Exploring the mechanism of Marsdenia tenacissima in the treatment of hepatocellular carcinoma based on network pharmacology / 药学实践杂志

Objective To investigate the material basis and antitumor mechanism of Marsdenia tenacissima (MT) on hepatocellular carcinoma (HCC) by bioinformatics, network pharmacology and molecular docking technology. Methods Active ingredients of MT were collected by literature search and screened by Swiss ADME website, which targets were predicted by Swiss Target Prediction. The chip data of HCC (GSE147888) were downloaded from the NCBI Gene Expression Omnibus (GEO) database. Differentially expressed genes were screened by R software. HCC-related targets were collected from the Genecards and OMIM databases. The Venny online tool was used to obtain the intersection of the herbal medicine targets and the disease targets. Subsequently, drug-target network and protein–protein interaction (PPI) network were constructed by Cytoscape software and String platform. GO enrichment analysis and KEGG pathway analysis were performed to analysis the functions and pathways enriched by key genes. The expression of key genes in HCC and its effect on survival were analyzed by the GEPIA database. The Human Protein Atlas (HPA) was used to analyze the immunohistochemical expression of key genes in HCC. Finally, molecular docking was carried out to investigate interactions between the top five targets and their related active compounds. Results A total of 50 active components were screened and 12 common targets were identified related to MT and HCC. Scutellarein-4-Methylether, Tenasogenin, Sinapic Acid, Dresgenin and Kaempferol were considered as the critical components. JUN, MMP9 and PTGS2 were recognized as key therapeutic targets. The GO analyses demonstrated that key targets mainly involved in the process of gene silencing and inflammatory response. KEGG analysis suggested that key targets were enriched in TNF signaling pathway and IL-17 signaling pathway. Survival analysis by the GEPIA showed significant differences in the expression of ESR1, MMP1, MMP9, JUN, and PPARG between high and low risk groups. Immunohistochemical results showed that ESR1 and MMP9 were differentially expressed in normal and hepatocellular carcinoma tissues. The molecular docking results verified that the drug active ingredient could be stably bound to the target protein. Conclusion This study reflected the multi-component, multi-target and multi-pathway characteristics of the MT in the treatment of HCC, which could provide a scientific basis for the clinical application of MT in HCC.

Mechanism of Marsdenia tenacissima against ovarian cancer based on network pharmacology and experimental verification / 中国中药杂志

The present study aimed to explore the main active components and underlying mechanisms of Marsdenia tenacissima in the treatment of ovarian cancer(OC) through network pharmacology, molecular docking, and in vitro cell experiments. The active components of M. tenacissima were obtained from the literature search, and their potential targets were obtained from SwissTargetPrediction. The OC-related targets were retrieved from Therapeutic Target Database(TTD), Online Mendelian Inheritance in Man(OMIM), GeneCards, and PharmGKB. The common targets of the drug and the disease were screened out by Venn diagram. Cytoscape was used to construct an "active component-target-disease" network, and the core components were screened out according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened out according to the node degree. GO and KEGG enrichment analyses of potential therapeutic targets were carried out with DAVID database. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock. Finally, the anti-OC activity of M. tenacissima extract was verified based on SKOV3 cells in vitro. The PI3K/AKT signaling pathway was selected for in vitro experimental verification according to the results of GO function and KEGG pathway analyses. Network pharmacology results showed that 39 active components, such as kaempferol, 11α-O-benzoyl-12β-O-acetyltenacigenin B, and drevogenin Q, were screened out, involving 25 core targets such as AKT1, VEGFA, and EGFR, and the PI3K-AKT signaling pathway was the main pathway of target protein enrichment. The results of molecular docking also showed that the top ten core components showed good binding affinity to the top ten core targets. The results of in vitro experiments showed that M. tenacissima extract could significantly inhibit the proliferation of OC cells, induce apoptosis of OC cells through the mitochondrial pathway, and down-regulate the expression of proteins related to the PI3K/AKT signaling pathway. This study shows that M. tenacissima has the characteristics of multi-component, multi-target, and multi-pathway synergistic effect in the treatment of OC, which provides a theoretical basis for in-depth research on the material basis, mechanism, and clinical application.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3β/STAT3 signaling axis / 中国天然药物

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKTSer473 and p-GSK3βSer9, and decreased the expression of p-STAT3Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3β, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.

