Cynanchum Auriculatum Research Articles

Cytotoxic and apoptosis-inducing properties of a C21-steroidal glycoside isolated from the roots of Cynanchum auriculatum

The present study aimed to investigate the anti-cancer effect of a C21-steroidal glycoside (CG) isolated from the roots of Cynanchum auriculatum. CG was able to inhibit the growth of human cancer cells (SGC-7901 cells) in a concentration and time-dependent manner in vitro. SGC-7901 cells exposed to CG (10.8 and 21.6 μM) exhibited typical morphological apoptosis characteristics, such as nuclear-chromatin condensation and apoptotic body formation. Flow cytometric analysis showed that after treatment with CG at 10.8 and 21.6 μM for 24 h, the percentage of apoptotic cells increased to 30.4 and 43.2%, respectively, while the number of cells in the G0/G1, S and G2/M phases of the cell cycle decreased (P<0.05). Furthermore, treatment with CG at a concentration of 21.6 μM for 24 h significantly increased the expression of caspase-3 and the activity of caspase-3 was increased ∼3-fold in SGC-7901 cells. These results suggest that CG is the active anticancer component of the total C21-glycosides of the roots of Cynanchum auriculatum which is able to inhibit the growth of cancer cells and induce cancer cell apoptosis through caspase-3-dependent pathways.

The antidepressant effect of Cynanchum auriculatum in mice

Context: Antidepressant effects of various plants are generally attributed to their anti-inflammation and antioxidant activities. Cynanchum auriculatum Royle ex Wight (Asclepiadaceae) is a traditional medicinal plant in China and India used for immunological regulation, anti-inflammation, and antioxidant purposes. However knowledge about its antidepressant activity has been poorly investigated.Objective:To investigate the antidepressant activities of the total glycosides of C. auriculatum (TGC) and its CHCl3/MeOH (10:1) fractions (TGC-D and TGC-E) in mice.Materials and methods: TGC, TGC-D and TGC-E (20, 40 and 80 mg/kg) were intragastrically administered to mice twice a day for 5 days. The tail suspension test, forced swimming test, and locomotor activity test in mice were used to evaluate the effect of C. auriculatum. The inhibition of [3H]-serotonin reuptake in rat brain synaptosomes was detected to investigate their mechanism.Results:TGC, TGC-D and TGC-E (80 mg/kg) decreased the immobility time by 61.7, 64.5, and 61.9% in tail suspension test. TGC (80 mg/kg), TGC-D (80 mg/kg) and TGC-E (20 mg/kg) decreased the immobility time by 32.6, 47.3, and 48.7% in forced swimming test. TGC (80 mg/kg) and TGC-E (20 and 40 mg/kg) decreased the crossing distances by 28.8, 29.5, and 36.2% in locomotor activity test. TGC, TGC-D and TGC-E (10 mg/L) inhibited serotonin reuptake by 7.4, 4.5, and 71.1% in rat brain synaptosomes, and IC50 value of TGC-E was 5.2 mg/L.Discussion and Conclusion:TGC, TGC-D and TGC-E have potential antidepressant activities. The antidepressive effect of TGC-E maybe attributed partly by the inhibiting effect on serotonin reuptake.

Cynandione A mitigates ischemic injuries in rats with cerebral ischemia

Cynandione A, an acetophenone from the roots of Cynanchum auriculatum and other species in the genus attenuates neurotoxicity of a variety of neurotoxic agents such as l-glutamate in vitro. In this study, we sought to further characterize the neuroprotective effects of cynandione A and other acetophenones from the roots of C. auriculatum in pheochromocytoma tumor cell line PC12 and investigate whether cynandione A protected against ischemic injuries in rats with experimentally induced cerebral ischemia. Viability assays using the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophen-yl)-2H-tetrazolium monosodium salt method and lactate dehydrogenase (LDH) release assays showed that cynandione A dose-dependently attenuated glutamate-induced cytotoxicity. Comparative proteomic analysis by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight MS/MS of PC12 cells treated with cynandione A showed 10 μM cynandione A caused broad changes in protein expression in PC12 cells including down-regulation of high mobility group box 1 (HMGB1) and dihydropyrimidinase-like 2 (DPYSL2). Immunoblotting studies showed that 10 μM cynandione A aborted glutamate-induced increase in DPYSL2 and HMGB1 levels in PC12 cells and 30 mg/kg cynandione A also attenuated the rise in HMGB1 levels and mitigated DPYSL2 cleavage in brain tissues of rats with cerebral ischemia. Furthermore, rats with cerebral ischemia treated with 30 mg/kg cynandione A exhibited markedly improved neurological deficit scores at 24 and 72 h compared with control and a 7.2% reduction in cerebral infarction size at 72 h (p < 0.05 vs. control). Our findings demonstrated that cynandione A mitigated ischemic injuries and should be further explored as a neuroprotective agent for ischemic stroke.

