The breast feeding has many advantages for delivery woman and infant, for example, it can reduce postpartum hemorrhage, reduce the onset of mammary cancer and oophoroma, and it has an incomparable superiority in the aspect of promoting the development of infant’s immune system [1]. However, many delivery women are often puzzled by hypogalactia. In the treatment of postpartum hypogalactia, the efficacy of Chinese herbal medicine treatment has been proved by the long-term clinical experience, which also widely accepted and used among the people. Medulla tetrapanacis is a kind of Araliaceae plant, one of the widely-used Chinese herbal medicines among the people and in the traditional Chinese medical application, and good at smoothing Qi and promoting lactation [2]. There are few researches about its medicinal mechanism at home and abroad, almost blank in the medicinal mechanism of promoting lactation and laboratory research. In this experiment, the animal experiment and in vitro experiment were conducted and the fluorescent quantitative RT-PCR method and Western technique were applied to study the pharmacological action of Medulla tetrapanacis, to preliminarily explore the approaches and acting mechanism of Medulla tetrapanacis to increase the secretion of milk and its influence on the composition of milk, to provide the theoretical basis for using Medulla tetrapanacis to treat the hypogalactia in clinical.
 
1 Materials and Method
1.1 Materials
1.1.1 Experimental animal
Crossbred clean-level ICR mouse, they are about 20g, total 16 mice, randomly divided into negative control group (8 mice) and Medulla tetrapanacis group (8 mice), each group is respectively settled in 36×48 plastic cage, to be fed under 12h of lighting and 12h of darkness, and the environment temperature is 20℃~25℃.
 
1.1.2 Experimental instrument
Biotek continuous spectrum multichannel microplate reader (BioTek Synergy H1 Hybizl Render), ABI Prism®7300 real-time fluorescent quantitative PCR instrument, CO-150 CO2 incubator, constant temperature water bath, TGL-16G high-speed centrifugal machine (Thermo), chemical lighting imager (BIO-RAD, ChemiDoc XRS+), PCR instrument (GeneAmp PCR System 9700), BIO-RAD small-size vertical electrophoresis trough (Mini-PROTEAN Tetra System).
 
1.1.3 Experimental Reagent
RPMI1640 culture medium(Gibco), FBS (Gibco), EGF (Sigma), insulin (Sigma), M-MLV reverse transcriptive enzyme (ABI), Trizol (Invitrogen), RIPA lysis solution (Pierce), Western lighting imaging reagent box (Pierce), BCA protein-concentration determination reagent box (Pierce), lactose determination reagent box (Biovision), fluorescent quantitative PCR reagent box (Takara).
 
1.2 Method
1.2.1 Concentration and Purification of Medulla tetrapanacis
The preparation of concentrated and purified Medulla tetrapanacis is as follows: after the selected Medulla tetrapanacis is identified, the water extraction and ethylalcohol deposition method shall be used to extract the effective components [3]. 1 kg Medulla tetrapanacis shall be soaked in the distilled water (20 times to the weight of herb) for 24 hours, then heated in 100℃ boiling water bath for 4 hours, the extracted solution shall be saved after filtration, the above steps shall be repeated twice, and the boiled liquid shall be gathered together. The 4-layer gauze shall be used to filter the impurities, and then it shall be centrifuged for 15 min at 3000 rpm, the supernatant fluid shall be collected to be evaporated and concentrated to 1/3 of the original volume, and then the water-free alcohol shall be added into the concentrated solution, to make the alcohol concentration of the concentrated solution to be up to 75%, and it shall be placed still for a night, and then it shall be centrifuged for 15 min at 3000 rpm, the supernatant fluid shall be collected to be evaporated and concentrated to 4g raw drug/ml, finally it shall be placed into the refrigerator at -20℃ for use.
 
