| This study was to compare the differences in chemical components and antioxidant activity of dioecious plants.The flower buds of Populus tomentosa Carri ere and the barks of Ginkgo biloba L.were research objects.The HPLC fingerprints were established by high performance liquid chromatography-diode array detector(HPLC-DAD).Similarity Evaluation System for Chromatographic Fingerprint of Chinese was used to calibrate the common peaks and evaluate the similarity.Based on the common peaks,SIMCA 14.1 software was used for multivariate statistical analysis,including principal component analysis(PCA),cluster analysis(HCA),and orthogonal partial least squares-discriminant analysis(OPLS-DA),to identify the female and male of the samples,and screened out the differential components.The differential components of the female and male samples were identified by ultra-high performance liquid phase-time-of-flight mass spectrometry(UPLC-Q-TOF/MS).Catechin was used as the control,and content of total flavonoid was determined by the aluminum nitrate colorimetric method.6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid(Trolox)was used as the positive control,and 1,1-diphenyl-2-(2,4,6trinitrophenyl)hydrazine(DPPH)and 2,2’-azinobis(3-ethylbenzothiazoline-6sulfonic acid ammonium salt)(ABTS)free radical scavenging methods were used to evaluate antioxidant activities in vitro.The t-test was used to compare female and male samples.The specific research results were as follows:1 Comparison of HPLC fingerprint and antioxidation between female and male of P.tomentosa1.1 HPLC fingerprint and multivariate statistical analysisThe fingerprints of 22 P.tomentosa flower buds were established by HPLC,and a total of 17 common peaks were calibrated.Compared with the reference substances,the peaks of 4,5,and 12 were siebolside B,isograndidentatin A,and sakuranetin,respectively.The similarity of 11 female flower buds ranged from 0.978 to 0.999,and the similarity of 11 male buds ranged from 0.943 to 0.985.Based on the peak area of the 17 common peaks,the 22 P.tomentosa flower buds were analyzed with multivariate statistical analysis by SIMCA 14.1 software,including PCA,HCA,and OPLS-DA.In the unsupervised mode,PCA results showed that the female and male flower buds were significantly divided into two groups along the longitudinal axis(R2X=0.753,Q2=0.564).HCA dendrogram showed that the female flower buds were located on the left and the male flower buds were located on the right.The two groups were well distinguished.In the supervised mode,OPLS-DA results showed that the female and male flower buds were significantly separated along the longitudinal axis(R2Y=0.980,Q2=0.970).The results of 200 permutation tests showed that the OPLS-DA model was not over-fitted.Four peaks of 4,9,14 and 15 were screened out according to S-plot(|p[1]|≥0.2,|p(corr)[1]|≥0.8),variable importance in the projection(VIP)(VIP>1)and P<0.05.The differential components were identified by UPLC-Q-TOF/MS.Combined with the reference substances and literature,two compounds were identified,namely siebolside B and tremulacin.Therefore,it can be considered that siebolside B and tremulacin may be potential gender markers of P.tomentosa.1.2 Determination of total flavonoid contentTaking catechin as the control,the total flavonoid content of 22 female and male flower buds of P.tomentosa was determined by the aluminum nitrate colorimetric method.The results showed that the total flavonoid content of the female flower buds of P.tomentosa was 24.30～27.56 mg/g and that of the male flower buds was 25.54～31.99 mg/g.The total flavonoid content of female and male samples was analyzed and compared with IBM SPSS statistics 25 software.The results showed that there was significantly different in the content of total flavonoid between the female and male flower buds(P<0.05),and the content of the male flower buds was higher than that of the female flower buds,indicating that the total flavonoid content of P.tomentosa was related to its gender.1.3 Evaluation of antioxidant activityTaking Trolox as the positive control,the antioxidant activity of female and male flower buds of P.tomentosa was compared by DPPH and ABTS free radical scavenging methods,the Trolox-Equivalent Antioxidant Capacity(TEAC)values of the female and male flower buds of P.tomentosa by DPPH method were 15.62～24.22 mmol/L and 20.13～29.03 mmol/L,respectively.The TEAC values of the female and male flower buds of P.tomentosa by ABTS method were 19.08～21.61 mmol/L and 20.90～26.34 mmol/L,respectively.The antioxidant activity of the female and male samples in vitro was analyzed and compared with IBM SPSS Statistics 25 software.The results showed that the TEAC values of the two methods were significantly different between the female and male flower buds(P<0.05).The TEAC values of male flower buds of DPPH and ABTS methods were higher than those of the female flower buds.The TEAC values were higher,the scavenging ability of DPPH and ABTS free radicals was stronger,and the antioxidant ability was stronger.The antioxidant activity of the male buds was higher than that of the female buds.Using IBM SPSS Statistics 25 software,the correlation was performed on the total flavonoid content of 22 female and male flower buds of P.tomentosa and their corresponding TEAC values determined by DPPH and ABTS method.The results showed that the correlation coefficients between the total flavonoid content and the TEAC values of DPPH and ABTS methods were 0.605 and 0.896,respectively.The TEAC values measured by the two methods were positively correlated with the content of total flavonoid,indicating