Functional characteristics of starches from the root of Cynanchum auriculatum Royle ex Wight grown in China

Starches isolated from two types of Cynanchum auriculatum Royle ex Wight roots grown in China (BW 201001 and HSW 6-18) were investigated and compared to commercial potato starch. The protein, lipid, ash and amylose content of BW 201001 and HSW 6-18 starches were 0.12%, 0.15%, 0.16%, 17.48%, and 0.15%, 0.13%, 0.11%, 21.10%, respectively. The starch granules were irregular with a round or polygonal shape and a size ranging between 5 and 15 μm. Compared to HSW 6-18 starch, BW 201001 starch had a higher solubility, water binding capacity, average chain length, and proportion of B1 chains, and a lower swelling power, syneresis, and proportion of A chains. Both starches showed the characteristic B type X-ray pattern. The degree of crystallinity and R(1047/1022) of BW 201001 and HSW 6-18 were 43.03%, 0.50, and 38.78%, 0.42, respectively. Gelatinisation temperatures followed the order of BW 201001 > HSW 6-18. The peak viscosity, setback, and final viscosity of BW 201001 were lower than those of HSW 6-18 starch. Both starches exhibited lower RDS and higher RS, which was related to their crystal and molecular structures.

The study of planting area on components of Baishouwu anti-tumor effective fraction byRP-HPLC

Baishouwu glycosides (CGB) is an anti-tumor effective fraction that has been extracted and isolated from Cynanchum auriculatum Royle ex Wight. To study the differences on components of CGB from 3 main planting areas, we extracted materia medica, isolated and purified CGB from the extract. Physical and chemical identification of CGB indicated that components in CGB had 2-deoxysugar steroids. The results of TLC proved that CGB of different planting areas had similar physical and chemical characteristics. However, the analytical results of RP-HPLC showed that the constituents of CGB from Gansu differed entirely from CGB produced Jiangsu and Shandong. In vitro, MTT results showed that CGB from Gansu had a strong effect on cytotoxicity; however, CGB from Jiangsu and Shandong almost had no effect of cytotoxicity. Constitutes of CGB from Gansu differed entirely from CGB produced Jiangsu and Shandong, this might induce the effect of greatly varied cytotoxicity. Key words: Baishouwu glycosides (CGB), Cynanchum auriculatum Royle ex Wight., anti-tumor effective fraction, tin layer chromatography (TLC), Reversed-phase high-performance liquid chromatography (RP-HPLC), microculture tetrazolium (MTT).

Three New Steroidal Glycosides from the Roots of Cynanchum auriculatum

AbstractThree new C21 steroidal glycosides with a cinnamoyl group at C(12) and a 2‐methylbutanoyl group at C(20), and a straight sugar chain at C(3), namely cyanoauriculosides C–E (1–3, resp.), together with three known steroidal derivatives, were isolated from the roots of Cynanchum auriculatum (Asclepiadaceae). Their structures were determined by spectroscopic analyses and chemical methods. The known constituents were identified as wilfoside K1N (4), cynanauriculoside II (5), and auriculoside IV (6).

Inhibitive effect of C-21 steroidal glycosides of Cynanchum auriculatum on rat glioma cells in vitro

To evaluate the inhibitive effect of C-21 steroidal glycosides from the root of Cynanchum auriculatum (CGB) on rat glioma C6 cells. 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. 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). CGB can inhibit the growth of rat glioma C6 cells, and induce apoptosis and G0/G1 cell cycle arrest.

