Identification of triterpenoid saponins from Anemone rivularis var. flore-minore and study on their antitumor activities / 中国药房

OBJECTIVE To study the triterpenoid saponins from Anemone rivularis var. flore-minore and their antitumor activities. METHODS The n-butanol extract of 70％ ethanol extract from rhizome of the plant was separated. The triterpenoid saponins were separated and purified by normal silica gel column chromatography ，reversed phase ODS column chromatography ， Sephadex LH- 20 gel column chromatography and semi-preparation high performance liquid chromatography. The structures of these saponins were identified by spectral analysis （NMR and MS ）and physical and chemical properties. MTT assay was used to test the proliferation inhibitory activity of the compounds against five kinds of human tumor cells （HL-60 cells，A549 cells，HepG2 cells，HeLa cells and U 87MG cells ）. The apoptosis inducing effect of compound 7 on U 87MG cells was evaluated by flow cytometric Annexin V-FITC/PI staining test. RESULTS：Sixteen triterpenoid saponins were obtained and identified as 3 β-O-β-D-xylopyranosyl-（1→2）-α-L-arabinopyranosyl-oleanolic acid-28-O-α-L-rhamnopyranosyl- （1→4） -β-D-glucopyranosyl-（1→6）-β-D-glucopyranoside（1），3β-O-L-arabinopyranosyl oleanolic acid- 28-O-β-D-glucopyranoside（2），saponin B （3）， 163.com oleanolic acid- 3β-O-β-D-glucopyranosyl-（1→2）-α-L-arabino- pyranoside（4），HN-saponin F （5），clematoside S （6），prosapogenin CP 4（7），cussonside B （8），pulsatilla saponin C （9）， clemastanoside D （10），3 β-O-β-D-glucopyranosyl-（1→2）-β-L-arabinopyranosyl-hederagenin-28-O-β-D-glucopyranoside（11）， ciwujianoside C 3（12），ciwujianoside A 1（13），huzhangoside D （14），kalopanaxsaponin B （15）and hederacolchiside E （16）. Compounds 3，4，6-9 displayed inhibitory activities on the proliferation of tumor cells to different extent ，and compound 7 had the strongest activity ；compound 7 induced the apoptosis of U 87MG cell so as to inhibit the proliferation of cancer cells in a time-dependent manner. CONCLUSIONS The obtained 16 saponins are all identified as oleanolane-type ，among which compound 1 is a new compound. The monodesmosidic saponins ，the sugar chain of which attached at C- 3 and a free carboxyl at C- 28， possess stronger antitumor activity than others.

Three new triterpenoid saponins from Aralia echinocaulis / 中草药·英文版

Objective: To study the active ingredients in the root bark of Aralia echinocaulis. Methods: Three triterpenoid saponins were separated from the 70% ethanol extracts and purified by column chromatography and their structures were determined by spectroscopic analysis. Compound 1 and 3 were evaluated for antioxidant activity by the in vitro DPPH free radical scavenging ability and the protective effect of OH

The glycosyltransferases involved in triterpenoid saponin biosynthesis: a review / 生物工程学报

Triterpenoid saponins are widely used in medicine, health cares, cosmetics, food additives and agriculture because of their unique chemical properties and rich pharmacological activities. UDP-dependent glycosyltransferases (UGTs) are the key enzymes involved in triterpenoid saponin biosynthesis, and play important roles in the diversity of triterpenoid saponin structures and pharmacological activities. This review summarized the UGTs involved in plant triterpenoid saponin biosynthesis based on the sources of UGTs and the types of receptors. Moreover, the application of UGTs in heterologous biosynthesis of triterpenoid saponins based on synthetic biology was also discussed.

Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Simultaneous quantitative analysis of nine saponins in Gynostemma pentaphyllum before and after heat processing based on UPLC-Q-Trap-MS / 中国中药杂志

Heat-processed Gynostemma pentaphyllum has strong biological activity, and saponins are the main components. To investigate the changes of saponins in G. pentaphyllum before and after heat processing, the present study determined and analyzed the content of nine saponins in G. pentaphyllum from Zhangzhou of Fujian and Jinxiu of Guangxi by ultra-high performance liquid chromatography with quadrupole ion-trap mass spectrometry(UPLC-Q-Trap-MS). The separation of the analytes was performed on an ACQUITY UPLC BEH C_(18) column(2.1 mm×50 mm, 1.7 μm) at 30 ℃, with acetonitrile and 0.1% formic acid in water as the mobile phase by gradient elution, and the flow rate was 0.3 mL·min~(-1). Quantitative analysis was performed using electrospray ionization source(ESI) in the multiple reaction-monitoring(MRM) mode. The results showed that the content of saponins with biological activities increased after heat processing. Specifically, gypenoside L, gypenoside LI, damulin A, damulin B, ginsenoside Rg_3(S), and ginsenoside Rg_3(R) in G. pentaphyllum produced in Zhangzhou of Fujian increased by 7.369, 8.289, 12.155, 7.587, 0.929, and 1.068 μg·g~(-1), respectively, while the content of ginsenoside Rd, gypenoside LVI, and gypenoside XLVI, which were abundant in the raw materials, decreased by 0.779, 19.37, and 9.19 μg·g~(-1), respectively. The content of gypenoside L, gypenoside LI, damulin A, damulin B, ginsenoside Rg_3(S), and ginsenoside Rg_3(R) in G. pentaphyllum produced in Jinxiu of Guangxi increased by 0.100, 0.161, 0.317, 0.228, 3.280, and 3.395 μg·g~(-1), respectively, while the content of ginsenoside Rd, gypenoside LVI, and gypenoside XLVI in the raw materials was reduced by 1.661, 0.014, and 0.010 μg·g~(-1), respectively. The results suggest that heat processing is an effective way to transform rare gypenosides. Furthermore, it is found that there are great differences in the content of gypenosides in different regions.

Chemical constituents of triterpenoid saponins from Glycyrrhiza glabra / 药学学报

Ten triterpenoid saponins were isolated and purified from the water extract of Glycyrrhiza glabra by polyamide resin combined with macroporous resin column chromatography, ODS medium pressure column chromatography and semi-preparative RP-HPLC. Their structures were elucidated by physicochemical properties, NMR and MS spectra, and determined as 3β-O-[β-D-glucuronpyranosyl-(1→2)-β-D-glucuronpyranosyl]-30β-O-β-D-glucuronpyranosyl-oleanane-11-oxo-12(13)-ene (1), 3β-O-[β-D-glucuronpyranosyl-(1→2)-β-D-glucuronpyranosyl]-30β-O-α-L-rhamnopyranosyl-oleanane-11-oxo-12(13)-en-22β,30-diol (2), uralsaponin C (3), licorice-saponin A3 (4), licorice-saponin P2 (5), 22β-acetoxyl-glycyrrhizin (6), macedonoside A (7), 29-hydroxyl-glycyrrhizin (8), licorice-saponin G2 (9), glycyrrhizin (10). Compounds 1 and 2 are two new compounds and named as licorice-saponin R3 and licorice-saponin S3.

Qualitative analysis of Gynostemma longipes for medicinal usage / 中国中药杂志

The Qinling-Daba Mountains area is the main producing areas of Gynostemma longipes for medicinal usage, and samples of wild whole plants in Pingli, Shaanxi Province and Qingchuan, Sichuan Province were collected. The ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS~E) was used to profile the chemical compositions and analyze the similarities and differences of G. longipes samples in these areas. Based on the accurate molecular weight and fragment information obtained from Q-TOF-MS~E, the structures of the main components were identified by combining with the mass spectra, chromatographic behaviors of reference standards and related literatures. The results showed that the components of wild G. longipes from different places among Qinling-Daba Mountains area were similar. Forty-five chemical components were identified in the whole plant of G. longipes from Pingli, Shaanxi Province, including 43 triterpenoid saponins and 2 flavonoids which contain all main peaks in its fingerprint. The main components are dammarane-type triterpenoid saponins, such asgypenoside ⅩLⅨ, gypenoside A and its malonylated product of glycosyl.

