Research progress on culture technologies,chemical components,and pharmacological activities of Saussurea involucrata cells / 中国中药杂志

Saussurea involucrata,a traditional Chinese medicinal material,is effective in the treatment of rheumatoid arthritis with cold-dampness blockage syndrome,cold pain in lower abdomen,and menstrual irregularities. However,due to the specific habitat,low natural reproduction rate,slow growth,and overexploitation,it is at the high risk of extinction. S. involucrata cells can be obtained through callus culture,suspension culture,and hairy root culture. This study highlighted the influences of reactor type,culture system,precursor,elicitor type, and light wavelength on the suspension culture of S. involucrate cells. The chemical components of S. involucrata cells mainly include phenylpropanoids,flavonoids,lignans,and steroids,among which phenylpropanoids are the most abundant. S. involucrata cells have multiple pharmacological activities of anti-inflammation,analgesia,activating blood and resolving stasis,immunoregulation,increasing bone density,lowering blood lipids,anti-hypoxia,anti-exercise fatigue,anti-radiation,anti-obesity,and anti-oxidation. Moreover,it has the potential of treating aplastic anemia. This study reviews the cell culture technologies,chemical components,and pharmacological activities of S. involucrata cells,laying a basis for the further research,development,and utilization.

Evaluation of the larvicidal potential of root and leaf extracts of Saussurea costus (Falc. ) Lipsch. against three mosquito vectors: Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus

Abstract INTRODUCTION: The larvicidal potential of Saussurea costus (Falc.) Lipsch. was studied against the early 4th instar larvae of Anopheles stephensi Liston., Aedes aegypti Linn.,and Culex quinquefasciatus Say. because of the emergence of mosquito resistance to conventional synthetic insecticides. METHODS: At concentrations of 12.5-200 ppm, larvicidal activities were studied under laboratory conditions. RESULTS: After 24 h of exposure, the methanol extract of the roots recorded the highest larvicidal activity against An. stephensi, with LC50 and LC90values of 7.96 and 34.39 ppm, respectively. CONCLUSIONS: We are developing potent larvicidal compound(s) from S. costus for controlling the mosquito larval population.

Research progress on terpenes and pharmacological effects of Saussurea lappa / 中国中药杂志

Saussurea lappa originates in India, and now mainly grow in Yunnan, Sichuan and other places in China. It is one of the commonly used traditional herbal medicines in Tibet and other minority regions, with effects in regulating qi to relieve pain and invigo-rating spleen to promote food. It has been used in clinic for gastrointestinal diseases, such as Qi stagnation syndrome of spleen and stomach, diarrhea and tenesmus. More than 200 compounds have been identified from S. lappa. Among them, sesquiterpenoids attracted much attention. In terms of the number of compounds, eudesmanetype is dominant, guaiane and germacranetypes have also been reported frequently. Pharmacological studies have involved extracts, volatile oils and monomeric components represented by dehydrocostus lactone. Anti-tumor, anti-inflammatory and anti-bacterial effects on digestive system have attracted great attention. However, due to the complex sources of S. lappa and widely used in clinical practice, there is few research progress on relevant chemical constituents and pharmacological activities. This paper systematically summarizes terpenes and the pharmacological effects of S. lappa, in order to provide basis for further studies and clinical applications.

Study on high throughput sequencing identification of Fructus Arctii and five counterfeit species mix power / 中国中药杂志

