| Objective: 1.Isolation and identification the secondary metabolites from tubers of B.striata.2.Screening secondary metabolites in B.striata liquid suspension cultured cells and establishing methods for measuring the content of these secondary metabolites.3.Curving cell growth and accumulations of secondary metabolites in suspension culture system.4.Optimizing the suspension culture system of B.striata.Methods: 1.To isolation and identification the secondary metabolites in tuber powder of B.striata,95% alcohol was initially applied for reflux extraction to separate the chemical constituents.The crude extract was leached in n-Butanol solution to get the fraction.The obtained was further processed by a series of chromatographic methods including silica gel,MCI column chromatographic and preparative HPLC to isolate and purify the extracts of Bletilla striata.The structures of the isolated compounds were identified by spectroscopic techniques which were NMR and MS.2.To establish a HPLC-DAD(High performance liquid chromatography-diode array detector)method for determination of secondary metabolites,the secondary metabolites in suspension cultured cells were screened by using the isolated secondary metabolites as standards,and the content of secondary metabolites was determined by analytical high performance liquid chromatography quantitative analysis.3.To characterize cell growth of suspension culturing callus,the matured seeds were used as experimental materials to obtain the loose callus through tissure culturing.The callus was harvested for estatment the suspension culture system as explants.Then the growth state of cells and the accumulation of secondary metabolites in the system were monitored to draw the variation curves.4.To optimize the suspension culturing system for obtaining more cells and more metabolites,the Box-Behnken design(BBD)experimental design of response surface method(RSM)were adopted to find the best values of several parameters at non-hormone level,respectively.Results: 1.Twenty secondary metabolites were isolated from the n-Butanol extraction fraction of the ethanol extract of B.striata tubers,including a new compound.Their structures are identified as 2,2?,2??,7,7?,7??-hexahydroxy-4,4?,4??-trimethoxy-[9,9?,9??,10,10?,10??]-hexahydro-1,8,1?,6??-triphenanthrene(1),gymnoside ?(2)? dactylorhin A(3)? militarine(4),2,7-dihydroxy-4-methoxy-9,10-dihydroxyphenanthrene(5),2,7-dihydroxy-3,5-dimethoxy-9,10-dihydrophenanthrene(6),2,3,7-trihydroxy-4-methoxy-9,10-dihydrophenanthrene(7),2,6-bis(p-hydroxybenzyl)-3?,5-dimethoxy-3-hydroxybibenzyl(8),3,3'-dihydroxy-2-(p-hydroxybenzyl)-5-methoxybibenzyl(9),2,6-bis(p-hydroxybenzyl)-3,3?-dihydroxy-5-methoxybibenzyl(10),3,3?-dihydroxy-5-methoxybibenzyl(11),3,3?-dihydroxy-2-(4-hydroxybenzyl)-5-methoxybibenzyl(12),3-hydroxy-5-methoxybibenzyl(13),3-hydroxy-5,3?-dimethoxybibenzyl(14),1-(p-hydroxybenzyl)-4,7-dimethoxyphenanthrene-2-ol(15),4,7,4?,7?-tetrahydroxy-2,2?-dimethoxy-1,1?-biphenanthrene(16),p-hydroxybenzaldehyde(17),4-hydroxybenzyl alcohol(18),p-hydroxybenzyl methyl ether(19),p-hydroxylbenzyl ethyl ether(20).2.Using the above 20 secondary metabolites as standards,four secondary metabolites were selected from suspension culture callus: 4-hydroxybenzyl alcohol(18),dactylorhin A(3),militarine(4),coelonin(5)as indicator components for the detection of secondary metabolites in the suspension culture systems.HPLC-DAD was used to detect the relationship between mass concentration and peak area and draw the standard working curve.The methodological investigation results showed that the relative standard deviations were all met the methodological requirements on the results of precision experiment,stability experiment,reproducibility experiment and sample recovery experiment.3.The light yellow loose callus subcultured twice was selected as the explant for drawing the curves of callus growth and the accumulation of secondary metabolites in suspension culture system.The Logistic function curve was found as the closest one with the measured data,by analyzing the cell growth curve of the suspension culture.And the cumulant detections of secondary metabolites in the suspension cells were shown that the cumulative amount of 4-hydroxybenzyl alcohol reached the maximum at 39 days after inoculation,dactylorhin A and militarine at 24 days,and the coelonin at 18 days.4.The RSM assays showed that in the suspension system,the cells could reach the maximum value of 0.7928 mg/g when the parameters were 21.74 mL of medium volume,0.92 g of initial callus addition and 28.77 g/L of sucrose,the content of 4-hydroxybenzyl alcohol.Likely,the content of dactylorhin A reached the maximum value of 7.7920 mg/g when the parameters were 40.13 mL of medium volume,0.82 g of initial callus addition and 30.73 g/L of sucrose.The content of militarine reached the maximum value of 9.4467 mg/g when the parameters were 38.35 mL of medium volume,1.40 g of initial callus addition and 38.32 g/L of sucrose.The content of coelonin reached the maximum value of 0.3451 mg/g when the parameters were 39.87 mL of medium volume,1.19 g of initial callus addition and 31.16 g/L of sucrose.Conclusion: We successfully established a suspension culture system in B.striata.20 kinds of secondary metabolites were isolated from the tubers.Moreover,the growth curves of the suspention callus and the accumulation of four secondary metabolites were established.Finally,the system of suspension culture was optimized by RSM,and the optimal combination of non-hormone level were obtained,indicating the suspension culture system with high accumulation of secondary metabolites was successfully established. |
PDF Full Text Request