Study on antitumor activity of triterpenoid saponins from Tujia ethnomedicine Kouziqi / 中草药

Objective: To study the active triterpenoid saponins of Tujia ethnomedicine Kouziqi (Panax japonicus var. major). The antitumor activity was screened and the relationship between the structure and activity of the compounds was discussed. Methods: The ethanol extract of Kouziqi was isolated by Silica gel, ODS and MCI column chromatograph and purified by preparative HPLC. The structures were elucidated on the basis of spectroscopic analysis and compared with literatures. Using MTT assay to detect the cytotoxicity of 14 compounds in BGC-823, HCT-116, Hela, HepG-2 cells. Results: A total of 14 known compounds were isolated from Tujia ethnomedicine Kouziqi and determined as chikusetsusaponin IVa methyl ester (1), chikusetsusaponin IVa butyl ester (2), chikusetsusaponin IV (3), chikusetsusaponin IVa (4), 28-desglucosylchikusetsusaponin IVa (5), oleanolic acid-3-O-β-D-(6'- methylester)-glucuronopyranoside (6), (24R)-majonoside R1 (7), (24R)-pseudoginsinoside F11 (8), (20S)-notoginsinoside-R2 (9), (20S)-ginsenoside Rg2 (10), ginsenoside Rg1 (11), ginsenoside Re (12), ginsenoside Rd (13) and chikusetsusaponin-V methyl ester (14). Among the 14 compounds, compounds 5 and 6 showed dose-dependent cytotoxicity to BGC-823, HCT-116, Hela and HepG-2 cells. Compound 5 had cytotoxicity in BGC-823 and HCT-116 cells with IC50 values of 9.94 and 14.17 μmol/L, respectively. Compound 6 had the best cytotoxicity in HepG-2 cells with IC50 value of 12.70 μmol/L. Conclusion: Compound 6 is isolated from Kouziqi for the first time and its spectral data were reported. The antineoplastic activity of Tujia ethnomedicine Kouziqi is based on the oleanolic acid-type triterpenoid saponins and related to the substituents of C-28, but the mechanism still needs to be deeply studied.

Chemical compositions and anticancer activity of Mollugo pentaphylla / 中草药

Objective: To investigate the chemical compositions and anticancer activity of Mollugo pentaphylla. Methods: These compounds were isolated by various technologies, such as silica gel, Sephadex LH-20 and high performance liquid chromatography. Their structures were elucidated on the basis of physicochemical properties and spectroscopic data. The cytotoxic activities of compounds 1-5 against five cancer cell lines were tested by MTT. Results: Six compounds were isolated from this plant and identified as mollugoside E (1), 3-O-[α-L-rhamnopyranosy1 (1→2)-α-L-arabinopyranosyl]-28-O-[β-D-glucopyranosyl (1→6)-β-D- glucopyranosyl]oleanolic acid (2), raddeanoside R8 (3), raddeanin A (4), mollugogenol A (5), and oleanolic acid (6). The cytotoxic activities results showed that compounds 1-5 had certain inhibitory effects on human prostate cancer DU145 cell lines, cervical cancer Hela cell lines and early-juvenile leukemia HL 60 cell lines, especially on HL 60 cells with IC50 value of 10.21, 38.43, 40.28, 20.59, and 83.16 μmol/L, respectively. Conclusion: Compound 1 is a new triterpenoid saponin and compounds 2-4 are isolated first time from M. pentaphylla. The cytotoxic activities results showed that compounds 1-5 had certain inhibitory effects on DU145, Hela and HL 60 cell lines.

