Preliminary Study On The Metabolism And Differential Expression Of Secondary Metabolites In Mulberry Leaves Before And After Frost

Mulberry leaves were first contained in the "Shen Nong’s Herbal Classic". It was dried leaves of Morus alba L. and had a long history. It is bitter, sweet, cold, and belongs to the lungs, the liver. So it has very important functions of evacuating wind-heat, clearing the lungs moistening and liver eyesight. It was mainly used for common cold, hyperactivity dry cough, dizziness, headache, red eyes dim and so on. Mulberry harvest have special requirements for the frost of the harvest, but the existing researches can not scientifically explain the rationality for the frost mulberry leaves.So the scientific connotation of the frost harvested mulberry leaves is worthy of further study. The key enzymes activity changes of the chemical and effective compositions combined with plant metabolomics and biological methods and techniques were analyzed and studied before and after frost. Correlation between activity of PAL, UFGT and temperature, flavonoids in mulberry leaves before and after Frost. This study provided a theoretical basis for elucidating the molecular mechanism of frost effect on flavonoids in Mulberry Leaves. The main contents and results are as follows:1. The dynamic changes of secondary metabolites contents in mulberry leaves before and after the frost. By using HPLC-MS, flavonoids in mulberry leaves were analyzed. And a method of using HPLC to simultaneously quantify the content of four main flavonoids compounds, namely rutin, isoquercitrin, astragalin, chlorogenic acid was established. The content of total flavonoids was determinated by using UV and the content of DNJ by using HPLC with fluorimetric detection with precolumn derivatization was determinated. The dynamic change rule of total flavonoids, four components, alkaloids(DNJ) content in mulberry leaves was discussed. The results showed that the content of flavonoids in mulberry leaves was low in July or August when the temperature was high. Flavonoids content reached a maximum in November or October after frost. The content of DNJ in mulberry leaves in July and August reached a maximum. Then DNJ content decreased in September or October when the temperature declined and it reached a minimum after frost. Correlation between contents of all components and temperature was analysed. The results showed that the content of flavonoids had significant negative correlation with the temperature and DNJ had significant positive correlation with the temperature.2. Study of activity of Phenylalanine Ammonia-lyase in mulberry leaves before and after Frost. The first key enzyme existing in biosynthetic pathways of flavonoids, namely phenylalanine ammonia lyase(PAL) was selected. Five parts containing the buffer, substrate concentration, bath temperature, bath time, and the amount of polyvinylpyrrolidone were considered. The results showed that Mulberry PAL activity was determined under the condition of PH 8.8(0.1 mol/L) borax hydrochloric acid buffer, adding 0.2 g polyethylene(PVP), substrate concentration of L-phenylalanine to 0.04 mol / L, 40 ℃ water bath reaction of 40 min. The results showed that PAL activity was low in April or May. Then PAL activity decreased when the average temperature increasd and it reached a minimum in August when the average temperature was high. In November or October, the PAL activity increased and reached a maximum after frost. The value of PAL activity in mulberry leaf in December was not detected.3. Study of activity of flavonoid glycosyltransferase enzyme in mulberry leaves before and after Frost. The last key enzyme existing in biosynthetic pathways of flavonoids, namely flavonoid glycosyltransferase(UFGT) was selected. And a method of using HPLC to quantify the UFGT activity in mulberry leaves was established. The results showed that UFGT activity was low in April or May. Then UFGT activity decreased when the average temperature increasd and it got a minimum in September when temperature was high. In November or October, the UFGT activity increased and reached a maximum after frost. The value of UFGT activity in mulberry leaf in December was not detected.4. Correlation between activity of PAL, UFGT and temperature, flavonoids in mulberry leaves before and after Frost. Correlation between activity of PAL, UFGT and temperature, flavonoids in mulberry leaves before and after Frost were analysed by using SPSS 20.0 software. The results showed that the activity of PAL and UFGT had very significant negative correlation with the temperature. There was significantly positive correlation between the flavonoids content and PAL, UFGT activity. Lower temperature had significant effect on accumulation of flavonoids coponents. The main reason may be that the low temperature could induce PAL, UFGT activity of mulberry biosynthesis pathway, and then increased activity of enzymes, thus promoting the accumulation of flavonoids.