Research progress on triterpenoids of Betula plants / 药学学报

The secondary metabolites of plants are important sources of natural drugs. Betula plants have abundant pharmacological value, complex mechanism and wide applications, which are closely related to the triterpenoids of theirs. Triterpenoids in Betula species are mainly divided into dammarane-type, ocotillol-type, oleanane-type, lupane-type and cycloaltunane-type. The extracts of Betula species have varieties of activities such as anti-tumor, anti-inflammatory, anti-oxidant, anti-bacterial, etc. And the biosynthetic pathways of triterpenoids after 2,3-oxidosqualene are split into four branches of dammarenediol-II, lupeol, cycloartenol and amyrin according to the different oxidosqualene cyclases. This review summarizes the chemical constituents, pharmacological activities and biosynthetic pathways of triterpenoids in Betula plants. It provides a reference for the research and development of new drugs and the production of these triterpenoids in microbial cell factories by synthetic biology methods.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-

Combinatorial mutation on the -glycosidase specific to 7--xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

Taxol is a "blockbuster" antitumor drug produced by species with extremely low amount, while its analogue 7--xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1-1 and LXYL-P1-2 can convert 7--xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1-2 is twice more active than LXYL-P1-1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1-1 to LXYL-P1-2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12 (A72T/V91S) was the most active and even displayed 2.8- and 3-fold higher than LXYL-P1-2 on -xylosidase and -glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7--xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant , the molecular chaperone gene and the bacterial hemoglobin gene . This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5K-P1-2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1-2 for industrial purpose.

HPLC-MS study of Aquilariae Lignum Resinatum / 中草药

Objective: HPLC-MS was used to analyze the characteristic constituent of Aquilariae Lignum Resinatum, providing basis for the establishment of quality evaluation system of agarwood. Methods: The analysis was carried out on a Dionex-Acclaim 120 C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile and water-acetic acid (1000.5) with the flow rate of 0.4 mL/min at 254 nm, and the separation was performed at 26 ℃. MS experiments were performed using an electrospray ionization tandem mass spectrometry mainly in positive-ion mode. The cluster analysis was performed by SPSS software. Results: A total of 14 common peaks of flidersia chromones were characterized by HPLC-MS analysis, and seven of them were identified by comparing their retention times and MS spectra with reference compounds. The 10 batch of samples were divided into five categories by cluster analysis. The result indicated that great difference existed between agarwood produced from living tree and dead tree, and the relationship between constituent and production place of “Huang-Shu” agarwood was obvious. Conclusion: The analytical method for common flidersia chromones established in this paper could be used to evaluate the quality of agarwood.

Anti-tumor activity and molecular mechanism of C3C12PPD in lung cancer / 中国药理学与毒理学杂志

OBJECTIVE The aim of the present study was to investigate the anti-tumor effect and mechanism of a novel compound, C3C12PPD, a bioactive unnatural ginsenoside by metabolically engi-neered yeasts based on a new UDP-glycosyl- transferase from Bacillus subtilis. METHODS MTT assay was used to analyze the anti-proliferation activity of C3C12PPD in vitro. The effect of anti-tumor activity was observed by mouse Lewis xenograft model in vivo.The effects of C3C12PPD on suppressing the angio-genesis and invasion of A549 cells were investigated in vitro using Transwell and tube formation assays. RNAseq was used to find tagets of C3C12PPD. Western blotting was performed to investigate the expres-sion level of proteins in tumor tissues treated with C3C12PPD. RESULTS C3C12PPD could inhibit the growth of lung cancer in vitro and in viv o. At the dosage of 10.0 mg·kg-1, C3C12PPD inhibited tumor growth by 40.0% (P<0.05) in tumor weight in mouse Lewis xenograft. The inhibition of tube formation was 77.5%(P<0.01)and 80.3%(P<0.01)following treatment with 1×10-4and 2×10-4mol·L-1C3C12PPD for 5 h, whereas the proliferation of EA.hy926 cells was not influenced under the above concentrations. Under the concentrations of 1×10-4mol·L-1,C3C12PPD inhibited invasive ability of A549 cells(P<0.05).The results of RNAseq susgested that antitumor activity of C3C12PPD were associated with epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, the proteins related to EMT, Raf/MEK/ERK and AKT/mTOR signal pathways were effected by C3C12PPD analysed by western blotting. CONCLUSION These data suggested that C3C12PPD was able to supress lung cancer through inhibit EMT, invision and angiogenesis.