Fructus Arctii is a traditional Chinese medicine. The main counterfeit species are the seeds of Arctium tomentosum, Onopordum acanthium, Silybum marianum, Saussurea costus, Amorpha fruticosa. Traditional identification methods or molecular barcoding techniques can identify Fructus Arctii and its counterfeit species. However, the identification of the mixture of it and its spurious species is rarely reported. In this paper, we sequenced the ITS2 sequences of Fructus Arctii and 5 kinds of spurious species mix powder by high-throughput sequencing to identify the mixed powder species and providing new ideas for the identification of Fructus Arctii mix powder. The total DNA in mixed powder was extracted, and the ITS2 sequences in total DNA was amplified. Paired-end sequencing was performed on the DNA fragment of the community using the Illumina MiSeq platform. The sequence was analyzed by the software FLASH, QIIME and GraPhlAn etc. The results showed that the high quality ITS2 sequences of 39910 mix samples were obtained from the mixed samples, of which the total ITS2 sequence of the samples genus was 34 935. Phylogenetic analysis showed that the samples contained Fructus Arctii, A. tomentosum, O. acanthium, S. marianum, S. costus and A. fruticosa. Using ITS2 sequences as DNA barcodes, high-throughput sequencing technology can be used to detect the Fructus Arctii and its spurious specie in mixed powder, which can provide reference for the quality control, safe use of medicinal materials of Fructus Arctii and the identification of mixed powder of traditional Chinese medicine.

Analysis of flavonoids and antitumor activity of transgenic Saussurea involucrate / 中国中药杂志

The aim of this paper was to investigate the flavonoids of callus of transgenic and non-transgenic Saussurea involucrate and its antitumor activity on the esophageal cancer CaEs-17 cells. The species and content of mono-phenols were detected by high performance liquid chromatography. The growth of human esophageal cancer CaEs-17 cells was detected using CCK-8 assay, apoptosis morphology observation and flow cytometry. Expression of related apoptotic genes Bax and Bcl-2 were determined by qPCR. The results showed that the content of total flavonoids in the transgenic callus was 2.72 times that of the non-transgenic callus. The cyanidin-galactoside was detected in the transgenic callus, but not in the non-transgenic callus. The inhibitory effect of the extracts from the transgenic callus on CaEs-17 cells was more significant than that of the non-transgenic callus, and the IC₅₀ value had a decreased of 26.4%. Flow cytometry analysis results showed that the apoptosis induction effect of the extracts from the transgenic callus on CaEs-17 cells was significantly better than that of the non-transgenic callus. Fluorescence quantitative PCR analysis results showed that the extracts from the transgenic callus could up-regulate the expression of proapoptotic gene Bax and down-regulate the expression of apoptotic gene Bcl-2, and the regulation effect of the transgenic callus was more significant. Therefore, compared with the non-transgenic callus, the antitumor activity of the extracts from the callus of transgenic S. involucrate on the esophageal cancer CaEs-17 cells was significantly increased, which was closely related to the accumulation of cyanidin-galactoside and its metabolism-related flavonoid compounds in the transgenic callus.

Anti-tumor Activity of Saussurea laniceps against Pancreas Adenocarcinoma

The purpose of this study was to confirm the anti-tumor activity of an ethanol extract of Saussurea laniceps against pancreatic tumor and to isolate the active compound from S.laniceps extract. Treatment with S.laniceps extract and hispidulin inhibited proliferation of pancreatic cell lines, such as Capan-1, Capan-2, Panc-1 and S2-013 in a dose-dependent manner using the hollow fiber assay. Hispidulin showed typical hallmarks of apoptotic cell death a significant anti-tumor activity on Capan-2 cells at a dose of 100 mg/kg and 200 mg/kg. S.laniceps has potential cytotoxic and apoptotic effects on human pancreatic carcinoma cells. Its mechanism of action might be associated with the apoptotic cell death through DNA fragmentation.

A new polyphenol, 1, 3-di-O-caffeoyl-5-O-(1-methoxyl-2-O-caffeoyl-4-maloyl)-quinic acid, isolated from cultured cells of Saussurea involucrata / 中国天然药物

The present study was designed to isolate the polyphenol constituents of cultured cells of Saussurea involucrata. The polyphenol type constituents were isolated using chromatography methods, and then characterized by spectral analysis. 1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)-hydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging were assayed using Vitamin C as the positive control. One new polyphenol 18, 1, 3-di-O-caffeoyl-5-O-(1-methoxyl-2-O-caffeoyl-4-maloyl)-quinic acid, together with 17 known compounds, was isolated and characterized. In conclusion, Compound 18 was a new caffeoyl maloyl quinic acid type polyphenol and showed desired vitro anti-oxidant activity. Compounds 1-5, 9, 10, 14, 15, and 17 were isolated from cultured cells of Saussurea involucrata for the first time.

