Impact of mannitol and poly ethylene glycol 6000 induced water deficit on plant biomass and major secondary metabolites in Centella asiatica (L.) Urb. in vitr

Low water potential related stresses are regulated by modifying water uptake and loss to avoid low water potential, accumulating solutes which in turn enhance active principles and its gene expressions. Present study examined effect of in vitro induced absorption of mannitol and PEG (poly ethelene glycol) 6000 in Indian pennywort, Centella asiatica (L.) Urb., neutraceutical plant, evidenced by phenotypic, molecular and phytochemical analyses. Both mannitol and PEG 6000 induce water deficit conditions in plants and retarded normal plant biomass in terms of fresh and dry weights. These effects were significantly less severe in plants subjected to mannitol, compared to PEG. PEG and mannitol imposed water deficit, resulted in decline in major active compound, asiaticoside evidenced by HPTLC of asiaticoside content. Differential expression of some selected key genes in the asiaticoside pathway including squalene synthase and ? amyrin synthase by qPCR, confirmed decrease in transcript level expression of asiaticoside, whereas upregulated transcript level expression was observed in cycloartenol synthase for synthesis of phytosterols. Estimation of total flavonoids and phenolics under different water deficit conditions were found declined. In conclusion, water deficit by mannitol and PEG 6000 can significantly affects processes associated with biomass growth and ability to synthesize secondary metabolites in C. Asiatica.

Identification and functional characterization of 2,3-oxidosqualene cyclase genes family in Tripterygium wilfordii / 药学学报

italic>Tripterygium wilfordii Hook. f. is a valuable medicinal plant, with anti-tumor, anti-inflammatory, immunosuppressive and other pharmacological activities. Triterpenoids are one of the main active components that exert pharmacological effects. However, the content of triterpenoids dominated by triptolide is very low in Tripterygium wilfordii, and the analysis of the biosynthetic pathway of triterpenoids in Tripterygium wilfordii provides an effective new idea for obtaining these compounds. 2,3-Oxidosqualene cyclases (OSCs) are the key enzyme that catalyzes the formation of triterpene skeleton diversity. Based on the genome and transcriptome data of Tripterygium wilfordii, 16 OSC genes were identified and analyzed. Phylogenetic analysis showed that 16 TwOSC proteins could be mainly classified as four groups. They are β-amyrin synthase group, friedelin synthase group, multifunctional amyrin synthase and cycloartenol synthase group. TwOSC6 was successfully cloned. Functional characterization analysis revealed that TwOSC6 can catalyze the formation of α-amyrin and β-amyrin. This indicates that TwOSC6 is a multifunctional amyrin synthase. This provides new gene resources for the diversity of Tripterygium wilfordii triterpenoids, as well as new gene elements for biosynthesis triterpenoids.

α, ß-Amyrin prevents steatosis and insulin resistance in a high-fat diet-induced mouse model of NAFLD via the AMPK-mTORC1-SREBP1 signaling mechanism

Nonalcoholic fatty liver disease (NAFLD), characterized by hepatosteatosis and steatohepatitis, is intrinsically related to obesity. Our previous study reported on the anti-obese activity of α,β-amyrin (AMY), a pentacyclic triterpene isolated from Protium heptaphyllum. This study investigated its ability to prevent fatty liver and the underlying mechanism using the mouse model of NAFLD. NAFLD was induced in male Swiss mice fed a high fat diet (HFD) for 15 weeks. The controls were fed a normal chow diet (ND). The mice were simultaneously treated with AMY at 10 and 20 mg/kg or fenofibrate at 50 mg/kg. Lipid levels along with metabolic and inflammatory parameters were assessed in liver and serum. The liver sections were histologically examined using H&E staining. RT-qPCR and western blotting assays were performed to analyze signaling mechanisms. Mice fed HFD developed severe hepatic steatosis with elevated triglycerides and lipid droplets compared with ND controls. This was associated with a decrease in AMP-activated protein kinase (AMPK) activity, an increase of mechanistic target of rapamycin complex 1 (mTORC1) signaling, and enhanced sterol regulatory element binding protein 1 (SREBP1) expression, which have roles in lipogenesis, inhibition of lipolysis, and inflammatory response. AMY treatment reversed these signaling activities and decreased the severity of hepatic steatosis and inflammatory response, evidenced by serum and liver parameters as well as histological findings. AMY-induced reduction in hepatic steatosis seemed to involve AMPK-mTORC1-SREBP1 signaling pathways, which supported its beneficial role in the prevention and treatment of NAFLD.

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.

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.

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.