Cynanauriculoside C–E, three new antidepressant pregnane glycosides from Cynanchum auriculatum

Based on the bioactive screening results, three new pregnane glycosides named as cynanauriculoside C–E ( 1– 3), were isolated from the roots of Cynanchum auriculatum Royle ex Wight (Asclepiadaceae), together with two known ones, otophylloside L ( 4) and cynauricuoside C ( 5). On the basis of detailed spectroscopic analysis and chemical method, the structures of new compounds were characterized to be qingyangshengenin 3- O-β- d-oleandropyranosyl-(1 → 4)-β- d-cymaropyranoside ( 1), qingyangshengenin 3- O-β- d-glucopyranosyl-(1 → 4)-β- d-glucopyranosyl-(1 → 4) -α- l-cymaropyranosyl-(1 → 4)-β- d-oleandropyranosyl-(1 → 4)-β- d-cymaropyranoside ( 2) and caudatin 3- O-β- d-glucopyranosyl-(1 → 4)-β- d-glucopyranosyl-(1 → 4)-β- d-cymaropyranosyl-(1 → 4)-β- d-oleandropyranosyl-(1 → 4)-β- d-digitoxopyranoside ( 3). In the despair mice models, these pregnane glycosides showed significant antidepressant activity at the dosage of 50 mg/kg (i.g.). The most potent one was cyanauriculatoisde D ( 2), which was close to the positive control fluoxetine (20 mg/kg).

Three new steroidal glycosides from the roots of Cynanchum auriculatum.

Three new steroidal glycosides, cyanoauriculosides F, G and H (1-3), were isolated from the roots of Cynanchum auriculatum (Asclepiadaceae) along with two known steroidal derivatives. On the basis of spectroscopic analysis and chemical methods, their structures were identified as 20-O-acetyl-8,14-seco-penupogenin-8-one 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside (1), 2′,3′-Z-gagaminine 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaro-pyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside (2), 17-O-acetyl-kidjoranin 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-cymaro-pyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-digitoxopyranoside (3), gagaminine 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-digino-pyranosyl-(1→4)-β-D-cymaropyranoside (4) and wilfoside D1N (5).

큰조롱과 넓은잎 큰조롱에서 신종 포티바이러스(큰조롱모자이크바이러스)의 동정

Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea(Received on October 5, 2010; Accepted on October 27, 2010)In 2006 fall, a preliminary survey of viruses in two important medicinal plants, Cynanchum wilfordii and C.auriculatum, was conducted on the experimental fields at the Agricultural Research and Extension Services ofChungbuk province in Korea. On each experimental fields, percentage of virus infection was ranged from 20to 80%, and especially an average of disease incidence propagated by roots was twice higher than that byseeds. The various symptoms were observed in Cynanchum spp. plants, such as mosaic, mottle, necrosis,yellowing, chlorotic spot and malformation etc. In electron microscopic examination of crude sap extracts,filamentous rod particles with 390-730 nm were observed in most samples. The virus particles were purifiedfrom the leaves of C. wilfordii with typical mosaic symptom, and the viral RNA was extracted from thissample containing 430-845 nm long filamentous rod. To identify the viruses, reverse transcription followed byPCR with random primers was carried out. The putative sequences of P3 and coat protein of potyvirus wereobtained. From a BLAST of the two sequences, they showed 26-38% and 62-72% identities to potyviruses,respectively. In SDS-PAGE analysis, the subunit of coat protein was approximately 30.3 kDa, close to the coatprotein of potyvirus. In bioassay with 21 species in 7 families, Chenopodium quinoa showed local lesion oninoculated leave and chlorotic spot on upper leave, but the others were not infected. RT-PCR detection usingspecific primer of C. wilfordii and C. auriculatum samples, all of 24 samples with virus symptom was positive,and five out of seven samples without virus symptom were also positive. On the basis of these data, the viruscould be considered as a new member of potyvirus. We suggested that the name of the virus was Keunjorongmosaic virus (KjMV) after the common Korean name of C. wilfordii.Keywords : Cynanchum wilfordii, Cynanchum auriculatum, Keunjorong mosaic virus, Medicinal plants,Virus disease

Caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside 1 induced apoptosis through caspase 3‐dependent pathway in human hepatoma cell line SMMC7721

Caudatin-2,6-dideoxy-3-O-methy-β-D-cymaropyranoside (CDMC), the C-21 steroidal glycoside recently extracted from the traditional Chinese medicinal plant, the root of Cynanchum auriculatum Royle ex Wight (Asclepiadaceae), has been shown to possess potent antitumor properties. However, the bioactivities of CDMC are still largely unknown, especially the antitumor effect and its mechanism. This study investigated the CDMC antitumor effects on human hepatoma cell line SMMC7721 cells by analysis of cell viability, cell cycle phases and apoptosis. The results showed that CDMC inhibited the growth of SMMC7721 cells in a time- and dose-dependent manner and resulted in cell cycle arrest in G(0)/G(1) phase. Furthermore, CDMC induced SMMC7721 cell apoptosis rather than necrosis through caspase 3 activation, and a caspase 3 inhibitor, Ac-DEVD-CHO, could attenuate the apoptosis induced by CDMC. The results suggested that the anticancer activity of CDMC could be attributed partially to its inhibition of cell proliferation and induction of apoptosis associated with caspase 3 activation.