1.2.2 Animal Experiment Design
The 16 postpartum ICR female mice were divided into 2 groups, the negative control group (8 mice) and the Medulla tetrapanacis group (8 mice). The female mice of Medulla tetrapanacis group were administrated 0.25ml extracted solution of Medulla tetrapanacis by gavage, while the mice of negative control group were fed the equal volume of normal saline. On the Day 1, 3, 8, 13, 18 of lactation period, the daily milking volume of female mouse can be calculated according to the weight of infant mouse. The specific steps are as follows: separate the female mouse from the infant mouse for 4 hours, then replace them in a same cage for feeding 2 hours, the weight of infant mouse before and after placing in a same cage shall be recorded, the milking volume of female mouse can be calculated through this formula: yield (g/pup/day)=0.0322+0.0667(weight)+0.877(gain/d), and then compare the two groups [4]. During the separation and combination, try to control the temperature and humidity in the lab, to avoid the stimulation of light and voice, to avoid shocking, reducing the influence on feeding.
 
1.2.4 Determination of Milk Protein and Lactose Content
Before taking the milk from mice, they shall be injected 2IU oxytocin by intraperitoneal injection, and the milk shall be collected by vacuum negative pressure for testing. The BCA protein detection kit and lactose detection kit are used to respectively measure the protein and lactose content in the milk of the negative control group and the Medulla tetrapanacis treatment group.
 
1.2.5 Culture of Mouse’s Mammary Epithelial Cells
The basic conditions for the culture of HC11 mouse’s mammary epithelial cells are as follows: RPMI1640 +10% FBS + insulin (10ug/ml) + EGF (10ng/ml). Before the treatment of cells, the cells shall be first digested and then shifted into the six-well plate, the cell concentration per well is 5 * 10 ^/ml, the culture medium shall be changed to the induction medium (RPMI1640 +10 ug/ml insulin). The cells shall be divided into the control group, the low dosage treatment group and the high dosage treatment group. The extracted solution of Medulla tetrapanacis shall be added into the culture medium, to make the concentrations of Medulla tetrapanacis in the low dosage treatment group and the high dosage treatment group to be up to 100ug raw drug/ml and 500ug raw drug/ml, and the same amount of ethanol shall be added into the culture medium of the control group. The cells shall be placed into CO2 incubator for 24 hours.
 
1.2.6 Extraction of Cell’s Total RNA and Fluorescent Quantitative PCR
The cells of the control group, the low dosage group and the high dosage group shall be extracted the total RNA by Trizol and transcribed into cDNA by the reverse transcriptase, and then the fluorescent quantitative PCR shall be conducted by the way of SYBR Green, to respectively amplify the internal reference GAPDH, and the milk casein gene and lactalbumin gene. The primers are as follows: Upstream primer of lactalbumin protein gene: 5'-CAAGAAGATCCTGGCTATCA-3 '; Downstream primer: 5'-AGATCATCTTCACAGGAATAGC-3'; Upstream primer of β-casein gene: 5'-TCACTCCAGCATCCAGTCACA-3'; Downstream primer: 5' -GGCCCAAGAGATGGCACCA-3'; Upstream primer of GAPDH gene: 5'-ACTCCCACTCTTCCACCTTC-3', Downstream primer: 5'-TCTTGCTCAGTGTCCTTGC-3 '.
 
1.2.7 Western Blot Analysis
The cells of the control group, the low dosage group and the high dosage group shall be extracted the total protein by RIPA lysates, and the Western method shall be used to detect the expression of intracellular casein, STAT5 and STAT phosphorylation, and please refer to the reference for the specific Western method [4].
 
1.3 Data Analysis
SPSS13.0 statistical software is used for data analysis and charting. Data were analyzed by t test or variance analysis, and if the P value is less than 0.05, the results of analysis can be considered as the criteria of statistical difference, if less than 0.01 it can be considered as the criteria of statistical significant difference.
 
2 Results
2.1 Influence of Medulla tetrapanacis on the Amount of Milk Secretion of Mice
The results show that the amount of milk secretion of mice is higher in the Medulla tetrapanacis group than the control group on Day 1, 3, 8, 13 and 18 of suckling period, but there is no statistical significance in the differences between Day 1 and 18 in the two groups, while the amount of milk secretion of mice in the Medulla tetrapanacis group is significantly higher than these of the control group (P <0.05), there are significant difference. Please see Figure 1 and Table 1.

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