Effect of total saponins from marsdenia tenacissima on the proliferative inhibition of human liver cancer HepG2 cells / 肿瘤研究与临床

Objective To investigate the proliferative inhibition effect and mechanism of total saponins from marsdenia tenacissima on human liver cancer HepG2 cells. Methods Human liver cancer HpeG2 cells cultured conventionally were divided into the marsdenia tenacissima saponins A control group, the experimental group and the blank control group. The experimental group was treated with different mass concentrations of total saponins from marsdenia tenacissima (0.14, 0.29, 0.58, 1.15, 2.13 mg/ml), while the marsdenia tenacissima saponins A control group was treated with marsdenia tenacissima saponins A (1.0 mg/ml), and the blank control group was established stimultaneously. The methyl thiazolyl tetrazolium (MTT) method was used to detect the effect of total saponins from marsdenia tenacissima on the activity of HepG2 cells, and the cell morphology was observed by using inverted microscopy. The cell apoptosis rate was detected by using flow cytometry from Annexin V-FITC/PI staining. The expressions of bcl-2, bax and p53 were analyzed by using Western blot after the drug effect. Results The results of cell activity test showed that total saponins from marsdenia tenacissima could inhibit the proliferation of HepG2 cells at the concentration of 0.29, 0.58, 1.15, 2.13 mg/ml, which was positively correlated with the concentration. The half maximal inhibitory concentration (IC 50) of cell growth inhibition of total saponins from marsdenia tenacissima on HepG2 cells was 0.75 mg/ml. Under inverted microscopy, the adherent cells were significantly reduced, the cells fell off into clusters and the debris was increased after the effect of total saponins from marsdenia tenacissima. Moreover, flow cytometry showed that total saponins from marsdenia tenacissima could increase the late apoptosis rate of HepG2 cells (P< 0.01). Western blot showed that the expression of bcl-2 was 0.62±0.16, 0.31±0.15, 0.84±0.09 and 1.00±0.11 respectively in the experimental group (total saponins from marsdenia tenacissima 0.58 mg/ml and 2.13 mg/ml), the marsdenia tenacissima saponins A control group and the blank control group;the expression of bax protein was 0.75±0.10, 0.83±0.12, 1.00±0.14 and 0.15±0.02, respectively in the above four groups; the expression of p53 protein was 0.63±0.08, 0.78±0.11, 1.00±0.13 and 0.18±0.02 respectively in the above four groups. The protein expression of bcl-2 was decreased and the protein expressions of bax and p53 were increased in the experiment group, and there was a statistical difference between the experiment group and the blank control group (P< 0.05). Conclusions Total saponins from marsdenia tenacissima can upregulate the expression of bax by upregulating the expression of p53 gene, and inhibit the expression of bcl-2, which would cause the cascade reaction to induce the apoptosis of HepG2 cells. Its inhibitory effect can be realized through mitochondrial pathmay to induce apoptosis.

Effect of Marsdenia tenacissima extract on G2/M cell cycle arrest by upregulating 14-3-3σ and downregulating c-myc in vitro and in vivo / 中草药·英文版

Objective: Marsdenia tenacissima extract (MTE) is a traditional Chinese herbal medicine with anti-cancer activity. In some previous studies, different mechanism actions of the anti-cancer effect of MTE have been revealed. In this study, we first observed that MTE exhibited G2/M cell cycle arrest on two different human breast cancer cell lines, MDA-MB-231 and MCF-7 by mediating 14-3-3σ and c-myc. Methods: The effect of MTE on G2/M cell cycle arrest was evaluated in MDA-MB-231 and MCF-7 cell lines. MTT assay was done for evaluation of cell viability. Flow cytometry was employed for cell cycle analysis. Western blotting analysis and immunohistochemistry were performed to analyze the expression of G2/M cell cycle-related key protein in cells and tissue samples. Animal studies have been conducted to elucidate the anti-tumor effect of MTE. Results: Cell cycle is the backbone for developing cancer. Cell cycle proteins play a major role in the progression of cell cycle and cell proliferation. However, some key protein directly or indirectly modulate the action of cell cycle protein that highly affect cell cycle regulation. In order to investigate cellular proliferation of cancer, we observed that MTE induced the upregulation of 14-3-3σ and downregulation of c-myc, and then reduced the expression of G2/M cell cycle associated key protein, leading to the inhibition of cellular entry into mitosis phase. We also confirmed that MTE exerted a significant antitumor effect on the MDA-MB-231 xenograft model in vivo. Conclusion: G2/M cell cycle arrest occurred by the action of MTE, mediated by the upregulation of 14-3-3σ as well as downregulation of c-myc in MDA-MB-231 and MCF-7 cell lines.