Cloning of flavone synthase (FNSII) gene and expression in three cell lines of Saussurea medusa / 生物工程学报

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.

Determination of skimmin, scopolin and umbelliferone in Tibetan medicine Saussurea hieracioides by HPLC / 中国中药杂志

This study is aimed to establish a high-performance liquid chromatography (HPLC) method for simultaneous determination of skimmin, scopolin and umbelliferone in Saussurea hieracioides. Samples were analyzed on a Wondasil C18-WR column (4.6 mm x 250 mm, 5 microm) with methanol (A) and water containing 0.1% phosphate (B) as mobile phases for gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength and column temperature were set at 325 nm and 35 degrees C, respectively, and the sample size was 10 microL. The results showed that skimmin, scopolin and umbelliferone were simultaneously achieved within 40 min under the above conditions. A good linearity was observed in the range of 0.18-5.6 microg (r = 1.000 0), 0.060-1.8 microg (r = 0.999 9), 0.032-0.97 microg (r = 0.999 8) for skimmin, scopolin and umbelliferone, respectively, with the average recoveries of 99.16% (RSD = 0.41%), 100.3% (RSD = 0.79%), 102.2% (RSD = 0.87%). The method is simple, accurate and reproducible and can be used for the quality control of S. hieracioides.

Regulation of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid biosynthesis in cell suspension cultures of Saussurea involucrata / 中国中药杂志

Syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid are three main bioactive ingredients in herbs of Saussurea involucrata with various pharmacological properties, while their contents are very low. In this study, the biosynthesis of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid in the cell suspension cultures of S. involucrata were regulated by feeding carbon sources and precursors, which resulted in a great increase of the contents and yields of the above three bioactive ingredients. After 16 days of fermentation, the yields of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid reached 339.0, 225.3, 512.7 mg x L(-1), respectively. Meanwhile, their contents increased up to 67.9, 1.9, 10.6 times of wild medicinal material, respectively. The results provided a solid basis for further studies on application of cell suspension cultures of S. involucrata for large-scale production of bioactive compounds syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid.

Protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats under constant pressure and closed conditions.</p><p><b>METHOD</b>The PESI dosage-dependent experiment for hypoxia rats was conducted under constant pressure and closed conditions by intraperitoneally injecting 125, 250, 500 mg x kg(-1) to finalize that the optimum dosage is the high dose of PESI. Afterwards, 90 Wistar rats were randomly divided into the hypoxic model group, the acetazolamide 250 mg x kg(-1) group and the PESI high dose group. Each group was further divided into three subgroups according to different hypoxia times, with 10 rats in each subgroup. Under the same hypoxia and administration conditions, the rats were sacrificed after 0, 3, 6 h respectively. Their brain samples were collected for common pathological observation and immunohistochemical staining of HIF-1alpha. Real-time RT-PCR was used to detect HIF-1alpha, EPO, HO-1 and Caspase-3 gene expressions. And the Western blot assay was adopted to detect HIF-1alpha protein expression.</p><p><b>RESULT</b>The brain tissues of the hypoxia model group were severely damaged with the increase in the hypoxia time. The acetazolamide group and the PESI high does group were damaged in a much lower degree. According to the gene expression and the Western blot assay, high dose of PESI could inhibit HIF-1alpha expression. According to the pure gene expression test, high dose of PESI could increase EPO and HO-1 mRNA expressions, but inhibit Caspase-3 mRNA expression.</p><p><b>CONCLUSION</b>PESI's protective mechanism for brain tissues of hypoxia rats under constant pressure and closed conditions may be related to its effects in inhibiting HIF-1alpha expression, increasing EPO expression and resisting cell apoptosis.</p>

