Identification,biological characteristics and fungicide screening of pathogen of southern blight in Cynanchum stauntonii / 中国中药杂志

During the high-temperature and rainy season from June to October in 2017-2019,serious southern blight broke out in the Cynanchum stauntonii planting area in Tuanfeng county,Hubei province,which had a great impact on the yield and quality of medicinal materials. In this study,the pathogen of C. stauntonii was isolated,purified,and identified,and the pathogenicity was tested according to Koch's postulates. Meanwhile,the biological characteristics of the pathogen were analyzed. On this basis,the effective fungicides were screened in laboratory. Finally,the pathogen( BQ-1) was identified as Athelia rolfsii( Deuteromycotina,Basidiomycota,anamorph: Sclerotium rolfsii). The optimum growth conditions for BQ-1 were 25-30 ℃,p H 5-8,and alternating light and dark.The effective chemical fungicides were lime-sulphur-synthelic-solution( LSSS) and flusilazole,and the effective botanical fungicide was osthole. BQ-1 was highly homologous to the pathogen HS-1 of peanut southern blight,with the similarity of 18 S r DNA and TEF sequences at 99. 09%. The southern blight in C. stauntonii might be resulted from that in peanut. In the production of C. stauntonii,the following measures should be taken: avoiding rotation or neighboring with peanut,draining water from June to October to reduce humidity,and reasonably applying fungicides.

Effect of C21 steroidal glycoside of Cynanchum auriculatum on liver and kidney fibrosis through TLR4 pathway / 中国中药杂志

The liver and kidney fibrosis model was established by thioacetamide(TAA) and unilateral ureteral obstruction(UUO) in SD rats. The rats were randomly divided into three groups: model group, low and high-dose groups of C21 steroidal glycosides of Cynanchum auriculatum. Another blank control group was set. Four weeks later, serum was taken to detect the biochemical indexes of liver and kidney function. Urine protein and urine creatinine were detected by kits. Liver and kidney tissue samples were stained with HE and Masson staining, and hydroxyproline content was detected. Western blot was used to detect expressions of fibrotic proteins, inflammatory factors and TLR4 signaling pathways, so as to observe the preventive and therapeutic effects of C21 steroidal glycosides from C. auriculatum on hepatic and renal fibrosis and explore its molecular mechanism. Four weeks later, serum biochemical results showed that liver and kidney functions were seriously damaged, and pathological sections showed that inflammatory cell infiltration, decrease of parenchymal cells, and increase of interstitial fibrosis in liver and kidney tissues. The results showed that low and high doses(150, 300 mg·kg~(-1)) of C21 steroidal glycosides could significantly reduce the collagen deposition and the pathological changes of liver and kidney fibrosis compared with the model group. At the same time, we found that the expression levels of TLR4 and MyD88 signaling pathway proteins were significantly increased in the liver and kidney tissues of the model group, and a large number of NF-κB signaling pathway proteins migrated into the nucleus. On the contrary, the expression levels of TLR4, MyD88 signaling pathway proteins and the nuclear migration of NF-κB were significantly inhibited in the low and high dose groups of C21 steroidal glycosides from C. auriculatum. Therefore, it was speculated that the mechanism of C21 steroidal glycoside for preventive and therapeutic effect on hepatic and renal fibrosis was related to inhibit TLR4/MYD88/NF-κB inflammatory pathway, thus preventing hepatic and renal fibrosis.

Effects of total C-21 steroidal glucosides from Cynanchum auriculatum on oxidative stress pathway in mice with liver injury / 中国中药杂志