Chemical profile characterization of Marsdenia tenacissima using HPLC-IT-TOF-MS / 中草药

Objective To in depth analyze the chemical profile of Marsdenia tenacissima using HPLC-IT-TOF-MS. Methods The pulverized materials were exacted with methanol in an ultrasonication manner and then separated on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Milford, MA, USA) that was eluted in gradient with 0.1% formic acid-acetonitrile. The data was collected using automatically triggered tandem mass spectrometric mode in positive/negative ionization polarities. The mass fragmentation patterns of polyoxypregnane derivatives were proposed using some authentic compounds. Results Six chlorogenic acid derivatives and 15 polyoxypregnane derivatives were definitely assigned by referring to reference components, whereas the other signals, including 100 polyoxypregnane derivatives, four flavonoids, and two chlorogenic acid derivatives were tentatively annotated via matching the acquired information with those achieved information and the proposed mass fragmentation rules. Conclusion The research efficiently and accurately analyzed the chemical profile of M. tenacissima using HPLC-IT-TOF-MS, which will provide meaningful information for the quality evaluation and the therapeutic mechanism investigation of M. tenacissima as well as its preparation Xiao-Ai-Ping.

Marsdenia tenacissima extract alters crucial metabolites in cancer, determined by 1 H NMR based metabolomics approach

ABSTRACT Altered metabolites level in the biosystems, is the potential cause of cancer, the primary reason of alteration of metabolism is change in nutrient consumption and waste excretion, as a result genetic mutation leads to cancer initiation and progression. Aberration of specific metabolites such as fumarate, succinate, 2-hydroxyglutarate may alter cell signaling. We collected liver and kidney samples and prepared for 1H NMR analysis, then executed NMR spectroscopy. We used a set of domestic R scripts to perform an unsupervised principal component analysis (PCA) and a supervised orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA). It signifies class discrimination for getting a clear separation, whereas PCA scores plot signifies the model group kept further away from the control group than drug group on the horizontal axis. In another PCA scores plots, most parts of the control group was overlapping with the drug group but was distant from the model group. Marsdenia tenacissima extract (MTE) (Chines name: Xiao-Ai-Ping, XAP) modulates level of crucial metabolites such as fumarate, lactate, succinate, determined by 1H NMR spectroscopy and their altered level contributes major role in cancer

Simultaneous Determination of 3 Kinds of Saponins in Marsdenia tenacissima by UPLC-MS/MS / 中国药房

OBJECTIVE:To establish the method for simultaneous determination of tenacissoside A,tenacissoside H and tenacissoside I in Marsdenia tenacissima. METHODS:UPLC-MS/MS method was adopted. The determination was performed on a Phenomenex Kinetex XB-C18column with mobile phase consisted of 0.1% formic acid solution-acetonitrile(gradient elution)at the flow rate of 0.2 mL/min. The column temperature was set at 40 ℃,and sample size was 5 μ L. Multiple reaction monitoring (MRM)mode was adopted with electrospray ion source as ion source,using positive ion scanning. Source jet voltage was 5 500 V,nebulizer pressure was 60 psi,heating pressure was 60 psi,curtain pressure was 20 psi and cone temp was set at 600 ℃. RESULTS:The linear ranges of tenacissoside A,tenacissoside H and tenacissoside I were 0.1-10 ng/mL(r=0.999 7),0.025-10 ng/mL(r=0.999 5),0.025-10 ng/mL(r=0.998 9),respectively;limited of quantation were 0.1,0.025,0.025 ng/mL,limited of detection were 0.05,0.012 5,0.012 5 ng/mL,respectively;RSDs of precision,stability and reproducibility tests were<4.0%. The recoveries were 97.67%-99.00%(RSD=0.47%,n=6),95.00%-101.67%(RSD=2.59%,n=6),96.67%-103.33%(RSD=2.83%, n=6). CONCLUSIONS:The method is simple,precise,stable and reproducible,and can be used for simultaneous determination of tenacissoside A,tenacissoside H and tenacissoside I in M.tenacissima.

