Evaluation of safety and efficacy of Maa-Lact in lactating Holtzman rats

Rohit Dhumal, N. A. Selkar, M. B. Chawda, K. S. Thakur, M. K. Vahalia, Venu Gopal Jonnalagadda, Geeta Vanage*

1National Centre for Preclinical Reproductive and Genetic Toxicology National Institute for Research in Reproductive Health, Parel, Mumbai-400012

2Shree Dhoothapapeshwar Ayurvedic Research Foundation (SDARF), Panvel, Navi Mumbai, Maharastra, India-410206

Evaluation of safety and efficacy of Maa-Lact in lactating Holtzman rats

Rohit Dhumal1, N. A. Selkar1, M. B. Chawda2, K. S. Thakur2, M. K. Vahalia2, Venu Gopal Jonnalagadda2, Geeta Vanage1*

1National Centre for Preclinical Reproductive and Genetic Toxicology National Institute for Research in Reproductive Health, Parel, Mumbai-400012

2Shree Dhoothapapeshwar Ayurvedic Research Foundation (SDARF), Panvel, Navi Mumbai, Maharastra, India-410206

ARTICLE INFO

Article history:

Received 1 November 2013

Received in revised form 15 December 2013

Accepted 16 December 2013

Available online 20 January 2014

Galactogogue

Objective: To evaluate the safety & efficacy of Maa-Lact granules for its galactogougue activity in Holtzman rats and its effect on suckling pups. Methods: Group I rats were treated as control, group II and III rats were treated with 500 mg/kg, 1 000 mg/kg of Maa-Lact granules for 21 days. Weekly body weights of dams and pups were collected, litter survivability for 22 days and ocular blood samples were collected on 1stday of parturition and 21stday of post parturition for the estimation of prolactin levels. On 21stday blood samples were collected from retro-orbital sinus for haemotological and biochemical estimations. On the same day of weaning rats were sacrificed and subjected to necropsy and individual organ weights were recorded. Results: No significant difference in weekly food weight consumption, body weights between control & treated groups with normal clinical signs. There is no mortaly in dams throught the study period with no significant difference in pups weights. The percentage mortality in pups was 14.43 %, 14.07 %, and 13.42% in group I, group II and group III, respectively. The histopathological finding has shown that treated groups have less convulution and adipose tissue deposition along with increase in length and branching of lactiferous duct and alveolar size. Conclusion: Based on above results, it can be concluded that Maa-Lact posseses significant galctogogue activity.

1. Introduction

Milk is the primary source nutrient for development and growth of the neonates in the weaning period. The composition of milk contains water, minerals, and organic nutrients to which baby have access. Colostrum is the first milk coming out from the mammary gland after parturition contains nutrient substances and further comes a mature milk which provides non-nutrient substances like antibodies and proteins. Low or lack of production of milk was one of the reason for discontinuation of breast feeding.

Galactogogues are the substances which enhances the milk production by assisting in initiation, augmentation, and maintainence of it [1]. Breast feeding offers a benefit to the child from sudden infant death symdrome and childhood leukaemia [2].

In Ayurveda, most of the formulations are herbomineral preparations, based on the ancient scripts of Charak samhitha, Sushruta samhitha, etc. Some of the herbal drugs having the galactogogue activity were Asparagus racemosus(A. racemosus)[3], Ipomea digitat(I. digitat)[4], Glycerrhiza glabra(G. glabra) [5], Leptadenia reticulate(L. reticulate), Bacopa monieri(B. monieri), Anethum sowa(A. sowa) [6], Centella asiatica(C. asiatica), fennel seeds, dill, borage, comfrey and Lamiaceae, etc [7,8]. Om Pharmaceuticals Limited, Bangalore, has developed a Maa-Lact preparation, a galactogogue, which contains Shatavari, Yashthimadhu, Shveta Sariva, Shunthi, Maricha , Pippali, Musta and Sharkara to stimulate the milk production, improve quality and quantity of it.

Compostion of Maa-Lact (10 g.) as follows: a. A. racemosus: 4 000 g; b. G. glabra:150 mg; c. Hemidesmus indicus(H.indicus): 150 mg; d. Zingiber officinalis (Z. officinalis): 50 mg; e. Piper nigrum: 50 mg; f. Piper longum: 50 mg; g. Cyperus rotundus (C. rotundus):150 mg; h. Sugar: q.s.