Pentacyclic triterpenes and other constituents in propolis extract from Melipona beecheii collected in Yucatan, México

ABSTRACT Thirteen pentacyclic triterpenes, methyl 3-oxours-12-en-23-oate, marsformosanone, taraxerone, β-amyrenone, α-amyrenone, lupenone, 24-methylencycloartan-3-one, moretenol acetate, β-amyrin acetate, germanicol acetate, 24-methylencycloartanyl acetate, β-amyrin, and α-amyrin were identified in a chloroform-methanol propolis extract from Melipona beecheii. Additionally, were identified in this propolis, hexadecanoic acid, methyl ester, octadecanoic acid, methyl ester and 1-triacontanol. The purification of the propolis extract was carried out using different chromatographic techniques, including vacuum liquid chromatography, gravity column chromatography and gel filtration chromatography Sephadex LH-20. The identification of the metabolites was performed using mass spectrometry.

Antinociceptive and anti-inflammatory activities of a triterpene-rich fraction from Himatanthus drasticus

Himatanthus drasticus (Mart.) Plumel belongs to the Apocynaceae family and the latex from its trunk bark (Hd) is known as "janaguba milk". This latex is widely used in Northeast Brazil, mainly in the Cariri region, for its gastroprotective, anti-inflammatory, and antitumor properties. The objective of this study was to investigate a triterpene-rich fraction (FJNB) from H. drasticus latex on acute models of nociception and inflammation and to clarify its mechanisms of action. Wistar rats or Swiss mice were subjected to the carrageenan-induced paw edema test or the formalin test, respectively, after the acute oral treatment with FJNB. The inflamed paws from the carrageenan-induced paw edema and formalin tests were processed for histological and immunohistochemical assays, respectively. The results were analyzed by ANOVA and considered significant at P<0.05. FJNB (10 mg/kg) decreased the paw edema by 25% at the 3rd h after the carrageenan injection. Indomethacin, used as reference, inhibited the paw edema by 59% at the same time-point. In the formalin test, FJNB inhibited the 1st phase by 27, 49, and 52% and the 2nd phase by 37, 50, and 67%, at the doses of 1, 5, and 10 mg/kg, respectively. In addition, FJNB significantly inhibited the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and the inflammatory cytokine tumor necrosis factor (TNF)-alpha. The histone deacetylase (HDAC) expression and the transcription factor nuclear factor kappa (NF-kB) were also inhibited at the same doses. In conclusion, the FJNB inhibitory actions on iNOS, COX-2, TNF-α, HDAC, and NF-kB could be involved with the drug anti-inflammatory activity.

Chemical constituents from Achyrocline satureioides (I) / 中草药

Objective To study the chemical constituents from Achyrocline satureioides. Methods Chemical constituents from A. satureioides were separated and purified by a variety of chromatographic techniques, and their structures were identified by various spectroscopic methods. Results Twenty-one compounds were isolated from ethyl acetate fraction of the plant, which were α-amyrin (1), ergosta-4,6,8,22-tetraene-3-one (2), (R)-24-ethylcholest-4-en-3,6-dione (3), clovandiol (4), caryolane-1,9β-diol (5), lepidissipyrone (6), 5,7-dihydroxy-3,6-dimethoxyflavone (7), quercetin-7-O-β-D-glucoside (8), pinocembrin (9), (2R,3R)-taxifolin (10), quercetin-4’-O-β-D-glucoside (11), helichrysetin (12), 3-[5,7-dihydroxy-2,2-dimethyl-8-(2-(S)-methyl-butanoyl)-2H-chromen-6- yl-methyl]-6-ethyl-4-hydroxy-5-methyl-pyran-2-one (13), quercetin (14), protocatechuic acid (15), (+)-(3R)-3-hydroxyl-4,4- dimethyl-4-butyrolactone (16), (4S,5R)-5-(4’-methyl-3’-pentenyl)-4-hydroxy-5-methyldihydrofuran-2-one (17), genkwanin (18), quercetin-3-methyl ether (19), galangin (20), and neosakuranin (21). Conclusion Compounds 1, 4, 5, 16, 17, and 21 are obtained from the genus for the first time, and compounds 1-12, 16-18, and 21 are obtained from the plant for the first time.

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.

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.

A new triterpene saponin from roots of Ligularia veitchiana / 中草药

Objective To study the chemical constituents from the roots of Ligularia veitchiana. Methods Some chromatographic methods were used to isolate the chemical constituents from the roots of L. veitchiana, their structures were elucidated on the basis of spectral data. Results Six compounds were isolated from roots of L. veitchiana and were identified as liguveitoside B (1), oleanolic acid (2), β-sitosterol (3), α-amyrin (4), α-amyrin-3-O-β-D-glucopyranoside (5), and β-daucosterol (6). Conclusion Compound 1 is a new compound, named liguveitoside B; Compounds 4 and 5 are isolated from L. veitchian for the first time.

