| Saffron (Crocus sativus L.) is a perennial herbaceous plant, which belongs to the Iridaceae family of Crocus. Its dry stigmas is a rare medicinal herbs, and traditional medicine as emmenagogue and so on. Recent studies show that crocin, crocetin and safranal, which are contained in saffron stigmas, have various biological activities, such as cytotoxic, antitumor, anticarcinogenie, antimutagenic activities, also have prevention and treatment action for cardiovascular and cerebrovascular diseases and neurological diseases, but little toxicity and side effect. Saffron is sterile triploid plants, propagated by vegetative production through the formation of a daughter corm. Under the conditions of traditional cultivation, the propagation rate of saffron was relatively low and the degradation of corms was serious. And saffron production requires labor-intensive harvesting, including flower picking and stigma separation, thus the produce of saffron corm and stigmas can’t satisfied the domestic market.In this dissertation, a primary study of saffron on biological characteristics of growth and development was carried out, and the system of in vitro regeneration of saffron was established. Meanwhile, optimizing the extraction process of active ingredients in saffron non-medicinal parts was carried out and its antioxidant activity was detected. This technique, therefore, offered an effective way for resolving the problem of saffron shortage and rational use of resources to provide some ideas. In this study, the main results obtained are as follows:(1) Corms main nutrients was starch, to276.52mg/g dry sample, which provides a source of nutrients for bulb dormancy, sprouting and flowering. Corm fresh weight has positive correlation with the leaf area. Growing trend of leaf area was arched growth and stabilize at the end of December. Sub-bulbs was rapidly expanded from the beginning of February to the end of March. Formational sub-bulbs from larger bulbs has a longer diameter and height, heavier weight. Thus, mother corms large enough can form larger sub-bulbs. In addition, the bulbs weight was also positively correlated with the number of leaves and flowers, and was also proportional to the saffron stigmas output. Saffron flowering date was from late October to early November, about20days. Light and humidity during flowering date has great influence to flowering. Humidity over the general will caused wilt, if light is too strong, buds will be leggy, which all can affect production. Nearly half of the petals’moisture content, moisture content of stigma was23.7%, so flowering air humidity should be suitable to meet the needs of flowering to water.(2) Saffron bulbs in vitro propagation was an effective way of solving the shortage of bulbs resources, how to shorten the tissue culture cycle and improve weight of tube bulb were an important part of this study. In callus induction phase, with bulbs cut as explants, after the explants sterilized60seconds by75%alcohol and15min by0.1%HgCl2. Using MS medium supplementing with0.2mg/L2,4-D and3mg/L6-BA+300mg/L casein hydrolyzate+20g/L sucrose+10%banana as a basic medium for callus induction, incubated at25±1℃and dark condition, it can significantly reduce callus induction time, and there was a high induction rate, callus grew well, showed dense and a yellow or milky-like. The optimal condition for regeneration shoot was the MS medium supplementing with3mg/L NAA and0.5mg/L6-BA, under23±1℃and12h/d light conditions culture, shoot induction rate reached86.7±2.7%. Using MS+0.5mg/L NAA+2mg/L6-BA as saffron buds proliferation medium, proliferation multiples can reach4.57±0.33. To shorten the induction time and improve weight of test tube bulb, using1/2MS+5mg/L NAA+5mg/L6-BA+0.5g/L AC+400mg/L casein hydrolyzate+30g/L sucrose+20%banana as optimal medium, under23±1℃and12h/d light conditions cultured five weeks it can get76.7±1.8%of the induction bulbs and corms average weight reached0.57±0.07g.1/2MS+400mg/L casein hydrolyzate+0.5mg/L IBA+0.5mg/L NAA+20%banana was suitable for saffron corms rooting induction. Choosing garden soil:peat:vermiculite:organic fertilizer ratio of2:2:1:0.1matrix as saffron corms transplanting matrix, test tube bulbs survival rate reached67.5±2.5%and growing well.(3) Using response surface methodology to optimize the extraction process of saffron petals polysaccharides, flavonoids and polyphenols, and studied its antioxidant activity. The results show: under the optimized conditions with extraction temperature80℃, extraction time1.40h, ratio of liquid to raw materials40ml/g, petals polysaccharide yield was1.902±0.093%(n=3). Extract petals flavonoids optimal conditions was:ethanol concentration of40%, ratio of liquid to raw materials65ml/g, extraction time2.5h and extraction temperature80℃, under these conditions, flavonoids content was15.3±0.5mg rutin equivalent/g dry sample. Optimal conditions for the extraction of polyphenols: ethanol concentration63%, ratio of liquid to raw materials55ml/g, extraction temperature78℃and extraction time1.5h, obtained polyphenols content was75.1±0.3mg gallic acid equivalent/g dry sample. Polysaccharides, flavonoids and polyphenols have shown a significant DPPH radical scavenging activity, flavonoids and polyphenols also showed significant ABTS radical scavenging activity and ferric reducing ability. Flavonoids has a stronger antioxidant activity than polyphenols.Ethanol extract of saffron non-medicinal parts was successively extracted with different polar organic solvent extraction, and the antioxidant activity of different polarity segments was detected. The results show that:the n-butanol and ethyl acetate ministry of stamens and petals has a strong antioxidant activity, antioxidant activity of the ministry of petroleum ether was extremely weak. Different polarity segments of lateral buds and bulbs hasn’t strong antioxidant activity. So, stamens and petals can be used as raw material source of natural antioxidants. |
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