| Semen Impatientis is the dried and mature seed of Impatiens balsamina L.,with many pharmaceutical effects,such as invigorating the circulation of blood,softening and resolving hard mass,removing food retention.It is widely used for curing abdominal diseases,amenorrhea,dysphagia and so on.Modern pharmacological studies have shown that it has a lot of bioactivities such as antibacterial,anti-tumor and anti-oxidation effects.The main chemical components in Semen Impatientis are saponins,flavonoids,naphthoquinones and volatile oils.Most of saponins obtained from Semen Impatientis belong to baccharane configuration,which have only been found in Impatiens balsamina L.,Actinostemma tenerum and Fatsia japonica.Thus,there are few reports on their chemical structures or pharmacological mechanisms.By far,pharmacological activities of total saponins in Semen Impatientis have been studied about effective components incompletely.Analysis of saponins by LC-MS in literatures showed that there were still many unknown saponins in Semen Impatientis,while only about ten saponins components were obtained by far.It is very important to ensure the chemical components in traditional Chinese medicine for pharmacodynamic basis and quality specification,especially for new biomedical applications.In this work,we investigated the components of saponins in Semen Impatientis and their biological activities.First of all,silica-gel column chromatography,octadecylsilyl silica gel column chromatography and high performance liquid chromatography were applied to separate saponins from the ethanol extract of Semen Impatientis.18 compounds were obtained and 7 of them were new.The compounds were identified as honsenkolA-3-O-?-D-glucopyranosyl-26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-O-?-D-glucopyranosyl?1?,?3S,4R,17R,20S?-17-hydroxy-3-O-?-D-xylopyranose?12?-?-D-glucopyranosyl-26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-21,24-epoxybaccharane?2?,hosenkolA3-O-?-D-glucopyranosyl?2?1?-?-D-glucopyranosyl-26-O-?-D-glucopyranoside?3?,?3S,4R,17R,20S?-17-hydroxy-3-O-?-D-glucopyranosyl?1?2?-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-21,24-ep oxybaccharane?4?,?3S,4R,17R,20S?-17-hydroxy-3-O-?-D-glucopyranosyl?1?2?-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-21,26-pentahydroxybaccharane?5?,hosenkol B 3-O-?-D-glucopyranosyl?2?1?-?-D-glucopyranosyl-26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranoside?6?,Hosenkoside A?7?,Hosenkoside B?8?,Hosenkoside C?9?,?-spinasterol?10?,Hosenkoside D?11?,Hosenkoside K?12?,Hosenkoside M?13?,Hosenkoside G?14?,Hosenkoside F?15?,?-spinasterol-3-O-?-D-glucopyranoside?16?,2-methoxy-1,4-naphthoquinone?17?,honsenkol B-26-O-?-D-glucopyranosyl?18?.Compounds 1,2,3,4,5,6,18 are the new compounds which have never been reported.The 13C NMR datas of Hosenkoside A-type and Hosenkoside B-type saponins were analysed in this work.The 13C NMR regular pattem of Hosenkoside A-type and Hosenkoside B-type saponins,even sugar connection positions was summarized,which would provide the scientific basis for the structural identification of Hosenkoside compotents in Semen Impatientis.In order to guarantee the safety of Chinese medicinal materials,it is important to establish the method for determination of active ingredients and index components.There are not any determination methods for Hosenkoside A and Hosenkoside K in Chinese Pharmacopoeia until 2015.In this paper,ionic liquid ultrasonic-assisted with solid phase extraction of Hosenkoside A?7?,Hosenkoside B?8?,Hosenkoside K?12?,Hosenkoside M?13?from Semen Impatientis was established for the first time.The optimization of experimental conditions was as follows:0.9 mL[C6mim]Br and 2.5 mL water were used as solvent to extract 0.1 g Semen Impatientis,the ultrasonic time was 30 min,load speed was 1.5 mL/min,0.9g CHP20/P120 macroporous resin was used as SPE and 1.5 m L methanol was as eluent.Compared with reflux extraction method,this method has the advantages of short time,safe,non-toxic and rapid extraction.This work provides a new index and method for further improving the quality standard of Semen Impatientis.It is an important method to apply the TLC identification and HPLC analysis in the quality standard of Chinese medicine.As a traditional Chinese medicine,Semen Impatientis is widely used in many prescriptions of Chinese medicine.However,there was no determination method for the quality standard of the Chinese medicine.The solid-phase extraction combined with HPLC-ELSD was used to determine the contents of the Hosenkoside A and Hosenkoside K in Xiao Yu