Various therapeutic uses have been established for A. racemosus such as galctogogue, aphrodisiac and demulscent activity[9]. G. glabra has hypolipidemic, anti-oxidant, antiinflammatory activity [10]. H. indicus for antinociceptive, hepatoprotective, antiallergic action [11]. Z. officinalis having antiemetic, cardioprotective, and immunomodulatory activity [12]. The present study was taken up to generate the preclinical data along with it’s safety to coordinate the clinical use and standardisation of the same.

2. Materials and methods

2.1. Chemicals

The chemicals were procured from the Sigma-Aldrich, Germany and serum prolactin ELISA kit was procured from IDS Ltd. USA.2.2. Animals

A total of 18 female Holtzmann rats were procured from animal house facility NIRRH, Mumbai; and transfered to the experimentation room, under controlled environmental conditions i.e (23±1) ℃ temperature and humidity (55±5) %, and in a 14 hr light/ 10 hr dark cycle with free access to feed containing crude protein, fiber and nitrogen free extract along with fresh purified water ad-libitum.

Rats were divided into 3 groups, group I as control receiving a 2 % CMC solution, group II & III were orally adiministered with 500 mg/kg and 1000 mg/kg of Maa-Lact for 21 days in a 2 % CMC solution. All the experimental procedure were performed in accordance with guidelines of CPCSEA and get the approval form IAEC before starting the experiments.

2.3. Observations

2.3.1. Feed consumption of female rats

The feed consumption was recorded every day starting from day 1 to day 22 and group average was calculated.

2.3.2. Body weight of females

Weight of the female rats was recorded weekly and the group average was calculated at the end of experiment.

2.3.3. Mean Body weight of pups

Average weight of surviving pups in each litter was recoreded every day from day 1 to day 22.

2.3.4. Litter survivability

Every day the litters were observed for any mortality. The percentage mortality at the end of the study (22 day) was also calculated.

2.3.5. Blood prolactin levels

Blood was collected on 1st day of delivery and on 22nd day of weaning; serum prolactin levels were estimated using the commercial ELISA kit.

2.4. Haemotology and serum biochemistry

After collecting the blood from the retro-orbital sinus, serum was separated and haemotological and biochemical estimations were performed.

They are haemoglobin (Hb), packed cell volume (PCV), total red cell count (RBC), total white cell count (WBC), absolute erythrocyte indices like mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), differential leuckocyte count and platelet counts using automatic haemotolgy analyzer (Abacus). Biochemical parameters like total protein, albumin, alanine amino transferase (ALT), aspartate amino transferase (AST), uric acid, creatinine, cholesterol, total bilirubin, direct bilirubin, and globulin levels by automatic biochemical analyzer using commercial kits.

2.5. Necropsy and histopathology

At the end of the study i.e on 22ndday rats were sacrificed using CO2asphyxiation and all the organs were properly weighed and collected in 10% neutral formalin sloution and subjected for histopathology. Mammary glands were subjected to histology and observations such as length and branching of lactiferous ducts, proliferation of alveoli, alveolar size, and adipose tissue in gland was performed..

2.6. Statistical analysis

Student “t” test was used to comapre treatment group with that of control andPvalue less than 0.05 was considered to be significant.

3. Results

3.1. Feed consumption & weekly body weights

There was no significant (P<0.05) difference in the weekly feed consumption between treated groups and that of control and there is no significant (P<0.05) difference in weekly ponderal changes between two treatment groups (Table 1).

3.2. Clinical signs & moratlity in dams

All the animals showed normal behaviour throughout the study. No mortality was observed in control as well as treated groups during the period of lactation till weaning.

3.3. Weekly male & female pups weight

No significant (P<0.05) weight difference was observed in the weekly body weights of male & female pups, and weights between two treatment groups and that of control group (Table 2).

Table 1Mean of weekly feed intake (g) & mean weekly body weights (g).

Table 2Average weekly male pup weights (g) & average weekly female pup weights (g).

3.4. Percentage mortality in pups

Mortality in pups in nornal control (Group I), Maa-Lact 500 (Group II), Maa-Lact 1 000 (Group III) was found to be 14.43 %, 14.07 % and 13.42 % respectively. There was no significant difference among control and treatmed groups (Table 3).

Table 3Average prolactin levels in blood (ng/mL) & percent mortality in pups (%)

3.5. Serum haemotological, clinical and prolactin levels

There was no significant (P<0.05) difference in various haemotological & clinical chemistry between control and treatment groups except WBC and creatinine values. In case of treatment groups WBC count was significantly (P<0.05) decreased, creatinine values were significantly (P< 0.05) increased as compared to control, but values were within normal range. On the day of delivery the serum prolactin values were comparable in control and treated groups. However on day of weaning i.e. on 21st day a significant decrease in prolactin levels were observed in treatment group as compared to control (Table 3 & 4).

