.



11.6 SIGNATURE





12.

12.1 REMARKS OF THE

CHAIRMAN & PRINCIPAL








12.2 SIGNATURE











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