Ding Tong ointment for the first time.This method had high sensitivity,good stability and good recovery,which provided a new idea for the analysis of Hosenkosides and provided a new basis for the establishment of the quality standards of preparation quality standard.According to the reports,Hosenkosides have a certain anti-tumor activity.However,based on preliminary experiments,the natural saponins with sugar chains had no obvious effect on tumor inhibition.There are some relationships between sugar chain and the biological activity of saponin compounds.For this reason,the degradation products of total saponins from Semen Impatientis were studied.The components were isolated and identified,and the biological activities of total saponins and degradation pruducts were investigated,respectively.First,the macroporous adsorption resin was used to prepare the total saponins,and its main components were studied by HPLC.Experiments showed that the best degradation conditions were as follows:1%tartaric acid was as catalyst,material liquid ratio was 1:200,the degradation temperature was 200?and degradation time was 60 min.The antitumor effect of degradation products of total saponins was significantly improved than the total ones.Then,in order to clarify the active constituents in the degradation products were isolated and identified.Five compounds were obtained,which are Hosenkol A?Z-1?,hosenkol A 26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranoside?Z-2?,Hosenkoside D?Z-3?,Hosenkoside C?Z-4?,honsenkol A-3-O-?-D-glucopyranosyl-26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-O-?-D-glucopyranosyl?Z-5?.Furthermore,hosenkol A 26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranoside?Z-2?is a new compound.By comparing the chromatographic fingerprint of liquid chromatography of degradation products and total saponins,the results showed that Hosenkoside K and Hosenkoside M which had more carbohydrate chains were degraded completely.The antitumor activity of Semen Impatientis were studied,antitumor cell proliferation of Hosenkoside A?7?,Hosenkoside B?8?,Hosenkoside K?12?,Hosenkoside G?14?,Hosenkoside M?13?and four new compounds?honsenkol A-3-O-?-D-glucopyranosyl-26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-O-?-D-glucopyranosyl?1?,?3S,4R,17R,20S?-17-hydroxy-3-O-?-D-xylopyranose?1?2?-?-D-glucopyranosyl-26-O-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-21,24-epoxyb accharane?2?,hosenkol A 3-O-?-D-glucopyranosyl?1?2?-?-D-glucopyranosyl-26-O-?-D-glucopyranoside?3?,?3S,4R,17R,20S?-17-hydroxy-3-O-?-D-glucopyranosyl?1?2?-?-D-glucopyranosyl-28-O-?-D-glucopyranosyl-21,24-epoxybaccharane?4??were investigated.The experimental results showd that antitumor activity of aglycone Honsenkol A?Z-1?was the best among them.The IC50 data of five kinds of tumor cells were HepG2 cells(IC50=0.0649?M),DU145 cells(IC50=1.890?M),DLD1 cells(IC50=0.179?M)and A375 cells(IC50=6.089×10-3?M).It showed that amount of carbohydrate and configuration had large difference in antitumor effect.The less amount of carbohydrate side chains is,the better antitumor effect it has.The antioxidant activity of hosikosides was also investigated.The experimental results showed that the total saponins had significant inhibitory effect on three kinds of free radicals,however,the scavenging capacity of the monomer hosenkosides was relatively weak.By studying the scavenging ability of O2-ˇfree radical,the results show that the scavenging ability is as follows:total saponins>degradation products>Hosenkol A>Hosenkoside G>Hosenkoside C>Hosenkoside M>Compound 2>Compound 4>Hosenkoside K>Hosenkoside A>Compound 1>Hosenkoside B>Compound 3.It was the same rank with the antitumor effect.The scavenging capacity of total saponins was stronger than that of monomers,which may be related to antioxidant active substances such as flavonoids and naphthoquinones.In summary,studies about the chemical composition and biological activities of saponins from Semen Impatientis were done in this work.7 kinds of new compound were obtained.Two new determination methods of saponins in Semen Impatientis and related prescription were established for Chinese medicine.Then the antitumor activity of total saponins,degradation products and monomers were studied thoroughly.The results in this work provide new research ideas,methods and scientific basis not only for the study of the pharmacodynamics basic research and the quality standard of the medicinal materials,but also for the development and utilization of Semen Impatientis. |
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