Two new steroidal glycosides from the root of Cynanchum auriculatum

Abstract Two new steroidal glycosides named cyanoauriculoside A (1) and B (2), have been isolated from the roots of Cynanchum auriculatum Royle ex Wight. The structure of cyanoauriculoside A (1) was determined to be 20-O-acetyl-penupogenin-3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopy-ranosyl-(1→4)-β-D-cymaropyranoside. Cyanoauriculoside B (2) was characterized as 20-O-acetylpenupogenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside. The structures of these compounds were elucidated based on the chemical and spectroscopic evidence.

Antitumor effects by Wilfoside C3N treatment in ECA109 cells

C21 steroidal glycoside (C21) is one of the most bioactive compounds of Cynanchum auriculatum Royle, known as Baishouwu, and possesses potent antitumor activity. Wilfoside C3N is one of the two most abundant and active C21 in it. The aim of this study was to further investigate the antitumor activity of C3N and to clarify its signaling pathway. The growth inhibition ofECA109 cells induced by C3N was assessed. Western blot analysis, reverse-transcription PCR, caspase-2 and Fas activity assay, and knockdown of caspase-2 with siRNA were used to study the apoptotic mechanisms. We showed that C3N inhibited the proliferation of ECA109 cells moderately in a dose and time-dependent manner and induced apoptosis in the ECA109 cell line through a mitochondrial pathway by triggered cytochrome c release from the mitochondria, with caspase-2 functioning upstream of caspase-9 rather than association with Fas and caspase-8. Furthermore, C3N-driven apoptotic events were associated with downregulation of Bcl-2. These results suggest that C3N-induced apoptosis of ECA109 cells in vitro was dependent on caspase-2 or mitochondria or caspase-9 and independent of the Fas-FasL or caspase-8 pathway.

Rapid molecular authentication of three medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum (Fallopia multiflorum), by the development of RAPD-derived SCAR markers and multiplex-PCR

Definitive identification of original plant species is important for standardizing herbal medicine. The herbal medicines Cynanchi Wilfordii Radix (Baekshuoh in Korean and Beishuwu in Chinese) and Polygoni Multiflori Radix (Hashuoh in Korean and Heshuwu in Chinese) are often misidentified in the Korean herbal market due to morphological similarities and similar names. Therefore, we developed a reliable molecular marker for the identification of Cynanchi Wilfordii Radix and Polygoni Multiflori Radix. We used random amplified polymorphic DNA (RAPD) analysis of three plant species, Polygoni multiflorum, Cynanchum wilfordii, and Cynanchum auriculatum, to obtain several species-specific RAPD amplicons. From nucleotide sequences of these RAPD amplicons, we developed six sequence characterized amplification region (SCAR) markers for distinguishing Polygoni Multiflori Radix and Cynanchi Wilfordii Radix. Furthermore, we established SCAR markers for the simultaneous discrimination of the three species within a single reaction by using multiplex-PCR. These SCAR markers can be used for efficient and rapid authentication of these closely related species, and will be useful for preventing the distribution of adulterants.

Structural characterization of pregnane glycosides from Cynanchum auriculatum by liquid chromatography on a hybrid ion trap time‐of‐flight mass spectrometer

A method coupling high-performance liquid chromatography with hybrid ion trap time-of-flight mass spectrometry (TOFMS) using an electrospray ionization source was firstly used to characterize ten major pregnane glycosides including one novel compound auriculoside IV from the roots of Cynanchum auriculatum Royle ex Wight. In the MS/MS spectra, fragmentation reactions of the [M+Na]+ were recorded to provide abundant structural information on the aglycone and glycosyl moieties. Experiments using TOFMS allowed us to obtain precise elemental compositions of molecular ions and subsequent product ions with errors less than 6 ppm. The pregnane glycosides in C. auriculatum were classified into two major core groups: one is caudatin characterized by the neutral loss of one ikemamic acid molecule (128 Da) from the precursor ion, and the other is kidjoranin characterized by the neutral loss of cinnamic acid (148 Da) from the precursor ion. Meanwhile, a series of sugar-chain fragment ions provided valuable information about the compositions of the sugar residues and the sequences of the sugar chain. Logical fragmentation pathways for pregnane glycosides have been proposed and are useful for the identification of these compounds in natural products especially when there are no reference compounds available.

