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OBJECTIVE To establis h the fingerprint of Cynanchum auriculatum，to conduct its chemical pattern recognition analysis，and to determine the contents of four components at the same time. METHODS High performance liquid chromatography （HPLC）method was adopted. The determination was performed on ACE UExcel C 18 column with mobile phase consisted of acetonitrile-0.1％ phosphoric acid solution （gradient elution ）at the flow rate of 1.2 mL/min. The determination wavelength was set at 210 nm，and the column temperature was 40 ℃. The sample size wa s 10 μL. Taking qingyang shengenin as the reference ，HPLC fingerprints of 16 batches of C. auriculatum medicinal materials were drawn and similarity was evaluated by using the Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine （2012 edition）， and the common peaks were determined. SPSS 26.0 software andSIMCA 14.0 software were used for cluster analysis ，principal component analysis and orthogonal partial least squares- discriminant analysis. The differential components affecting the quality of C. auriculatum were screened by taking the value of variable importance in projection （VIP）greater than 1 as the standard ；same HPLC method was used to determine the contents of syringic acid ，acyl asclepiadelenin ，baishouwubenzophenone and qingyang shengenin. RESULTS There were 29 common peaks in 16 batches of C. auriculatum ，with a similarity of 0.723-0.998. Four common peaks were identified ，namely syringic acid （peak 7），acyl asclepiadoidin （peak 9），baishouwubenzophenone（peak 13）and qingyang shengenin（peak 15）. The results of cluster analysis showed that 16 batches of C. auriculatum could be clustered into three categories ，among which S 1 were grouped into one category，S3 were grouped into one c ategory，S2，and S 4-S16 were grouped into one category. The results of principal component analysis showed that the cumulative variance contribution rate of the five principal components was 88.706％，and the classification results were consistent with the results of cluster analysis. The results of orthogonal partial least squares-discriminant analysis showed that the common peaks （from large to small ）with VIP value greater than 1 were peak 20，peak 10，peak 25，peak 12， peak 15（qingyangshengenin），peak 21，peak 14，peak 16，peak 26，peak 22 and peak 17. The linear ranges of syringic acid，acyl asclepterin，baishouwubenzophenone and qingyangshengenin were 0.715 3-45.778 0，2.379 4-152.281 0，0.642 0- 41.085 0，14.541 6- 930.662 0 µg/mL respectively （all R 2＞0.999）. The quantitative limits were 0.357 7，0.475 9，0.642 0 and 2.423 6 μg/mL；the detective limits were 0.146 0，0.164 1，0.248 8 and 0.833 3 μg/mL，respectively. RSDs of precision ，repeatability and stability （24 h）tests were less than 3％；the average recoveries were 99.11％（RSD＝2.00％，n＝9），98.54％（RSD＝2.21％，n＝9）， 96.33％（RSD＝2.54％，n＝9）and 95.96％（RSD＝2.93％，n＝9）；the contents were 17.12-147.80，95.23-583.10（S8 below the quantitative limit ），16.91-210.88 and 211.68-3 587.15（S1 below the quantitative limit ）μg/g，respectively. CONCLUSIONS Established HPLC fingerprint and the method of content determination are stable ，reliable，accurate and reproducible. Combined with analysis of chemical pattern recognition ，it can be used for the quality control of C. auriculatum .
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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.
Subject(s)
Animals , Rats , Cynanchum , Fibrosis , Glycosides , Kidney/pathology , Liver , NF-kappa B/genetics , Rats, Sprague-Dawley , Toll-Like Receptor 4/geneticsABSTRACT
Objective: To investigate the effects of C-21 steroidal glucosides from Cynanchum auriculatum on renal fibrosis in rats caused by unilateral ureteral ligation and explore its mechanisms. Methods: A total of 15 rats were randomly chosen as sham operation group (Sham), while the remaining rats underwent unilateral ureteral ligation. The rats after the operation were randomly divided into four groups, namely the Sham operation group, the model group, the positive control group (20 mg/kg), C. auriculatum high group (400 mg/kg), C. auriculatum low group (200 mg/kg), 15 per group, and the rats in each group were administrated with corresponding drugs. After the intervention of 28 d, all the rats were sacrificed and the kidneys were then removed. The content of hydroxyproline was measured. HE and Masson staining were conducted to assess kidney pathological changes and renal fibrosis. The protein expression of TGF-β1, α-SMA, and E-cadherin were tested with immunohistochemistry and Western blotting. The mRNA expression of collagen-I and collagen-III was evaluated using qRT-PCR. Results: Compared with the model group, C-21 steroid glycosides significantly alleviated the kidney pathological injury and renal fibrosis, and reduced fibrous tissue and collagen proliferation. C-21 steroid glucosides from C. auriculatum can significantly reduce the kidney/body weight ratio and the content of hydroxyproline in kidney tissue (P < 0.05, 0.01), and it showed in a dose-dependent manner. Compared with the model group, the expression of α-SMA and TGF-β1 was decreased significantly, and the expression of E-cadherin was increased significantly (P < 0.05, 0.01) in C. auriculatum group. Moreover, compared with the model group, C-21 steroidal glucosides significantly down-regulated the mRNA expression of collagen-I and collagen-III mRNA (P < 0.01). Conclusion: C-21 steroid glycosides from C. auriculatum can effectively attenuate renal fibrosis in rats with unilateral ureteral ligation and its underlying mechanisms may be related to inhibiting the over-expression of collagen-I and collagen-III, down-regulating the expression of α-SMA and TGF-β1, and up-regulating the expression of E-cadherin. By regulating the tubular epithelial-mesenchymal transition, it exerts its anti-fibrotic effect.