Seed quality test methods of Marsdenia tenacissima / 中草药

Objective: To optimize the testing methods for seed quality of Marsdenia tenacissima, and provide basis for establishing seed quality standard of M. tenacissima. Methods: The seed quality of M. tenacissima from different producing areas was measured based on the International Seed Testing Protocol made up by ISTA and Rules for Agricultural Seed Testing issued by China. Results: The samples weight of M. tenacissima were at least 900 g for purity analysis and were at least 90 g for testing. The 1 000-seed weight was determined by 500-seed method, and the water content was carried out by higher temperature (133 ± 2)℃ for 6 h. After soaking in water for 24 h, M. tenacissima seeds were cultured in wet sand at 30℃ for 1-8 d under illumination for germination testing. Seed viability was tested by TTC method in 35℃ for 3 h. Conclusion: The seed testing methods for quality items of M. tenacissima have been initially established.

Optimization of Ultrasonic Extraction of Total Saponins from Marsdenia tenacissima by Box-Behnken Re-sponse Surface Method and Its Purification Technology / 中国药房

OBJECTIVE:To optimize the ultrasonic extraction and purification technology of total saponins from Marsdenia te-nacissima,and to prepare purified total saponins from M. tenacissima. METHODS:Using the extraction rate of total saponins from M. tenacissima as index,ethanol volume fraction,solvent-solid ratio and extraction temperature as factors,ultrasonic extraction technology of total saponins from M. tenacissima was optimized by single factor test and Box-Behnken response surface design. Us-ing the content of total saponins in elution,the effects of ethanol elution volume fraction on purification technology of total sapo-nins by D101 type macroporous adsorption resin was investigated. RESULTS:The optimal ultrasonic extraction technology was as follows as ethanol volume fraction of 85%,solvent-solid ratio of 18.5∶1 (ml/g),extraction temperature of 80 ℃. In verification test,the average extraction rate of total saponins from M. tenacissima was 4.80%(RSD=1.06%,n=3);it was close to predicted value 4.89%,and the deviation was 1.84%. The optimal purification technology was as follows as 45% ethanol as eluant,90%ethanol for purification. Verification test showed that the average content of total saponins from M. tenacissima was 62.45%(RSD=0.88%,n=3). CONCLUSIONS:The optimized technology can be used for the extraction and purification of total saponins from M. tenacissima. The technology is stable and reliable.

Studies on germination condition of Marsdenia tenacissima seeds / 中草药

In order to identify the germination behavior of Marsdenia tenacissima seeds. We studied on ground substance, soaking time, temperature, illumination, and pH value, which have the effects on the germination of M. tenacissima seeds. The results showed that M. tenacissima seeds can germinate in different ground substances especially in sandy soil, the effect of soaking time on the seed germination of M. tenacissima is not significant, the seeds could germinate well during 25-30℃. Based on these appropriate conditions, the effect of illumination is not significant but the illumination should be the key factor under inappropriate temperature. The effect of acidic soil is lighter than that of alkaline soil. The optimal germination condition of M. tenacissima seeds is 24 h of soaking at the temperature of 30℃ with sandy soil by which pH value is 7.0-7.5 under illumination.

