Cynanchum Auriculatum Research Articles

Preparation, characterization, antioxidant activity and protective effect against cellular oxidative stress of polysaccharide from Cynanchum auriculatum Royle ex Wight

In the present study, a novel polysaccharide fraction (CAP2-1) from Cynanchum auriculatum Royle ex Wight was obtained by hot water extraction, ethanol precipitation and sequential purification through anion-exchange and size-exclusion chromatography. CAP2-1 was a homogeneous heteropolysaccharide composed of mannose, rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose, with an average molecular weight of 830.93 kDa. FT-IR and NMR spectra of CAP2-1 exhibited typical characteristic signals of polysaccharide. For antioxidant activity evaluation in vitro, CAP2-1 showed effective scavenging capacities against ABTS, DPPH and superoxide anion radical in a dose-dependent manner, with IC50 values at 0.1232, 0.5543 and 0.5881 mg/mL, respectively. At cellular level, CAP2-1 provided a significant protective effect against hydrogen peroxide-induced oxidative stress in HepG2 cells by a compositive oxidation defense mechanism. CAP2-1 could reduce oxidative stress by significantly enhancing the contents of antioxidant enzyme SOD and non-enzymatic antioxidant GSH in oxidative damaged cells, in addition to scavenging ROS directly and improving cell viability and membrane integrity, consequently achieving the intracellular antioxidant activity. The results unveiled that CAP2-1 could be explored as a promising natural antioxidant for application in functional foods.

Preparation, characterization, digestibility and antioxidant activity of quercetin grafted Cynanchum auriculatum starch

In this study, a novel method was developed to conjugate quercetin with Cynanchum auriculatum starch. Quercetin was first succinylized and then grafted onto C. auriculatum starch through a N,N′‑carbonyldiimidazole mediated reaction. The obtained water soluble quercetin grafted starch (quercetin-g-starch) was characterized by several instrumental methods. UV–vis spectrum of quercetin-g-starch aqueous solution exhibited two absorption bands at around 300 and 430nm. Fourier-transform infrared spectrum of quercetin-g-starch showed characteristic bands at 1731 and 1568cm−1, assigning to CO stretching of esterified carboxyl group and CC stretching of aromatic ring, respectively. Proton nuclear magnetic resonance spectrum of quercetin-g-starch showed partial proton signals of starch (3.30–5.47ppm) and quercetin (6.19–7.68ppm). All above results suggested quercetin was successfully grafted onto C. auriculatum starch. Besides, quercetin-g-starch particles were irregular in shape and were in an amorphous state. Notably, the thermal stability, resistant starch content as well as antioxidant activity of C. auriculatum starch was greatly enhanced by grafting with quercetin. This indicated the potential of quercetin-g-starch in the development of a novel resistant starch with antioxidant activity.

Preliminary Quality Evaluation and Characterization of Phenolic Constituents in Cynanchi Wilfordii Radix.

A new phenolic compound, 2-O-β-laminaribiosyl-4-hydroxyacetophenone (1), was isolated from Cynanchi Wilfordii Radix (CWR, the root of Cynanchum wilfordii Hemsley), along with 10 known aromatic compounds, including cynandione A (2), bungeisides-C (7) and –D (8), p-hydroxyacetophenone (9), 2′,5′-dihydroxyacetophenone (10), and 2′,4′-dihydroxyacetophenone (11). The structure of the new compound (1) was elucidated using spectroscopic methods and chemical methods. The structure of cynandione A (2), including a linkage mode of the biphenyl parts that remained uncertain, was unambiguously confirmed using the 2D 13C–13C incredible natural abundance double quantum transfer experiment (INADEQUATE) spectrum. Additionally, health issues related to the use of Cynanchi Auriculati Radix (CAR, the root of Cynanchum auriculatum Royle ex Wight) instead of CWR have emerged. Therefore, constituents present in methanolic extracts of commercially available CWRs and CARs were examined using UV-sensitive high-performance liquid chromatography (HPLC), resulting in common detection of three major peaks ascribed to cynandione A (2), p-hydroxyacetophenone (9), and 2′,4′-dihydroxyacetophenone (11). Thus, to distinguish between these ingredients, a thin-layer chromatography (TLC) method, combined with only UV irradiation detection, focusing on wilfosides C1N (12) and K1N (13) as marker compounds characteristic of CAR, was performed. Furthermore, we propose this method as a simple and convenient strategy for the preliminary distinction of CWR and CAR to ensure the quality and safety of their crude drugs.