Identification of Chemical Components of Yangzheng Xiaoji Capsule by GC-MS and UPLC-Q-TOF-MS / 中国药学杂志

OBJECTIVE: To study the chemical components of Yangzheng Xiaoji capsule. METHODS: The liposoluble constituents and water-soluble constituents of Yangzheng Xiaoji capsule were analyzed by GC-MS and UPLC-Q-TOF-MS. The GC-MS conditions were as follows, PH-5 capillary column(0.25 mm×30 m,0.25 μm) for column chromatography, temperature-programmed and EI were used, the scanning range of quality was m/z 40-650; the UPLC-Q-TOF-MS conditions were as follows, ACQUITY UPLC HSS T3(2.1 mm×100 mm,1.8 μm) with mobile phase of 15 mmol·L-1ammonium formate solution and acetonitrile under gradient elution, with the ESI source. The scanning range was m/z 50-1 500. RESULTS: Twenty liposoluble constituents were identified by GC-MS, including volatile oils,sesquiterpenoids, phenols and lipids, et al. These components were mainly derived from Rhizoma Curcumae, Cynanchum Paniculatum and Tuckahoe. And 38 water-soluble constituents were identified by UPLC-Q-TOF-MS, including triterpenes, triterpenoid saponins, flavonoids, iridoid glycoside and phenolic acid, mainly derived from Panax ginseng, Astragalus membranaceus and Ligustrum lucidumait. CONCLUSION: This study provides a fast and effective method to understand the chemical components of Yangzheng Xiaoji capsule comprehensively, and makes a qualitative analysis and theoretical support for pharmacodynamic substance foundation, quality control and pharmacological mechanism study.

Study on triterpenoid glycosides from roots of Clematis manshurica / 中草药

Objective: To study the chemical constituents from the roots of Clematis manshurica. Methods: Ten compounds were separated and purified by silica gel, Sephadex LH-20, and preparative HPLC. The structures were identified by physicochemical properties and spectral analyses. Results: A total of ten oleanane-type triterpenoid saponins were isolated and identified as clematomandshurica saponin L (1), clematichinenoside A (2), clematochinenoside F (3), clematernoside A (4), clematichinenoside B (5), clematichinenoside C (6), clematomandshurica saponin B (7), clematomandshurica saponin D (8), clematomandshurica saponin C (9), and huzhangoside B (10), respectively. Conclusion: Compound 1 is a new oleanane-type triterpenoid glycoside, and compounds 2-6 are isolated from this plant for the first time.

Chemical Constituents from Caulophyllum robustum (Ⅱ) / 中国药学杂志

OBJECTIVE: To isolate the chemical constituents from Caulophyllum robustum and confirm their chemical structures. METHODS: The chemical constituents were isolated by MCI gel, repeated silica gel chromatography, preparative liquid chromatography.and their structures were elucidated by NMR and MS etc. RESULTS: The structures of compounds 1-10 were identified as echinocystic acid (1), oleanolic acid-3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside (2), hederagenin-3-O-β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside (3), hederagenin-3-O-β-D-glucopyranosyl-(1→2) [β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside (4), 3-O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl echinocystic acid-28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (5), 3-O-α-L-arabinopyranosyl hederagenin-28-O-(4-O-acetyl)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (6), (6R, 7E, 9R)-9-hydroxy-4, 7-megastigmadien-3-one-9-O-β-D-glucoside (7), (9R)-9-hydroxy-4, 6-megastigmadien-3-one-9-O-β-D-glucoside (8), maltose (9), and sucrose (10). CONCLUSION: Compounds 1-10 are firstly isolated from the genus Caulophyllum except 5.

Triterpenoid saponins from roots of Phytolacca acinosa / 中国中药杂志

A new triterpenoid saponin named esculentoside U(1), along with the five known compounds, was isolated and characterized from the roots of Phytolacca acinosa, a commonly used traditional Chinese medicine with anti-inflammatory and anti-rheumatoid activities. The structure of the new saponin was elucidated as 3-O-[β-D-glucopyranosyl-(1→4)]-β-D-xylopyranosyl]-2, 23-dihydroxyolean-11, 13(18)-diene-28, 29-dioic acid 29-methyl ester(1). The assignment of all NMR signals of 1 was performed by means of 2D-NMR experiments.

