Galactagogue Effect of Euphorbia hirta (Euphorbiaceae) Aqueous Leaf Extract on Milk Production in Female Wistar Rats

Background: Populations of Gontougo (Côte d’Ivoire) commonly used Euphorbia hirta (Euphorbiaceae) leaves as galactagogue to boost milk production in lactating women. Our study was carried to verify this traditional belief via evaluations of milk production, serum prolactin levels, body weight and mammary lobuloalveolar proliferation induced by the aqueous leaf extract of this medicinal plant in Wistar rats. Methods: 6 groups of 6 virgin rats and 6 groups of 6 lactating rats were used. Each lactating rat was isolated with its six pups. These groups of animals were treated orally with distilled water, Metoclopramide (Metocl 5 mg/kg) and E. hirta aqueous leaf extract (EHae 100, 200, 400 and 600 mg/kg) daily during 17 days of lactation. The indirect method which assimilates the weight gain of untreated pups to the quantity of milk produced by lactating rats was used. At the end of the treatment, histological sections of the abdominal mammary glands of lactating rats were observed. Results: EHae administration induced an increase of milk production in lactating rats. This action of EHae was stronger at the dose of 200 mg/kg. of the lobuloalveolar system of the mammary glands in treated female rats. This galactagogue property could partly justify the traditional use of this plant to boost milk production in lactating women.

finding makes breastfeeding a real public health problem on a global scale and more specifically in Africa with social and economic implications. There are still many constraints to its total adoption by populations. These include socioprofessional, cultural and/or aesthetic constraints [7] [14].
In developing countries and especially in rural areas where breast milk is the main infant food, hypogalactia remains the major constraint related to breastfeeding. Many mothers wanting to breastfeed their children are affected by this problem. They have low breast milk production [15]. These women cannot get the pharmacy products because of their high costs. In modern therapeutic, several medications have been used as galactagogues. Galactagogues are medications or substances to assist initiation, maintenance and augmentation of maternal milk production. Metoclopramide, domperidone, and the antipsychotics sulpiride and chlorpromazine are some galactagogues used in modern medicine [16]. They use medicinal plants to increase the production of breast milk. Previous works have shown the galactagogue activity of some plants including Leptadenia reticulate, Ipomoea digitata, Glycerrhiza glabra, Centella asciatica, Bacopa monnieri, Anethum sowa, fenugreek etc. [17]. Bako et al. [18] showed the galactagogue activity of Hibiscus sabdariffa (Malvaceae). The ethyl acetate fraction of the flowers significantly increases milk production and PRL synthesis.
According to Datti et al. [19] and Lompo-Ouedraogo et al. [20], the leaves of Hippocratea obtusofolia (Alismataceae) and Acacia nilotica ssp adansonii (Fabaceae) would be capable of inducing galactagogue activities in guinea pigs and rats, respectively. The work of Gbadamosi and Okolosi [21] helped to safeguard traditional knowledge about the use of several galactagogue plants. In Côte d'Ivoire, 95% of women admit to having used galactogenic plants including Euphorbia hirta (Euphorbiaceae) to induce milk production [22]. Many traditional medicinal practices commonly use Euphorbia hirta (Euphorbiaceae) to stimulate milk production in nursing mothers in Gontougo Region (Côte d'Ivoire). Gontougo located in the east is one of the very poor regions of Côte d'Ivoire.
Thus, the objective of this study was to verify the traditional belief regarding galactagogue property of E. hirta. This belief says that E. hirta increase milk production in lactating women. For this, the quantities of milk produced by lactating rats as well as weight gains of their pups were measured during 17 days.
Histological sections of mammary glands of lactating females were observed.
Serum PRL concentration was also measured in virgin females. According to this method, the milk yield produced is given by the difference in weight of the pups before and after the feeding sessions. Weight gains are considered to be the milk yield ingested by the pups. However during each feeding session, it is noted use of ingested milk for the production of energy for respiration and movement of pups. This milk yield is therefore evaluated by the correlation coefficient of the weight loss. The milk yield measured is given by the following equation:

Preparation of Euphorbia hirta Aqueous Extract
: Weight gain of the pups after lactating.
( ) : Correlation coefficient of the weight loss.

Histological Study of Mammary Gland
At J18 of post-partum, experimental lactating rats were anesthetized with ethyl urethane and sacrificed [30]. Then the upper abdominal pairs of the mammary glands were taken for histological study according to the standard method described by Bouagnon et al. [31] and Zougrou et al. [32]. Sections were taken from abdominal tissues, dehydrated in gradual ethanol, cleared in toluene and infiltrated in paraffin using automatic tissue processor. After routine processing, paraffin sections of each tissue were cut into 5 μm thickness, stained with haematoxylin and eosin, and observed under light microscope.

