| Solanum is the largest genus in the Solanaceae family.More than 20 kinds of traditional Chinese medicines derived from this genus are widely used in China,among which Solanum nigrum(S.nigrum)and Solanum lyratum(S.lyratum)are two common ones.S.nigrum and S.lyratum have a long history of medicinal use.They have the effects of clearing away heat and detoxification,reducing swelling and dispersing knots,and are also commonly used as clinical anti-tumor Chinese medicines.The efficacy of traditional Chinese medicine is closely related to its chemical components.Studies have found that steroids in S.nigrum and S.lyratum,especially steroidal alkaloids,are the key chemical components that exert anti-tumor activity.Steroids are a class of natural products with a cyclopentane-polyhydrophen anthrene parent nucleus,widely present in nature.Steroids can play important physiological functions,common steroid drugs,such as steroid hormones and c ardiac Glycosides play an important role in medical applications.Alkaloids are a class of nitrogen-containing organic compounds,most of which have special and significant biological activities,for example,morphine has analgesic effect,ephedrine has antitussive and antiasthmatic effect,atropine has antispasmodic ef fect,paclitaxel,camptothecin and vincristine have anti-tumor effects.As a class of alkaloids with special structures,steroid alkaloids have a variety of significant pharmacological activities,and have received more attention in recent years.Studies have shown that both S.nigrum and S.lyratum contain much of steroid alkaloids.Although there are a lot of reports on the antitumor activity of steroid alkaloids in S.nigrum,they are limited to a few common alkaloids.The steroidal alkaloids in S.nigrum are extremely complex,and there has been a lack of systematic research on these components,and also their anti-tumor material basis has not been fully elucidated.In addition,our research group has systematically studied the steroidal alkaloids and anti-tumor activities in S.lyratum previously,and it has been confirmed that S.lyratum contains a large number of steroidal alkaloids with similar parent nucleus structures,and this kind of alkaloids have significant anti-non-small cell lung cancer activities.At the same time,the study further proved that steroid alkaloids had the antitumor mechanism by agglutinating tumor vascular endothelial cell membrane lipid raft cholesterol and interfering with lipid raft morphology and function.Based on the research progress at home and abroad and our group’s research foundation on steroid alkaloids and anti-tumor components of S.lyratum,this paper expands the study object to S.nigrum,which belongs to the same family as S.lyratum.Firstly,this paper systematically studies the components of alkaloids in S.nigrum and evaluates the antitumor activity of the steroidal alkaloids,in order to comprehensively reveal the composition of alkaloids in S.nigrum,clarify the structure of each alkaloid,and then explore the antitumor material basis and action mechanisms of steroidal alkaloids.At the same time,the structure-activity relationship of steroid alkaloids was summarized and studied based on the pharmacodynamic results.In addition,since the previous experimental results showed that there were still a lot of unknown alkaloids in S.lyratum,this paper further studies the alkaloids in S.lyratum,aiming to fill in the blank of the previously unseparated part,and more comprehensively clarify the composition of alkaloids in S.lyratum,and clarify its new structure of alkaloids.This paper is divided into four chapters,of which the second,third and fourth chapters are the main research content of this paper.The main innovative research content and results are as follows:Chapter 2 Study on the steroidal alkaloids of S.nigrum and S.lyratumThis chapter systematically studies the steroidal alkaloid constituents in S.nigrum,and S.lyratum.Firstly,the 70%ethanol extracts of S.nigrum,and S.lyratum were enriched with D151 weakly acidic cation exchange resin column,and the total alkaloids of S.nigrum,and S.lyratum were obtained respectively.Then,silica gel column chromatography and reversed-phase ODS column chro matography were used to separate and purify the total alkaloids,and 50 compo unds were isolated from the total alkaloids.Combined with mass