Cloning and bioinformatic analysis of β-AS gene from Panax japonicus / 中草药

Objective: The key enzyme of triterpene saponin metabolism was cloned and its sequence, structure and function were analyzed by bioinformatics. Methods: RNAs were extracted from the leaves of Panax japonicus The full-length cDNA sequences of β-AS were cloned by utilizing RT-PCR method, and the sequence was connected to the pMDTM18-T for cloning and sequencing.β-AS protein characteristics in transplanted species and cultivated species of P. japonicus were predicted and compared by bioinformatics analysis and the phylogenetic tree of β-AS protein was constructed. Results: The β-AS sequences in transplanted species and cultivated species of P. japonicus were obtained, which had 2 286 bp ORF and encoded 761 amino acids. There were little differences between the two varieties of β-AS proteins in physicochemical properties, secondary structure, tertiary structure, and phosphorylation sites, which may lead to show difference in catalytic activity. Conclusion: This work also obtained the full-length of cDNA sequence of β-AS gene in transplanted and cultivated varieties of P. japonicus, and provided a systemic sequence analysis of β-AS proteins, which can provide the useful information for β-AS studies in the future.

Molecular cloning and functional characterization of multiple ApOSCs from Andrographis paniculata / 中国天然药物

Triterpenoids have been described in Andrographis paniculata. Oleanolic acid exhibits high biological activity and is widely used in the clinic, and β-sitosterol not only has good biological activity but also plays an important physiological role in plants. However, analysis of the biosynthetic pathway of triterpenoids in Andrographis paniculata has not been reported. Here, we provide the first report of the isolation and identification of nine 2, 3-oxidosqualene cyclases (ApOSC3 to ApOSC11) from A. paniculata. The results showed that ApOSC4 represented a monofunctional synthase that could convert 2, 3-oxidosqualene to β-amyrin. ApOSC5 as a bifunctional 2, 3-oxidosqualene cyclases, could transfer 2, 3-oxidosqualene to β-amyrin and α-amyrin. ApOSC6 to ApOSC8 composed the multifunctional 2, 3-oxidosqualene cyclases that could convert 2, 3-oxidosqualene to β-amyrin, α-amyrin and one or two undetermined triterpenoids. This study provides a better understanding of the biosynthetic pathway of triterpenoids in A. paniculata, and the discovery of multifunctional 2, 3-oxidosqualene cyclases ApOSC5 to ApOSC8 of the facilitates knowledge of the compounds diversity in A. paniculata.

Regulation of β-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells / 中国中药杂志

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in β-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the β-amyrin production. Compared with the control strain, the production of β-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the β-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of β-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

Synthesis of triterpenoid saponins from Aesculus chinensis based on transcriptome data / 中国中药杂志

Aesculus chinensis belongs to Hippocastanaceae family,bears medicinal and ornamental values. The oleanane type triterpenoid saponin aescin is regarded as active ingredient and accumulated in seed. In order to understand its molecular basis of the triterpenoid biosynthesis,we used high-throughput sequencing under Illumina Hi Seq 2000 platform to obtain the transcriptome data of seed and flower from A. chinensis to further mine the genes involved in its metabolic pathway. Unigene's de novo splicing was performed using Trinity software; the transcriptome results were annotated with KEGG database to predict the specific pathways of the aescin triterpenoid metabolism. Terpenoid and triterpenoid pathways were found from transcriptome data,and forty seven and twenty seven corresponding genes were uncovered respectively. It was found that there are eight kinds of enzymes related to the terpenoid metabolism pathway precursors and three kinds of enzymes related to the triterpenoid metabolism pathway. In this study,five genes corresponding to triterpene cyclase were analyzed in A. chinensis for the first time,which may participate in the synthesis of triterpenoid. It' s revealed that there were thirty three differential genes associated with the ko00900 and ko00909 pathways by analysis on the difference in transcriptome expression between seeds and flowers; seventeen unigenes were up-regulated and sixteen unigenes were down-regulated in the seeds relative to flowers. In this study, qRT-PCR experiments were used to verify the expression of three key enzyme genes of SQE( Unigene25806),HMGS( Unigene36710),and β-AS( Unigene33291). The results of qRT-PCR were consistent with the transcriptome data. The candidate genes related to triterpenoid saponin aescin synthesis in A. chinensis found in this study can provide theoretical basis for the metabolism synthesis and regulation of aescin.

Study of heterologous efficient synthesis of β-amyrin and high-density fermentation / 中国中药杂志

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.

Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of glycyrrhiza uralensis / 中草药·英文版

Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene (GuBAS). Methods: Tobacco root-specific promoter TobRB7 and GuBAS cDNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G. uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC. Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of GuBAS in these transgenic hairy roots was intended by qRT-PCR to be 3, 7, and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots. Conclusion: Over-expressing GuBAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

Chemical constituents from flowers of Gentiana dahurica / 中草药

Objective: To investigate the chemical constituents of the flowers of Gentiana dahurica. Methods: All compounds were isolated and purified by silica gel, Sephadex LH-20, ODS, and MCI column chromatography. Their structures were determined by physicochemical properties and spectral data. Results: Twenty compounds were isolated from the flowers of G. dahurica. Among them, eight triterpenoids were identified as roburic acid (1), 3β-acetoxy-28-hydroxy-12-ene-oleanane (2), 28-hydroxy-α-amyrin (3), 28-hydroxy-β-amyrin (4), α-amyrin (5), β-amyrin (6), ursolic acid (7), and oleanolic acid (8). Twelve flavonoids were identified as 1-hydroxy-3,7,8-trimethoxyxanthone (9), kaempferol (10), naringenin (11), apigenin (12), luteolin (13), (2S)-naringenin-7-O-β-D- glucopyranoside (14), apigenin-7-O-β-D-glucopyranoside (15), luteolin-7-O-β-D-glucopyranoside (16), isoorientin (17), isovitexin (18), saponarin (19), and lutonarin (20). Conclusion: Seventeen compounds 1-11, 13-16, 19, and 20 are found from flowers of G. dahurica for the first time; Compounds 2-7, 9-11, 13-16, 19, and 20 are isolated from this species for the first time.

Triterpenes and sterols from Hoya diversifolia Blume.

Chemical investigation of the dichloromethane extracts of Hoya diversifolia Blume led to the isolation of β-amyrin cinnamate (1), squalene (2), β-sitosterol (3), a mixture of β-amyrin (4a), α-amyrin (4b) and lupeol (4c) in a 4:2:1 ratio and saturated hydrocarbons from the leaves; and 2, taraxerol (5), lupeol cinnamate (6), and a mixture of 3 and stigmasterol (7) in a 2:1 ratio from the stems. The structures of 1-7 were identified by comparison of their NMR data with those reported in the literature.

Chemical constituents in whole herb of Euphorbia lunulata / 中草药

Objective: To study the chemical constituents in the whole herb of Euphorbia lunulata. Methods: The compounds were separated and purified by silica gel and Sephadex LH-20 column chromatography. The structures of the compound were identified on the basis of chemical and spectral methods. Results: Seventeen compounds were isolated from 90% ethanol extract from the whole herbs of E. lunulata and identified as 1-octacosanol (1), β-sitosterol (2), octadecyl caffeate (3), cycloart-23Z-en-3β, 25-diol (4), scopoletion (5), 24-methylenecycloartanol (6), stigmasterol (7), vifolin (8), o-phthalic acid bis-(2-ethyl decyl)-ester (9), ferulic acid hexacosyl ester (10), scopoletin-7-glucoside (11), β-amyrin (12), gallic acid (13), sucrose (14), 17-hxdroxyjolkinolide A (15), jolkinolide A (16), and jolkinolide B (17). Conclusion: Compounds 1,3,8-12 and 14-17 are isolated from this plant for the first time, and compound 9 is isolated from the plants of Euphorbia L. for the first time.

Chemical Constituents of Hoya buotii Kloppenb.

Chemical investigation of the dichloromethane extracts of Hoya buotii Kloppenb. afforded taraxerone (1), taraxerol (2), a mixture of β-sitosterol (3a) and stigmasterol (3b) in about 2:1 ratio, and a mixture of α-amyrin cinnamate (4a) and β-amyrin cinnamate (4b) in about 1:2 ratio from the stems; 1, 2, and 3a from the roots; a mixture of 4a and 4b in about 3:2 ratio from the flowers; and 3a, squalene (5) and saturated hydrocarbons from the leaves. The structures of 1-5 were identified by comparison of their NMR data with those reported in the literature.

Triterpenes and Acylglycerols from Canarium ovatum.

Chemical investigations of the dichloromethane extracts of the leaves of Canarium ovatum Engl. afforded β- amyrin (1a), α-amyrin (1b), epi-β-amyrin (2a), epi-α-amyrin (2b), epi-lupeol (2c), β-carotene (3) and lutein (4); while the twigs yielded 1a-1b. The dichloromethane extracts of the fruits of C. ovatum yielded triacylglycerols (5); the mesocarp also afforded 1a, 1b, 1,2-dioleylglycerol (6), and monounsaturated and saturated fatty acids; the nutshell also provided 6; and the kernel also yielded monounsaturated and saturated fatty acids. The structures of 1-6 and the fatty acids were identified by comparison of their 1H and/or 13C NMR data with those reported in the literature.

Terpenoids and Sterols from Hoya multiflora Blume.