Evaluation of Serum PRL
36 virgin rats were divided into 6 groups of 6 animals. Group 1 received distilled water. Group 2 received Metocl 5 mg/kg. The other groups (3,4,5,6) were treated with EHae at 100, 200, 400 and 600 mg/kg respectively. Drugs were administered daily to the animals in a single dose. At the days 0, 7 and 14 of experimental period, animals were starved (16 hours), anaesthetized with ethyl urethane [30] [33]. Blood samples were collected from the animals through caudal puncture. Blood samples were centrifuged at 3000 rpm for 10 min [34]. Serum was separated from the clot with pasteur pipette and dispensed into clean tube for the measurement of prolactin according to standard methods using the MiniVidas auto-analyser (Biomérieux, France). An immuno-enzymatic method was used for this purpose [35].

Statistical Analysis
All the data were expressed as Mean ± Standard Error of Means (SEM). Statistical analyses were performed by one way analysis of variance (ANOVA) and differences between means were determined by Turkey's Multiple Comparison test using Graph Pad Prism 7.0 program (Microsoft, San Diego California, USA).
A value of P < 0.05 was considered significant.

Milk Production
Milk yields produced daily by the female rats before and after administration of the test substances were measured. The data revealed that milk yield produced by the rats in each experimental group were not proportional to the days of lactation ( Figure 1). It has been observed periods of high and low increase of milk production. An increase of milk production was observed at intervals D4-D7 and D10-D14 when lactating rats were treated separately with Metocl 5 mg/kg and EHae. In contrast, a decrease production was observed at intervals D7-D10 and D14-D17. Figure 2 shows the milk yields produced by the rats from D3 to D17. Metocl 5 mg/kg induced a milk yield of 40.9 ± 2.2 g. With EHae, the most active dose was 200 mg/kg. The rats receiving this dose produced 39.38 ± 1.5 g of milk. This value is substantially equal to that obtained with Metocl 5 mg/kg (P > 0.05).
Compared with control rats (28.05 ± 0.57 g), Metocl 5 mg/kg and EHae 200 mg/kg induced highly significant increases of 45.78% (P < 0.0001) and 40.36% (P < 0.0001). Quantities of 36.32 ± 1.2 g and 34.12 ± 1.3 g were obtained with EHae at 400 and 600 mg/kg respectively, corresponding to increases of 28.43% and 22.63%. 28.1 ± 1 g of milk were produced when EHae 100 mg/kg was used, an increase of 0.17%. EHae at this low dose did not affect the production of milk in lactating rats (P > 0.05).

Body Weight of Pups
From day 1 of postpartum to day 17 of the experiment, the weights of the pups receiving exclusively breast milk were raised. On day 1 of the post partum, the weight of the six rats in the control group was estimated at 36.5 ± 0.101 g. Those of the pups whose mothers were treated with Metocl 5 mg/kg and the doses of EHae (100; 200; 400; 600 mg/kg) were 36.3 ± 02; 35 ± 3.1; 36 ± 1.2; 34.7 ± 0.38 and 38.4 ± 2.3 g respectively. Comparing the weight of pups of different groups showed no significant difference at post partum D1 (P > 0.05). At the end of the experiment, weights were 129.2 ± 2.3 g and 127.7 ± 1.4 g respectively for pups whose mothers had received Metocl 5 mg/kg and EHae 200 mg/kg. These two recorded values were greater than that of the pups whose mothers had been treated with distilled water (control). The weight of these pups was 107.79 ± 0.8 g. Metocl 5 mg/kg and EHae 200 mg/kg promoted very significant growth of pups (P < 0.0001). These two doses induced respective increases of 19.86% and 18.47% (Figure 3).

Histology of Mammary Tissue
The galactagogue activity induced by EHae could also be due to the ability of the extract to promote the development of lobuloalveolar system. To test this hypothesis, histological sections were performed on samples from mammary glands of lactating rats that were treated daily with the test substances ( Figure 4).

Serum PRL Release
In order to justify the galactagogue effect induced by E.

