Preliminary Establishment Of Cell Suspension System Of Lonicera Japonica Thunb And Study On The Inhibition Of Nitrosation By Chlorogenic Acid

Chlorogenic acid is one of the main functional components of the traditional medicinal materials---Lonicera japonica Thunb,which have pharmacological effects of anti-cancer,anti-oxidation,lowering blood sugar,lowering blood lipids,and so on.It is widely used in the fields of medicine,food and daily chemical industry.The way of extracting chlorogenic acid by planting Lonicera japonica Thunb is far from satisfying people's needs.Plant cell suspension culture is a new method for the production of secondary metabolites,It has the advantages of being free from natural conditions,short production period and high efficiency.Therefore,it is of practical significance to study the efficient accumulation of chlorogenic acid in plant cell suspension culture system.Lonicera japonica Thunb plant was used as the material in this study.Firstly,The callus was induced by tissue culture technology.Secondly,Lonicera japonica Thunb cell suspension culture system was established by exploring the conditions suitable for the growth of Lonicera japonica Thunb cells.Finally,the ability of chlorogenic acid---Lonicera japonica Thunb cells' secondary metabolites to inhibits nitrosation reaction were studied.The main results showed as follows:(1)Study on conditions of callus induction and subculture,using callus status and growth rate as indicators.The optimal explant types,explant sterilization method,culture time in dark and hormone ratio were obtained through single factor tests and orthogonal tests.Lonicera japonica Thunb buds were treated with 70% alcohol for 30 s and then soaked for 10 s with 5% sodium hypochlorite solution.The light yellow color,loose,fragile callus was induced in the MS medium with 1.5 mg/L NAA,1.0 mg/L 2,4-D,0.75 mg/L KT and 0.15 mg/L 6-BA after 9 days' dark culture.Callus growth curve for the S-type.After 24 days of culture,the weight reached a maximum.The chlorogenic acid was extracted from the dry callus by HPLC at a concentration of 57 mg/100 g.The optimal hormone combination for the subculture of Lonicera japonica Thunb callus was 6-BA1.5 mg/L + NAA 0.2 mg /L + 2,4-D 0.1 mg/L by orthogonal design.(2)Study on conditions of cell suspension culture of Lonicera japonica Thunb,with the calluses induced in the above experiment used as materials.The inoculation amount,sucrose concentration,initial pH and hormone ratio were tested through single factor tests and orthogonal tests to optimized the culture conditions of Lonicera japonica Thunb cell suspension system.Inoculated the callus into MS liquid medium,after 7-10 days culture and the cell suspension culture system was established,Callus' inoculation amount is 0.2 g/mL,sucrose concentration is 30 g/L,initial pH is 5.8,MS medium supplemented with 1.4 mg/L 6-BA,0.5 mg/L NAA and 0.7 mg/L 2,4-D.There are three periods during the growth of Lonicera japonica Thunb suspension cells: lag phase,growth phase and decay phase.The fresh weight of the suspension cells reached the maximum on the 10 th day,with 2.512 g per 5mL medium.The suspension cells cultured for 7 to 10 days were vigorous and can be subcultured.Through the inverted microscope,most of the cells are in the form of 2 to 10 cell clumps during the growing period and a small number of cells were single.Determined the content of chlorogenic acid in dry cells was 826.7 mg/100 g by HPLC.(3)Study on the inhibition of nitrosation by chlorogenic acid.The effect of chlorogenic acid,which are the main components of Lonicera japonica Thunb,on removing nitrite and blocking synthesis of nitrosamine increased with the increase of chlorogenic acid concentration,reaction time and temperature,and it decreased with the increase of pH.The optimum concentration,pH,reaction time and temperature of chlorogenic acid in removing nitrite were 5 ?g/?L,3.4,40 min and 47 °C.The optimum concentration,pH,reaction time and temperature of chlorogenic acid in blocking synthesis of nitrosamine were 3 ?g/?L,2.8,20 min and 47 °C.The optimum conditions for the inhibition of nitrosation were as follows: concentration of sample solution was 5 ug/uL,pH was 2.8,reaction time was 40 min and temperature was 47°C.on the inhibition nitrosation reaction,chlorogenic acid and Vc have the same effect,weaker than isogenic acid A,The ability of the three is weaker than rutin.Chlorogenic acid and iso-chlorogenic acid A have good ability to remove nitrite and blocking synthesis of nitrosamine,and the ability is different with the reaction environment changs.