Five New C21 -Steroidal Sapogenins from the Acid Hydrolysate of Cynanchum otophyllum Roots.

Five new C21 -steroidal sapogenins (1-5) named cynotogenins J-N, were isolated from the acid hydrolysate of Cynanchum otophyllum roots. Their structures were established by extensive spectroscopic analysis (UV, IR, HR-ESI-MS, and NMR). Most notably, compounds 1-3 harboring a rare 5ß,6ß-epoxy group in the C21 -steroidal skeleton of Cynanchum plants. All compounds were evaluated for their cytotoxicities against multiple cancer cell lines, in which compounds 5 showed weak cytotoxicity against HepG2 cancer cells with IC50 values of 44.90 µM.

Cynanotophyllosides E-F, two minor pregnane glycosides from the roots of cultivated Cynanchum otophyllum.

Cynanotophyllosides E-F, two new minor pregnane glycosides were isolated from the antidepressant active fraction of cultivated Cynanchum otophyllum, and their structures were determined as 12-O-vanilloyl-deacetylmetaplexigenin 3-O-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-digitoxopyranoside, and 12-O-nicotinoyl-deacetylmetaplexigenin 3-O-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranoside respectively, with the combination of spectroscopic and chemical analysis.

New Immunomodulating Polyhydroxypregnane Glycosides from the Roots of Cynanchum otophyllum C.K.Schneid.

Seven new polyhydroxypregnane glycosides, named cynotophyllosides P-V, together with three known analogs were isolated from the roots of Cynanchum otophyllum C.K.Schneid. Their structures were elucidated by a variety of spectroscopic techniques, as well as acid-catalyzed hydrolysis. All isolates were tested for their immunological activities in vitro against Con A- and LPS-induced proliferation of mice splenocytes. Immunoenhancing (for 1, 9) and immunosuppressive (for 2) activities were observed. Furthermore, cynotophylloside R (3) showed immunomodulatory as it enhanced the proliferation of splenocytes in low concentration and suppressed immune cells in concentration more than 1.0 µg/ml.

C21 steroidal glycosides with cytotoxic activities from Cynanchum otophyllum.

Eight new C21 steroidal glycosides, namely cynanotins A-H (1-8), together with fifteen known analogues, were isolated from the roots of Cynanchum otophyllum. Their structures were elucidated by spectroscopic analysis and chemical methods. In this study, all of isolates were tested for their vitro inhibitory activities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480). Compounds 3-15 showed moderate cytotoxic activities against HL-60 cell lines with IC50 values ranging from 11.4 to 37.9 µM. Compounds 5, 9, and 10 showed marked or moderate cytotoxic activities against five human tumor cell lines with IC50 values ranging from 11.4 to 36.7 µM. Compound 11 displayed moderate cytotoxic activities against HL-60, SMMC-7721, MCF-7 and SW480 cell lines with IC50 values of 12.2-30.8 µM. Compared to the positive control (IC50: 35.0 µM), compounds 5, 9-11 exhibited more potential inhibitory activity against MCF-7 cells (IC50: 16.1-25.6 µM).

Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases.

The herbaceous plant Cynanchum otophyllum Schneid. is widely used as a milk coagulant to make a Chinese traditional milk product, milk cake. However, the milk-clotting compounds and their mechanism remain unclear. In this study, crude proteases were extracted from the dried leaves of Cynanchum otophyllum Schneid. using citric acid-phosphate buffer and then partially purified by weak anion exchange chromatography. Two proteases, QA and QC, with molecular weights of 14 and 27 kDa, respectively, were shown to exhibit milk-clotting activity. A study of the effects of pH and temperature on the milk-clotting activity and proteolytic activity of the proteases showed that they exhibited good pH stability from pH 5.5 to 7.5 and good thermal stability at temperatures from 50 to 70°C. The QA and QC were the cysteine proteases, able to hydrolyze ß-casein and κ-casein completely, and α-casein partially. The cleavage site on κ-casein determined by Orbitrap (Thermo Fisher Scientific, San Jose, CA) analysis showed that QA and QC could cleave κ-casein at Ser132-Thr133. Overall, the results suggest that the Cynanchum otophyllum Schneid. proteases are a promising milk-clotting enzyme that could be used for manufacturing milk cake and cheese.

[Chemical constituents of ethnic medicine Cynanchum otophyllum].