Chemical constituents from flowers of Chrysanthemum indicum / 中草药

Objective To study chemical constituents from the flowers of Chrysanthemum indicum. Methods The chemical constituents of C. indicum were isolated and purified by various chromatographic techniques, including thin-layer chromatography, silica gel, ODS reversed-phase silica gel, and Sephadex LH-20 for column chromatography, and their structures were identified by NMR spectral analysis. Results Fourteen compounds were isolated and identified as stigmata-4-ene-3-one (1), calenduladiol-3β-O- palmitate (2), 16β,22α-dihydroxypseudotaraxasterol-3β-O-palmitate (3), α-amyrin (4), urs-12-ene-3β,16β-diol (5), 3β-hydroxyurs- 12-ene-11-one (6), arnidiol (7), maniladiol (8), 3β-hydroxyolean-12-ene-11-one (9), luteolin (10), apigenin (11), apigenin-7,4’- dimethyl ether (12), genkwanin (13), and 1-linoleic acid glycerate (14). Conclusion Compounds 1-6, 10-12, and 14 are isolated from the flowers of C. indicum for the first time.

Chemical constituents from branches and leaves of Viburnum sargentii / 中草药

Objective: To study the chemical constituents from the branches and leaves of Viburnum sargentii collected at Mountain Tai. Methods: The chemical constituents were isolated and purified by chromatographic methods, including silica gel, ODS, Sephadex LH-20 columns, and RP HPLC. The structures of the isolated compounds were identified by ESI-MS, 1D-NMR, and 2D-NMR data analyses, and compared with the literature data. Results: Thirteen compounds were obtained from the 95% ethanol extract of branches and the leaves of V. sargentii, and determined as α-amyrin (1), uvaol (2), 3α-ursolic acid (3), 11,12-dehydroursolic acid lactone (4), 3-O-acetyloleanolic aldehyde (5), oleanolic acid (6), betulinic acid (7), magnolin (8), (+)-eudesmin (9), (-)-epieudesmin (10), vibsanol (11), 3,4'-dimethoxylvibsanol (12), and α-asarone (13), respectively. Conclusion: Compound 12 (3,4'- dimethoxylvibsanol) is a new natural product, and compounds 1-11 and 13 are isolated from V. sargentii for the first time.

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.

Mesua ferrea stem bark extract induces apoptosis and inhibits metastasis in human colorectal carcinoma HCT 116 cells, through modulation of multiple cell signalling pathways / 中国天然药物

Considering the great potential of natural products as anticancer agents, the present study was designed to explore the molecular mechanisms responsible for anticancer activities of Mesua ferrea stem bark extract against human colorectal carcinoma. Based on MTT assay results, bioactive sub-fraction (SF-3) was selected for further studies using HCT 116 cells. Repeated column chromatography resulted in isolation of less active α-amyrin from SF-3, which was identified and characterized by GC-MS and HPLC methods. α-amyrin and betulinic acid contents of SF-3 were measured by HPLC methods. Fluorescent assays revealed characteristic apoptotic features, including cell shrinkage, nuclear condensation, and marked decrease in mitochondrial membrane potential in SF-3 treated cells. In addition, increased levels of caspases-9 and -3/7 levels were also observed in SF-3 treated cells. SF-3 showed promising antimetastatic properties in multiple in vitro assays. Multi-pathway analysis revealed significant down-regulation of WNT, HIF-1α, and EGFR with simultaneous up-regulation of p53, Myc/Max, and TGF-β signalling pathways in SF-3 treated cells. In addition, promising growth inhibitory effects were observed in SF-3 treated HCT 116 tumour spheroids, which give a hint about in vivo antitumor efficacy of SF-3 phytoconstituents. In conclusion, these results demonstrated that anticancer effects of SF-3 towards colon cancer are through modulation of multiple molecular pathways.

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.

Study on chemical constituents from Gnaphalium affine and their anti-oxidative activities / 中草药

Objective: To study the chemical constituents from Gnaphalium affine and their anti-oxidative activities. Methods: Chemical constituents were isolated by column chromatography and semi-prepared HPLC, the structures were elucidated by spectral data and physicochemical properties. DPPH free radical scavenging activities of compounds 1,3-11, and 16 were determined. Results: Seventeen compounds were isolated and respectively identified as apigenin (1), dihydroapigenin (2), luteolin (3), chrysin (4), wogonin (5), stimasterol (6), β-sitosterol (7), ursolic acid (8), oleanolic acid (9), 19α-hydroxyl-oleanolic acid (10), 2α, 3α, 19α-trihydroxy-28-norurs-12ene (11), α-amyrin acetate (12), β-amyrin acetate (13), patriscabratine (14), aurantiamide acetate (15), 4'-hydroxydehydrokawain (16), and isovanillin (17). Compounds 1 and 3-5 had significant anti-oxidative activities. Conclusion: Compounds 2,4,5,11-15, and 17 are isolated from G. affine for the frist time.