| Tribuli fructus is the dry fruit of Tribulus terrestris L. in which many chemicalconstituents such as saponins, flavonoids, alkaloid, polysaccharide and othercompounds have been isolated and identified. This herb has the functions such asflat liver, activating blood, nominal itching etc and it is mainly used for thetreatment of headache and dizziness, breast close and breast pain, itchy rubella, etc.Pharmacological studies have shown that the fruits of Tribulus terrestris L. haveantifungal, antitumor and diuresis bioactivities, especially the prominent curativeeffect in the aspect of prevention and cure of disease of heart and cerebral vessels.In this paper,12kinds of furostanol saponins were isolated from30Kg driedfruits of Tribulus terrestris L. by the extraction of decoct with water and the isolationof column chromatography ofAB-8macroporous adsorption resin, silica gel, ODSand prep-HPLC. The12compounds were obtained and six of them were identified byNMR and MS spectroscopy methods combined with the structure of the physical andchemical properties.This paper has also established HPLC-ELSD method of content determination ofcompound JYH–D in dry fruits of Tribulus terrestris L.Extraction and isolationThirty kilograms of dry fruits of Tribulus terrestris L. were decocted with10times water for three times (1.5h,1h,0.5h). The extraction solution wasconcentrated to1/3volume and adsorbed by AB-8macroporous adsorption resin,and then eluted with deionized water to colorless, eluted with40%and90%ethanolsolution respectively. The40%ethanol elution was collected and recovered,280gtotal steroidal saponins was obtained. Total steroidal saponins (280g) was subjected to column chromatography onsilica gel and eluted with EtOH-EtOAc-H2O(2:1:0.3) to give3frs.(1、2、3). Fr.2was subjected by chromatography on silica gel and eluted with EtOH-EtOAc-H2O(5:4:0.5) into Fr.4and Fr.5. Fr.5was chromatographied on ODS and eluted withacetone-H2O(1:2~1.2:1) to give fr.1-fr.10. Fr.3was performed by prep-HPLC andgave compound F (JYH-F) and G(JYH-G). Fr.5-10were subjected by prep-HPLC（Acetonitrile–water in different proportion as mobile phase）or ODS columnchromatography（eluted by different proportion acetone–water, seven monomersmarked JYH-K, JYH-L, JYH-A, JYH-J, JYH-I, JYH-H and JYH-E were obtainedrespectively.IdentificationSix compounds were identified by NMR and MS spectra as3-O-{[β-D-xylopyranosyl(1'3)][β-D-xylopyranosyl-(1'2)]-[β-D-glucopyranosyl(1'4)]}[α-L-rhamnopyranosyl(1'2)]-[β-D-galactopyransyl]-26-O-β-D-glucopyranosyl-5α-furost-12-one-25(R)-3β,22,26-trio（lJYH-A）,3-O-{[β-D-xylopyranosy(1'3)][β-D-galactopyransyl-(1'2)]-[β-D-glucopyranosyl(1'4)]}-[β-D-glucopyranosyl]-26-O-β-D-glucopyranosyl-5α-furost--12-noe-25(R)-3β,22,26-triol（JYH-D）,3-O-{[β-D-xylopyranosy(1'3)][β-D-xylopyranosy-(1'2)]-[β-D-galactopyransy(1'4)]}[α-L-rhamnopyranosyl(1'2)]-[β-D-galactopyransyl]-26-O-β-D-glucopyranosyl-25(R)-5α-furost--3β,-22α-26-triol(JYH-E),3-O-[β-D-glucopyranosyl(1'2)]-[β-D-glucopyranosyl(1'4)]-[β-D-galactopyransy]-26-O-β-D-glucopyran-osyl-25(S)-5α-furost-12-noe-2α，3β，22α，26-terraol（JYH-F）,3-O-[β-D-glucopyranosyl(1'2)]-[β-D-glucopyranosyl(1'4)]-[β-D-galactopyransy]-26-O-β-D-glucopyranosyl]-25(R)-5α-furost-12-noe-2α，3β，22α，26-terraol（JYH-G）and3-O-{[β-D-xylopyranosyl(1'3)][β-D-galactopyransyl-(1'2)]-[β-D-glucopyranosyl(1'4)}-[β-D-glucopyranosyl]-26-O-β-D-glucopyranosyl-25(R)-5α-furost-3β-22α-26-triol(JYH-J). Compound JYH-J is a new compound. Determination of compounds JYH-D in the fruits of Tribulusterrestris L. byHPLC-ELSDInstruments and Conditions:HPLC was performed on an Agilent1200HPLC instrument and COSMOSILC18-MS-II column (4.6×250mm,5μm), which was gradient eluted by acetonitrile-water in the ratio of15.5:84.5~40:60with the flow rate of1ml/min at25°C，Thegas(air) pressure of ELSD was2.15Bar and the temperature of drift tube formeasurement was104°C.ResultsI. Linearit The range of the linearity was4.00μg～20.00μg for compound JYH-D.The regressive equation is y=1.5170x+0.2567with R2was0.9994.II. Precision Solutions of compound JYH-D was injected into HPLC for six times,the values of RSD as the results of precision tests was0.51%.(n=6).III. Repeatability The repeatability tests for six samples showed their RSD was0.47%.(n=6).IV. Stability The stability of compound JYH-D was good in ten hours, because theRSD was0.51%.V. Recovery The average recovery (n=6) for compound JYH-D was97.92%，RSD=1.05%(n=6).VI Contents determination: The samples of. Tribulus terrestris L. from Bozhouwere determined, the results show the average content of compound JYH is0.027%. |
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