| Dioseorea zingiberensis C.H. Wright (D. zingiberensis) is a perennial herb plant, whose genus and famlily belong to Dioscorea and Dioscoreaceae, respectively. It is also named Huangjiang and earlier recorded in Shanhaijing medicine book in Spring and Autumn Period. Its specialties are dry and cool, and has bitter flavor. As a Traditional Chinese Medicine, it could clear lung cough, promote dampness, treat stranguri, stimulate meridian, and activate collaterals. Due to its pharmacological effect, the rhizome of this plant is often used to treat these symptoms such as skin purulent infection, cough caused by lung heat, bee biting, soft tissue injury, pain, injuries (caused by falls, fractures, contusions and strains), and sprain, etc. The chemical ingredients in rhizome are mainly included starch, cellulose, saponins, alkaloid, and flavones, among which steroid saponins are the chief bioactive constituents. The diosgenin is extracted from its rhizome of D. zingiberensis as a primary botanic source. The water-soluble steroid saponins have evident effects on curing and preventing the cardiovascular disease.The relative researches contained isolating the single compound, illustrating the structures, establishing qualitative and quantitative analytical methods, exploring new pharmacological activity, are carried out and aimed at the steroid saponins which are major bioactive compounds in D. zingiberensis in this paper.(1):Extracting and separating the pharmacological constituents from D. zingiberensisWe established a mode, which the traditional column chromatograph technology and modern separating chromatograph technology was combined together, to systemically isolate the steroid saponins from the rhizome of D. zingiberensis, the main bioactive ingredients of this plant. Eluting with solvent composed in different volume ratios, the steroid saponins were separated into different parts according to their various polarities on the traditional column chromatography at first, then these obtained parts with different polarities were further isolated in detail by HSCCC and preparative HPLC.5 compounds were obtained by HSCCC, while 20 compounds by preparative HPLC. The chemical structures were elucidated by spectroscopy methods such as NMR and MS.25 compounds are in the following: 1:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6),20(22)-dien-3β,26-diol-3-O-β-D-glucopyranosyl-(1â†'3)-β-D-glucopyranosyl-(1â†'4)-α-L-rhamnopyranosyl-(1â†'2)-β-D-glucopyranoside; 2:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22a,26-triol-3-O-β-D-glucopyranosyl-(1â†'3)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 3:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1â†'4)-β-D-glucopyranoside; 4:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6) 20(22)-dien-3β, 26-diol-3-O-β-D-glucopyranosyl-(1â†'3)-β-D-glucopyranosyl-(1â†'2)-[α-L-rhamnopyranosyl-(1â†'4)]-β-D-glucopyranoside; 5:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6),20(22)-dien-3β,26-diol-3-O-β-D-glucopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 6:25(R)-spirost-â–³5(6)-en-3-O-α-L-rhamnopyranosyl-(1â†'2)-β-D-glucopyranoside; 7:25(S)-spirost-â–³5(6)-en-3-O-α-L-rhamnopyranosyl-(1â†'2)-α-D-glucopyranoside; 8:25(R)-spirost-â–³5(6)-en-3-O-α-L-rhamnopyranosyl-(1â†'2)-[α-L-rhamnopyranosyl-(1â†'4)]-β-D-glucopyranoside; 9:25(R)-spirost-â–³5(6)-en-3-O-α-L-rhamnopyranosyl-(1â†'2)-[β-D-glucopyranosyl-(1â†'4)]-β-D-glucopyranoside; 10:25(R)-spirost-â–³5(6)-en-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-glucopyranoside; 11:25(R)-spirost-â–³5(6)-en-3-O-β-D-glucopyranosyl-(1â†'3)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 12:25(S)-spirost-â–³5(6)-en-3-O-α-L-rhamnopyranosyl-(1â†'2)-[β-D-glucopyranosyl-(1â†'4)]-β-D-glucopyranoside; 13:25(R)-spirost-â–³5(6)-en-3-O-β-D-glucopyranosyl-(1â†'2)-[a-L-rhamnopyranosyl-(1â†'3)]-β-D-glucopyranoside; 14:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1â†'4)-[a-L-rhamnopyranosyl-(1â†'2)]-p-D-glucopyranoside; 15: 25(S)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 16:25(S)-spirost-â–³5(6)-en-3-O- a-L-rhamnopyranosyl-(1â†'2)-[α-L-rhamnopyranosyl-(1â†'4)]-β-D-glucopyranoside; 17: 25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6),20(22)-dien-3β,26-diol-3-O-α-L-rhamnopyranosyl-(1â†'3)-α-L-rhamnopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2) β-D-glucopyranoside; 18:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 19:25(R)-spirost-â–³5(6)-en-3-O-α-L-rhamnopyranosyl-(1â†'4)-α-L-rhamnopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 20:25(R)-spirost-â–³5(6)-en-3-O-β-D-glucopyranosyl-(1â†'3)-β-D-glucopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 21:25(S)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 22: 