Discussion
Measuring the milk yield produced by a rat is usually very difficult. However, according to Lompo-Ouedraogo et al. [20], it is possible to estimate this quantity of milk produced by carrying out different sessions of weighing of the pups. This method considers the pups as powerful vacuum cleaners capable of completely emptying the contents of the mammary glands. It is commonly used to evaluate the galactagogue property of vegetable products [29]. In this study, different test substances were administered daily to lactating rats. Comparison of milk yields produced by the treated rats with that of the control group showed that Metocl 5 mg/kg and aqueous extract (200 mg/kg) increased milk production. These increases, highly significant (P < 0.0001), were 45.78% and 40.36% respectively.
The results are similar to those of Hosseinzadeh et al. [36] obtained with 500 mg/kg of Nigella sativa (Ranunculaceae) seeds extract.  40.36% respectively. While the increase in milk production induced by the ethanolic extract of N. sativa seeds (500 mg/kg) was estimated at 37%. The galactagogue effects induced by plant species can be explained by their composition of bioactive substances capable of increasing the biosynthesis of milk [18].
Bioactive substances suspected to be at the origin of the galactagogue activity of medicinal plants are flavonoids, tannins, saponins and alkaloids. Gaya et al. [37] have shown that flavonoids, tannins, saponins and alkaloids are the major causes of galactagogue activity of Hibiscus sabdariffa (Malvaceae) leaves. These phytochemicals would be recognized dopamine antagonists by many authors. Dopamine is a competitive antagonist of PRL. Thus, an inhibition of dopamine synthesis by bioactive substances would lead to an increase of milk synthesis [38].
The differences in effects observed between doses of EHae (100; 200; 400 and 600 mg/kg) would be explained by a variation of the quantity of bioactive molecules responsible of the galactagogue activity contained in each dose. These bioactive compounds present in the E. hirta leaves have been identified [39] [40] [41] [42].
Analysis of milk production versus days of lactation showed that the milk yield produced by the rats of each experimental group was not proportional to the days of lactation. The same observations were made during the evaluation of the galactagogue activity of the leaves of Musa x paradisiaca (Musaceae) in the rat [43]. The non-linearity of milk production versus days of lactation is explained by the fact that the mammary glands undergo an alternation of proliferation and apoptosis of mammary cells during lactation [44]. Therefore, there is a variation in the kinetics of milk biosynthesis depending on the period of lactation. The cell proliferation rate being greater than the rate of apoptosis in early lactation, there has been an increase in milk production from D1 to D7 of lactation for all rats. After the peak of milk production observed at D7 of lactation, the equilibrium was reversed, that is to say, the rate of apoptosis becomes higher than the rate of proliferation of mammary cells. This had the effect of reducing milk production from D7 to D10 of lactation for almost all experimental groups.
During the experiment, the pups gained weight. This body weight increase of the pups would be exclusively dependent on the quantity and quality of the milk Journal of Biosciences and Medicines produced by the lactating rats. This is in agreement with Hamed et al. [45].
These authors note that weight gains of the pups are indicators of milk production in female rats. The highly significant growths (19.86% and 18.47%) of the pups receiving milk from female rats treated with Metocl 5 mg/kg and EHae 200 mg/kg, respectively, would militate in favor of galactagogue activity of these two substances.
In order to identify the mechanism of induced galactagogue activity, serum PRL assays were performed after treatment of virgin rats at D0, D7, and D14 of the experiment. The results revealed that at D0, none of the substances administered was able to elicit a PRL secretion greater than 0.5 ng/ml. This result is comparable to that obtained by Simelane et al. [46] [47]. The increased of PRL secretion induced by leaves of E. hirta as those caused by other plants would be related to the galactagogue activity of this plant. After parturition, the initiation and maintenance of milk production are mainly controlled by continuous PRL secretion [48]. PRL increases the proliferation of receptors on the surface of alveolar cells.
It promotes the entry of water and nutrients into alveolar cells for the synthesis of milk [49] [50].
The galactagogue activity induced by E. hirta leaves could also be explained by the ability of the extract to promote development of the lobuloalveolar system. Metocl 5 mg/kg had no effect on the development of mammary glands [46]. The aqueous extract of Acacia nilotica ssp adansonii (Mimosaceae) stimulates the development and differentiation of the lobuloalveolar system [20]. During lactation, mammary glands undergo an alternation of proliferation and apoptosis of mammary alveolar cells [44]. Moreover, consumption of plants rich in antioxidants such as flavonoids and phenolic compounds may have some beneficial effect on slowing the aging of mammary alveolar cells [51]. This would have the advantage of increasing the number of alveolar cells and the production of milk [20].

Conclusion
In conclusion, the aqueous extract of Euphorbia hirta (Euphorbiceae) leaves showed a galactagogue activity in female rats. Euphorbia hirta aqueous extract induced an increase of milk production and prolactin secretion. This property could be attributed to the substances contained in this plant aqueous extract.
These substances would promote a slowing of programmed death of mammary alveolar cells. These results could militate in favor of its use by the Gontougo populations to increase milk production in lactating women. However, in view of the safe and efficient use of this medicinal plant, it would be interesting to carry out further studies. These works will include the evaluation of the effects of aqueous leaf extract on the growth, hematological and biochemical parameters of pups and lactating rats.