Studies on the enzymatic synthesis of a new PPT-type ginsenoside via UDP-glycosyltransferase PgUGT74AE2 from Panax ginseng / 药学学报

UDP-glycosyltransferase PgUGT74AE2 from Panax ginseng can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxadiol (PPD) to produce ginsenoside Rh2. However, no report demonstrates that PgUGT74AE2 can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxatriol (PPT) to produce a PPT-type ginsenoside. In this study, the expression plasmid pET-32a-PgUGT74AE2 was constructed for expression of the recombinant protein and transferred into Escherichia coli Transetta (DE3) to generate the recombinant strain Transetta-PgUGT74AE2. The recombinant enzyme PgUGT74AE2 was expressed by induction of isopropyl-β-D-thiogalactoside (IPTG). An in vitro enzymatic reaction system was established with the recombinant enzyme PgUGT74AE2 and the substrate PPT. PgUGT74AE2 catalyzed the glycosylation of the free C-3 hydroxyl of PPT to produce 3-O-β-D-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol, a new PPT-type ginsenoside. This study provides an efficient approach for the biosynthesis of a new PPT-type ginsenoside through in vitro enzymatic reaction, which may pave a way to produce promising lead in drug discovery.

Screening indices and their cut-off values for full-term neonates carrying β-thalassemia gene / 中国当代儿科杂志

OBJECTIVE@#To investigate the screening indices and their cut-off values for full-term neonates carrying β-thalassemia gene.@*METHODS@#A retrospective analysis was performed for the clinical data of 1 193 full-term neonates who underwent β-thalassemia screening (hemoglobin analysis with dried blood spots on neonatal heel blood filter paper and mutation detection of 17 β-globin genes). A multivariate logistic regression analysis was used to investigate the association between screening indices and β-thalassemia gene, and the receiver operating characteristic (ROC) curve was used to analyze the value of screening indices in determining the presence or absence of β-thalassemia gene.@*RESULTS@#Of the 1 193 neonates, 638 carried β-thalassemia gene. Of the 1 193 neonates, 637 (53.39%) had no HbA, among whom 310 carried β-thalassemia gene and 327 did not carry this gene; 556 (46.61%) had HbA, among whom 328 carried β-thalassemia gene and 228 did not carry this gene. As for the neonates without HbA, the β-thalassemia gene group had a significantly lower HbA level and a significantly higher HbF level than the β-thalassemia gene-negative group (P1.4 had the largest AUC in determining the presence or absence of β-thalassemia gene, with a sensitivity of 91.38% and a specificity of 91.89%.@*CONCLUSIONS@#HbA and HbA/HbA ratio are effective indices for screening out full-term neonates carrying β-thalassemia gene.

Studies on site-directed mutagenesis of BmK AngM1 from scorpion venom and its anti-inflammatory activity / 药学学报

Scorpion toxin BmK AngM1 has been reported to have a strong analgesic effect. However, its anti-inflammatory activity was unknown. In this study, the recombinant BmK AngM1 (rBmK AngM1) was expressed in Escherichia coli BL21 trxB (DE3). The purified rBmK AngM1 was obtained efficiently through the IMPACTTM-TWIN system. The anti-inflammatory activity of the recombinant protein was investigated. In order to improve the anti-inflammatory activity of rBmK AngM1, the potential active sites (Y5, Y42, R58) were substituted with different amino acids. The results showed that rBmK AngM1 and its mutants all have significant anti-inflammatory activity. The activities were significantly increased in the single mutant R58N and mutants Y5F/R58N, Y42F/R58N over the wild type protein. The data suggest that position 58 in BmK AngM1 plays a functional role in the anti-inflammatory activity. This study lays a foundation for the protein engineering design of BmK AngM1 to improve its pharmacological activity.

