| Object Lepisorus ussuriensis?Regel et Maack?Ching is Lepisorus genus of Polypodiaceae family of Fern plant which contains chlorogenic acid as the main active component that has a variety of roles involving antioxidant,antitumor,antibacterial,anti-inflammatory,antiviral,immunomodulatory,hypoglycemic and so on.Therefore,this thesis selects Lepisorus ussuriensis as research object which aims to obtain culture system of high chlorogenic acid through the training of organization,to provide a new method for culturing chlorogenic acid fast and largely,it intends to screen out tissue culture system which breeds rapidly,in order to provide scientific foundation for the applications of tissue culture as well as the further development and sustainable utilization of it.Methods Take fresh Lepisorus ussuriensis as experimental material,using tissue culture technique,to induce callusof laminaand rhizomerespectively.To investigate the factors of three kinds of plant growth regulators,each factor is selected in three levels to designexperiments,to have orthogonal test L9?33?,to measure the number of chlorogenic acid in callusby HPLC and UV points spectrophotometric method for the determination of the optimum culture systemof chlorogenic acid.To study the scavenging effects of DPPH free radical and superoxide anion radical by using antioxidation experiment in vitro.At the same time,to study the function of antibacterial activity in vitro of callus for bacillus subtilis,escherichia coli,staphylococcus aureus and pseudomonas aeruginosa by paper method,and the analgesic effect of the callus by the method of acetic acid and hot plate in mice.Results The condition of inducing callus of lamina and rhizome is 6-BA0.5mg·L-1?2,4-D 3mg·L-1?IBA1mg·L-1 as medium growth hormone,culture medium is agar 7.0g·L-1,sucrose 30.0g·L-1,and the value of p H is 5.8±0.2.Meanwhile,it is put in the circumstance of 25±2?in dark.To optimise the design of orthogonal experiment,it shows that the optimum growth regulator to get chlorogenic acid is151.6?g g-1 when callus of lamina is 2,4-D 1mg·m L-1;and 3.318mg g-1 when callus of rhizome are 6-BA 0.5mg·L-1?IBA 0.6 mg·L-1?2,4-D 2mg·L-1.When Lepisorus ussuriensis of callus tissue in alcohol extract minimum inhibitory concentration is0.02g·m L-1 of Bacillus subtilis,Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa Bacillus 0.04g·m L-1,callus DPPH radical scavenging rate reaches 77.35%.The highest scavenging rate of callus extract on superoxide anion free radical is 59.25%,weaker than the vitamin C;from the results of hot plate test and acetic acid writhing method experiment,it shows that the high concentration of Lepisorus ussuriensis callus tissue ethanol extraction liquid,low dose group has good analgesic effect,it has obvious discrimination compared with blank control group after having low dose group drug in 60mins on the hot plate test?P<0.01?,and compared with its anterior pain domain,aspirin group and high dose group,low dose group have obvious difference?P<0.01,P<0.05?.Compared with the blank control group,it has obvious difference on the analgesic effect of high dose group?P<0.001,P<0.005?,the analgesic effect of the positive control group has significant difference?P<0.01?.Compared with the positive control group,there is no significant difference between the analgesic effect of high dose group?P>0.05?.Conclusions To use laminaand rhizome as explants culture tissue.Moreover,to induce the callus of laminaand rhizome proliferate the amount and test the number of chlorogenic acid.After that it has been screened out that the optimal culture condition to get chlorogenic acid is the callus of rhizome which is 6-BA 0.5mg·L-1?IBA 0.6mg·L-1?2,4-D 2mg·L-1,agar7.0g·L-1,sucrose30.0g·L-1,and the value of p H is 5.8±0.2.The callus of Lepisorus ussuriensis has the functions of antibacterial activity,antioxidation and analgesic effect. |
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