Flavonoids contents and expression analysis of related genes in red cell line of Saussurea medusa / 生物工程学报

Saussurea medusa is a rare traditional Chinese medicinal herb. Besides anti-inflammatory and analgesic activities, it has effects of disinhibiting cold, dispelling dampness and promoting blood circulation. Flavonoids are the main medicinal compounds in S. medusa. Contents of flavonoids and expression of flavonoids biosynthesis related genes in white and red (induced by low temperature, high sucrose and high light) callus were analyzed. The results showed that the total flavone in red line was 3.60 times higher compared to white line. The accumulation of rutin in red line (0.25% of dry weight) was 2.40 times higher compared to white line. Anthocyanins were abundant in red line, with the contents of cyanidin 3-O-glucosidechloride and cyanidin 3-O-succinyl glycoside 0.12% and 0.19% of dry weight respectively. CHS, F3'H, FNS, FLS, DFR and ANS genes were highly expressed in red line compared to white line. Expression of three transcription factors (MYB, bHLH and WD40) in red line was significantly higher than that in white line, especially the expression of MYB (19.70 times higher compared to white line). These results indicated that high expression levels of transcription factors induced high expression of structural genes in red line, thereby enhancing the flavonoids biosynthesis. The expression of bHLH and WD40 was similar, whereas it was significantly different from that of MYB, indicating that bHLH and WD40 could form a binary complex to regulate expression of structural genes and flavonoids biosynthesis.

Identification and characterization of a flavonoid-3-O-glucosyltransferase gene from Saussurea involucrata / 生物工程学报

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.

Chemical constituents from a portion of ethanolicextract of Saussurea lappa roots / 中国中药杂志

<p><b>OBJECTIVE</b>To study chemical constituents from the roots of Saussurea lappa.</p><p><b>METHOD</b>Chemical constituents were separated and purified by various techniques such as silica gel column chromatography, Sephadex LH-20 and reversed phase RP-18 column chromatography. Their structures were identified on the basis of spectral data.</p><p><b>RESULT</b>Seventeen compounds were separated and identified as ascleposide E(1), (+)-1-hydroxypinoresinol-4"-O-methyl ester-4'-beta-D-glucopyranoside (2), (+)-1 -hydroxypinoresinol-4"-O-beta-D-glucopyranoside(3), (+)-1-hydroxypinoresinol-1-O-P-D-glucopyranoside (4), phenyl-beta-D-glucopyranoside (5), benzyl-beta-D-glucopyranoside (6), n-butyl-beta-D-glucopyranoside (7), ilicic alcohol (8), beta-cyclocostunolide (9), reynosin (10), 11beta, 13-dihydroreynosin (11), arbusculin A(12), 1beta-hydroxy-arbusculin A (13), santamarin (14), dehydrocostuslactone (15), 11beta, 13-dihydro-3-epizaluzanin C(16)and costunolide (17).</p><p><b>CONCLUSION</b>Compounds 1-2 were separated from this genus for the first time,and compounds 3,5-7 were separated from this plant for the first time.</p>

Study on chemical constituents from the roots of Saussurea lappa / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents roots of Saussurea lappa C. B. Clarke.</p><p><b>METHOD</b>Isolation and purification were carried out by silica gel, Sephadex LH-20 and RP-18 column chromatography. The chemical structures of constituents were elucidated on the basis of spectral data.</p><p><b>RESULT</b>Eight compounds were isolated and identified as: 1beta-hydroxycolartin (1), 5alpha-hydroxy-beta-costic acid (2), 11alpha,13-dihydroxydehidrocostuslactone (3),11,13-dihydro-7,11-dehydro-13-hydroxy-3-desoxyzaluzanin C (4), 8alpha-hydroxyl-11betaH-11,13-dihydrodehydrocostuslactone (5), Soulangianolide A (6), Syringaresinol (7), Scopoletin (8).</p><p><b>CONCLUSION</b>Compounds 1-4, 6-8 were isolated from the genus Saussurea for the first time, and compound 5 was isolated from this plant for the first time.</p>

