| Vaccaria segetalis （Neck） Garcke （Caryophyllaceae） is an annual herb.The dried seeds of this plant, well-known as Wang-Bu-Liu-Xing in traditional Chinese medicine, have been frequently used for promoting milk secretion in China. Vaccarin, a flavonoid glycoside, is considered one of the major active constituents of the seeds of Vaccaria segetalis, and it is regarded as the representative substance for quality control of the medicine in Chinese Pharmacopoeia 2010 edition.There is little data available about the extraction of vaccarin in the seeds of V. segetalis and its bioactivity as well as the effects of enviromental factors （such as light intensity, soil water content, mineral nutrient, salinity-alkalinity, etc.） on the development of V. segetalis and the accumulation of vaccarin. These results hindered to some extent the development and utilization of the plant resource of V. seglistis.To strengthen the quality control of V. segetalis, RP-HPLC analysis method of vaccarin was established. The microwave assisted extraction （MAE） of vaccarin in the seeds of V. segetalis was sysmetically studied, and Sanning electron microscope （SEM） was applied to evaluate the extraction efficiency. Followed by isolation and identification of dairy cow mammary epithelial cells （DCMECs）, in vitro bioactivitiy of the promoting lactation of vaccarin extract and its possible mechanism were also explored. The effects of enviromental factors such as light intensity, soil water content, mineral nutrient and salinity-alkalinity on the development of V. segetalis and the changes of content and yield of vaccarin in V. segetalis were studied by pot and field experiment.The main results were listed below:1. RP-HPLC method of vaccarin in the seeds and V. segetalis plant was developed as follows:chromatographic column HIQ SIL C18V （250 mm×4.6 mm I.D.）, mobile phase of methanol-0.6% phosphoric acid solution （30:70, v/v）, flow rate of 1 mL·min-1, wavelength at 332 nm, injection volume of 10 μL, and column temperature at 30 ℃. The determination method of vaccarin showed high precision, repeatability and stability.2. Compared with the conventional extraction methods, maceration extraction （ME）, ultrasonic-assisted extraction （UAE） and heat reflux extraction （HRE）, microwave-assisted extraction （MAE） possessed higher efficiency for the extraction of vaccarin. MAE of vaccarin from the seeds of V. segetalis was optimized in this study. The MAE conditions including methanol concentration, temperature, liquid/solid ratio, and microwave power were studied by single factor experiment and further optimized by RSM. The maximum extraction rate of vaccarin reached 0.52%, under the optimal conditions:methanol concentration 57%, temperature 65 ℃, liquid/solid ratio 50 mL/g, and microwave power 400 W. The order of factors influencing the response value of the extraction rate of vaccarin was as follows: temperature> liquid/solid ratio>microwave power> methanol concentration. At these optimal extraction parameters, the maximum extraction rate of vaccarin obtained experimentally was found to be very close to its predicted value.3. Dairy cow mammary epithelial cells （DCMECs） were established through tissue culture combined with pancreatic enzyme digestion method, the effects of vaccarin extract on vitality and proliferation ability of DCMECs were studied by in vitro experiment. Vaccarin extract could enhance the vitality and proliferation ability of DCMECs cells at the best concentration of 5μg·mL-1; Secretion capacity of lactose, β-casein, and TG in DCMECs were improved by vaccarin extract, respectively. Lactation function of DCMECs appears to be improved. The expression level of P-casein gene in DCMECs treated with vaccarin extract was enhanced through the activation of Stat5 lactation signal transduction pathways.4. The effects of environmental factors such as light intensity, soil water content, mineral nutrient and salinity-alkalinity on the development of V. segetalis and accumulation of vaccarin in V. segetalis were conducted by pot and the test of cultivation in woodland. The results were as follows:（1）Full light intensities were beneficial to the biomass accumulation in different organs like roots, stems, leaves and seeds of V.segetalis. However, moderate shade can improve the contents of vaccarin in different organs of V.segetalis. Yields of vaccarin in different organs of V.segetalis under full light conditions were highest when compared with other light treatments.（2）There were significant effects on the bimass, content and yield of vaccarin in different organs like roots,stems,leaves and seeds of V. Segetalis under the treatment of different canopy density.The biomass of roots, stems, leaves and seeds of V. Segetalis had the greatest accumulation under full light treatment, which was gradually reduced with the increase of canopy density; The proper crown density treatment helps to improve vaccarin content in different organs of V.segetalis;Yields of vaccarin under the treatment of different canopy density were lower than those of control.The effect of biomass of V.segetalis on yield of vaccarin was greater than that of content of vaccarin.（3）Compared with moderate soil water content （the water regime of 70%～75% of field capacity）, severe soil drought, moderate soil drought and excessive soil moisture would lower the biomass of different organs of V. segetalis. Vaccarin content in different organs of V.segetalis can be improved significantly in moderate soil drought condition. The best biomass and vaccarin yield of V. segetalis were obtained when soil water was kept under the condition of moderate soil water content.（4）The effects of single mineral nutrient （N, P and K） and NPK complex fertilizer on the biomass of V.segetalis and the accumulation of vaccarin were different. Among various mineral nutrient treatments, the effects of NPK complex fertilizer on the accumulation of the biomass and vaccarin of V.segetalis were most significant followed by N, P and K. When it comes to yields of vaccarin in different organs of V.segetalis, seeds ranked first which are followed by stems, leaves and roots.（5） V.segetalis plants are tolarent of mild salinity-alkalinity stress to some extent.With the increase of salinity-alkalinity stress concentraion, the biomass of roots, stems, leaves, and seeds of V.segetalis was gradually decreased. Vaccarin contents in different organs of V. segetalis can be improved under low salinity-alkalinity condition, and inhibited under high salinity-alkalinity condition. The yields of vaccarin in V. segetalis decreased with the increase of concentrations of salinity-alkalinity stress. The contents of soluble sugar, praline and MDA were improved 5-15 days after the treatment of salinity-alkalinity stress, respectively.（6） Comprehensive consideration of the effects of enviromental factors on the development of V. segetalis and the accumulation of vaccarin, V. segetalis plants were proposed to grow under full light condition, the water regime of 70%～75% of field capacity, and applying NPK compound fertilizer during the growing stage of plant. V. segetalis can be grown under forest and developed as a kind of mild salinity -and alkalinity-tolerant plants. |
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