The study aimed to investigate the mechanism of hepatoprotective effect of C-21 steroidal glucosides from Cynanchum auriculatum( Baishouwu) on oxidative stress in mice with liver injury. Mice were randomly divided into normal group,model group,positive control group,Baishouwu high group and Baishouwu low group. The liver injury model was induced by intraperitoneal injection of CCl4 peanut oil solution. All mice were sacrificed to collect blood and liver specimens. The activities of serum levels of ALT and AST were detected. The content of MDA and the activity of SOD in liver homogenate were examined by colorimetry method. Tissues were stained with hematoxylin-eosin for histological examination. The hepatic protein expressions of NF-κB p65,p-IκBα,i NOS and COX-2 were detected by Western blot. The mRNA expressions of TNF-α and IL-6 were determined by RT-PCR. It was found that treatment with C-21 steroidal glucosides from Baishouwu successfully attenuated liver injury induced by CCl4,as shown by decreased levels of serum biochemical indicators( AST,ALT)( P<0. 01). Administration of total C-21 steroidal glucosides enhanced the activity of SOD( P<0. 01) and decreased the content of MDA( P<0. 01) in liver homogenate. Microscopic features suggested that treatment with C-21 steroidal glucosides from Baishouwu was effective in inhibiting CCl4-induced hepatocyte edema and degeneration. Further studies showed that NF-κB p65 overexpression induced by CCl4 was decreased by C-21 steroidal glucosides,leading to the markedly down-regulated protein expression levels of p-IκBα,i NOS and COX-2,as well as the depression of TNF-α and IL-6 mRNA expressions. In conclusion,total C-21 steroidal glucosides from Baishouwu exhibited potent effect on oxidative stress pathway in mice with liver injury induced by CCl4,with enhanced activity of SOD,decreased content of MDA,and down-regulated levels of NF-κB p65,p-IκBα,i NOS and COX-2.

Two new C steroidal glycosides isolated from Cynanchum komarovii / 中国天然药物

The present study was designed to further investigate the C steroidal glycosides in Cynanchum plants. Two new steroidal glycosides based on a 13, 14:14, 15-disecopregnane-type aglycone, komaroside P (1) and komaroside Q (2), together with three known compounds (3-5) were isolated from the whole herbs of Cynanchum komarovii. The aglycones of compounds 1 and 2 were two new disecopregnane. Their structures were elucidated on the basis of 1D, 2D NMR spectroscopic data and acid hydrolysis. All the compounds (1-5) showed potent inhibitory activities against human leukemia cell lines (HL-60) with IC values ranging from 16.6 to 26.3 μmol·L, compared to the positive control 5-fluorouracil (6.4 μmol·L).

Two new C steroidal glycosides isolated from Cynanchum komarovii / 中国天然药物

The present study was designed to further investigate the C steroidal glycosides in Cynanchum plants. Two new steroidal glycosides based on a 13, 14:14, 15-disecopregnane-type aglycone, komaroside P (1) and komaroside Q (2), together with three known compounds (3-5) were isolated from the whole herbs of Cynanchum komarovii. The aglycones of compounds 1 and 2 were two new disecopregnane. Their structures were elucidated on the basis of 1D, 2D NMR spectroscopic data and acid hydrolysis. All the compounds (1-5) showed potent inhibitory activities against human leukemia cell lines (HL-60) with IC values ranging from 16.6 to 26.3 μmol·L, compared to the positive control 5-fluorouracil (6.4 μmol·L).

Recent advances in phytochemistry and pharmacology of C21 steroid constituents from Cynanchum plants / 中国天然药物

Cynanchum is one of the most important genera in Asclepiadaceae family, which has long been known for its therapeutic effects. In this genus, 16 species are of high medicinal value. The extracts of the root and/or rhizome parts have been applied in traditional Chinese medicines (TCM) for the prevention and treatment of various illnesses for centuries. C21 steroids, as the typical constituents of Cynanchum species, possess a variety of structures and pharmacological activities. This review summarizes the comprehensive information on phytochemistry and pharmacology of C21 steroid constituents from Cynanchum plants, based on reports published between 2007 and 2015. Our aim is to provide a rationale for their therapeutic application, and to discuss the future trends in research and development of these compounds. A total of 172 newly identified compounds are reviewed according to their structural classifications. Their in vitro and in vivo pharmacological studies are also reviewed and discussed, focusing on antitumor, antidepressant, antifungal, antitaging, Na(+)/K(+)-ATPase inhibitory, appetite suppressing and antiviral activities. Future research efforts should concentrate on in vitro and in vivo biological studies and structure activity relationship of various C21 steroid constituents.

