CC BY-NC-ND
0Document heading doi: 10.1016/S2305-0500(13)60177-3
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 Jonnalagadda , Geeta Vanage *
'National 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
ABSTRACT
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 1st day of parturition and 21st day of post parturition for the estimation of prolactin levels. On 21st day 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.
ARTICLE INFO
Article history:
Received 1 November 2013
Received in revised form 15 December 2013
Accepted 16 December 2013
Available online 20 January 2014
Keywords: Galactogogue
Histopathology of mammary gland
Prolactin
Shathavari
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,
*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).
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) °C 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 22nd day rats were sacrificed using CO2 asphyxiation 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 and P value 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
3.3. Weekly male & female pups weight
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.
Table 1
Mean of weekly feed intake (g) & mean weekly body weights (g).
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).
Group Mean of weekly feed intake Mean weekly body weights
1st week ~nd i ~rd i 2 week 3 week 1st week nd rd 2 week 3 week 4th week
Group I 33. 603±4.556 49.946±5.406 55.969±11.173 275.883±26.482 285.550±22.885 282.967±17.967 285.00±14.913
Group II 38.469±2.473 53.081±5.725 62.671±3.783 283.150±27.535 301.700±28.847 303.883±30.361 301.783±30.779
Group III 33.278±6.407 46.850±11.763 58.346±16.011 298.800±19.233 307.017±15.212 308.667±15.769 307.550±21.586
Table 2 Average weekly male pup weights (g) & average weekly female pup weights (g).
Group Average weekly male pups weights Average weekly female pups weights
1st day 1st week 2nd week 3 rd week 1s day 1st week 2nd week 3rd week
Group I 7.216±1.020 Group II 80.152±1.461 Group III 7.344± 0.517
14.653±2.207 15.968±3.969 15.270±2.032
27.532±3.996 28.825±6.488 27.557±4.601
40.625±5.654 43.971±9.410 44.631±7.879
7.038±1.000 7.411±1.708 7.128±2.866
14.423±2.311 15.816±3.952 15.556±2.018
26.795±3.372 27.906±6.537 25.812±4.776
42.392±8.490 40.700±8.235 43.868±7.236
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 3
Average prolactin levels in blood (ng/mL) & percent mortality in pups
Group Average prolactin levels in blood (ng/mL) Percent mortality in
0th day 21st day pups (%)
Group I 23.800±6.544 35.830±9.075 14.439±14.544
Group II 24.030±23.909 23.910±7.027 14.078±10.637
Group III 31.190±13.402 14.780±5.526 13.420±12.339
P< 0.05 and considered as significant when compared with control
group.
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 4
Average haemotological & biochemical values (g).
Parameter Group I Group II Group III
Haemoglobin (g/dL) 15.583±0.840 15.100±0.587 15.050±0.898
RBC (1012/L) 9.395±0.508 9.180±0.583 9.073±0.509
PCV (%) 48.767±2.056 50.383±2.344 48.600±2.160
MCV (pg) 51.933±1.421 54.950±1.550 53.633±2.039
MCH (fL) 16.567±0.242 16.467±0.497 16.600±0.089
MCHC (g/dL) 31.967±1.113 30.033±0.0674 31.000±1.185
WBC (109/L) 8.267±1.391 7.133±3.836 5.467±2.199*
Lymphocytes (%) 66.033±3.369 71.117±2.084 66.767±3.717
Neutrophils (%) 31.483±3.864 25.383±3.485 31.300±4.334
Monocytes (%) 1.900±0.885 3.167±1.389 1.300±1.026
Platelets (X10'/L) 461.833±23.216 515.500±99.198 347.167±192.805
Total Protein (g/dL) 8.684±0.491 9.072±0.423 9.250±0.590
Albumin (g/dL) 3.128±0.198 3.282±0.070 3.176±0.251
Globulin (g/dL) 5.556±0.304 5.790±0.379 6.074±0.405
SGPT (IU/L) 155.920±46.188 183.760±53.018 154.076±67.293
SGOT (IU/L) 152.660±18.234 175.240±24.615 188.680±37.277
Bilirubin (mg/dL) 0.082±0.031 0.086±0.029 0.086±0.017
Direct Bilirubin (mg/dL) 0.046±0.017 0.054±0.0.41 0.046±0.015
Indirect Bilirubin (mg/dL) 0.036±0.015 0.032±0.015 0.040±0.007
Cholesterol (mg/dL) 119.350±19.840 131.260±14.832 126.060±10.417
Uric Acid (mg/dL) 2.730±0.433 2.976±1.299 3.056±1.121
Creatinine (mg/dL) 0.616±0.019 0.696±0.047* 0.724±0.095*
P< 0.05 considered as significant when compared with control group.