Discrimination of Cynanchum wilfordii and Cynanchum auriculatum by terahertz spectroscopic analysis.

Precise identification of botanical origin of plant species is crucial for the quality control of herbal medicine. In Korea, the root part of Cynanchum auriculatum has been misused for C. wilfordii in the herbal drug market due to their morphological similarities. Currently, DNA analysis using the polymerase chain reaction (PCR) method is employed to discriminate between these species. In order to develop a new analytical tool for the rapid discrimination of C. wilfordii and C. auriculatum, terahertz (THz) spectroscopy was employed. Authentic samples of C. wilfordii and C. auriculatum were provided from the National Institute and standardized pellets for each species were prepared to get optimum results with terahertz time-domain spectroscopy (THz-TDS) in frequency range 0.2-1.20 THz. The C. wilfordii pellet showed longer time delay compare to the sample of C. auriculatum and this was due to the difference in permittivity. The pellet samples of C. wilfordii and C. auriculatum showed a permittivity difference of about 0.08 at 0.2-1.20 THz. The experimental results indicated that THz-TDS analysis can be an effective and rapid method for the discrimination of C. wilfordii and C. auriculatum, and this application can be expanded for the discrimination of other similar herbal medicines.

Chemical constituents from endophytic fungus Plectosphaerella cucumerina YCTA2Z1 of Cynanchum auriculatum

ObjectiveTo study the chemical constituents from the EtOAc extract of endophytic fungal Plectosphaerella cucumerina YCTA2Z1. MethodsThe chemical constituents were isolated and purified by silica gel column, Sephadex LH-20, and reverse-phase C-18 column chromatography as well as crystallization. ResultsThirteen compounds were isolated from the EtOAc extract of the fungal strain YCTA2Z1. Their chemical structures were elucidated according to the spectral evidence. They were identified as caudatin (1), baishouwubenzophenone (2), cynandione B (3), asterbatanoside A (4), p-hydroxyphenethyl-O-β-D-glycoside (5), caffeic acid (6), ferulic acid (7), 2,5-dihydroxyacetophenone (8), protocatechuic acid (9), vanillic acid (10), stearic acid (11), azelaic acid (12), and succinic acid (13). ConclusionsIt is the first chemical study on endophytic fungi from Cynanchum auriculatum and all the compounds are obtained from the species, the genus, as well as the family Plectosphaerellaceae for the first time.

Antiangiogenic properties of caudatin invitro and invivo by suppression of VEGF‑VEGFR2‑AKT/FAK signal axis.

Tumor angiogenesis provides essential nutrients and oxygen to the tumor microenvironment, which is important in tumor growth, progression and metastasis. Inhibition of tumor angiogenesis represents one of the most promising strategies in tumor therapy. The authors previously demonstrated that caudatin, one species of C-21 steroidal from Cynanchum auriculatum (C. auriculatum), effectively inhibits human glioma growth in vitro and in vivo through triggering cell cycle arrest and apoptosis. However, little information regarding the antiangiogenic properties of caudatin in human glioma is available. Based on the author's previous study, the antiangiogenic effect of caudatin against human glioma was explored, and the underlying mechanism was investigated. The results suggested that caudatin treatment significantly inhibited HUVEC human umbilical vein endothelial cell proliferation, blocked the HUVECs migration, invasion and capillary-like tube formation by disturbing the vascular endothelial growth factor (VEGF)-VEGFR2-protein kinase B (AKT)/focal adhesion kinase (FAK) signal axis. Notably, caudatin treatment abolished the glioma cell growth by suppression of the in vivo angiogenesis, which involved FAK and AKT dephosphorylation and inhibition of VEGF expression. The findings validated the antiangiogenic potential of caudatin in hunting human glioma.

Antitumor evaluation and multiple analysis on different extracted fractions of the root of Cynanchum auriculatum Royle ex Wight.

