Study On The Dynamic Variation Of The Flavonoids Contents And The Inlfuences Of The Elicitor On The Flavonoids Contents And Related Enzyme Activities In Rhododendron L.

China is the country with richest species of Rhododendron Linn., it is the main distributioncenter of the Rhododendron Linn.in the world. Ii is the largest genus of the Ericaceae, it is notonly with extremely high appreciation value, but also has good medicinal value, so that it hasbroad prospects of its application in the fields such as foods, medicine. Flavonoids is the mostimportant secondary metabolites in the plants. The flavonoids compounds widely exist in theRhododendron Linn., which is a broad class of substances with bioactivity, so it is a kind of thepotential natural resources. Nowadays the main ways to research the Rhododendron Linn.arecentered on the feral breed, but the blind use of resources lead to a seriously storage of availablenatural resources of the Rhododendron Linn.. And at present there is not a systematical researchwith flavonoids so that it restricts peoples to develop and utilize them further. This paper usedthe Rhododendron pulchrum Sweet. and the Rhododendron mucronatum (BL.) which weresuccessfully grown by artificial cultivation as the materials, to research the flavonoidscompounds scientifically. Consequently, it can provide the important theoretical and techniquefoundation for further utilizing the flavonoids compounds in Cultivated Rhododendron.Simultaneously, it also can provides a guarantee for enlarging the flavonoids production.The major research contents of this paper were:(1) By combining BP artificial neuralnetwork with traditional orthogonal tests, optimiz the extraction processes for flavonoids inRhododendron and then conducted the antibacterial experiments.(2) Analysed flavonoids in theleaves and flower of Rhododendron with color reaction and HPLC-MS.(3) Established a HPLCmethod for determination the major compounds of flavonoids and study the dynamic variation ofthe contents of flavonoids in the leaf and stem of Rhododendron.(4) By studying on theinfluences of the different exogenous elicitor on the content of flavonoids and related enzymeactivities of Rhododendron, selected the exogenous elicitor with fit types and concentration toincrease the content of flavonoids. The main results were as follows:1. On the base of the orthogonal tests, apply BP artificial neural network to determine theoptimal conditions for extraction of total flavonoids in Rhododendron pulchrum Sweet. was thatthe solid-liquid ratio of1:20,40min of extraction time,50℃of extraction temperature,40%ofethanol concentration. Thwas optimal process was better than traditional orthogonal tests, it canreduce the cost and the energy consumption. The results show artificial neural network combinedwith traditional orthogonal design constitute the baswas on which a new method of test data analyzing and processing was put forward and we also can use it to optimize the extractionprocess of flavonoids in other plants.2. The flavonoids of Rhododendron pulchrum Sweet. and Rhododendron mucronatum(BL.) had almost no bacteriostaswas to molds such as penicillin sp. and aspergillus niger, buthad different inhibition effects to bacteria. The antibacterial rate of the extraction reach100%.The inhibition effects on staphylococcus aureus was the best, and next were escherichia coli andbacillus subtilwas. The higher concentration of the flavonoids extraction, the more inhibitioneffects increased. The inhibition effects of the extracts in Rhododendron mucronatum (BL.) wasbetter than Rhododendron pulchrum Sweet.. Thwas could be due to the content of theflavonoids. The MIC of the flavonoid extracts from Rhododendron pulchrum Sweet. were asfollows:0.03125g/ml for staphylococcus aureus and escherichia coli;0.0625g/ml for bacillussubtilwas. The MIC of the flavonoid extracts from Rhododendron mucronatum (BL.) was0.03125g/ml for staphylococcus aureus, escherichia coli and bacillus subtilwas.3. In thwas paper, the separation was performed on Waters C18column (4.6mm×250mm,5μm) by gradient elution(0~24min,31%~42%B;24~30min,42%~50%B;30~35min,50%~60%B;35~40min,60%B) using methanol(B) and water(B) as the mobile phase. Thedetection wavelength was356nm and the flow rate was0.7mL/min with column temperature at30℃. By the HPLC-MS/MS with electrospray ionization, five constituents were identified asquercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-0-arabinoside,quercetin-3-rhamnoside, quercetin in the leaf and six constituents were identified asmalvidin-pentoside, myricetin-rhamnoside, quercetin-3-galactoside, quercetin-3-0-arabinoside,quercitrin, quercetin in the flower of Rhododendron pulchrum Sweet..4. The seasonal dynamic variation of the contents of the total flavonoids and the maincomponent