Isoprenoids and phenylpropanoids from Saussurea deltoidea / 中国中药杂志

To investigate the chemical constituents of Saussurea deltoidea, 10 compounds were isolated from the title plant by various chromatography methods such as silica gel, RP-18 silica gel, Sephadex LH-20 column chromatography, HPLC, et al. Their structures were elucidated by spectral analysis. Five isoprenoids and Five phenylpropanoids were isolated and elucidated as (3R, 6R, 7E) -3-hydroxy-4, 7-megastigmadien-9-one (1), (3S, 5R, 6S, 7E) -5, 6-epoxy-3-hydroxy-7-megastigmen-9-one (2), 3-hydroxy-beta-damascone (3), S-(+) -dehydrovomifoliol (4), megastigman-5-ene-3beta, 9R-diol (5), coniferaldehyde (6), beta-hydroxypropiovanillone (7), 3-hydroxy-1-(4-hydroxy-3, 5-dimethoxyphenyl) -1-propanone (8), dihydrosyringenin (9), 4-[(1S) -3-hydroxy-1-methoxypropyl]-2, 6-dimenthoxyphenol (10). All the compounds were isolated from S. deltoidea for the first time.

Cloning, expression and charaterization of chalcone synthase from Saussurea medusa / 生物工程学报

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.

Effects of phytohormones on plant regeneration and production of flavonoids in transgenic Saussurea involucrata hairy roots / 生物工程学报

We investigated the plant regeneration and production of flavonoids in three high-yield flavonoids transgenic Saussurea involucrata hairy roots C17, C27 and C46 by quantification of two phytohormones GA3 and IAA. The results showed that GA3 concentration at more than 1.0 mg/L could induce adventitious shoots in the hairy root lines. The highest shoot regeneration rate, about 82%, was obtained when the hairy roots C17 were cultured with 2.0 mg/L GA3. The results on HPLC and UV spectrophotometry showed that exogenous application of both GA3 and IAA increased the content of flavonoids in the hairy roots. The contents of flavonoids and apigenin in the hormone-treated hairy roots and regenerates were higher comparing with those in the untreated hairy roots and the regenerates. However, the content of flavonoids was not related to tissue weight, and was negatively related to the regeneration efficiency.

Formulation optimization of gel matrix of Saussurea involucrate by orthogonal design / 中国中药杂志

<p><b>OBJECTIVE</b>To optimize the formulation of gel matrix of Saussurea involucrate.</p><p><b>METHOD</b>The formulation of gel containing carbopol, azone, trimethylene glycol and alcohol was optimized by orthogonal design with evaluation indexes such as consistency in room temperature, consistency of average temperature of skin, yield value and rheopexy in room temperature.</p><p><b>RESULT</b>The optimal proportion of matrix components was made from carbopol-azone-trimethylene glycol-absolute alcohol (2. 0: 2. 0:15.0 : 30.0).</p><p><b>CONCLUSION</b>The optimal formulation of S. involucrate gel based the optimal formulation is well-distributed and transparent, it has proper consistency and well rheopexy.</p>

Comparison of rutin and syringin content between tissue culturing seedlings and botanical drug of Saussurea involucrata / 中国中药杂志

<p><b>OBJECTIVE</b>To compare the rutin and syringin content in tissue culturing seedlings and in botanical drug of Saussurea involucrata.</p><p><b>METHOD</b>The HPLC with Hydro-RP C18 (4.6 mm x 250 mm, 5 microm) column was used, a mixture of acetonitrile-water (5:95) was used as a mobile phase, with flow rate of 1 mL x min(-1), column temperature at 25 degrees C and detection wavelength at 220 nm.</p><p><b>RESULT</b>The effective constituents of tissue culturing seedlings were almost similar to the botanical drug. And syringin in tissue culturing seedlings was increased 4.35 times.</p><p><b>CONCLUSION</b>It has a good prospect to acquire high-quality S. involucrata by tissue culturing seedlings.</p>