that the antioxidant activity of P.tomentosa flower buds was positively correlated with the content of total flavonoid.2 Comparison of HPLC fingerprint and antioxidation between female and male of G.biloba2.1 HPLC fingerprint and multivariate statistical analysisThe fingerprints of 10 male and female barks of G.biloba were established by HPLC,and a total of 9 common peaks were calibrated.The similarity of 5 female barks ranged from 0.981 to 0.993,and the similarity of 5 male barks ranged from 0.955 to 0.992.Based on the peak area of the 9 common peaks,the 10 barks of G.biloba were analyzed with multivariate statistical analysis by SIMCA 14.1 software,including PCA,HCA,and OPLS-DA.In the unsupervised mode,PCA results showed that the female and male barks were significantly divided into two groups(R2X=0.779,Q2=0.674).HCA dendrogram showed that the female barks were on the left and the male barks were on the right,which were clearly separated.In the supervised mode,OPLS-DA results showed that female and male barks were significantly separated along the longitudinal axis(R2Y=0.951,Q2=0.900).The results of 200 permutation tests showed that the OPLS-DA model was not over-fitted.Three peaks of 4,6 and 8 were screened out according to S-plot(|p[1]|≥0.4,|p(corr)[1]≥)0.6),VIP value(VIP>1)and P<0.05.The differential components were identified by UPLC-Q-TOF/MS.Combined with the literature,three compounds were identified,namely piscidic acid,olivil 4,4’-di-O-β-Dglucopyranoside,and olivil 4-O-β-D-glucopyranoside.Therefore,these three compounds may be potential gender markers of G.biloba.2.2 Determination of total flavonoid contentTaking catechin as the control,the total flavonoid content of female and male barks of 10 G.biloba was determined by the aluminum nitrate colorimetric method.The results showed that the total flavonoid content of the female barks of G.biloba was 4.72～5.35 mg/g and that of the male barks was 5.86～8.94 mg/g.The total flavonoid content of the female and male samples was analyzed and compared with IBM SPSS statistics 25 software.The results showed that there was significant difference in the total flavonoid content between the female and male barks(P<0.05),and the content of the male barks was higher than that of the female barks,indicating that the total flavonoid content of G.biloba was related to its gender.2.3 Evaluation of antioxidant activityTaking Trolox as the positive control,the antioxidant activity of female and male barks of G.biloba were evaluated and compared by DPPH and ABTS free radical scavenging methods.The results showed that the TEAC values of the female and male barks of G.biloba by DPPH method were 2.08-3.92 mmol/L and 4.11-8.69 mmol/L,respectively.The TEAC values of the female and male barks of G.biloba by ABTS method were 3.28～5.46 mmol/L and 6.35～9.63 mmol/L,respectively.The antioxidant activity of the female and male samples in vitro was analyzed and compared by IBM SPSS statistics 25 software.The results showed that the TEAC values of the two methods were significantly different between the female and male barks(P<0.05).The TEAC values of the male barks of DPPH method and ABTS methods were higher than those of the female barks.The TEAC values were higher,the scavenging ability of DPPH and ABTS free radicals was stronger,and the antioxidant ability was stronger.The antioxidant activity of the male barks was higher than that of the female barks.The correlation analysis was carried out between the total flavonoid content of the female and male barks and their corresponding TEAC values determined by DPPH and ABTS methods with IBM SPSS statistics 25 software.The correlation coefficients between the total flavonoid content and the TEAC values of DPPH and ABTS methods were 0.801 and 0.920,respectively.The TEAC values measured by the two methods were positively correlated with the content of total flavonoid,indicating that the antioxidant activity of G.biloba barks was positively correlated with the content of total flavonoid.3 ConclusionThere were differences in the chemical components of the female and male flower buds of P.tomentosa.HPLC fingerprint combined with multivariate statistical analysis can be used to identify male and female of P.tomentosa.The total flavonoid content and antioxidant activity of the male flower buds of P.tomentosa were higher than those of the female flower buds,and there were significant differences between the female and male flower buds,which may be the reason why male flower buds were selected for medicine.There were differences in the chemical components of the female and male barks of G.biloba.HPLC fingerprint combined with multivariate statistical analysis can be used to identify male and female of G.biloba.The total flavonoid content and antioxidant activity of the male barks of G.biloba were higher than those of the female barks,and there were significant differences between the female and male barks,which can increase the development and utilization of male barks of G.biloba in practical applications.In this study,HPLC fingerprint combined with multivariate statistical analysis was used to identify female and male G.biloba for the first time.For the first time,the total flavonoid content and antioxidant activity of the flower buds of P.tomentosa and the barks of G.biloba were measured,and the correlation analysis was carried out.It enriches the connotation of the material basis of gender differences on dioecious plants and provides a reference for the further development and utilization of P.tomentosa and G.biloba. |
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