In order to isolate and purify the reference compounds and improve the quality standard of ethnic medicine of Radix of Cynanchum otophyllum, the ethanol extracts were isolated by column chromatography onsilica gel, C18 reverse-phase silica gel, and semi-preparative HPLC. Twelve compounds were isolated and their structures were elucidated as 2,4-dihydroxy-6-methoxyphenylethanone(1), 4,6-dihydroxy-2-methoxyphenylethanone(2), p-hydroxyacetophenone(3), baishouwubenzophenone(4), 2,4-dihydroxyacetophenone(5), 2,5-dihydroxyacetophenone(6), otophylloside A(7),otophylloside B(8), caudatin-3-O-ß-D-cymaropyranosyl-(1→4)-ß-D-digitoxopyranoside(9),caudatin-3-O-ß-D-glucopyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-digitoxopyranoside(10),qingyangshengenin-3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranoside(11),caudatin-3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside(12) on the basis of spectral analysis. Compounds 1 and 2 were isolated from the genus Cynanchum for the first time, and compounds 3-4, 9-12 were obtained from this plant for the first time.These compounds are main active components of Radix of C.otophyllum and can be used as reference substances for the quality control of this ethnic medicine.

Antiepileptic C21 steroids from the roots of Cynanchum otophyllum.

In order to discover more natural products possessing potentially antiepileptic activities, three C21 steroids, including a new one, characterized as caudatin-3-O-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside (1), and two known analogs, otophylloside B (2) and caudatin-3-O-ß-D-oleandropyranosyl-(1 → 4)-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranoside (3), were isolated from the chloroform extract of the roots of Cynanchum otophyllum and evaluated for their antiepileptic activities by pentylenetrazole (PTZ)-induced zebrafish larval locomotor assay. The results showed that all of them had marked activities of suppressing PTZ-induced seizure behaviors in larval zebrafish at the dose of 10 µg/ml.

Two new steroidal glycosides from Cynanchum otophyllum Schneid.

Two new C21 steroidal glycosides were isolated from Cynanchum otophyllum Schneid. Their structures were elucidated as qinyangshengenin-3-O-ß-d-digitoxopyranoside (1) and rostratamine-3-O-ß-d-cymaropyranosyl-(1 â†’ 4)-ß-d-digitoxopyranoside (2) on the basis of chemical and spectroscopic methods, including 1D and 2D NMR experiments.

The complete plastome genome sequence of Cynanchum otophyllum (Asclepiadaceae), a unique medicinal species in China.

Cynanchum otophyllum Schneid is an important medicinal plant in China. In this paper, the chloroplast genome of C. otophyllum was sequenced based on high-throughput technology, and the chloroplast genome structure characteristics and phylogenetic relationship of C. otophyllum were analyzed. The results showed the complete plastome genome size of C. otophyllumis 160,874bp, including one small single copy (SSC, 19,851bp) and one large single copy (LSC, 92,009bp) regions isolated by a pair of inverted repeat regions (IRs, 24,507bp). The whole plastome genome including 84 protein encoding genes, 8 rRNA and 37 tRNA. Based on the phylogenetic topologies, C. otophyllum shows close association with additional Gomphocarpus and Asclepias genus. This study contributes to an enhanced understanding of the genetic information of C. otophyllum and provides a theoretical basis for the development of molecular markers and phylogeographic of the species, as well as for constructing the phylogenetic tree of Asclepiadaceae.

Cancer Pain Management with Traditional Chinese Medicine: Current Status and Future Perspectives.

Cancer pain, especially the moderate-to-severe pain experienced by patients with advanced cancer, is still one of the most challenging clinical problems. The current mainstream pharmacological treatment for cancer pain involves applying opioid medications and other pain-killing drugs. However, analgesic drugs have many adverse effects such as addiction, tolerance, and other formidable clinical and social issues. Thus, finding a new therapeutic approach to treat cancer pain is essential. Traditional Chinese medicine (TCM) has been increasingly applied in clinical practice because of its good efficacy and few side effects. However, its mechanisms of action in treating pain are still under investigation. The most important mechanism of cancer pain is that a large amount of pain-causing substances are secreted from cancer cells and promote their growth and invasion. The physical and chemical stimulations of these substances exist along with the cancer growth, leading to constantly increased pain sensation. Whether cancer pain can be alleviated by inhibiting cancer cells from releasing the substances and changing the microenvironment around the cancer mass, or even by eliminating pain-causing substances, is largely unknown. Based on TCM theory, this study reported that the aforementioned approach could effectively manage different cancer pains by tonifying qi, clearing and activating channels and meridians, and strengthening body resistance. The TCM therapies activate blood circulation, remove blood stasis, and nourish the heart. Commonly used Chinese herbal drugs include Corydalis yanhusuo, Angelica dahurica, and Ligusticum chuanxiong. Instead of using conventional analgesics to reduce pain, we should focus on using TCM modalities to alleviate cancer pain and increase the quality of life in patients suffering from cancer pain. TCM should provide us with a new strategy for managing cancer pain.