spectrometry,infrared,nuclear magnetic resonance and other spectroscopic techniques,the str uctures of 43 compounds were identified,including 35 steroidal alkaloid compo unds(1～20,29～43),7 aliphatic alkaloid compounds(22～28)and 1 aliphatic hy drocarbon non-alkaloid compound(21).Among them,there are 13 new compou nds,all of which are steroid alkaloids,they are respectively:(3β,22αN,25R)-s pirosol-5(6)-en-12β,27-dihydroxy-3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyra noside(1);(3β,22αN,25R)-spirosol-5(6)-en-12β,27-dihydroxy-3-O-β-D-galactop yranosyl-(1→3)-β-D-glucopyranoside(2);(3β,22αN,25R)-spirosol-5(6)-en-27-hy droxy-3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside(3);(22αN,25R)-spi rosol-4(5)-en-12β,27-dihydroxy-3-one(4);(22αN,25R)-spirosol-5(6)-en-12β,27dihydroxy-3,7-dione(5);(22αN,25R)-spirosol-4(5),6(7)-dien-12β-hydroxy-27-a cid-3-one(6);3β-hydroxypregn-5(6)-en-22αN-carbonylamide-3-O-α-L-rhamnopyra nosyl-(1→4)-β-D-glucopyranoside(7);3β-hydroxy-pregnen-5(6)-en-22αN-carbonyl amide-3-O-α-L-rhamnopyranosyl-(1→2)-[O-α-L-rhamnopyranosyl-(1→4)]-O-β-D-gl ucopyranoside(8);(22αN,25R)-spirosol-12β,27-dihydroxy-3,6-dione(9);Solan igrine J(10);3β-pregn-5(6)-en-20-carbonyl-16-amidogen-3-O-α-L-rhamnopyranos yl-(1→4)-β-D-glucopyranoside(12);3β-pregn-5(6),16(17)-dien-20-carbonyl-16-a midogen-23-carboxyl-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-βD-galacopyranoside(13);3β-pregn-5(6),16(17)-dien-20-carbonyl-16-amidogen-23-carboxyl-3-O-β-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyran osyl-(1→4)-β-D-galactopyranoside(14).In addition,compounds 21～28 were isol ated from this plant for the first time.Chapter 3 Study on the Antitumor Activity of alkaloids in S.nigrumIn order to study the antitumor activity and material basis of steroidal alkaloids isolated from S.nigrum,new compounds 1～9 and known compound 19(solamargine)were screened for the in vitro antitumor activity in this chapter.Five human tumor cell lines were selected,including human non-small cell lung cancer cell line H1299,human liver cancer cell line HepG2,human cervical cancer cell line HeLa,human colon cancer cell line HCT116,and human breast cancer cell line MCF7.The effect of each compound on the proliferation of different tumor cells was detected by the CCK-8 method.The results showed that the compounds could inhibit the proliferation of five tumor cell lines to varying degrees.Among them,compound 5(Solanigrine E)showed the strongest cytotoxic activity against H1299,HCT116 and MCF7 cells,and its IC50 value were 11.88 μM,30.00 μM and 18.87 μM,respectively,which deserved further research.The compound has carbonyl substitution at the 7-position and forms an α,βunsaturated carbonyl structure,so it is speculated that this structure may be an important group for its antitumor activity.Chapter Four Study on the antitumor mechanisms of compound 5In order to further evaluate the antitumor pharmacological mechanism of the new steroidal alkaloids,we selected the best monomeric compound 5 obtained by the in vitro activity screening and its corresponding sensitive tumor line H1299 cells.By means of Transwell,scratch,Flow cytometry and Western blot,the possible anti-tumor mechanisms were explored from the aspects of apoptosis and metastasis.The results show that compound 5 can significantly(P<0.001)promote the apoptosis of H1299 cells,and significantly(P<0.001)reduce the mitochondrial membrane potential.Compound 5 significantly(P<0.001)increases the expression of apoptosis-related proteins Casepase3/8/9,FADD,FAS,BID and Cytochrome C.At the same time,compound 5 can significantly(P<0.001)inhibit the migration and invasion of H1299 cells,and study further proves that compound 5 significantly(P<0.001)increases the expression of metastasis-related proteins E-cadherin and ZO-1,and reduces the expression of metastasis-related proteins N-cadherin,Vimentin and Snail 2,which regulates the epithelial-mesenchymal transition process,thereby inhibiting tumor metastasis.In conclusion,a total of 50 compounds were isolated from S.nigrum and S.lyratum,of which 13 were new compounds.The antitumor activity of the isolated new alkaloids was evaluated,and it was found that compound 5 was the alkaloid with the best antitumor activity.Further research proved that compound 5 exerted antitumor activity by promoting apoptosis and inhibiting metastasis. |