Chemical investigation of the dichloromethane extracts of Hoya multiflora Blume led to the isolation of lupeol (1a), α-amyrin (1b), β-amyrin (1c), lupeol acetate (2a), α-amyrin acetate (2b), and β-amyrin acetate (2c) from the stems; and 1b, bauerenol (3), squalene (4), lutein (5), β-sitosterol (6a), and stigmasterol (6b)from the leaves. The structures of 1-6 were identified by comparison of their 1H and/or13C NMR data with those reported in the literature.

Gene cloning and expression level of bAS and their correlation with content of saponins in Eleutherococcus senticosus / 中草药

Objective: To clone β-amyrin synthase (bAS) gene from Eleutherococcus senticosus and analyze the effect of its expression on saponin contents. Methods: The sequence of cDNA of E. senticosus bAS was cloned by homologous cloning strategy. Quantitative real time PCR was developed to analyze the expression pattern of E. senticosus bAS gene. And E. senticosus saponin contents were measured by spectrophotometry method. Results: Length 1 223 and 1 226 bp of E. senticosus bAS1 and bAS2 genes were cloned. The results showed that bAS1 and bAS2 were expressed in the each growth period and every organ of E. senticosus, and the expression differed significantly (P < 0.05). bAS1 showed the highest expression when the plants were grown in germination stage, then rapidly depressed, and changed slightly in the end. The expression of bAS2 showed the characteristic of low-high-low. The expression of bAS1 in different organs of E. senticosus was constant, but the highest content of the expression of bAS2 was in the leaves. With the treatment of methyl jasmonate (MeJA), bAS2 expression has been significantly improved and bAS1 without a significant changing. There exists significantly positive correlation (P < 0.01) between the content of E. senticosus saponins and the expression levels of bAS2, and bAS1 without a significant difference. Conclusion: bAS2 may be a key enzyme gene in the biosynthesis of triterpenoid saponins.

Cloning, prokaryotic expression, and functional identification of β-amyrin synthase cDNA of Psammosilene tunicoides / 中草药

Objective: To clone, express, and characterize the full-length cDNA of β-amyrin synthase provided an important basis for the study on the key role in the biosynthetic pathway of triterpenoid saponins and secondary metabolism engineering applied to Psammosilene tunicoides. Methods: The full-length cDNA fragment of P. tunicoides was isolated by the method of RT-PCR and rapid amplification of cDNA ends (RACE). The fragment was transformed into Escherichia coli expression strain BL21, which was induced by IPTG, and the crude recombinant enzyme was purified from E. coli cell. In the presence of 2, 3-oxidosqualene and other substances, 2, 3-oxidosqualene was converted into β-amyrin efficiently. The catalytic product of P. tunicoides β-amyrin synthase was detected by high-performance liquid chromatography (HPLC) and identified as β-amyrin. Results: The full-length cDNA fragment of P. tunicoides is 2 882 bp and contains an open reading frame of 2 284 bp nucleotides, which codes for 760 amino acids. Conclusion: The full-length cDNA can produce β-amyrin synthase by prokaryotic expression. The expression product has the catalytic activity of 2, 3-oxidosqualene into β-amyrin, which would provide an important basis for the secondary metabolism engineering to P. tunicoides.

Cloning of β-amyrin synthase gene from Panax quinquefolius and its bioinformatics analysis / 中草药

Objective: To obtain the full-length cDNA of β-amyrin synthase (bAS) involved in triterpene saponin biosynthesis in Panax quinquefolius and provide the reference for saponin biosynthesis and the regulation of secondary metabolism in P. quinquefolius. Methods: Based on large-scale ESTs sequencing and RACE technology, the full-length cDNA of P. quinquefolius bAS (PqbAS) was obtained. Results: The full-length cDNA of PqbAS (GenBank No. JX185490) was 2 309 bp which included an open reading frame (ORF) code of 631 amino acid peptide. Common conserved domains of oxidosqualene cyclases (OSCs) were found in PqbAS including active sites and conserved sequence. Singal P4.0 analysis showed that PqbAS was a non-secreted protein. Tmhmm 2.0 analysis showed that PqbAS was a non-transmembrane protein. Real-time fluorescence quantitative PCR analysis showed PqbAS gene expressed in various organs, higher in flowers and stems while relatively lower in roots and leaves. Conclusion: The full-length of bAS is first cloned, which could provide the foundation for the investigation on expression characteristics and its role in synthesis of saponin.