The root of Cynanchum auriculatum (C. auriculatum) Royle ex Wight has been shown to possess various pharmacological effects and has recently attracted much attention with respect to its potential role in antitumor activity. The C-21 steroidal glycosides are commonly accepted as the major active ingredients of C. auriculatum. In this study, the antitumor abilities of different extracted fractions of the root bark and the root tuber of C. auriculatum were investigated by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human cancer cell lines HepG2 and SMMC-7721. The results showed that thechloroformand ethylacetate fractions of the root tuber suppressed tumor cell growth strongly. To identify and characterize the chemical constituents of different active fractions, an ultra high performance liquid chromatography with triple-quadrupole tandem mass spectrometry method was developed forthe simultaneous quantitation of eight C-21 steroidal glycosides. The analysis revealed that the C-21 steroidal glycosides were concentrated in thechloroformand ethylacetate fractions, and the total contents of different fractions in the root tuber were significantly higher than those of corresponding ones in the root bark. Furthermore, the C-21 steroidal glycosides based on different types of aglucones were prone in different medicinal parts of C. auriculatum.

C21 steroidal glycosides from Cynanchum auriculatum and their neuroprotective effects against H2O2-induced damage in PC12 cells

Twenty-three undescribed previously C21 steroidal glycosides, named saccatols D–K and cynsaccatols I−W, together with six known analogs were isolated from the roots of Cynanchum auriculatum Royle ex Wight (Apocynaceae). Their structures were elucidated by a variety of spectroscopic techniques, as well as HRESIMS and GC method. All isolated compounds were tested their neuroprotective effects on the model of H2O2-induced damage to PC12 cells. Except saccatols D–F, J, cynsaccatol V and wilfoside A, other twenty-three isolates produced significantly activities against H2O2-induced cell damage (P < 0.001). Furthermore, cynsaccatols I, N, O and S can obvious inhibition of damaged PC12 cell apoptosis at their dosages of 1 μM by Annexin V-FITC/PI double staining assay with flow cytometry.

GC-MS, FTIR and Raman Analysis of Antioxidant Components of Red Pigments from Stemphylium lycopersici.

Many microorganisms can generate pigments with different colours and structures during the growth process. In this study, an endophytic fungus producing red pigments was isolated from Cynanchum auriculatum Royle ex Wight tissue. PCR amplification sequencing was conducted, and phylogenetic analysis was performed on the ITS region sequences. Combined with morphological observation, the fungus was identified as Stemphylium lycopersici. The antioxidant activities of the pigments were evaluated in vitro and showed good antioxidant properties. Ultraviolet (UV), Raman, and Fourier transform infrared (FTIR) spectroscopy and gas chromatography-mass spectroscopy (GC-MS) were used to analyse the pigments' components, which were shown to contain phenolics and anthraquinones. Most of these components have not been previously reported in Stemphylium lycopersici, especially physcione. This study is the first report of Stemphylium lycopersici secondary metabolites and their potential use as red pigments and antioxidants. Further optimisation of the culture conditions of this fungal strain might permit its application for pigment production.

Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight

Effect of acid hydrolysis on the morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight was investigated in this study. The hydrolysis degree of C. auriculatum starch rapidly increased to 63.69% after 4days and reached 78.67% at the end of 9days. Morphology observation showed that the starch granules remained intact during the first 4days of hydrolysis. However, serious erosion phenomenon was observed after 5days and starch granules completely fell into pieces after 7days. During acid hydrolysis process, the crystal type of hydrolyzed starch changed from original CB-type to final A-type. Small-angle X-ray scattering patterns showed the semi-crystalline growth rings started to be hydrolyzed after 4days. The proportions of single helix and amorphous components as well as amylose content in starch gradually decreased, whereas the proportion of double helix components continuously increased during acid hydrolysis. However, the contents of rapidly digestible starch, slowly digestible starch and resistant starch were almost constant during acid hydrolysis process, indicating the in vitro digestion property of C. auriculatum starch was not affected by acid hydrolysis. Our results provided novel information on the inner structure of C. auriculatum starch granules.

First report of powdery mildew caused by Podosphaera sp. on Cynanchum auriculatum

Powdery mildew symptoms were observed on the upper surface of leaves of Cynanchum auriculatum in Eumseong, Korea in October 2015. Microscopic observations and molecular characteristics determined this fungus as Podosphaera sp., which is reported for the first time on Cynanchum auriculatum.