3.6. Terminal body weights & absolute organ weights

No significant difference in terminal body weight was observed in female rats of treated groups and there is no significant difference in absolute organ weights of treated and control groups except weight of ovary in group III was significantly (P<0.05) increased (Table 5).

3.7. Histopathology of mammary gland

Terminal sacrificed animals didn’t show any gross pathological changes except lesions in liver, periportal mononuclear cell infiltration, cytoplasmic vaculization and hyperplasia of bronchus associated lymphoid tissue (BALT) in lungs. Histopathology of mammary gland didn’t shown any signs of toxicity related pathological changes. There is a increase in increase in involution of mammary gland & adipose tissue deposition in normal control group (Figure 1). Incase of treated groups there is less involution of mammary gland & adipose tissue deposition along with increase in length and branching of lactiferous duct and alveolar size (Figure 2, 3). These results indicate of prolong lactation/milk production in treatment group as compared with control group .

Table 4Average haemotological & biochemical values (g).

Table 5Terminal body weight and absolute organ weight (g).

Figure 1. Group I (Vehicle control) showing involution of mammary gland and with decreased active alveoli.

Figure 2. Group II (Maa Lact -500) showing active mammary gland and with increased active alveoli and alveolar space.

Figure 3. Group III (Maa Lact -1000) showing active mammary gland and with increased active alveoli and alveolar space & increased length and branching of alveolar length.

4. Discussion

Lactation is a natural and multiple complex process which serves as an invaluable food to baby. Several herbal medications have been used as a galactogogue along with modern medicine. These include domperidone, metoclopamide, antipsychotics sulpiride and chlorpromazine which acts through blocking dopamine receptors and subsequently increasing prolactin levels [13]. Meanwhile, some herbs like Fenugreek (Trigonalla foenum graecum), Galega (Goat’s rue, Galega officinalis), Silymarin (Milk thistle, Silybum marianum) and Shathavari (Stanya, Asperagus racemosus) has been used as a galactogogues in traditional medicine [14].

Galactogogues were mainly reported that they increase the proliferation of lactiferous tubules, there by increase the milk production [15]. Moreover some herbs increase the incraese the synthesis of lactogenic hormones such as growth hormone, prolactin, cortisol and B-casein in mammary glands [16]. Another proposed mechanism of action to increase the milk production was by inhibiting the dopamine pathway [17]. Almost all species share the common pattern of milk productioni.e., after parturition prolactin acts on mammary glands to increase the epithelial and secretory cells of it [18].

The main aim of the present study was to determine the galactogoue activity of Maa-Lact granules in rats, by means of pup weight gain and involution in mamary gland. There is no significant differences in feed consumption and ponderal changes were observed, along with no differences in male & female pups body weights and pups mortality. Meanwhile, there is no significant differences in the serum biochemical & clinical parameters excluding WBC levels i.e., levels were significantly decreased in treated group as (5.467 ±2.199) in group III when comapred with group I as (8.267±1.391). Where as, creatinine levels were significantly (P< 0.05) increased in treated groups i.e., group II and group III as (0.696±0.047) and (0.724±0.095) respectively. As expected, milk production in treated groups was increased due to less involution of mammary gland & adipose tissue deposition along with increase in length and branching of lactiferous duct and alveolar size supported by histopathological examination.

Results of the present study explained taht the milk production was significantly increased in treated groups which can be ascribed to the presence of A. racemosus [9] and supports the Maa-Lact as a galactogogue.

Mother’s milk is very important to the child as it contains plenty of nutrients required for nurturing the child. Mother’s feel lack of insufficient milk production, and face numerable number of hurdles in breastfeeding mainly due to physical, mental and emotional stresses.To conclude that, our research findings corroborate and validate the galactogoue activity of Maa-Lact, a polyherbal formulation using scientifically proven methods.

Conflict of interest statement

We don’t have potential conflicts of interest.

Acknowledgement

Authors would like to say their gratitude of thanks to Solumiks Herbaceuticals Limited (SHL), Mumbai for providing funds for the execution of this project (Grant no: SHL/ 2009/ 04).

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ment heading

10.1016/S2305-0500(13)60177-3

*Corresponding author: Dr. Geeta Vanage, National Centre for Preclinical Reproductive and Genetic Toxicology, National Institute for Research in Reproductive Health, Mumbai - 400 012, India.

E-mail: vanageg@nirrh.res.in

Foundation Project: This study was funded by Solumiks Herbaceuticals Limited (SHL), Mumbai (Grant no: SHL/ 2009/ 04).

Histopathology of mammary gland

Prolactin

Shathavari