Nonflavonoid constituents from leaves of Apocynum venetum / 中草药

Objective: To study the nonflavonoid constituents in the leaves of Apocynum venetum, the medicinal halophyte. Methods: The constituents were isolated and purified by silica gel and Sephadex LH-20 column chromatography as well as preparative HPLC, and their structures were elucidated by means of physicochemical properties and spectrosocpic analyses. Results: Fifteen nonflavonoid compounds were isolated from the methanol extract from the leaves of A. venetum, and identified as scopoletin (1), esculetin (2), methyl chlorogenate (3), chlorogenic acid (4), grasshopper ketone (5), benzyl-O-β-D-glucopyranoside (6), 2-phenylethyl-O-β-D-glucopyranoside (7), 1-β-O-benzoyl-D-glucopyranoside (8), tyrosol (9), isovanillic acid (10), vanillic acid (11), protocatechuic acid (12), lupeol (13), β-amyrin (14), and α-linolenic acid (15). Conclusion: Compounds 2, 3, 5-10, 14, and 15 are isolated from this plant and the plants in Apocynum Linn. for the first time.

Enhancement of erectile function of sexually na?ve rats by β-sitosterol and α-β-amyrin acetate isolated from the hexane extract of Mondiawhitei

Objective: The aim of the study was to investigate the prosexual activity of two compounds isolated from the hexane extract of the dried roots of Mondia whitei in rats. Methods: β-sitosterol and the mixture of α and β-amyrin acetate were isolated and characterised using several reagents, chromatography, mass spectrometry and physical analysis (RMN, 1H and 13C). Sexually inexperienced adult male rats were orally treated with these compounds at doses of 0mg/kg (control), 10mg/kg or 50mg/kg. 1h after the treatment, mount and intromission latencies and frequencies, penile erection, ejaculation latency and post-ejaculatory interval were measured for 60 minutes. Results: β-sitosterol and the mixture of α and β-amyrin acetate significantly increased the mount frequency (p0.05) when compared to control. The two purified compounds were efficient at the lower dose (10 mg/kg b.w) with β-sitosterol being the most potent. Conclusion:Results of the present work give added value to the aphrodisiac property of Mondia whitei and further justify its popular use as a sex stimulant.

Chemical and biological studies of Kalanchoe pinnata (Lam.) growing in Bangladesh

Objective: To isolate compounds from K. pinnata and elucidate their structures and to explore preliminary antioxidant, antimicrobial, cytotoxic and thombolytic activities of extractives of the plant. Methods: The methanol extract of whole plant of K. pinnata has been subjected to different chromatographic separation and purification processes to isolate the secondary metabolites. The structures of the isolated compounds have been elucidated by extensive NMR studies. The free radical scavenging activity of the crude extract and its different Kupchan fractions were determined on stable radical DPPH. In vitro antimicrobial activity was determined by the disk diffusion method. Cytotoxicity screening has been performed against Artemia salina. Total phenolics content, membrane stabilizing activity and thombolytic activities were assessed by following established protocol. Results: The isolated compounds were identified as glut-5(6)-en-3-one, taraxerone, 3β-friedelanol, β-amyrin-3-acetate, 3,5,7,3',5'-pentahydroxyflavone and β-sitosterol. The chloroform soluble fraction showed potent antioxidant activity of (IC50=80.0 μg/mL) and significant cytotoxicity, while the crude extract demonstrated noticeable total polyphenol content (149.24 mg of GAE/gm of extractive), moderate membrane stabilizing activity and inhibition of clot lysis of blood. Conclusions: The obtained results rationalize the folkloric use of the plant and can be further investigated to isolate the active compounds responsible for the biological activities.

β-Amyrin from Ardisia elliptica is more potent than aspirin in inhibiting collagen-induced platelet aggregation.

Ardisia elliptica Thunberg (Myrsinaceae) is a medicinal plant traditionally used for alleviating chest pains, treatment of fever, diarrhoea, liver poisoning and parturition complications. The objectives of the study were to investigate the effect of A. elliptica on collagen induced platelet aggregation and to isolate and purify potential antiplatelet components. Fresh A. elliptica leaves were extracted using methanol (70% v/v) by Soxhlet extraction and the extract was analysed for its inhibition of collagen-induced platelet aggregation. Inhibition of platelet aggregation was assessed by incubating the extracts with rabbit blood and collagen in a whole blood aggregometer and measuring the impedance. The leaf extract was found to inhibit platelet aggregation with an IC50 value of 167 µ/ml. Using bioassay guided fractionation, β-amyrin was isolated and purified. The IC50 value of -amyrin was found to be 4.5 µ/ml (10.5 µM) while that of aspirin was found to be 11 µ/ml (62.7 µM), indicating that β-amyrin was six times as active as aspirin in inhibiting platelet aggregation. This paper is the first report that β-amyrin isolated from A. elliptica is more potent than aspirin in inhibiting collagen-induced platelet aggregation. In conclusion, A. elliptica leaves were found to inhibit collagen-induced platelet aggregation and one of the bioactive components responsible for the observed effect was determined to be β-amyrin.