New C21-steroidal aglycones from the roots of Cynanchum otophyllum and their anticancer activity.

Naturally occurring C21-steroidal aglycones from Cynanchum exhibit significant antitumor effects. To expand the chemical diversity and get large scale C21-steroidal aglycones, the extracts of the roots of Cynanchum otophyllum were treated with 5% HCl in aqueous and the resulting hydrolysate was investigated. Nine new C21-steroidal aglycones (1-9) namely cynotogenins A-I, along with seventeen known analogous (10-26), were isolated from the hydrolysate. The structures of compounds 1-9 were elucidated by spectroscopic analysis (IR, HR-ESI-MS, 1D and 2D NMR) and comparison of observed spectroscopic data with those of reported in the literature. Aglycones 2-5 with rare cis-cinnamoyl group as well as 8 and 9 with 5ß,6ß-epoxy group were found from the genus of Cynanchum for the first time. The cytotoxicities of compounds 1-26 toward human cancer HeLa, H1299, HepG2, and MCF-7 cells were evaluated and preliminary structure-activity relationship (SAR) was discussed. Moreover, compound 20 inhibits HepG2 cell apoptosis and induces of G0/G1 phase arrest in a dose dependent manner.

Hepatoprotective steroids from roots of Cynanchum otophyllum.

Seven undescribed C21 steroids, namely cynanchin A-G, together with thirteen known analogues, were isolated from the roots of cynanchum otophyllum. Their structures were elucidated by 1D, 2D NMR and MS spectra, as well as chemical methods. Meanwhile, all of isolates were tested for their anti-hepatic fibrosis activity. Among them, compounds 4-6, 10-12 and 14-17 showed moderate or significant inhibitory effects for the proliferation of hepatic stellate cells (HSCs) induced by transforming growth factor-ß1 (TGF-ß1) in vitro.

Effects of Achyranthis bidentatae -Cynanchum otophyllum on oxidative stress of cerebral tissue in Parkinson ’s disease mice with syndrome of hyperactivity of liver -Yang / 中国药房

OBJECTIVE To explore the effects of Achyranthis b identatae-Cynanchum otophyllum as core couplet medicinals of Zhengan xifeng decoction on oxidative stress of cerebral tissue in Parkinson ’s disease （PD）mice with syndrome of hyperactivity of liver-Yang. METHODS C57BL/6 mice were randomly divided into normal control group ，model group ，Zhengan xifeng decoction group，A. bidntatae group，C. otophyllum group and couplet medicinals of A. bidentatae-C. otophyllum group，with 10 mice in each group. PD model of hyperactivity of liver -Yang was established by intragastric administration of Aconitum carmichaelii decoction（4 g/kg） and intraperitoneal injection of 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine （25 mg/kg）. The behavioral changes and ultrastructure of substantia nigra neurons in mice were observed . The expressions of tyrosine hydroxylase （TH）positive neurons in substantia nigra were detected . The expressions of total antioxidant capacity （T-AOC），superoxide dismutase （SOD） and malondialdehyde（MDA）in substantia nigra as well as mRNA and protein expressions of nuclear factor -erythroid 2-related factor 2 （Nrf2），thioredoxin reductase 1（Trxr1），thioredoxin interacting protein （Txnip）were also determined . RESULTS Compared with model group ，PD behavior and ultrastructure of substantia nigra neurons were all improved in administration groups . The expressions of TH positive neurons ，T-AOC（except for C.otophyllum group）and SOD （except for C.otophyllum group），mRNA relative expression and protein expression levels of Nrf 2 and Trxr 1 were all increased significantly ；the expression of MDA （except for C.otophyllum group）and mRNA relative expression and protein expression levels of Txnip were all decreased significantly （P＜0.05）. Intervention effect of couplet medicinals of A. bidentatae-C. otophyllum group was better than that of A.bidntatae group and C. otophyllum group（P＜ 0.05），and the effect was similar to that of decoction group （P＞0.05）. CONCLUSIONS The couplet medicinals of A. bidentatae-C. otophyllum can inhibit the level of oxidative stress in the cerebral tissue of PD mice with hyperactivity of liver -Yang by targeting Nrf 2，and play a protective role on the brain neurons . Its effect is better than that of A.bidntatae and C.otophyllum，and it plays the same role as that of the formula in tonifying the kidney ，softening the liver and suppressing the Yang .

