Bioactivity-guided isolation of cyclooxygenase-2 inhibitors from Saussurea obvallata (DC.) Edgew. Using affinity solid phase extraction assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea obvallata (DC.) Edgew. is a traditional Tibetan medicine used for the treatment of inflammation-related diseases, but the scientific validation was very limited. AIM OF THE STUDY: This study aimed to rapid screen and targeted isolate cyclooxygenase-2 (COX-2) inhibitors from S. obvallata extract. MATERIALS AND METHODS: An efficient ligand-fishing method based on affinity solid phase extraction (A-SPE) combining with HPLC was developed. The identified COX-2 inhibitors were separated using preparative liquid chromatography. In vitro COX-2 inhibition assays were employed to confirm the inhibitory activities of the isolated compounds. In addition, the effect of the isolated compounds on the production of prostaglandin E2 (PGE2) and the expression of COX-2 in LPS-induced RAW 264.7 were evaluated. RESULTS: A total of four phenylpropanoids, isolariciresinol, syringaresinol, pinoresinol and balanophonin were targeted isolated as COX-2 inhibitors with IC50 values of 36.4 ± 2.6 µM, 23.1 ± 1.8 µM, 3.6 ± 0.3 µM and 12.1 ± 0.9 µM, respectively. The isolated compounds significantly inhibited LPS-induced NO production in a dose-dependent manner. And, the results of the inhibitory effect on the release of PGE2 and the expression of COX-2 in LPS-induced macrophages were consistent with A-SPE analysis. CONCLUSION: The present work demonstrated that the developed A-SPE-HPLC method could successfully targeted isolated COX-2 inhibitors from S. obvallata extract. And, the isolation results indicated that the therapeutic effect of S. obvallata on inflammation-related diseases was partly based on the COX-2 active ingredients.

Identification and characterization of SlbHLH, SlDof and SlWRKY transcription factors interacting with SlDPD gene involved in costunolide biosynthesis in Saussurea lappa.

The genes involved in costunolide biosynthesis in Saussurea lappa have been identified recently by our lab. However, the study of transcriptional regulators of these genes was lacking for better opportunities for engineering the pharmacologically important biosynthetic pathway. Therefore, we cloned the promoter region of diphosphomevalonate decarboxylase gene (DPD) and analyzed its cis-acting regulatory elements to reveal the potential transcription factor (TF) binding sites for Dof, bHLH and WRKY family proteins in the gene promoter. The transcriptome study approach followed by the hidden Markov model based search, digital gene expression, co-expression network analysis, conserved domain properties and evolutionary analyses were carried out to screen out seven putative TFs for the DPD-TF interaction studies. Yeast one-hybrid assays were performed and three TFs were reported, namely, SlDOF2, SlbHLH3 and SlWRKY2 from Dof, bHLH and WRKY families, respectively that interacted positively with the DPD gene of the costunolide biosynthetic pathway. The tissue specific relative gene expression studies also supported the linked co-expression of the gene and its interacting TFs The present report will improve the understanding of transcriptional regulation pattern of costunolide biosynthetic pathway.

[Cloning of flavone synthase (FNSII) gene and expression in three cell lines of Saussurea medusa].

Saussurea medusa is a rare traditional Chinese medicinal herb, of which luteolin is the niain active medicinal compound for cancer prevention and treatment. A full-length FNSII gene, namely SmFNSII (GenBank Accession No. KF170286), was obtained from green cell line of Saussurea medusa by RT-PCR and RACE-PCR. Sequence analysis indicated that SmFNSII is 1 710 bp in full length, containing a 34 bp 5'-untranslated region (5'-UTR), a 125 bp 3'-UTR, and a 1 551 bp open reading frame (ORF) encoding 516 amino acid residues. Amino acid sequence analysis indicated that SmFNSII belonged to subfamily CYP93B of plant cytochrome P450. Sequence alignment and phylogenetic analysis revealed that amino acid sequences of SmFNSII shared 87% homology with the protein in Hieracium pilosella. Quantitative real-time PCR analysis indicated that SmFNSII expression is the highest in red cell line and the lowest in white cell line, corresponding to quantitative analysis of luteolin concentration. pET-SmFNSII, a prokaryotic expression recombinant plasmid, was constructed and transferred into Escherichia coli, and the expressed protein band was the same size with predicted protein. Saussurea medusa cultivars with high anti-inflammatory, anti-cancer activities and health care function would be cultivated through filtering cell lines and plants with high expression level of FNSII gene and luteolin accumulation.

[Identification and characterization of a flavonoid-3-O-glucosyltransferase gene from Saussurea involucrata].

The flavonoid-3-O-glucosyltransferas (3GT) is one of the most important enzymes for biosynthesis of plant secondary metabolites. In this paper, the homology analysis and gene special primers design were used. With the methods of modern molecular biology, the full-length gene of 3GT (GenBank Accession No. JN092127) which was cloned from Saussurea involucrata Kar. et Kir by RT-PCR and RACE. The cDNA sequence of 3GT consisted of 1548 bp open reading frame (ORF) encoding 516 amino acid, the deduced 3GT protein shared 91% and 89% identities with that of Fragaria x ananassa GT6, Manihot esculenta anthocyanidin 3-O-glucosyltransferase. Homology analysis showed that deduced 3GT protein has a glycosyltransferase signature domain PSPG-box. The transcripts of 3GT members were found mainly in leaves and callus. 3GT gene of S. involucrata was under the control of the cauliflower mosaic virus (CaMV) 35S promoter, homologous transformation used an Agrobacterium rihizogenes-mediated transformation system. The results on UV spectrophotometry showed S. involucrata callus after suspension culture that an average of total flavonoids on transgenic callus was 2.06 times higher than non-transgenic callus.

Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa.

Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomyces cerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similarity with previously characterized plant DFRs and phylogenetic analysis showed that it belonged to the plant DFR super-family. SmDFR also possessed flavanone 4-reductase (FNR) activity and can catalyze the conversion of eridictyol to luteoforol. Real-time PCR analysis showed that the expression level of SmDFR was higher in flowers compared with both leaves and roots. This work greatly enhances our knowledge of flavonoid biosynthesis in S. medusa and marks a major advance that could facilitate future genetic modification of S. medusa.

[Cloning, expression and charaterization of chalcone synthase from Saussurea medusa].

A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1313 bp, containing an open reading frame (1170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.

Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco.

Chalcone isomerase (CHI; EC 5.5.1.6) is a key enzyme in the flavonoid biosynthesis pathway. We isolated a CHI gene (SmCHI) from a cDNA library derived from Saussurea medusa (Asteraceae) cell cultures. The cDNA and genomic sequences of SmCHI are the same; in other words, this gene is intronless. The coding region of the gene is 699 bp long, and its deduced protein consists of 232 amino acids with a predicted molecular mass of 24 kDa and a pI of 4.7. The deduced amino acid sequence of SmCHI shares 79.3% identity with CHI from Callistephus chinensis, a familial relative to S. medusa; this homology is higher than those with CHI's from any other plant species. A functional bioassay for SmCHI was performed by transforming Nicotiana tabacum plants in the sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI produced up to fivefold total flavonoids over wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic tobacco plants with antisense SmCHI accumulated smaller amounts of flavonoids; this is apparently brought about by suppressed expression of the endogenous CHI gene. CHI activities also positively correlated with the amounts of total flavonoids accumulated in the transgenic plants. It is concluded that overexpression of SmCHI can be used as a useful approach to increase flavonoid production in transgenic plants.

Cloning and characterization of a flavanone 3-hydroxylase gene from Saussurea medusa.

Flavanone 3-hydroxylase (F3H) is a key enzyme in the flavonoid biosynthetic pathway, providing a branching point for the biosynthesis of different flavonoids, including the formation of 3-deoxy and 3-hydroxy flavonoids found in the silks of maize. Here, we report the cloning and characterization of a F3H gene (Smf3h) from a cDNA library derived from a red line callus of Saussurea medusa, a traditional Chinese medicinal plant. The cDNA contains a 1032 bp open reading frame (ORF) encoding a protein of 343 amino acid residues, a 149 bp long 5'untranslated regions (UTR) and a 163 bp long 3'UTR containing three putative polyadenylation signals (AATAAA) and an ATTTA element. The secondary structure of the mRNA predicted by MFOLD is very complex, suggesting a role in a post-transcriptional mechanism of regulation of Smf3h. The genomic structure of Smf3h includes four exons and three introns within the coding region, with all the splice donor/acceptor site sequences in accordance with the "GU-AG" consensus rule. The deduced SmF3H protein is 343 amino acid residues in length and has 40% and 39% identity and 60% and 58% similarity to the F3H of Arabidopsis and rice, respectively. Strikingly, the identity of SmF3H is higher to the H6H (hyoscyamine 3beta-hydroxylase, 45%) from Atropa belladonna. However, the analysis of the active center and the predicted protein secondary structure are more related to F3H than H6H. Together, our studies provide the first identification of a S. medusa flavonoid gene and its similarities to metabolic enzymes from other plants.

[Effects of cold-hardening on freezing tolerance and antioxidant enzyme activities in plantlets of Saussurea laniceps Hand.-Mazz].

Living conditions for plants in the mountains become increasingly less favorable with increasing altitude. In the alpine region, the plants are commonly exposed to daily rather than seasonal temperature fluctuations and by frequent freezing temperature. To elucidate the freezing tolerance mechanism of alpine plants, Saussurea laniceps Hand.-Mazz. was used as a model plant. It is a perennial herbal plant distributed in alpine regions of Yunnan and Tibet of China. It can survive on mountains with elevations over 4000 m. Wild S. laniceps plants are propagated only by seeds in the alpine areas. Micropropagation of S. laniceps through seed was a desirable method to get enough seedlings for freezing research. Micropropagation through plantlets derived from germinated S. laniceps seeds collected from Tibet was achieved successfully. Activities of antioxidant enzyme and solute contents were investigated in plantlets of S. laniceps. Freezing tolerance in plantlets increased after 7 or 15 d of cold-hardening (Table 1). Cold-hardening (2 degrees C) increased the activities of SOD, peroxidase, and catalase (Fig.1) in plantlets. A similar increase was also observed in the protein and proline content (Fig.1), whereas soluble carbohydrates changed little (Fig.1). These results obtained suggest that the higher activities of SOD, peroxidase, and catalase, as well as the higher protein and proline content may be biochemical adaptation for freezing toleranc in cold-hardened S. laniceps plantlets. Interestingly, deacclimation was slow; even after the plants were placed again under a temperature of 21-23 degrees C for 5 d, the higher freezing hardiness, enzyme activities, protein and proline content acquired after cold acclimation remained. In conclusion, our plantlet cultures have proved to be good materials for experimentation on freezing resistance in study of freezing-resistance mechanism in the alpine plant S. laniceps.