A phytochemical and pharmacological advance on Ilex asprella / 中国中药杂志

Ilex asprella is one of representative medicinal plants in South of the Five Ridges of China. The roots and rhizomes of I. asprella have the effects of clearing heat and detoxifying, stimulating salvia, and reducing thirst, which has been used to treat wind-heat cold, acute and chronic pharyngitis, and sore throat. Contemporary studies showed that I. asprella contains the major triterpenoids and glycosides, phenolic acids, and minor steroids. The extracts and compounds show activities of anti-inflammatory, antiviral, anti-tumor, and regulating lipid metabolism.The present paper summarizes a phytochemical and pharmacological advance on this species to provide reference for clarification of its pharmacologically active ingredients, quality evaluation, and further explorations.

Cloning and ectopic expression analysis of squalene epoxidase 1 gene from Pseudostellaria heterophylla / 中草药

Objective: To clone the full-length cDNA encoding squalene epoxidase 1 (SQE1), a key enzyme of triterpenes biosynthesis, from Pseudostellaria heterophylla and to perform functional analysis. Methods: With the total RNA as template, the full-length cDNA of SQE1 in P. heterophylla was cloned via RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The bioinformatics of the cloned SQE1 gene was performed. The target gene was transfered into tobacco by Agrobacterium-mediated transformation. Results: The full-length cDNA (2 038 bp) of SQE1 gene was obtained with an open reading frame of 1 554 bp, encoding 517 amino acid polypeptides, which had higher homology with the known SQEs in other medicinal species. The calculated relative molecular mass was 5.67 × 104, the isoelectric point was 8.8. The deduced protein sequence exhibited FAD-binding domains and four transmembrane regions. The content of total triterpenes was increased in transgenic tobacco plants. Conclusion: This is the first report that the full-length cDNA encoding SQE1 from P. heterophylla is cloned. The ectopic expression of SQE1 could promote to increase the content of total triterpenes in transgenic plant. This work provides a foundation for exploring the biosynthetic pathway of triterpenes in P.heterophylla and their applications in bioengineering.

Triterpenoid saponins from Bupleurum marginatum var. stenophyllum / 中国中药杂志

Twelve compounds were obtained by phytochemical investigation of 70% EtOH ( containing 0.5%NH3•H2O )extract of the roots of Bupleurum marginatum var. stenophyllum. Based on comparison of their spectral data, including HR-ESI-MS, ¹H-NMR, ¹³C-NMR data, with those of the literature, their structures were elucidated as saikosaponin b2 (1), saikosaponin a(2), saikosaponin b1(3), saikosaponin d (4), hydroxysaikosaponin a (5), saikosaponin b3 (6), saikosaponin c(7),saikosaponin i (8), saikosaponin f (9), chikusaikosides Ⅱ(10), saikosaponin s (11), and saikosaponin I(12). All compounds belong to olean-type triterpenoid saponin and compounds 1, 3, 5, 8-9,11, and 12 were isolated from this plant for the first time. At a concentration of 20 μmol•L⁻¹, compounds 2, 4, 6, 8, 11 and 12 showed strong inhibition activity against influenza virus WSN33 with the inhibition rate of 91.3%,88.6%,53.4%,61.3%,77.3% and 57.4%,respectively.