Pregnane glycosides from the antidepressant active fraction of cultivated Cynanchum otophyllum.

Based on the bioactive screening results, four new pregnane glycosides, namely cynanotophyllosides A-D (1-4) were isolated from the anti-depressant active fraction of cultivated Cynanchum otophyllum, along with thirteen known compounds (5-17). The new compounds were characterized as qingyangshengenin 3-O-ß-D-glucopyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside (1), qingyangshengenin-3-O-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-α-L-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside (2), caudatin-3-O-ß-D-glucopyranosyl-(1→4)-ß-D-thevetopyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-digitoxopyranoside (3) caudatin-3-O-ß-D-glucopyranosyl -(1→4)-ß-D-thevetopyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside (4), by detailed spectroscopic analysis and acidic hydrolysis.

Protective Effects of Otophylloside N on Pentylenetetrazol-Induced Neuronal Injury In vitro and In vivo.

Approximately 30% of epileptic patients worldwide are medically unable to control their seizures. In addition, repeated epileptic seizures generally lead to neural damage. Pentylenetetrazol (PTZ) is a clinical circulatory and respiratory stimulant that is experimentally used to mimic epileptic convulsion in epilepsy research. Here, we systematically explore the neuroprotective effects of a pure compound isolated from Cynanchum otophyllum Schneid (Qingyangshen), Otophylloside N (OtoN), against PTZ-induced neuronal injury. We used three models: in vitro primary cortical neurons, in vivo mice, and in vivo zebrafish. Our results revealed that OtoN treatment may attenuate PTZ-induced morphology changes, cell death, LDH efflux in embryonic neuronal cells of C57BL/6J mice, and convulsive behavior in zebrafish. Additionally, our Western blot and RT-PCR results demonstrated that OtoN may attenuate PTZ-induced apoptosis and neuronal activation in neuronal cells, mice, and zebrafish. OtoN may reduce PTZ-induced cleavage of poly ADP-ribose polymerase and upregulation of the Bax/Bcl-2 ratio and decrease the expression level of c-Fos. This study is the first investigation of the neuroprotective effects of OtoN, which might be developed as a novel antiepileptic drug.

Cytotoxicity of pregnane glycosides of Cynanchum otophyllum.

Fourteen new pregnane glycosides, including nine caudatin glycosides (1-9), three qinyangshengenin glycosides (10-12), one kidjoranin glycosides (13) and one gagaminin glycosides (14), along with twelve known analogs (15-26) were isolated from roots of Cynanchum otophyllum Schneid. Their structures were deduced by detailed analysis of 1D and 2D NMR spectra, as well as HRESIMS. In this study, all pregnane glycosides obtained (1-26) were evaluated for their cytotoxic activities using three cancer cell lines (HepG2, Hela, U251). As results, except 6 and 10, other twenty-four pregnane glycosides showed cytotoxicities at different degrees against three cell lines.

Neuroprotective polyhydroxypregnane glycosides from Cynanchum otophyllum.

Five new polyhydroxypregnane glycosides, namely cynanotosides A-E (1-5), together with two known analogues, deacetylmetaplexigenin (6) and cynotophylloside H (7), were isolated from the roots of Cynanchum otophyllum. Their structures were established by spectroscopic methods and acid hydrolysis. The neuroprotective effects of compounds 1-7 against glutamate-, hydrogen peroxide-, and homocysteic acid (HCA)-induced cell death were tested by MTT assay in a hippocampal neuronal cell line HT22. Compounds 1, 2, and 7 exhibited protective activity against HCA-induced cell death in a dose-dependent manner ranging from 1 to 30µM, which may explain the Traditional Chinese Medicine (TCM) use of this plant for the treatment of epilepsy.