C21 steroids from roots of Cynanchum otophyllum / 中国中药杂志

Eleven C21 steroids were isolated from chloroform extract of roots of Cynanchum otophyllumby silica gel, MCI, ODS columns, and semi-preparative HPLC. Their structures were determined by spectroscopic data analysis as otophylloside B(1), caudatin-3-O-beta-D-cymaropyranosyl-(1-->4)-beta-D-oleandropyranosyl-(1-->4)-beta-D-cymaropyranosyl-(1-->4)-beta-D-cymaropyranoside (2), caudatin-3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-oleandropyranosyl-(1-->4)-beta-D-cymaropyranosyl-(1-->4)-beta-D-cymaropyranoside (3), caudatin-3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-digitoxopyranosyl-(1-->4)-beta-D-cymaropyranoside (4), otophylloside O (5), gagamine-3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-cymaropyranosyl-(1-->4)-beta-D-cymaropyranoside (6), sinomarinoside B (7), mucronatosides C (8), wallicoside J (9), stephanoside H (10), and qinyangshengenin-3-O-beta-D-oleandropyranosyl-(1-->4)-beta-D-cymaropyranosyl-(1-->4)-beta-D-digitoxopyranoside (11). Among them, compounds 2-3, and 6-11 were separated from the roots of this plant for the first time.

Separation and identification of endophytic fungi from desert plant Cynanchum komarovii / 中国中药杂志

<p><b>OBJECTIVE</b>The research aimed to investigate the entophytic fungal community of Cynanchum Komarrovii, including the biodiversity in different organs and the correlations with ecological environment. Endophytic fungi with patent bioactivity were also rapidly screened.</p><p><b>METHOD</b>PDA medium was used to isolate and purify the endophytic fungi from C. komarovii living in Shaanxi and Ningxia district, respectively. The strains were identified based on the morphological characteristics of the fungi and similarity of 5.8S gene and internal transcribed spacer (ITS) sequence. Pyriculaia oryzae model was applied to preliminarily screen the active fungi.</p><p><b>RESULT</b>Ninety-four strains of endophytic fungi were isolated and identified to 9 species, 13 genera, 9 families and 6 orders, among them, 47 strains were from the plants living in Ningxia. And then, 5 of them were isolated from roots, 14 from branches, and 28 from leaves. They were identified belonging to 8 species, 9 genera, 5 families and 4 orders. Additionally, 47 strains were from the plants living in Shaanxi. 16 were isolated from the roots, 18 from branches, 13 from leaves. They were identified belonging to 5 species, 8 genera, 6 families and 4 orders. By preliminary screening, 18 strains of endophytes completely inhibited the germination of conidium, which showed a potential bioactivity for these fungi. Both N4 and S17 strains had stronger growth inhibition effect.</p><p><b>CONCLUSION</b>Endophytic fungi from desert plant C. komarovii have the feature of diversity. Different geographical environment and type of organizations lead to the significant difference on the quantity and the species composition. Most of fungi in Ningxia C. komarovii distribute in leaves. However, most of those in Shaanxi C. komarovii distribute in stems and leaves. It also indicated that endophytes from C. komarovii had a strong antifungal activity.</p>

Anti-HIV activity and mechanism of Cynanchum otophyllum glucan sulfate in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To study anti-HIV activity and mechanism of Cynanchum otophyllum glucan sulfate in vitro.</p><p><b>METHOD</b>Anti-HIV-1 activity was detected with syncytial formation assay and quantitative P24 enzyme-linked immunosorbent assay (ELISA); cytotoxicity was tested with MTT colorimetric assay. Antiviral mechanism was investigated by fusion inhibition, time of addition and pre-treatment experiments.</p><p><b>RESULT</b>The 50% inhibition concentrations (IC50) of PS20 for HIV-1(IIIB), HIV-1(Ada-M), and HIV-1(Bal), were 0.26, 0.46, 0.90 micromol x L(-1), respectively. Studies on antiviral mechanism of PS20 showed that target molecule may be viral envelope protein.</p><p><b>CONCLUSION</b>The results suggested that PS20 had high anti-HIV activity and was worth to be studied further.</p>