25(R)-26-0-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,26-diol-22-methosy-3-O-β-D-glucopyranosyl-(1â†'3)-β-D-glucopyranosyl-(1â†'2)-[α-L-rhamnopyranosyl-(1â†'4)]-β-D-gluc opyranoside; 23:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-7-keto-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1â†'3)-β-D-glucopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside; 24:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1â†'4)-α-L-rhamnopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)-β-D-glucopyranoside; 25:25(R)-26-O-β-D-glucopyranosyl-furost-â–³5(6)-en-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1â†'6)-β-D-glucopyranosyl-(1â†'3)-β-D-glucopyranosyl-(1â†'4)-[α-L-rhamnopyranosyl-(1â†'2)]-β-D-glucopyranoside. (2):Establishing the method to anlyze the D. zingiberensisSteroid saponins were the bioactive ingredients of D. zingiberensis, and two methods were established to comprehensively evaluate its quality.The qualitative analysis and contents of these 5 steroid saponins named hujiangsu A, zingiberensis new saponins, deltonin, dioscin, and gracillin in D. zingiberensis were performed and determined by HPLC-ELSD.7 batches of D. zingiberensis from different places were evaluated by this method after investigating its methodology.Based on the above method, the standard fingerprint of total steroid saponins from 10 batches of D. zingiberensis from different places and harvest time was established by HPLC-ELSD. The similarity evaluation was performed on the sample fingerprint by fingerprint analytical software,and it could compare the difference between different batches of samples according to this indicator..The aforementioned two methods were combined together to detect the D. zingiberensis and could obtain reliable results, which was used to identify the authenticity of medicine materials and evaluate the quality. (3) Summarizing the ion fragmental characteristics of steroid saponinsThe chemical structures of steroid saponins in the rhizome of D. zingiberensis were characterizing by HPLC-ESI-Q/TOF-MS/MS.12 reference standards were firstly analyzed by this established method. The mass behaviours of steroid saponins attached with different types of aglycones were summarized through observing and analyzing their ion fragments after elimination in the next step. There were 68 chromatographic peaks on the MS,12 of which were illuminated according to the reference standards, while the other remaining 56 peaks were tentatively identified based on the summarized mass behaviours and previously reported data. (4):Exploring new pharmacological activitySteroid saponins displayed better effect on curing cardiovascular disease. The medicine made from this kind of compounds like Di’aoxinxuekang capsules, Weiaoxin, Dunyeguanxinning tablets, etc. has been used in clinic, and they proved to satisfactory results in fact. There was specific inner relationship between cerebrovascular disease and cardiovascular disease in some extent. The steroid saponins in the rhizome of D. zingiberensis was investigated whether it exhibited promising neuroprotection or not when the brain was subjected to cerebral ischemia reperfusion (I/R, also named brain stroke, a kind of cerebrovascular disease).The rats were exposed to MCAO (Middle cerebral artery occlusion) for 90 min, then the brain was restored the blood flow for 24 h to execute the reperfusion step. Until now, the disease mode was successfully established. The rats were previously administrated of steroid saponins by intragastric, and performed the intervened therapy. The pathology and related serum indicators were examined by different methods. The results revealed that the death rate, neurological deficit score, cerebral infarction, and brain water edema were remarkably reduced in the group where the rats were pretreated with the steroid saponins, when compared with model group. The abnormal morphology of neuros caused by ischemia was alleviated. The oxidant stress (CAT, SOD, MDA, NO and iNOS) and inflammatory cytokines (IL-1β, IL-6, IL-1, and TNF-a) were also restored to their level in normal physiological situation. The corresponding apoptotic genes (Bcl-2, Bax, and Caspase-3) were regulated in some extent. The activated signaling pathways such as NF-κB and ERK 1/2 by injury brain tissue were inhibited. The above-mentioned results demonstrated that it could protect the injury brain induced by I/R after pre-treatment of steroid saponins. The underlying mechanisms might be related to the anti-edema, anti-oxidant, anti-inflammatory, and anti-apoptotic of steroid saponins. These producing mechanisms were probably involved with NF-κB and ERK 1/2 signaling pathways. |
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