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Cynanchi Bungei Radix is a traditional Chinese herbal medicine which has been used as a tonic agent owing to its primary abilities of tonifying liver and kidney, nourishing blood and replenishing semen, strengthening tendons and bones, promoting hair growth, and prolonging life. Pharmacological effects and material basis of Cynanchi Bungei Radix have been extensively investigated. This paper reviews the recent research progress of Cynanchi Bungei Radix based on the difference in the pharmacological effects and content of different chemical constituents in different species of Cynanchi Bungei Radix. It provides a scientific basis for further rational application of the medicinal value of different varieties of Cynanchi Bungei Radix.
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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.
Subject(s)
Animals , Mice , Carbon Tetrachloride , Chemical and Drug Induced Liver Injury , Drug Therapy , Cynanchum , Chemistry , Glucosides , Pharmacology , Hepatocytes , Liver , Oxidative Stress , Random AllocationABSTRACT
Objective: To study the chemical constituents from the EtOAc extract of endophytic fungal Plectosphaerella cucumerina YCTA2Z1. Methods: The chemical constituents were isolated and purified by silica gel column, Sephadex LH-20, and reverse-phase C-18 column chromatography as well as crystallization. Results: Thirteen 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). Conclusions: It 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.
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Objective To investigate the chemical constituents of Cynanchum auriculatum. Methods Compounds were isolated and purified by using a combination of various chromatographic techniques including D-101 macroporous adsorptive resins, silica gel, Sephadex LH-20, and other methods. Their structures were elucidated by MS, NMR, and other modern spectroscopies. Results Fifteen compounds were isolated and identified as isocopoletin (1), isofraxidin (2), deacetylmetaplexigenin (3), vomifoliol (4), 4,4-dimethyl heptanedioic acid (5), (+)-isolariciresinol (6), 3-hydroxypyridine (7), 3-hydroxy-2-methylpyridine (8), 5-hydroxyl-2-hydroxymethylpyridine (9), 2-methyl-6-(2’,3’,4’-trihydroxybutyl)-pyrazine (10), kiwiionol (11), picein (12), adenosine (13), cynanoneside B (14), and cynanoneside A (15). Conclusion Compounds 1-2, 4-13 are isolated from the plants of genus Cynanchum Linn for the first time, while compound 15 is firstly obtained from C. auriculatum.
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Objective To identify Cynanchum auriculatum and its closely related species using the ITS2 barcode. Methods A total of 54 samples of C. auriculatum and its related species were collected. Genomic DNAs were extracted from these samples. The ITS2 sequences of these samples were amplified and bidirectional sequenced by PCR. The obtained sequences were submitted to the Gen Bank and the ITS2 sequences of 47 samples belonging to 15 species were downloaded from the GenBank, and ITS2 sequences were annotated by ITS2 database. A total of 101 ITS2 sequences were aligned and the intraspecific and interspecific distances were calculated using the MEGA 5.0. Identification analyses were performed using the similarity search method and nearest distance method, and were presented intuitively by constructing neighbor-joining (NJ) tree. Results The length of all ITS2 sequences of C. auriculatum was 249 bp, which was a haplotype and was close to Cynanchum. There was a significant difference between the interspecific and intraspecific genetic distances of the ITS2 sequences. The NJ tree showed that C. auriculatum obviously differed from its closely related species, which showed high monophyly. According to the secondary structure of ITS2, it was also possible to distinguish between C. auriculatumi and Asclepiadaceae related species. Conclusion As a DNA barcode, ITS2 sequences can stably and accurately distinguish C. auriculatum from its closely related species and also provide a new technique to ensure the clinical safety in utilization of Chinese materia medica.
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Objective To study the chemical constituents of Cynanchum auriculatum. Methods Two steroidal glycosides were purified by column chromatography and their structures were elucidated by spectroscopic analysis. Results and Conclusion Compound 1 is a new steroidal glycoside, named kidjoranin-3-O-β-D-cymaropyranoside. Compound 2 is isolated from this plant for the first time.
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OBJECTIVE:To study the pharmacological action and mechanism of Cynanchum auriculatum(CYA)on the gastrointestinal tract.METHODS:Using atropine sulfate to inhibit mice's intestinal propulsive activity and the effect of CYA on the intestinal propulsive activity of mice was observed.Using clipping tail stimulation method to induce functional dyspepsia model in rats and the effect of CYA on nitrogen monoxidum(NO)level in gastric mucous membrane of the model rats was observed.Using cold and bitter rhubarb lax method to induce rat spleen-deficiency diarrhea model and the effect of CYA on levels of gastrin(GAS)and motilin(MTL)in the model rats was observed.RESULTS:CYA can apparently increase the intestinal propulsive activity of mice,markedly decrease the level of NO in gastric mucous membrane of functional dyspepsia model rats,and markedly increase levels of GAS in serum and MTL in plasma of spleen-deficiency diarrhea model rats.CONCLUSION:CYA can improve intestinal propulsive activity,which might be attributed to the decreased NO level in gastric mucous membrane and the elevated levels of GAS in serum and MTL in plasma.