Evaluation of Cynanchum otophyllum glucan sulfate against human immunodeficiency virus and herpes simplex virus as a microbicide agent.

OBJECTIVE: The root of Cynanchum otophyllum-also known as Qing Yang Sheng-is a traditional ethnical Chinese medicine. The objective of this study was to evaluate in vitro activities and safety of C. otophyllum glucan sulfate (PS20) against Human Immunodeficiency Virus (HIV) and Herpes Simplex Virus (HSV). MATERIALS AND METHODS: Anti-HIV activity was detected with syncytial formation assay and quantitative P24 Enzyme-Linked Immunosorbent Assay (ELISA). Anti-HSV activity was detected with plaque reduction assay; cytotoxicity was tested with MTT colorimetric assay; and anti-bacterial activity was tested with microdilution method. Anti-HIV mechanism was investigated with fusion inhibition, time of addition, and pretreatment. RESULTS: The 50% Inhibition Concentration (IC(50)) of PS20 for HIV-1(IIIB), HIV-(Ada-M), HIV-1(Bal), HSV-I, and -II were 0.26 ± 0.02 mM, 0.46 ± 0.02 mM, 0.90 ± 0.04 mM, 3.45 ± 0.85 µM, and 0.70 ± 0.22 mM, respectively. Selectivity Indices (SI) were 653, 50, 39, 85, and 362, respectively. Studies on anti-HIV mechanism of PS20 showed that the target molecule should be the envelope protein. The 50% Cytotoxicity Concentrations (CC(50)) of PS20 for HeLa and ME-180 cell lines and human foreskin fibroblast cells was more than 70 µM. The Minimum Inhibitory Concentration (MIC) for vaginal lactobacilli was more than 1000 µM. CONCLUSION: PS20 possesses anti-HIV and HSV effect and low cytotoxicity to epithelium cells and vaginal lactobacilli. It may be considered as a potential microbicide agent for further investigation.

Three New C21 Steroidal Glycosides from Roots of Cynanchum otophyllum / 中草药·英文版

Objective: To investigate the chemical structures of glycosides in the roots of Cynanchum otophyllum (Asclepiadaceae) and to find new glycosides. Methods: The total glycosides in the roots of C. olophyllum were separated by silica gel column chromatography. The structures of the resulting compounds were determined by NMR and FAB-MS spectra. Results: Three C21 steroidal glycosides were separated. Their structures were determined as caudatin 3-O-(4-O-methyl-β-D-cymaropyranosyl)-(1→4)-α-D-oleandropyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranoside (1), caudatin 3-O-β-D-digitoxopyranosyl-(1→4)-α-D-oleandropyranosyl-(1→4)-β-Ddiginopyranosyl-(1→4)-β-D-glucopyranoside (2), and caudatin 3-O-β-D-diginopyranosyl-(1→4)-α-D-oleandropyranosyl-(1→4)-α-D-oleandropyranosyl-(1→4)-β-D-glucopyranoside (3), respectively. Conclusion: Glycosides 1-3 are new compounds.

Chemical constituents of ethnic medicine Cynanchum otophyllum / 中国中药杂志

In order to isolate and purify the reference compounds and improve the quality standard of ethnic medicine of Radix of Cynanchum otophyllum, the ethanol extracts were isolated by column chromatography onsilica gel, C₁₈ reverse-phase silica gel, and semi-preparative HPLC. Twelve compounds were isolated and their structures were elucidated as 2,4-dihydroxy-6-methoxyphenylethanone(1), 4,6-dihydroxy-2-methoxyphenylethanone(2), p-hydroxyacetophenone(3), baishouwubenzophenone(4), 2,4-dihydroxyacetophenone(5), 2,5-dihydroxyacetophenone(6), otophylloside A(7),otophylloside B(8), caudatin-3-O-β-D-cymaropyranosyl-(1→4)-β-D-digitoxopyranoside(9),caudatin-3-O-β-D-glucopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-digitoxopyranoside(10),qingyangshengenin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside(11),caudatin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside(12) on the basis of spectral analysis. Compounds 1 and 2 were isolated from the genus Cynanchum for the first time, and compounds 3-4, 9-12 were obtained from this plant for the first time.These compounds are main active components of Radix of C.otophyllum and can be used as reference substances for the quality control of this ethnic medicine.