Inhibitory Effect Of Polyketides In Cortinarius Sp.on Biological Enzyme Activity

Cortinarius sp.is a precious wild fungal resource with great research value.Its secondary metabolites have diverse structures and various biological activities,such as antitumor activity,antioxidant activity,antibacterial activity and insecticidal activity.The most abundant compounds in Cortinarius sp.are polyketides,which are the main source of its active substances.In this study,glutathione S-transferase(GST)and glutamate dehydrogenase(GDH)were chosen as targets to study the insect resistance and antitumor effects of polyketides from Cortinarius sp.Among them,GST is an insect detoxification enzyme,which can protect insects from insecticide poisoning;the reduction of GDH activity may have insect resistance effects,and it can inhibit the growth of tumors.In this study,the Cortinarius sp.picked from Changbai Mountain was soaked using ethanol under ultrasonic.Then,three extraction phases and raffinate aqueous were obtained via extraction using petroleum ether,ethyl acetate and n-butanol.Mutually.The inhibitory effect of 4 extracts on glutathione S-transferase(GST)and glutamate dehydrogenase(GDH)was determined by the enzyme kinetics method,and the ethyl acetate phase was confirmed to exhibit the strongest inhibitory effect against GST and GDH.Three polyketides were separated and purified from the ethyl acetate phase using gel chromatography,silica gel chromatography,reversed-phase column chromatography and TLC thin-layer chromatography.On the basis of high-resolution electrospray ionization mass spectrometry(HR-ESI-MS),1D and 2D nuclear magnetic resonance(NMR)and electronic circular dichroism(CD)data,their structures were identified to be rufoolivacin E(1),viocristin B(2)and 1-Hydroxy-3,6,8-trimethoxyanthraquinone(3).It is noted that 1 is a new polyketide compound,2 and 3are natural products isolated for the first time.Subsequently,sixteen compounds including 1-3 and thirteen known compounds previously obtained in our laboratory from Cortinarius sp: emodin(4),1-Omethylemodin(5),uestinol(6),rufoolivacin A(7),rufoolivacin C(8),endothianin(9),cassiamin B(10),anhydrophlegmacin-9,10-quinone-8'-O-meth-yl ether(11),sinapiquinone(12),2,2''-birufoolivacin B(13),laccaic acid D(14),endocrocin(15),SEK 15b(16),were assigned to evaluate their inhibitory effects on GST and GDH by enzyme kinetics experiments.The results showed that four polyketides exhibited inhibitory effect against GST,and the inhibitory effect is: 11>16>1>15.Eight compounds had inhibitory effect on GDH,of which 4 and 9 exhibited extremely strong inhibitory effects.The inhibitor type of 4 and 9 towards GDH were analyzed by fitting the double reciprocal curve of the Michaelis equation,the result showed that these two compounds are both GDH competitive inhibitor with Ki values were 2.09 ± 0.73 ?M and 5.39 ± 1.99 ?M,respectively.The other six compounds also show inhibition on GDH,the inhibitory effect is: 1>8>11>5>15>10.Molecular docking was utilized to simulate the binding of compounds to GST and GDH to infer their binding sites,and their mechanism was explained by auxiliary enzyme kinetics.In summary,Three polyketides were separated and purified from Cortinarius sp.It is noted that a new polyketide compound was separated,two natural products isolated for the first time.four polyketides showed inhibitory effects on GST and two polyketides exhibited potential as GDH competitive inhibitors.The research of GST and GDH inhibitors and their inhibition mechanism in this paper will prove the application prospects of polyketides in pest control and the potential to develop into anti-tumor drugs.This paper will provide a new insight for application of natural products in pest control and medicine development.