Table 5
Terminal body weight and absolute organ weight (g).
Organ Group I Group II Group III
Body weights 285.000±14.913 301.833±30.675 307.500 ±21.603
Heart 1.217±0.170 1.179±0.090 1.368±0.177
Liver 12.861±2.044 15.239±1.690 14.134±2.097
Kidney 2.101±0.266 2.211±0.320 2.269±0.217
Adrenal 0.112±0.024 0.100±0.022 0.124±0.034
Spleen 0.701±0.090 0.718±0.102 0.716±0.068
Brain 1.887±0.074 1.917±0.111 1.960±0.118
Ovary 0.145±0.030 0.176±0.037 0.205±0.054*
Uterus 0.508±0.208 0.554±0.147 0.594±0.285
P< 0.05 considered as significant when compared with control group
Figure 2. Group II (Maa Lact -500) showing active mammary gland and with increased active alveoli and alveolar space.
Figure 1. Group I (Vehicle control) showing involution of mammary gland and with decreased active alveoli.
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 production i.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
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).
References
[1] Sjolin S, Hofvander Y, Hillervik C. Factors related to early termination of breast feeding: A retrospective study in Sweden. Acta Paediatr Scand 1977; 66: 505-511.
[2] Stuebe A. The risks of not breastfeeding for mothers and infants. Rev Obstet Gynecol 2009; 2: 222-231.
[3] Goyal RK, Singh J, Lal H. Asparagus racemosus-An update. Indian J Med Sci 2003; 9: 407-414.
[4] Moharana D. Shatavari, jastimadhu and aswagandha .the ayurvedic therapy. Bhuvaneswar: Orissa Review; 2008, p. 72-77.
[5] Kokate CK, Purohit AP, Gokhale SB. Drugs containg glycosides. In: Pharmacognosy. 39th ed. Pune: Nirali Prakashan; 2007, p. 212-220.
[6] Sumanth M, Narasimharaju K. Evaluation of galactogogue activity of Lactovedic: A polyherbal formulation. Int J Green Pharm 2011; 5: 61-64.
[7] Dog TL. The use of botanicals during pregnancy and lactation. Altern Ther Health Med 2009; 15: 54-59.
[8] Zapantis A, Steinberg JG, Schilit L. Use of herbals as galactagogues. J Pharm Pract 2012; 25: 222-231.
[9] Sharma K, Bhatnagar M. Asparagus racemosus (Shatavari): A versatile female tonic. Int J Pharm Bio Arch 2011; 2: 855-863.
[10]Vispute S, Khopade A. Glycyrrhiza glabra Linn. - "Klitaka": A review. Int J Pharm and Bio Sci 2011; 2: 42-51.
[11]Austin A. A review on Indian sarsaparilla, Hemidesmus indicus (L.) R.Br. J Bio Sci 2008; 8: 1-12.
[12]Mishra RK, Kumar A, Kumar A. Pharmacological activity of Zingiber officinale. Int J of Pharm Chem Sci 2012; 1: 1073-1078.
[13]Gabay MP. Galactogogues: Medications that induce lactation. J Hum Lact 2002; 18: 274-249.
[14]Zuppa AA, Sindico P, Orchi C, Carducci C, Cardiello V, Romagnoli C. Safety and efficacy of galactogogues: substances that induce, maintain and increase breast milk production. J Pharm Pharm Sci 2010; 13(2): 162-174.
[15]Lompo-Ouedraogo Z, van der Heide D, van der Beek EM, Swarts HJ, Mattheij JA, Sawadogo L. Effect of aqueous extract of Acacia nilotica ssp adansonii on milk production and prolactin release in the rat. J Endocrinol 2004; 182: 257-266.
[16]Sawadogo L, Houdebine LM, Thibault JF, Rouau X, Olivier-Bousquet M. Effect of pectic substances on PRL and growth hromone secretion in the ewe and on the induction of casein synthesis in the rat. Reprod Nutr Dev 1988; 28: 293-301.
[17]Dog TL. The use of botanicals during pregnancy and lactation. Altern Ther Health Med 2009; 15: 54-59.
[18]Hadsell D, George J, Torres D. The declining phase of lactation: Peripheral or central, programmed pathological? J Mammary Gland Biol Neoplasia 2007; 12: 59-70.
We don't have potential conflicts of interest.