Separation and Purification of Baishouwubenzophenone, 4-Hydroxyacetophenone and 2,4-Dihydroxyacetophenone from Cynanchum auriculatum Royle ex Wight by HSCCC

A preparative high-speed counter-current chromatography method for isolation and purification of three acetophenones, baishouwubenzophenone, 4-hydroxyacetophenone and 2,4-dihydroxyacetophenone from the Chinese medicinal plant Cynanchum auriculatum Royle ex Wight was successfully established. With a two-phase solvent system composed of light petroleum (b.p. 60–90 °C)–ethyl acetate–methanol–water (4:9:6:6, v/v), about 20.2 mg compound 1, 35.0 mg compound 2 and 8.3 mg compound 3, each at over 95% purity as determined by LC, were obtained in one-step elution from 400 mg of the ethanol extract. The structures of these compounds were identified by UV, IR, ESI-MS, 1H NMR and 13C NMR spectroscopy. Among them, compounds 2,4-dihydroxyacetophenone and 4-hydroxyacetophenone were obtained from C. auriculatum for the first time.

Molecular Authentication of the Traditional Medicinal PlantFallopia multiflora

The root of Fallopia multiflora is one of the most widely used traditional Chinese medicines. However, it is often confused and substituted with the roots of F. multiflora var. ciliinervis, Pteroxygonum giraldii, Cynanchum auriculatum, and Stephania cepharantha. To establish a DNA polymorphism-based assay for the identification of F. multiflora, the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) regions of six Fallopia species were sequenced and analyzed. Based on the diversity of ITS regions among the species the diagnostic primers PMITS28 and PMITS545, which amplified an expected 517-bp DNA fragment from F. multiflora DNA, were designed. No amplified product was observed when DNA from other species was used. This method can be used for the authentication of F. multiflora.

Antitumor activity of C-21 steroidal glycosides from Cynanchum auriculatum Royle ex Wight

The antitumor activities of six C-21 steroidal glycosides isolated from the root tuber of Cynanchum auriculatum Royle ex Wight were performed according to a microculture tetrazolium (MTT) method on human tumor cell lines SMMC-7721, MCF-7 and Hela. Of these compounds, caudatin-2,6-dideoxy-3-O-methy-β-D-cymaropyranoside and caudatin were found to be of the highest effects against human tumor cell line SMMC-7721 with IC(50) values of 13.49 and 24.95 μM, respectively. Then the in vivo assay further showed that caudatin-2,6-dideoxy-3-O-methy-β-D-cymaropyranoside and caudatin significantly inhibited the growth of transplantable H(22) tumors in mice.

Two New Cytotoxic Pregnane Glycosides from Cynanchum auriculatum

Two new pregnane glycosides, kidjoranin 3- O- alpha-diginopyranosyl-(1-->4)- beta-cymaropyranoside (1) and kidjoranin 3-O-beta-digitoxopyranoside (2), together with one known compound caudatin 3 -O-beta-cymaropyranoside (3), were isolated from the roots of Cynanchum auriculatum. Their structures were established on the basis of NMR analyses. Compounds 1 - 3 were tested for their in vitro inhibitory activity against the growth of human tumor cell lines SMMC-7721, HeLa and MCF7; all of them displayed marked cytotoxic activities against cells SMMC-7721 and HeLa with IC (50) values ranging from 8.6 microM to 58.5 microM, yet no activity against the cell line MCF7 was detected.

Cytotoxic and Apoptosis‐Inducing Properties of Auriculoside A in Tumor Cells

The C21-steroidal glycoside auriculoside A (1), recently isolated from the roots of Cynanchum auriculatum, was found to inhibit the growth of several human tumor cell lines and to induce apoptosis in human breast cancer (MCF-7) cells. Compound 1 was evaluated for its in vitro cytotoxicity against MCF-7, HO-8910, and Bel-7402 cells, and for its in vivo antitumor effects on implanted sarcoma-180 (S180) tumors in mice. It showed significant, concentration-dependent inhibition of the cancer cells, both in vitro and in vivo. MCF-7 Cells exposed to 1 displayed typical morphological apoptosis characteristics such as cytoplasm contraction and nuclear-chromatin condensation. Flow-cytometric analysis showed that the MCF-7 cell cycle was arrested at the G0/G1 phase. When treated with 40 microg/ml of 1 for 24, 48, and 72 h, respectively, the apoptotic rates of the cells were ca. 5, 8, and 18.5%, respectively.