Morphology, structural and physicochemical properties of starch from the root of Cynanchum auriculatum Royle ex Wight

The root of Cynanchum auriculatum Royle ex Wight is a traditional Chinese herbal medicine and healthy food. Although C. auriculatum has already been processed into starch for human consumption in China, the structural characterizations of C. auriculatum starch is still unknown. Therefore, the morphology, structural and physicochemical properties of C. auriculatum starch were investigated in this study. C. auriculatum starch exhibited both spherical and polygonal shapes with granule size ranging from 2 to 12μm. Some void cavities and serpentine channels were observed in the inner of starch granules. X-ray powder diffraction pattern revealed that C. auriculatum starch was a CB-type with relative crystallinity of 25.19%. Small-angle X-ray scattering spectrum indicated C. auriculatum starch had a lamellar repeat distance of 9.21nm. The proportions of single helix, double helix and amorphous components in C. auriculatum starch were 3.42%, 27.11% and 69.47%, respectively. The amylose content of C. auriculatum starch was 28.0% with the gelatinization temperature ranging from 59.3 to 70.1°C. The maximum weight loss rate of C. auriculatum starch appeared at 309°C. In addition, C. auriculatum starch showed higher swelling power than other starches tested. Our results suggest C. auriculatum starch will have wide applications in food industry.

Fast and Simple Chemical Fingerprinting Analysis of Medicinal Herbs by Paper Cone Spray Ionization Mass Spectrometry (PCSI MS)

Paper cone spray ionization (PCSI), an extractive ambient ionization method that is specialized for raw solid material analysis, was applied to chemical fingerprinting analysis of raw medicinal herbs. Experimental parameters of PCSI mass spectrometry (MS) were first optimized against Salvia miltiorrhiza Bunge (danshen). Our results showed that simple alkyl alcohols, such as methanol and 2‐propanol, generally worked well in both polarities and could generate representative chemical fingerprints instantly for a given herbal sample. In addition, in situ extraction from untreated herbal slices during PCSI MS was found to be enough to generate characteristic chemical signatures of a given medicinal herb. After optimization, representative fingerprints of various medicinal herbs, including Sophora flavescens (kushen) and Paeonia lactiflora Pall (shaoyao), were successfully obtained by PCSI MS. In addition, morphologically similar herbs, Cynanchum auriculatum and Cynanchum wilfordii, could be differentiated from each other by their chemical signatures by PCSI MS.

Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum.

Identification of plant species is important for standardizing herbal medicine. Cynanchum wilfordii (Baekshuoh in Korean) and Polygonum multiflorum (Hashuoh in Korean) are important oriental medicinal herbs in Korea, Japan, and China. Cynanchum auriculatum is a faster growing and more productive plant than C. wilfordii; and, it is not recognized as a medicinal plant in the Korean Pharmacopoeia. C. wilfordii, P. multiflorum, and C. auriculatum are often misidentified in the Korean herbal medicine marketplace due to their morphological similarities and similar names. In this study, we investigated molecular authentication of these three medicinal plants using DNA sequences in the TrnL-F chloroplast intergenic region. Specific species identification was achieved by detecting allelic variations of single nucleotide polymorphisms (SNPs) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and high resolution melting curve analysis. Our results demonstrate that the intraspecific genetic distance between C. wilfordii and C. auriculatum is relatively low. We also developed a quantitative PCR assay using species-specific TrnL-F primers, which allowed us to estimate the ratio of C. wilfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. Additionally, to identify species in hybrid plants produced by cross-fertilization, we analyzed nuclear ribosomal DNA internal transcribed spacer regions in C. wilfordii and C. auriculatum by ARMS-PCR. Our results indicate that SNP-based molecular markers, usable to barcode tools could provide efficient and rapid authentication of these closely related medicinal plant species, and will be useful for preventing the distribution of products contaminated with adulterants.

The complete chloroplast genome sequence of Cynanchum auriculatum Royle ex Wight (Apocynaceae)

Cynanchum auriculatum is a climbing vine belonging to the Apocynaceae family and shows very similar morphology to Cynanchum wilfordii, a medicinal plant. The complete chloroplast genome of C. auriculatum was generated by de novo assembly using the small amount of whole genome sequencing data. The chloroplast genome of C. auriculatum was 160 840 bp in length and consisted of four distinct regions, such as large single copy region (91 973 bp), small single copy region (19 667 bp), and a pair of inverted repeat regions (24 600 bp). The overall GC contents of the chloroplast genome were 37.8%. A total of 114 genes were predicted and included 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that C. auriculatum is most closely related to Cynanchum wilfordii, a medicinal plant.

Biotransformation and metabolic profile of caudatin-2,6-dideoxy-3-O-methy-β-d-cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time-of-flight mass spectrometry.