in the leaf of Rhododendron pulchrum Sweet.had some similarities. With the springtemperatures rebound and the development of the bud, the content of flavonoids increasedsignificantly. In3~6months, the content of flavonoids was in a high standard, and then it fellrapidly. In September, the contents started to climb back up. When it turned cold in November, itfell rapidly again. In the four components of the flavonoids, the most abundant content wasquercetin-3-glucoside, and then was quercetin, wasoquercitrin was lease. The seasonal dynamicvariation of the contents of the total flavonoids in the stem was different from leaf. It didn't enterthe second growth in September. Thwas may be related to the low contents in stem which had noobvious change. The content in stem was lower than leaf dwastinctly. The seasonal dynamicvariation of the contents of the total flavonoids in the leaf of Rhododendron mucronatum (BL.)and Rhododendron pulchrum Sweet. was similar, but the content of the former was higher than the latter.5. Spraying the Rhododendron pulchrum Sweet with SA of different concentration, thecontents of the total flavonoids and the main component all reached the maximum value on theeighth day. The low concentration of SA (50μg/mL) had the most dwastinct influence on thecontent of flavonoids. The high concentration of SA had an inhibitory effect. The PAL activityincreased with the induction processing time increased. On the eighth day, it reached the highestvalue. On the4th and8th day, PAL activity was in a higher standard. So using SA can make thePAL preserve high activity in a longer period. With100μg/mL concentration of SA, the PALactivity had the biggest increase. The activity of PPO could reach a high level on the8th day.When the concentration of SA was50μg/mL or100μg/mL, the activity had a higher increase.The was phenomenon explained that the concentration ranged50μg/mL to100μg/mL of SAcould increase the activity of PAL and PPO. The PPO activity could reach a higher value on the4th day. It may be correlated with the respond to external stimuli.Thwas results indicateddifferent concentration of SA was a better elicitor for improving POD activity.6. Spraying the Rhododendron pulchrum Sweet with MeJA of different concentration, thecontents of the total flavonoids and the main component were affected observably. The highconcentration of MeJA (150μg/mL) had the most dwastinct influence on them, while the lowconcentration (50μg/mL) of MeJA had almost no effect. The was phenomenon indicated that thehigher concentration of MeJA has an stronger effect on the contents of flavonoids. When theconcentration of MeJA ranged from100μg/mL to200μg/mL, the activity of PAL improvedmarkedly, so spraying rhododendron with the range of the concentration of MeJA could improvethe activity of PAL observably. Using100μg/mL concentration of MeJA, the activity of PPOimproved most obviously. The POD activity could reach maximum value on the4th day, thenthey came down rapidly. The activity of POD increased substantially with differentconcentration of MeJA, so it was also a better elicitor for improving POD activity.7. Using GA3of different concentration dwasposed Rhododendron pulchrum Sweet.,when the concentration was100μg/mL, the influence on the contents of the total flavonoids wasmost obvious. The high concentration of GA3(200μg/mL)had an inhibitory effect. Theinfluence on the contents of the4main components was different with different concentration ofMeJA. The best concentration for quercetin3-galactoside and quercetin-3-rhamnoside was100μg/mL. The best concentration for isoquercitrin and quercetin was150μg/mL. Using MeJA of50μg/mL or100μg/mL concentration, the activity of PAL changed much obviously The activityof PAL sustained at a high level for a long time. The was phenomenon indicated that theconcentration ranged from50μg/mL to100μg/mL of GA3had a greater impact on the activity of PAL. with the100μg/mL concentration of GA3, the activity of PPO improved most obviously.With the lower concentration of GA3(50μg/mL), the activity of POD increased by a widermargin after the4th day, and other concentrations actually reduced the activity.8,Using different concentration of ABA dwasposed Rhododendron pulchrum Sweet.,when the concentration was150μg/mL, the influence on the contents of the total flavonoids andthe main components was obvious. When the concentration was low (50μg/mL), the activity ofthe PAL had a biggest augment. With the100μg/mL concentration of ABA, the activity of PPOimproved most obviously. When the concentration of ABA was200μg/mL and150μg/mL, theinfluence on the activity of POD was bigger. With50μg/mL and100μg/mL, the activity ofPOD was reduced. So the high concentration of ABA could increase the activity of POD, and thelower had an inhibitory action.