Optimization of the hydrolysis process for C21 steroidal glycoside of bai shou wu by acetic acid with multi-target orthogonal design / 中国中药杂志

<p><b>OBJECTIVE</b>To study the optimizal hydrolysis process for C21 steroidal glycoside of Bai Shou Wu by acetic acid.</p><p><b>METHOD</b>The effects of acetic acid concentration, reaction temperature and reaction time had been investigated using orthogonal design and the contents of kidjoranin 3-O-beta-digitoxopyranoside, caudatin, kidjoranin 3-O-alpha-L-diginopyranosyl-(1 --> 4)-beta-cymaropyranoside and caudatin 3-O-beta-cymaropyranoside as response indexs were determined by the high performance liquid chromatography.</p><p><b>RESULT</b>The factors influencing acetic extraction efficiency were as follows: A > B > C (A. reaction temperature; B. reaction time; C. acetic acid concentration). The optimal hydrolysis condition obtained was: refluxing for 6 hours with 5.0% dilute CH3COOH solution at 100 degrees C.</p><p><b>CONCLUSION</b>The content of antitumor active ingredients under the optimum hydrolysis condition is raised obviously and has a great part in studying this antitumor drug.</p>

Inhibitive effect of C-21 steroidal glycosides of Cynanchum auriculatum on rat glioma cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the inhibitive effect of C-21 steroidal glycosides from the root of Cynanchum auriculatum (CGB) on rat glioma C6 cells.</p><p><b>METHODS</b>C6 cells were treated with CGB for 24, 48,72 h at concentration of 30, 60, 120 mg/L, respectively. MTT assay was used for evaluating cell viability; fluorescence-activated cell sorting analysis after Annexin V/propidium iodide staining or single propidium iodide staining was used to test cell apoptosis and cell cycle.</p><p><b>RESULTS</b>CGB at 30, 60, 120 mg/L concentration-dependently decreased C6 cell viability (P<0.001). CGB at 60 and 120 mg/L induced C6 cell apoptosis and cell cycle arrest. The fraction of G0/G1 cells was increased (P<0.05) and that of S phase cells was decreased (P<0.01).</p><p><b>CONCLUSION</b>CGB can inhibit the growth of rat glioma C6 cells, and induce apoptosis and G0/G1 cell cycle arrest.</p>

Sequence analysis of rDNA-ITS of Bai Shouwu from different species / 中国中药杂志

<p><b>OBJECTIVE</b>To study the genetic diversity of rDNA ITS sequences in different species of Bai Shouwu, utilize the molecular diversity of ITS sequences to authenticate the different species of Bai Shouwu.</p><p><b>METHOD</b>Firstly, total DNA was extracted from the different species of Bai Shouwu. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and sequenced after cloning and purification.</p><p><b>RESULT</b>From four species the complete sequence of ITS and 5.8 S rDNA, the partial sequences of 18S rDNA and 26S rDNA were obtained. The rDNA ITS sequences of Cynanchum bungei (sign in No. GU198970 and No. GU479037) were obtained. Ten variable sites among the sequences were found.</p><p><b>CONCLUSION</b>ITS sequence could be used to authenticate the species. The method could be used to identify germplasm resources and authenticate.</p>

Effects of general glycosides in Cynanchum auriculatum of Jiangsu province on liver fibrosis of rats / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of general glycosides from Cynanchun auriculatum of Jiangsu on liver fibrosis of rats.</p><p><b>METHOD</b>Seventy-two Wistar rats were randomly divided into normal group, model group, BSW three doses treated group and hydrocortisone treated group. CCl4 (50%, 2 mL x kg(-1)) was orally administraeated twice a week for 8 weeks. The liver and spleen indices were observed. The level of serum GPT, GOT, and HA, PCIII and the level of SOD, HyP, MDA in liver homogenates was also measured. The histopathologic change in hepatic of rats was examined.</p><p><b>RESULT</b>The elevation of serum GPT, GOT, HA, PCIll, MDA and HyP and the content of liver homogenates were attanuated remarkably by BSW treatment. BSW groups also increased the level of SOD of liver homogenates, and make the fibrotic liver better.</p><p><b>CONCLUSION</b>The general glycosides in C. auriculatum of jiangsu province have an anti-hepatic fibrosis ettect on CCl4-induced fibrosis rats, the mechanisms might be associated with its anti-oxidative action.</p>