Gene cloning, sequence, and expression analysis of GpSS1 and its regulation by MeJA in Gynostemma pentaphyllum / 中草药

Objective: To clone Gynostemma pentaphyllum squalene synthase gene (GpSS1) and analyze its sequence and expression pattern as well as its regulation in response to MeJA. Methods: Primers were designed based on the sequence of GpSS (GenBank accession numbers: FJ906799) and GpSS1 was cloned by using RT-PCR method. Physicochemical properties and transmembrane regions of the deduced GpSS1 protein were predicted via Protparam and Tmpred programs, respectively. Conserved domains involved in catalytic activity of SS enzyme were identified using MotifScan and multiple sequence alignment was achieved using the BioEdit software. Expression pattern of GpSS1 and its regulation by MeJA were analyzed by quantitative real-time RT-PCR. Results: GpSS1 contains an open reading frame of 1 254 bp encoding a putative protein of 417 amino acids. The protein has an aspartate-rich motif and an endoplasmic reticulum membrane anchoring region in addition to three conserved domains involved in catalytic activity of SS enzyme. The expression level of GpSS1 in young leaves was the highest, followed by that in old leaves, and the lowest was in rhizomes. The expression of GpSS1 was significantly upregulated in G. pentaphyllum leaves sprayed with different concentration of MeJA. The greatest upregulation of GpSS1 occurred in G. pentaphyllum leaves treated with 50 μmol/L MeJA. In both young and old leaves, the transcription of GpSS1 gradually increased and then decreased to varying degrees at 6-96 h after MeJA treatment. However, the expression level of GpSS1 was always higher in young leaves than that in old leaves. Conclusion: The cloning of GpSS1 and analysis on the expression regulated by MeJA will be helpful for further elucidating the function of GpSS1 and its mechanism regulated by MeJA, as well as for improving the quality and content of gypenosides.

Analysis on chemical constituents in Hedera nepalensis by UPLC/Q-TOF-MS/MS / 中草药

Objective: Using UPLC/Q-TOF-MS/MS to analyze and identify the chemical constituents from the stems and leaves of Hedera nepalensis. Methods: The analysis was performed on an Welch C18 reverse phase column (100 mm×2.1 mm, 1.7 μm). The mobile phase consisted of acetonitrile (B) and 0.1% formic acid (A) was used as gradient elute. The flow rate was 0.3 mL/min gradient elution and column temperature was 40℃, the injection volume was 5 uL, MS conditions were ESI, positive and negative ion mode scanning. Results: Under the optimized condition, based on the database of Scifinder, MS/MS of standards and compared with reference results, 43 compounds were identified, including triterpenoid saponins, flavonoid glycosides, phenylpropanoids, and nucleotides. Conclusion: By using UPLC/Q-TOF-MS/MS method the main chemical constituents from H. nepalensis can be rapidly and accurately identified.

Research progress on chemical constituents and biological activities of plants from Hemsleya Cogn / 中草药

Plants in genus Hemsleya Cogn. are rich in triterpenoid saponins, and are got more attention for their significant antitumor and anti-inflammatory activity from all walks of life. This paper summarizes the progress of plant sources for nearly 30 years, including chemical constituents, spectral characteristics, and biological activity, and provides the direction for further study.

Transcriptomic Analysis and Systematic Mining of Genes Involved in Biosynthetic Pathway of Triterpenoid Saponins in Ilex Asprella / 世界科学技术-中医药现代化

This study was aimed to screen candidate genes involved in the triterpenoid saponins biosynthetic pathway of the Ilex asprella root. The Illumina platform was applied to perform transcriptomic sequencing of I. asprella root, followed by a series of bioinformatics analysis. The results showed that a total of 272 candidate unigenes were anno-tated to be involved in the biosynthetic pathway of terpenoid in the transcriptome of I. asprella root, including 72 u-nigenes for the upstream pathway and 26 unigenes for cyclization, oxidation and glycosylation in the downstream pathway. Phylogenetic analysis was carried out to further analyze the evolution relationship of some candidate uni-genes and their homologous genes. Two genes IaA S1 and IaA S2 were proved to be mixed amyrin synthases in yeast expression system. Moreover, IaA S1 was identified to one of the rare ASs with α-amyrin as the major product. It was concluded that a series of candidate genes, which might be involved in the biosynthetic pathway of triterpenoid saponins, were screened out from the transcriptome of I. asprella root. Further investigation of these candidate genes will provide insight into their actual functions in the triterpenoid saponins biosynthetic pathway in I. asprella.