Studies On The Exploitation And Utilization Of Medical Plant Ardisia Crenata Sims

Ardisia crenata Sims was one of the two kinds of medical plant which belongs to Ardisia spp., recorded in China Pharmacopoeia(1977 edition). It had nearly 500 years history since it first appeared in《ben cao gang mu》of ming dynasty. The research shows that it has distinct physiologically-active and pharmaceutical effects. So it has a great value of exploitation. In this paper, we studied the tissue culture technology of Ardisia crenata Sims, extraction, purification, content determination and structural identification of Ardicrenin. The main results were as follows:The results of the study on tissue culture technique were as follows.1. The period from Mar. to Jun. was the optimal time for explants collection in cause of the pollution rate nearly 10ï¼…. Continuously sterilize twice(4+4) min, then treated with mercuric chloride was the suitable sterilizing condition for stem segment. The(3+3) min was the suitable sterilizing condition for leaf. The pollution rate and browning rate could controled about 10ï¼….2. Axillary bud multiplication regeneration technique:1) Induction of axillary bud: The stems of A. crenate were taken as explants, the optional culture medium for their initiation was MS+6-BA 0.5mg/L+NAA 0.1mg/L.The startup rate was 97.53ï¼….2) Multiplication of axillary bud: MS+6-BA 2.0mg/L+NAA 0.1mg/L+KT 0.5mg/L was the suitable culture medium for multiplication of axillary bud. The propagation coefficient was over 3.0 after 60d.3) Rooting culture: The best medium for the rooting of shoots was 1/2 MS+IBA 0.2mg/L; the rooting rate was about 90ï¼…. Adding 0.2ï¼…Ac can accelerate growth of root, get high rooting rate and rooting quantity.4) Transplanting: Plantlets were transferred to the medium with ratio of 1: 1: 1 of sands and ruby mica and vermiculite or 1: 1 of ruby mica and vermiculite. The survival rate was 80ï¼…above.3. Organ regeneration technique:1) Induction of callus: Stem segment was the optimum explants for callus culture of Ardisia crenata Sims. MS+2, 4-D 2.0mg/L+NAA 0.5mg/L+6-BA 0.2mg/L was the optima culture medium for callus induction. The induction rate was over 90ï¼….2) Type of callus: There were two kinds of callus——the first was moist, loose and ivory-white or yellowy; the second was dry, compact and white, damask or turquoise. The first callus was suitable for cell culture and the second callus was suitable for differentiation.3) Multiplication of callus: Loose callus was the best explants for multiplication. While the optima medium was MS supplemented with 6-BA 0.2mg/L, NAA0.2 mg/L and 2, 4-D 0.5mg/L. The multiplication rate was over 300ï¼…after 30d.4) Differentiation of callus: Compact callus was the best explants for differentiation cultivation. Callus on the medium MS+6-BA1.0mg/L+NAA0.1mg/L, could differentiate adventitious bud and root. But adventitious bud couldn't germinate.5) Effects of light: The results showed that light are each useful to the multiplication and differentiation of callus.Through study on separation, purification process, content determination and structural identification of Ardicrenin, we got the results below:1. The extraction technology of the Ardicrenin was accquried. The process was as follows: The volume of methanol and treatment time is 8time(3h), 6 times(2h), 4 times(1h) at 70℃extrated with reflux 3 times using 80ï¼…methanol. The crude samples were obtained by decompress concentration, centrifugal, chromatography with macroreticular resin AB-8, washed by 80ï¼…methanol and vacuum dried under 70℃. The yield ration of Ardicrenin extraction was about 5ï¼….2. It was the first report for preparation technique of standard substance of Ardicrenin. The extraction of Ardicrenin was carried out through silica gel column analysis. Then the detergent of dichloromthane-ethanol-ethyl acetate(4: 1.5: 1, V/V/V) was used to elute propargite. TLC method was used for identification of Ardicrenin. The standard substance of Ardicrenin was obtained by collecting eligible products, concentrating at 70℃and recrystallization by ethanol. The content of Ardicrenin was about 98.58ï¼….3. The TLC method for analyzing of Ardicrenin was established as below, using silica gel GF₂₅₄ thin layer plate and dichloromthane-ethanol-ethyl acetate(4: 2: 1, V/V/V) as the developing solvent, 10ï¼…vitriol-ethanol solution as chromogenic agent and drying at 100℃. The TLC atlas was clear, the spot was distinct with good separation and R_f value was about 0.6 with good reproducibility.4. The RP-HPLC method for determination of Ardicrenin was built up. SHIM-PACK VP-ODS C₁₈(150 mm×4.6 mm, 5μm) column was selected and acetonitrile-water (37: 63, V/V) was used as mobile phase at a flow-rate of 1.0mL/min. The detection condition was as follows, wave length of UV at 205nm, column temperature at 40℃and sensitive 0.16AUFS. In this condition, the apex was perfect and retention time was 12.2min. The average recovery was 94.14～99.38ï¼…, and RSD was 0.48～1.97ï¼…(n=3). The results showed that the method of determination was convenient, steady, accurate and repeatable which was suitable for measuring the content of Ardicrenin.5. The content of Ardicrenin in wild plants and tissue culture was measured. The result showed that the content of Ardicrenin in root was higher than that those in wild plants. So the tissue culture was the suitable part for extraction of Ardicrenin. Ardicrenin content in the root of tissue culture plantlets is higher than anywhere else, which was equal to 54.62ï¼…of nature plant. Between the callus from stem, loose callus has more Ardisiacrispin content, it is equal to natural content.6. Mass spectrum and analyse MRI revealed that the molecular weight of Ardicrenin is 1074 and the molecular formula is C₅₃H₈₆O₂₂. The detail strcture is cyclamiretinA-3-O-[alpha-L-rhamnopyranosyl-(1→4)-beta-D-glucopyranosyl-(1→4)][beta-D-glucopyranos yl-(1→2)]-alpha-L-arabinopyranoside.