Effects of three C21 steroidal saponins from Cynanchum auriculatum on cell growth and cell cycle of human lung cancer A549 cells / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the inhibition of three C2 steroidal saponins from Cynanchum auriculatum on the cell growth and cell cycle of human lung cancer A549 cells.</p><p><b>METHOD</b>A549 cells were exposed to three C21 steroidal saponins of different concentrations (5, 10, 20, 40, 60, 80, 100 micromol L(-1)) for 48 hours. After 48 h, MTT assay was used to evaluate the inhibiting effects of three C21 steroidal saponins on the proliferation of the A549 cells. Exponential growth phase A549 cells were treated with 47, 34, 60 micromol x L(-1) of three C21 steroidal saponins respectively for 12, 24, 48 h, then the changes of cell cycle were analyzed by flow cytometry.</p><p><b>RESULT</b>The three C21 steroidal saponins could inhibit the growth of A549 cells in dose-dependent manner and IC50 is 46. 07 mol x L(-1), 33.02 mol x L(-1), 59.92 mol x L(-1) respectively. The cell cycle analysis showed that wilfoside C1N and wilfoside C3N increased the percentage of G0/G1 phase cells and decreased G2/M and S-phase cells while the proportion of cells in S-phase was lower and in G2/M phase the proportion was higher than control when the cells were cultivated with wilfoside K1N (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>Three C21 steroidal saponins could inhibit the proliferation of A549 cells in dose-dependent manner and the mechanism may be related to its arresting the cell cycle.</p>

Study on C21 steroidal glycosides of Cynanchum atratum by LC-MS / 中国中药杂志

High performance liquid chromatography coupled with on-line electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) was used to identify C21 steroidal glycosides in the roots of Cynanchum atratum. The structures of C21 steroidal glycosides were deduced from mass fragments features in positive and negative mode. The constituents of C. atratum were separated and detected. 7 compounds were identified by comparing their ESI-MS/MS data with the reference compounds and 2 compounds were inferred solely by the ESI-MS/MS data. The method is sensitive, and provides good separation and rapid qualitative characterization of C21 steroidal glycosides in the roots of C. atratum.

Enrichment of saponins in Radix et Rhizoma Cynanchi Atrati with macroporous resin / 中国中药杂志

<p><b>OBJECTIVE</b>To develop an approach to the determination of saponins in Radix Cynanchi Atrati, and to optimize the parameters for purified the preparation of total saponins by macroporous resin column chromatography.</p><p><b>METHOD</b>Using cynanversicoside A as a reference, the determination of saponins was performed; according to the elution rate and the purity of the products, the preparation performance of total saponins by macroporous resin was investigated, and its parameters were optimized.</p><p><b>RESULT</b>The saponins in Radix Cynanchi Atrati were successfully determined at 518 nm by vanillin-perchloric acid as spray reagent. The macroporous resin HP-20 showed static absorption ratio of 59. 3 mg x g(-1); the 70% ethanol extraction of Radix Cynanchi Atrati was eluted from column of macroporous resin HP-20 by water and 30% ethanol, and the saponins were concentrated in 90% ethanol solution. The content of saponin part eluted from HP-20 column was 77.62%.</p><p><b>CONCLUSION</b>The proposed approach allows convenient and efficient preparation and purification of saponin in Radix Cynanchi Atrati.</p>

