| Steroid saponins are a structurally and biologically diverse class of glycosides that are distributed in many kinds of plants and have a good deal of biologic activities such as hypocholesterolemic, antitumour, antidiabetes, antiniflamma- tory, inhibitory activities against platelet aggregation and antifungal. The structural diversity of saponins lies mainly in their sugar moieties. The same things of Paris polyphylla SM.var. yunnanensis (Chong-Lou in Chinese) and many kinds of saponins are distributed in it. There are many articles about the isolation of steroid saponins from the rhizomes of these plants. Among these saponins there are several kinds of diosgenyl and pennogenyl saponins which are connections of diosgenin and pennogenin with different sugars chains at 3-hydroxyl group. They have been used as haemostatic agents and promoter for shrinkage of uterus in clinic. They also exhibit antibiotic and antitumor activity. As valuable components, very few pennogenyl saponins can be gained from nature and it is very important to extract those saponins from Paris polyphylla SM.var. yunnanensis with an efficient methodAs a novel extraction method, the technology of Ultrahigh Pressure Extract (UPE) comes from high pressure processing of food and has sprung up in these years. Besides food processing, the technology of high pressure also has been used in antisepsis, microbial inactivation, proteins denaturalized and bacterin manufactur. As an effective technology, it will be widely in use of extracting natural medicament. A plenty of pharmic components such as flavone, saponins and alkaloid can be extracted from Epimedium koreanum, propolis and ginseng by UPE. And UPE show a promising future because of its many advantages such as higher extraction yield, higher bioactivity, shorter processing time, lower power consumption and more safety.In this paper the ultrahigh pressure extract of steroid saponins from Paris polyphylla SM.var. yunnanensis root was studied. The operation parameters of UPE, such as solvent kind, solvent concentration, extraction time, extraction pressure, the ratio of solvent to material, were examined emphatically. On this basis we introduce another parameter of temperature and the ultrahigh pressure extract in diverse temperature was studied. The extraction results of UPE were compared with methods of marination at rt, ultrasound, soxhlet and heat reflux extraction.The main contents of research and results in this pape are as follow:1. The essential components of two kinds of products which obtained by the UPE and the conventional reflux extraction were analyzed by HPLC, FAB-MS and TLC respectively. As a result, they contain totally same saponins molecules. So it could be deduced that the extraction of steroid saponins from Paris polyphylla SM.var. yunnanensis root by UPE is reasonable.It is the first time to determine the effect of ultrahigh pressure on the molecule structure of the essential components of plant extractions, and it is confirmed that the product of UPE has no different of conventional extraction by chemical evidence and spectrum analysis.2. The process of ultrahigh pressure extraction had been studied roundly, the results are as follow:①The 70% ethanol-water (v/v) is the best suitable extraction solvent to extract steroid saponins from Paris polyphylla SM.var. yunnanensis boot. Within 0～70% of ethanol concentration, the extraction yield of saponins is increase as increasing of the ethanol concentration. When the concentration of ethanol is larger than 70% (v/v), the extraction yield of saponins would decrease slowly with increasing of the ethanol concentration.②The optimal pressure to extract saponins is 500MPa. The extraction yield of saponins would increase with increasing of pressure. When the pressure is beyond 500 MPa, the yield will increases slightly. The lower pressure should be chosen to save the investment of ultrahigh pressure equipment.③The optimal ratio of solvent to material of ultrahigh pressure extraction of saponins is 40/1. The extraction yield of saponins increases with increasing of the ratio of solvent to material, but more solvent would be consumed while the ratio is large. When the ratio of solvent to material is higher than 40/1, the extraction yield of saponins increases slowly with increasing of the ratio of solvent to material.