A new sesquiterpene from Chinese agarwood induced by artificial holing / 中国中药杂志

In order to study the chemical constituents of n-butanol fraction of ethanol extract from Chinese agarwood induced by artificial holing, various chromatographic techniques were carried out to isolate compounds, and the structures of compounds were determined through a combined analysis of physicochemical properties and spectroscopic evidence. Seven compounds were obtained and identified as selina-3,11-dien-9,15-diol (1), aquilarone D (2), 5α,6β,7α,8β-tetrahydroxy-2-[2-(2-hydroxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone (3), 6,7-dimethoxy-2-[2-(4-methoxyphenyl)ethyl]chromone (4), syringin (5), methyl (Z)-p-coumarate (6), and 4'-methoxycinnamic acid (7), among which compound 1 was a new compound and compounds 5-7 were isolated from agarwood for the first time. The bioactivity assay results concluded that compounds 6 and 7 showed certain nematicidal activity against Panagrellus redivivus, and compounds 4, 6 and 7 exhibited cytotoxicity against BEL-7402, SGC-7901 and A549 carcinoma cell lines.

Expression, subcellular localization and characterization of dammarenediol-Ⅱ synthase of Panax ginseng in Saccharomyces cerevisiae / 药学学报

To study the expression and subcellular localization of recombinant dammarenediol-Ⅱ synthase (DS) in Saccharomyces cerevisiae, the dammarenediol-Ⅱ synthase gene ds was cloned from Panax ginseng, and the gene ds was fused with the gene of green fluorescent protein to obtain the fusion gene ds-gfp. The recombinant expression plasmids pESC-HIS-DS and pESC-HIS-DS-GFP were constructed and transformed into S. cerevisiae INVSc1 to obtain recombinant strains INVSc1-DS and INVSc1-DS-GFP. Microsomes of recombinant strains were prepared by differential centrifugation and observed by fluorescence microscope. The green fluorescence was only detected in INVSc1-DS-GFP microsomes, which indicated that DS was a membrane protein. It was also proved that dammarenediol-Ⅱ was produced from the cyclization of 2, 3-oxidosqualene catalyzed by DS through in vitro enzymatic reaction. In addition, our results revealed that the fusion expression of ds with gfp significantly improved the production of dammarenediol-Ⅱ from 7.53 mg·g-1 to 12.24 mg·g-1. This study provides a new strategy in the optimization of the pathway of ginsenosides biosynthesis in S. cerevisiae.

Downregulation of lanosterol synthase gene expression by antisense RNA technology in Saccharomyces cerevisiae / 药学学报

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.

The basic strategies and research advances in the studies on glycosyltransferases involved in ginsenoside biosynthesis / 药学学报

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.

The expression of BmK AngM1 in Mut(s) and Mut(+) recombinants of Pichia pastoris / 药学学报

BmK AngM1 is a long-chain scorpion toxin purified from the venom of Buthus martensii Karsch. It has been reported to exhibit evident analgesic effect and low toxicity, and has the potential to be a novel analgesic drug. The BmKAngM1 gene was transformed into Pichiapastoris GS115. Mut+ and Mut(s) recombinant strains were screened by phenotype and Mut+ recombinant strains were used to detect BmK AngMl gene copy number in the real-time PCR. Expression of BmK AngM1 in the Mut+ recombinant strain was compared with that of the Mut(s) recombinant strain with the same single copy of BmK AngM1 gene under the same condition. The results indicated that the transcription level of BmK AngM1 gene in the Mut(s) recombinant strain was 2.7 fold of that in the Mut recombinant strain in the real-time PCR, and the expression of BmK AngM 1 in the Mut(s) recombinant strain was 1.5 fold of that in the Mut+ recombinant strain. Therefore, Mut(s) recombinant strain showed better ability to express BmK AngM1 than Mut+ recombinant strain.

HPLC fingerprint of Aquilariae Resinatum Lignum in China / 中草药

Objective: To establish an HPLC method for the fingerprint analysis of Aquilariae Resinatum Lignum (ARL), so as to provide evidence for the identification and quality control of ARL. Methods: The analysis was carried out on a Dionex-Acclaim 120 C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile and water-acetic acid (99.5:0.5) with the flow rate of 0.4 mL/min at 254 nm and the separation was performed at 26℃. The similarity was analyzed with "Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica 2004A" and the cluster analysis was performed by SPSS software. Results: The HPLC characteristic fingerprint of ARL has been established. A total of 24 common peaks were characterized, and nine of them were identified by comparing their retention time with reference subslances. The values of similarity evaluation mostly agreed with the result of cluster analysis. Conclusion: It is the first time to establish the HPLC fingerprint of ARL. The method is simple and quick, and reflects the information of chemical composition of ARL comprehensively, which provides the scientific basis for the identification and quality evaluation of ARL.