Effect of C21 steroidal glycoside from root of Cynanchum auriculatum on D-galactose induced aging model mice / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of C21 steroidal glycoside (CSG) from the root of Cynanchum auriculatum from Jiangsu on D-galactose (D-gal) induced aging model mice.</p><p><b>METHOD</b>D-gal aging mouse model was established by cervicodorsal region subcutaneous injection with D-gal once a day for eight successive weeks. The mice in the normal control group (NCG, non-modeled) and the model control group (MCG, modeled but untreated) were treated with 1% CMC-Na. The model mice in the low, middle and high-dose CSG and Vitamin E treated groups were treated with a dose of (10, 20, 40, 100 mg x kg(-1) per day, respectively. The SOD activity, MDA content and telomerase activity in serum, heart, liver and brain tissues of mice were measured.</p><p><b>RESULT</b>CSG could obviously increase the SOD activity and decrease the MDA level in serum, heart, liver and brain tissues in D-gal aging mice (P < 0.01). There was no significant difference between three CSG treated groups and Vitamin E treated groups. In comparison of telomerase activity between MCG and the treated groups, it was shown that there was a significant increase in serum in middle and high dose group, and in heart tissues in CSG and Vit E treated groups, but was not in liver and brain tissue.</p><p><b>CONCLUSION</b>This study demonstrates that CSG can antagonize free radical injury, increase the SOD activity and decrease the MDA content of serum, heart, liver and brain in D-gal aging mice, and increase the telomerase activity in serum and heart tissues but not in liver and brain tissue.</p>

Studies on chemical constituents from root of Cynanchum atratum / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents from the roots and rhizome of Cynanchum atratum.</p><p><b>METHOD</b>The chemical constituents were isolated and repeatedly purified by silica gel chromatography and the structures were elucidated by the NMR spectra and physicochemical properties.</p><p><b>RESULT</b>Twelve compounds were obtained and nine of them were identified as 2, 4-dihyroxyacetophe none (1), 2, 6-dihyroxyacetophenone (2), 4-hydroxybenzenemethanol (3), benzoic acid (4), beta-amyrin acetate (5), palmitic acid (6), beta-sitosterol (7), beta-daucosterol (8), glaucogenin C-3-O-alpha-D-oleandropyranosyl-(1 --> 4)-beta-D-digitoxopyranosyl-(1 -->4)-alpha-D-oleandropyranoside (9).</p><p><b>CONCLUSION</b>Seven compounds were obtained from C. atratum for the first time.</p>

Studies on chemical constituents of Cynanchum forrestii / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of Cynanchum forrestii.</p><p><b>METHOD</b>Chromatographic techniques were applied to isolated chemical constituents. The structures were identified on the basis of physico-chemical constants and spectroscopic data.</p><p><b>RESULT</b>Eight compounds were isolated from the 95% ethanol extract of the roots of C. forrestii and elucidated as ( + ) -5'-methoxyisolariciresinol 3a-O-beta-D-glucopyranoside (1), hexahydroxycholest-7-en-6-one (2), tylophorinidine (3), sucrose (4), palmitic acid (5), beta-sitosterol (6), daucosterol (7), nonanedioic acid (8).</p><p><b>CONCLUSION</b>Compounds 1-3 from this genus, and compounds 4-8 from the plant were obtained for the first time.</p>

Hypnotic effect of Chinese materia medica Cynanchum chinese in mice / 中国中药杂志

<p><b>OBJECTIVE</b>To study the central pharmacological effect of the water and chloroform-extract compounds from C. chinese in mice.</p><p><b>METHOD</b>The independent activity test and the hypnotic synergism test by sub-threshold hypnotic dosage of pentobarbital were employed to evaluate the central pharmacological effect of the extract-compounds, and the minimal neurotoxicity was evaluated by the rotorod test.</p><p><b>RESULT</b>the extract-compounds exhibited significant dose-related inhibition effect of the spontaneous motor activity in mice after intraperitoneal administration. And the two extract-compounds promoted the hypnotic effect by sub-threshold hypnotic dosage administration of pentobarbital, and produced ED50 value of 2.36 g kg (-1) and 0.75 g kg(-1), respectively. Also, both extract-compounds showed no neurotoxicity in the experiment.</p><p><b>CONCLUSION</b>The extract compounds from C. chinese showed inhibitional effect on CNS.</p>