Study On The Chemical Constituents And Biological Activity Of Degradative Products Of Diterpene Esters From Euphorbiae Semen

Euphorbia Semen is the dry and mature seed of Euphorbia lathyris L..Euphorbia Semen mainly has the effects of diarrhea,water removal,blood breaking and swelling reduction.Scholars at domestic and foreign have isolated a variety of chemical components from Euphorbia Semen,among which diterpene esters are the main functional components,which have pharmacological effects such as anti-tumor,anti-inflammatory,removing freckles,whitening and so on.However,studies have also shown that both the extracts of Euphorbia Semen and Euphorbia Semen have certain toxicity.It has been reported in the literature that the toxic site of Euphorbia Semen is in its fatty oil,and it has been preliminarily confirmed that several diterpene esters have certain toxicity.It shows that the diterpene esters in Euphorbia Semen are likely to be both functional and toxic components.Therefore,in this paper,these diterpene esters components will be degraded by chemical measures,and some or all of the ester bonds in the molecule will be broken to obtain degradation products,and the chemical components of the degradation products will be separated,identified then study their biological activities,in order to find chemical composition with high and low toxicity,and provide scientific basis for the development of its new medical applications.First of all,the main diterpene esters components in Euphorbia Semen were studied in this paper.Using conventional column chromatography and preparative chromatography separation techniques,combined with nuclear magnetic resonance and other spectroscopic methods,ten main diterpene esters were isolated and identified as Euphordracunculin C,5,15,17-O-tri-acetyl-3-O-nicotinoyllathyol-6,12-diene-14-one,（2S,3S,4S,5R,7R,9S,11R,15R）-7,15-diacetoxy-3-benzoyloxy-5-hydroxy-14-oxolathyra-6-（17）,12E-diene,Euphorbia factor L₈,Euphorbia factor L_1,Euphorbia factor L₉,Euphorbia factor L₂₇,Euphorbia factor L₂₂,Euphorbia factor L₃₁,Euphorbia factor L₃.Secondly,this paper established the optimal conditions for the alkali degradation of diterpene esters through single factor experiments.The experimental results showed that the optimal degradation conditions are as follows:add methanol extract of Euphorbia Semen oil（the main component is diterpene alcohol ester compounds）into methanol at a ratio of 1:5,adjust the p H to 10 with 2M sodium hydroxide methanol solution,degrade it in a water bath at 100℃ for 6 hours,then cool it down,the p H was adjusted to neutral with with 6M HCl solution,and then the solvent was recovered under reduced pressure to dryness,to obtain the degradation product of diterpene esters of Euphorbia Semen.Then,the above-mentioned degradation products were separated and structurally identified by column chromatography and preparative HPLC.The results showed that three main components were obtained,namely,lathyrol,17-hydroxyjolkinol and 7-hydroxylathyrol.All three are the products of the corresponding diterpene esters degrading acetyl group,benzoyl group or cinnamoyl group.Thirdly,in order to clarify the content of lathyrol,17-hydroxyjolkinol and7-hydroxylathyrol in the degradation product of diterpene esters of Euphorbia Semen,the HPLC method for the determination of these three components was established and the methodology was investigated.The results showed that the injection mass of lathyrol,17-hydroxyjolkinol and 7-hydroxylathyrol had good linear relationship in the range of 0.04～9.86μg,0.038～9.70μg and 0.04～9.90μg,respectively;The limits of detection for lathyrol,17-hydroxyjolkinol and 7-hydroxylathyrol were 7.0 ng,4.0ng,6.0ng and the limits of quantification were 20.0 ng,10.0 ng,16.0 ng respectively;The RSDs values of the intra-day precision,inter-day precision,repeatability,stability,and sample recovery for each compounds were all less than 3%.It showed that the method has high accuracy,good precision and high sensitivity.The results of content determination showed that among the degradation products of diterpene esters of Euphorbiae Semen,the content of lathyrol is the highest,with an average content of 17.93 mg/g,followed by 17-hydroxyjolkinol,with an average content of 5.62 mg/g,and the lowest content is 7-hydroxylathyol,with an average content of 3.32 mg/g.The sum of the average content of the three is 26.87mg/g.Finally,this paper explored the inhibition of tyrosinase by methanol extracts of Euphorbia Semen oil,the degradation product of diterpene esters of Euphorbia Semen,Euphorbia factor L₃ and lathyrol.The experimental results showed that the two components and the two compounds are all had strong inhibition rates on tyrosinase at different concentrations,and there was no significant difference in the inhibition rates between them.When the concentration was 0.003 mg/m L～0.025 mg/m L,the inhibition rate increased significantly;when the concentration was 0.025 mg/m L～1.00mg/m L,the inhibition rate increased slowly;when the concentration was greater than or equal to 1.00 mg/m L,the inhibition rate tends to be constant,reaching about 90%.Among them,the tyrosinase inhibition rate of lathyrol was only slightly higher than that of Euphorbia factor L₃ at some individual concentrations,but there was no significant difference on the whole.It can be seen that the diterpene esters in Euphorbia Semen can effectively inhibit the activity of tyrosinase,but there is no significant difference in the inhibition rate of tyrosinase activity before and after the degradation of these compounds.To sum up,this paper isolated and identified the main diterpene esters in Euphorbia Semen oil,and established the best degradation method for these components.Then,the chemical components of the degradation product of diterpene esters of Euphorbia Semen were separated,structurally identified,content determined and the inhibitory effect of these components on the activity of tyrosinase was investigated.The research results of this paper provide a new scientific basis for the development and utilization of diterpenoids in Euphorbia Semen.