In our previous studies, caudatin-2,6-dideoxy-3-O-methy-β-d- cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technique combined with Metabolynx(TM) software was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC-Q-TOF-MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant.

Pharmacokinetics and tissue distribution study of caudatin in normal and diethylnitrosamine-induced hepatocellular carcinoma model rats.

Caudatin is a potential antitumor agent isolated from the traditional Chinese medicine “baishouwu”, which was the root tuber of Cynanchum auriculatum Royle ex Wight. In our previous studies, caudatin showed selectivity on human hepatoma cell line SMMC7721 among several different tumor cell lines, and further in vivo tests validated the inhibitory action of caudatin against hepatic cancer using an H22 solid tumor model in mice, but to our knowledge, the biopharmaceutical properties of caudatin are largely unknown. In this study, a simple, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of caudatin in rat plasma and tissues, which kept the run time to detect one sample within 4 min, was developed and validated. Pharmacokinetics and tissue distribution studies of caudatin in conventional rats and hepatocellular carcinoma (HCC) model rats were then conducted for the first time. Statistically significant differences were observed between conventional rats and diethylnitrosamine (DEN)-induced HCC rats with respect to pharmacokinetic parameters, including maximum concentration (Cmax), time to reach Cmax (Tmax), half-life (t1/2), area under the concentration-time curve (AUC0-t, AUC0-∞), mean residence time (MRT0-t and MRT0-∞), and oral clearance (CL/F). Increased exposures of caudatin were found in the plasma and livers of HCC model rats, which would be helpful for a better understanding of pharmacological effect of caudatin in treating HCC disease.

Monitoring and identification of Cynanchum wilfordii and Cynanchum auriculatum by using molecular markers and real-time polymerase chain reaction

Cynanchum wilfordii (Asclepiadaceae) is widely distributed throughout Korea, Japan, and China. Dried roots of this plant have been used as a tonic to promote renal function. Due to the morphological similarities of the dried roots of this plant to those of Cynanchum auriculatum, which is used as a substitute herbal medicine for C. wilfordii, distinguishing these two species is extremely difficult. The present study was conducted to develop molecular markers to distinguish C. wilfordii and C. auriculatum by using conventional polymerase chain reaction (PCR) and realtime PCR analyses. Comparative analysis based on the sequence of the trnL-trnF intergenic spacer revealed 4 base-pair variations, and the inter-individual sequences of the 2 species separately showed 100% homology. According to these results, the variations were divided into 2 groups. The 2 species were further distinguished using a sequence-characterized amplified region marker developed based on a randomly amplified polymorphic DNA-PCR product, and then a single nucleotide polymorphism marker was designed based on the trnL-trnF intergenic spacer for more efficient detection in real-time PCR. The results showed that speciesspecific molecular markers might allow accurate discrimination of C. wilfordii and C. auriculatum.

Appetite suppressing pregnane glycosides from the roots of Cynanchum auriculatum

In the search for plant alternatives to Hoodia gordonii containing P57, a pregnane glycoside with potential appetite suppressant effect, the roots of Cynanchum auriculatum were investigated. As a result, 15 pregnane glycosides including nine never previously reported were isolated. Their structures were elucidated on the basis of extensive spectroscopic analyses and chemical methods. Appetite suppressant effect and body weight loss were observed when tested with the most abundant pregnane glycoside, wilfoside K1N, in an in vivo test with rats.

Quality assessment and discrimination of the roots of Cynanchum auriculatum and Cynanchum wilfordii by HPLC–UV analysis

Cynanchum auriculatum and Cynanchum wilfordii are widely used as folk medicine in Eastern Asia. However, the indeterminacy in the authentic original plant material has resulted in the same appellative name being given to the two plants, and they are commonly misused. Therefore, it is necessary to establish an analytical method for discrimination as well as quality control of the two species. This study was to develop HPLC-UV methods for quality assessment of C. auriculatum and C. wilfordii and discrimination between the two species. Two HPLC methods to analyze eight marker compounds were established and validated. The first method analyzed seven marker compounds simultaneously on a reversed-phase column, while the second method analyzed a single marker compound, conduritol F, which exists only in C. wilfordii, on a Si-column. Thirty-nine batches of C. auriculatum and nineteen batches of C. wilfordii that were collected from different geographical regions of South Korea were analyzed by these methods. The constructed data matrix was subjected to principal components analysis and hierarchical cluster analysis in order to classify the samples. The established methods offer a potential strategy for authentication and differentiation of the two species.