Study On The Chemical Constituents And Bio-activities Of The Stems And Leaves Of Tribulus Terrestris L.

Tribulus terrestris L.belongs to the Tribulus genus of Zygophylaceae family,whose roots,stems,leaves,flowers and fruits may be used for medicine.Modern pharmacological studies show that Tribulus terrestris L.has anti-aging,regulation of blood lipid,hypoglycemic,antibacterial,anti-tumor,inhibition of tyrosinase activity and other pharmacological effects.According to literatures,the root,stem,leaf,flower and fruit of Tribulus terrestris L.contain steroidal saponins,which are the main functional components.At present,the research on the fruit of Tribulus terrestris L.is more in-depth and the total saponins extracted from it have been used as cude drug in Xinnaoshutong capsules.However,there are few reports on the root,stem,leaf and flower of Tribulus terrestris L..Therefore,the chemical constituents and biological activities of the stem and leaves of Tribulus terrestris L.were studied in this paperIn this paper,40%ethanol reflux was used to extract.Fourteen monomeric compounds(Z-l～Z-14)were separated by column chromatography and preparative high performance liquid chromatography from the extract and they were identified by 1D-NMR,2D-NMR,HR-MS,IR spectral method combining with the physical and chemical properties,including 4 new compounds and 8 compounds obtained from the stems and leaves of Tribulus terrestris L.for the first time.The new compounds are(25R)-5a-spirostan-12-ketone-3β,24-diol-24-O-β-D-glucopyranoside(Z-5),(25R)-5a-f-urostan-12-ketone-3β,22a,26-triol-26-O-β-D-glucopyranoside(Z-12),26-O-β-D-gluco-pyranosyl-(25R)-furostan-5(6)-alkene-3β,22a,26-triol-3-O-{[β-D-xylopyranosyl-(1→3)][β-D-xylopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→4)]}[a-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside(Z-13),(25R)-furostan-4(5)-alkene-3-ketone-22a,26-tri-ol-26-O-β-D-glucopyranoside(Z-14).For the first time,compounds were isolated from the stems and leaves of Tribulus terrestris L.are Hecogenone(Z-1),(25R)-tigogenin-3-O-β-D-xylopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glu-copyranosyl-(1→4)-[a-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside(Z-3),hecogenin-3-O-β-D-glucopyranosyl-(1→4)]-[β-D-galactopyranoside(Z-4),26-O-β-D-glucopyranosyl-22-methoxyl-(25R)-5α-furostan-3 β,26-diol-3-O-[β-D-xylopyranosyl-(1→3)]-[β-D-xylopyranosyl-(1→2)]-β-D-glucopyranosyl-(1→4)-[α-L-rhamopyranos-yl-(1→2)]-β-D-galactopyranoside(Z-6),26-O-β-D-glucopyranosyl-(25R)-5α-furostan-12-ketone-3β,22a,26-triol-3-O-{α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-galactopyranoside(Z-8),26-O-β-D-glucopyranosyl-(25R)-5α-furostan-2α,3β,22a,26-tetraol-3-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside(Z-9),26-0-β-D-glucopyranosyl-(25R)-5α-furostan-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-galactopyranoside(Z-10),(25R)-furostan-4(5)-alkene-3,12-diketone-22a,26-diol-26-O-β-D-glucopyranoside(Z-11)。On the basis of the above studies,a method for the determination of three furostanol saponins in the stems,leaves and fruits of Tribulus terrestris L.was established by solid phase extraction-high performance liquid chromatography-evaporative light scattering detector(SPE-HPLC-ELSD).These three furostanol saponins were isolated in this paper and they are compound Z-7,compound Z-11,compound Z-12.Firstly,the optimal extraction method was established through the investigation of the influencing factors such as extraction solvent,amount of solvent,extraction method,extraction times,SPE types and elution conditions.Secondly,the HPLC conditions were determined and the methodological investigation was completed.Experimental results show that the peak area y(1gS average)and the sample mass x(lgl)of the compounds Z-7,Z-11 and Z-12 show a good linear relationship in the range of 0.40～39.22 μg,0.20～19.68 μg and 0.16～15.72 μg,respectively.The RSDs of precisions,repeatabilities,stabilities and of compound Z-7,Z-11 and Z-12 are all less than 2.0%(n=6),indicating that the analytical method established in this experiment has the advantages of high sensitivity,high precision and high accuracy.The results show that the contents of Z-7,Z-l1 and Z-12 in the stems,leaves and fruits of Tribulus terrestris L.are different,and the contents of the stems are the highest,0.1208 mg·g-1(Z-7),0.0372 mg·g-1(Z-11),0.0166 mg·g-1(Z-12).Leaves were followed by 0.0740 mg·g-1(Z-7),0.0057 mg· g-1(Z-11)and 0.0061 mg· g-1(Z-12).The fruits contents are very low,lower than the low value of linear range.The literatures showed that the ethanol extract from the fruits of Tribulus terre.stris L.and the total saponins of furostanol and spirostanol had whitening effects.Therefore,this paper prepared total saponins furostanol and spirostanol from the stems and leaves of Tribulus terrestris L.by macroporous adsorption resin method,and investigated the whitening effect of them inhibiting the activity of tyrosinase.The results show that both total furostanol saponins and total spirostanol saponins could inhibit the activity of tyrosinase.The inhibition rate increases with the increase of the sample concentration between 0 and 3.00 mg·mL-1.These results suggest that two kinds of steroidal saponins from the stems and leaves of Tribulus terrestris L.can inhibit the activity of tyrosinase.To sum up,the chemical constituents of the steims and leaves of Tribulus terrestris L.are further studied in by this paper.A method for the content determination of three furostanol saponins in the stems and leaves of Tribulus terrestris L.is established by SPE-HPLC-ELSD method.The inhibitory effects of total furostanol saponins and total spirostanol saponins from the stems and leaves of Tribulus terrestris L on the activity of tyrosinase are studied.The results of this study provide a new scientific basis for the further study of the phannacodynamic material in the stems and leaves of Tribulus terrestris L.and the development and utilization of steroidal saponins in the stems and leaves of Tribulus terrestris L..