Construction of Saccharomyces cerevisiae haploid mutant deficient in lanosterol synthase gene / 药学学报

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.

Research advances of the influence factors of high level expression of recombinant protein in Pichia pastoris / 药学学报

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.

Advances in the biosynthesis research of ginsenosides / 药学学报

Ginsenosides are the main active components of medicinal herbs including Panax ginseng and Panax quinquefolium, which have potent effects of anti-tumor, anti-inflammatory, antioxidant and apoptosis inhibition. But the low content of ginsenosides limits its development and usage. At present, how to improve the production of ginsenosides by biological technology has been a new research focus. Some advances in the biosynthesis of ginsenosides by tissue culture and biotransformation have been made in recent years. So far at least twenty genes related to the biosynthesis of ginsenosides from Panax genus plants have been cloned and functionally identified, which has laid a good foundation for the study on the synthetic biology of ginsenosides. This review outlines recent advances in several aspects and is expected to provide a theoretical support to the thorough research of the pathway and regulation of ginsenosides biosynthesis.

Codon optimization and eukaryotic expression analysis of the analgesic peptide gene BmK AngM1 from Buthus martensii Karsch / 药学学报

Codon bias is an important factor which influences heterologous gene expression. Optimizing codon sequence could improve expression level of heterologous gene. In order to improve the expression level of BmK AngM1 gene encoding the analgesic peptide from Buthus martensii Karsch in Pichia pastoris, the codon-optimized BmK AngM1 gene according to its cDNA sequence and the preference codon usage of P. pastoris were cloned into expression vector pPIC9K and then transformed into P. pastoris. The expersion of recombinant BmK AngM1 (rBmK AngM1) was inducced by methanol in the medium, and the expression level of the optimized BmK AngM1 gene was 3.7 times of the native one. These results suggested that the expression of BmK AngM1 in P. pastoris could be successfully improved by codon optimization.

Measurement of eosinophils and interleukin-17 in nasopharyngeal secretions of children under 5 years old with wheezing / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the value of eosinophils (EOS) and interleukin-17 (IL-17) in nasopharyngeal secretions in the evaluation of progress of wheezing in children under 5 years old.</p><p><b>METHODS</b>Fifty-three children under five years old who had recurrent wheezing were classified into two groups: wheezing group I with atopic body (n=27) and wheezing group II without atopic body (n=26). Twenty pre-surgical children with non-infectious disease were used as the control group. Nasopharyngeal secretions were collected. Inflammatory cells in nasopharyngeal secretions were counted under the microscope. IL-17 levels in supernatants were measured using ELISA.</p><p><b>RESULTS</b>EOS counts in nasopharyngeal secretions in wheezing group I were significantly higher than those in wheezing group II and the control group (p<0.05, p<0.01, respectively). There were no significant differences in EOS counts between wheezing II and the control groups. The IL-17 levels in both wheezing groups were significantly higher than those in the control group (p<0.01), and the wheezing group I had increased IL-17 levels than wheezing group II (1 474+/-974 pg/mL vs 788+/-132 pg/mL; p<0.05). The IL-17 level was positively correlated with the EOS counts in wheezing group I (r=0.62, p<0.05).</p><p><b>CONCLUSIONS</b>EOS counts and IL-17 levels in nasopharyngeal secretions may be used as indices for identifying the tendency to develop asthma in children under 5 years old with wheezing.</p>

Optimization on the production of analgesic peptide from Buthus martensii Karsch in Pichia pastoris / 药学学报

The technology of liquid fermentation for producing the recombinant analgesic peptide BmK AngM1 from Buthus martensii Karsch in Pichia pastoris was studied by single-factor and orthogonal test. The results showed that the optimal culture conditions were as follows: 1.2% methanol, 0.6% casamino acids, initial pH 6.0, and three times of basal inoculation volume. Under the above culture conditions, the expression level of recombinant BmK AngM1 in Pichia pastoris was above 500 mg x L(-1), which was more than three times of the control. The study has laid a foundation for the large-scale preparation of BmK AngM1 to meet the needs of theoretical research of BmK AngM1 and development of new medicines.