④The optimal extracting time of UPE is 2 min. Under the high pressure the diffusion speed is very great, and in a very short time the solubility and diffusion in the inner and outer cells get balance. And the solubility will be the maximum value.3. Orthogonality Experiment design is used to study the effects of extraction pressure, concentration of solvent, and the ratio of solvent to material and their interactive effects on extraction yield of steroid saponins. Experimental results showed that the effect of concentration of solvent on extraction yield is the most significant factor. The optimum extraction conditions to extract saponins by UPE are as follows: (I) Pressure is 500MPa; (II) The ethanol concentration is 70%; (III) The ratio of solvent to material (ml/g) is 40/1; (Ⅳ)Extracting time is 2min.4. We introduce the important parameter of temperature and make univariate analysis of ultrahigh pressure extraction in diverse temperature. Orthogonality Experiment design is also used to study the effects of extraction pressure, concentration of solvent, the ratio of solvent to material, extracting temperature and their interactive effects on extraction yield of steroid saponins. The optimum extraction conditions of saponins by UPE in diverse temperature are as follows: (I) Pressure is 350MPa; (II) The ethanol concentration is 70%; (III) The ratio of solvent to material(ml/g) is is 40/1; (Ⅳ) Extracting time is 6min; (Ⅴ) extraction temperature is 70℃.Same extraction yields are achieved at the higher temperature whith lower pressure than UPE at room temperature. It means that the cheaper equipment could be used in UPE.5.Comparing the extraction methods of UPE with the others, we could find that the methods of UPE(including UPE at rt and UPE in diverse temperature) and soxhlet extraction were excellent. But the extraction method of soxhlet has the shortcomings of long timeconsuming(4hours) and big energyconsuming and the process of UPE can be finished in 2 of 6 minutes. The extraction yields of extraction process of heat reflux , ultrasound, soakage which also have the shortcomings of timeconsuming and energyconsuming are less than those of forementioned methods. So the best extaction method is UPE.There are very small quantity of compounds of pennogenyl saponins in nature because of the sparsity of Chinese traditional medicine Chong-Lou which reanimates us to synthesize these kinds of compounds.Firstly we made the research on how to get the sapogenin of pennogenin largely. And it was the first time to extract and separate diosgenin and pennogenin from Chong-Lou at one time using the method of direct acid hydrolysis. According to the research and control of hydrolysis conditons, we could improve the extraction ratio of two kinds of sapogenin. Petroleum ether-ethyl acetate(5:2) was chosen to elute column and the pure sapogenin could ben gained (diosgenin:1.74%;pennogenin:0.05%). The method of this article provided a feasible approach to using Chong-Lou and preparation of pennogenin in large scale.Then we explored the microbiological transformation of pennogenin from diosgenin using microorganism of Trichoderma viride(AS3.3711), which is the only fungiform bought by us . And some important factors including culture medium ,the method and time of adding substrate, the concentration of substrate and PH of system etc. But we had not got the aim compound. The research is worth of going on.The essential purpose of synthesis of steroid saponin is to get rid of a molecule of H2O, HBr, HF, EtSH, etc. between anomeric centre of donor and acceptor subtly which is called glycosylation. A pennogenyl saponin has a characteristic that there is a glycosidic bond between pennogenin (3-OH) and sugar donor however there is no this kind of bond at 17-OH of pennogenin. So the fact whether the reaction of glycosidation can happen or not at 17-OH in synthesis of pennogenyl saponins is very important: if no reactions occur can we get a lot of convenience in synthesis of pennogenyl saponins. Through observation and analysis of pennogenin , we could find that 17-OH is t-hydroxide and we supposed the activity of 17-OH was very low because of steric hindrance at this site.We synthesized pennogenyl saponins using three important methods of glycosylation. And six correlative compounds were synthesized firstly. As donors, glycosyl halide, trichloroimidate and thioglycoside were chosen to research into the results that they reacted with the acceptor pennogenin. In these reactions the difference of steric hindrance between 3-OH and 17-OH of pennogenin was utilized skillfully and only 3-hydroxyl group of pennogenin could be connected with each kind of donors selectively and there was no reaction at 17-hydroxyl group which had no protection. The characteristic above makes it convenient to synthesize compounds of pennogenyl saponins.On this basis , we synthesized two natural pennogenyl saponins whose sugar or sugar chain is oligosaccharide and disaccharide at the condition of NIS/TfOH and room temperature successfully. In order to diminishα-isomer between sugar chain and sapogenin we chosed thioglycoside of sugar chains as donors.
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