Hydrolysis Of Gypenosides By Recombinase And Anti-Melanogenesis Effect Of Resulting Rare Gypenosides

Gynostemma Herba was the dried whole grass of Gynostemma pentaphyllum(Thunb.)Makino.Its main chemical component is gypenoside,which has anti-cancer,anti-inflammation,anti-aging and anti-oxidation pharmacological effects.The number and configuration of glycosyl in the structure of gypenoside are related to its biological activity.Studies have shown that secondary glycosides have stronger anti-tumor activity and higher bioavailability than gypenoside,but secondary glycosides in plant origin is of very low content or they even don’t exist,and it is difficult to prepare them in large quantities by conventional separation methods.Therefore,enzyme conversion with mild conditions and strong specificity is adopted to obtain rare secondary glucosides.Studies have shown that ginsenosides have anti-aging,whitening and hydrating effects.As the only medicinal plant rich in ginsenosides except ginseng,Gynostemma Herba contains triterpenoid saponins similar to ginsenosides in structure,but its effect on melanin generation has not been studied.Melanin generation and skin pigmentation are important protective factor to deal with UV radiation damage,but abnormal melanin will lead to serious facial beauty and skin diseases,such as acanthosis nigricans,melasma,skin cancer,etc.Tyrosinase plays a crucial role in regulating the metabolic pathway of melanin formation.In this research,based on recombinant enzymatic hydrolysis technology,gypenoside Ⅲ(GypⅢ)was transformed specifically to obtain more active natural products,namely gypenoside ⅩⅦ(Gyp ⅩⅦ),gypenoside LⅩⅩⅤ(Gyp LⅩⅩⅤ)and ginsenoside CK(Gin CK),gypenoside Ⅷ(Gyp Ⅷ)was transformed specifically to gypenoside Ⅻ(Gyp Ⅻ)and ginsenoside CK(Gin CK).In addition,in vitro enzyme inhibition and cell experiments were conducted to investigate the effect of rare gypenosides on melanin production,which provided solid support for expanding the application of those rare gypenosides.The research includes the following four parts:(1)β-D-glucosidase was heterologous expressed by E.coli expression system,and its enzymatic properties were characterized.The recombinant enzyme was used to hydrolyze Gyp Ⅲand Gyp Ⅷ.Crude enzyme solution was separated by GST 4B chromatography column,and the recombinant enzyme of high purity was obtained,with molecular weight of about 97 kDa.The optimum pH and temperature of the recombinant enzyme were 7.0 and 37℃ respectively.Common metal ions had certain inhibitory effect on enzyme activity,but the enzyme had good tolerance to NaCl.When pNPG was used as substrate,the Km of recombinant β-D-glucosidase was 2.342 mM,and the Vm was 9.217 μM/min.In addition,gynostemma triterpenoid saponins and epimedium flavones can be hydrolyzed by this enzyme and the corresponding secondary products were produced.Especially,its hydrolysis efficiency on gypenosides is higher than that of commercial enzymes,which provides a scientific basis for the use of this recombinant enzyme to hydrolyze gypenosides to obtain rare secondary glycosides.(2)The key technology of obtaining Gyp ⅩⅦ,Gyp LⅩⅩⅤ,Gyp Ⅻ and Gin CK by recombinant enzymatic hydrolysis of Gyp Ⅲ and Gyp Ⅷ was established.The results of single factor experiment showed that when the enzyme activity was 100 U/mL,the buffer pH was 7.0,the hydrolysis time was 15 min,and the reaction temperature was 37℃,the conversion rate of Gyp Ⅲ and Gyp Ⅷ was 65%and 75%,respectively.After 0.5-h hydrolysis,the conversion of Gyp Ⅲ and Ⅷ reached 100%,and the obtained hydrolyzed products were Gyp ⅩⅦ and GypⅫ.With the hydrolysis time extended to 6 h,Gyp LⅩⅩⅤ was obtained.After 36-h hydrolysis,the obtained products were Gin CK.In addition,the scale-up experiment of recombinant enzyme hydrolysis of Gynostemma pentaphyllum saponins was completed.The results showed that GypⅢ and Gyp Ⅷ could be completely hydrolyzed.At the same time,the reaction products formed precipitate during the hydrolysis process,and secondary gypenoside could be easily separated from the hydrolyzed system by centrifugation and other subsequent operations.The results of this chapter laid a foundation for the preparation of secondary glycosides of Gynostemma Herba by enzymatic hydrolysis of primary glycosides in industrial practice.(3)The effects of Gyp Ⅲ,Gyp Ⅷ and its secondary glycosides on tyrosinase activity were investigated by in vitro enzyme inhibition experiments,and the inhibitory mechanism,inhibitory type and inhibitory constant of gypenoside triterpenoid were clarified by kinetic experiments.The results showed that compared with Gyp Ⅲ and Gyp Ⅷ,Gyp LⅩⅩⅤ,Gyp Ⅻ and Gin CK could reduce the activity of tyrosinase,especially the ginsenoside CK at 1200μM could significantly inhibit the activity of tyrosinase,and the secondary glucoside of gypenoside had reversible inhibition on tyrosinase.Gyp ⅩⅦ had a mixed activation effect on tyrosinase,while Gyp LⅩⅩⅤ,Gyp Ⅻ and Gin CK were competitive inhibitors.Molecular docking method was used to simulate the binding mode and binding force of Gynostemma Herba triterpenoid saponins and tyrosinase.The results showed that the interaction between gypenoside and tyrosinase was lower than that between Gyp Ⅲ and Gyp Ⅷ,so it was easier to bind to tyrosinase.The results of this chapter provide scientific basis for expanding the application of gynostemma pentaphylside in medicine,cosmetics and other fields.(4)Using α-MSH-induced mouse B16F10 melanoma cells as models,the effects of Gyp Ⅲ,Gyp Ⅷ and its secondary glycosides on cell viability,intracellular tyrosinase activity,intracellular and intracellular melanin content,and MITF protein expression were investigated.The α-MSH concentration used for modeling was determined to be 100 nM,and the triterpenoid saponins with concentrations of 50 μM,150 μM and 300 μM were determined by MTT method to have little effect on the activity of the model cells,and were used for subsequent experiments.In vitro experiments showed that 300μM Gyp ⅩⅦ,Gyp LⅩⅩⅤ,Gyp Ⅻ and Gin CK significantly reduced the intracellular and extracellular melanin content(P<0.01),and significantly inhibited tyrosinase activity(P<0.05);Gype Ⅲ and Gyp Ⅷ had no significant effects on melanin content and tyrosinase activity.In addition,Gyp ⅩⅦ,Gyp LⅩⅩⅤ,Gyp Ⅻ and Gin CK at the concentration of 300 μM could inhibit the expression of MITF,and the inhibitory effect of GypⅩⅦ and Gin CK on the expression of MITF was significantly stronger than that of Gyp Ⅲ and Gyp Ⅷ administration groups(P<0.05),these results indicated that the inhibitory effect of secondary glucoside on melanin formation may be realized by down-regulating melanin-related proteins and inhibiting tyrosinase activity.The research in this chapter shows that the rare secondary glucosides obtained by enzymatic hydrolysis have better anti-melanin activity than the gypenosides.