Marsdenia tenacissima extract induces G0/G1 cell cycle arrest in human esophageal carcinoma cells by inhibiting mitogen-activated protein kinase (MAPK) signaling pathway / 中国天然药物

Marsdenia tenacissima extract (MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal cancer and other cancers in the digestive tract. In the present study, the potential mechanism for MTE's activity in esophageal cancer was explored. The effects of MTE on the proliferation of human esophageal cancer cells (KYSE150 and Eca-109) were investigated by the MTT assay, the BrdU (bromodeoxyuridine) incorporation immunofluorescence assay, and flow cytometric analysis. MTE inhibited cell proliferation through inducing G0/G1 cell cycle arrest in KYSE150 and Eca-109. Western blot analysis was employed to determine protein levels in the MTE treated cells. Compared with the control cells, the expression levels of the cell cycle regulatory proteins cyclin D1/D2/D3, cyclin E1, CDK2/4/6 (CDK: cyclin dependent kinase), and p-Rb were decreased significantly in the cells treated with MTE at 40 mg·mL(-1). In addition, MTE had an inhibitory effect on the MAPK (mitogen-activated protein kinase) signal transduction pathway, including ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38MAPK. Moreover, MTE showed little additional effects on the regulation of cyclin D1/D3, CDK4/6, and p-Rb when the ERK pathway was already inhibited by the specific ERK inhibitor U0126. In conclusion, these data suggest that MTE inhibits human esophageal cancer cell proliferation through regulation of cell cycle regulatory proteins and the MAPK signaling pathways, which is probably mediated by the inhibition of ERK activation.

Pregnane glycosides from stems of Marsdenia tenacissima / 中草药

Objective: To study the chemical constituents from the stems of Marsdenia tenacissima. Methods: The chemical constituents were isolated and purified by various column chromatographic methods. Their structures were identified on the basis of spectral data and chemical analysis. Results: Two pregnane glycosides were isolated from the stems of M. tenacissima and identified as 3-0-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-allopyranosyl- (1→4)-β-oleandropyranosyl-11α-O-tigloyltenacigenin B, named as tenacigenoside I (1); 3-O-β-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl- β-allopyranosyl-(1→4)-β-oleandropyranosyl-11α, 12β-di-O-acetyltenacigenin B (tenacigenoside K, 2). Conclusion: Compound 1 is a new compound and compound 2 is obtained from the M. tenacissima for the first time.

Pregnane Glycosides from Stems of Marsdenia tenacissima / 中草药·英文版

Objective To study the chemical constituents from the stems of Marsdenia tenacissima.Methods The chemical constituents were isolated by various column chromatography and their structures were identified by spectral and chemical analysis.Results Two pregnane glycosides were isolated from the stems of M.tenacissima and identified as3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 →4)-β-D-oleandropyranosyl-11α-Otigloyltenacigenin B,named as tenacigenoside I(1)and 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-Dallopyranosyl-(1→4)-β-D-oleandropyranosyl-11α,12β-di-O-acetyltenacigenin B,named as tenacigenoside K(2).Conclusion Compound 1 is a new compound,1H-NMR and 13C-NMR data of compound 2 are reported for the first time.

Stability of Chlorogenic Acid in Marsdenia Tenacissima Extract / 中国药房

OBJECTIVE:To study the factors influencing the stability of Chlorogenic acid in Marsdenia tenacissima extract.METHODS:The contents of Chlorogenic acid in Marsdenia tenacissima extract in different solvents,different lighting and different time under the heat were determined by HPLC.RESULTS:The Chlorogenic acid in Marsdenia tenacissima extract in organic solvents was more stable,in addition,lighting and heating temperature were important factors affecting the stability of the Chlorogenic acid.CONCLUSION:The stability of the Chlorogenic acid in Marsdenia tenacissima extract changes in different conditions;and the study results serve as a reference for the preparation,storage,analysis and production of Marsdenia tenacissima extract.

One new C_(21) steroid from canes of Marsdenia tenacissima / 中草药

Objective To study chemcial constituents in the canes of Marsdenia tenacissima.Methods Chemical constituents were isolated and purified by silica gel and ODS column chromatography.The structures were identified by means of physico-chemical and spectral data.Results From the 70% ethanol extract of the material,four compounds were isolated.Their structures were identified as 11?,12?-di-O-2-methylbutyryl-tenacigenin B (Ⅰ),11?-O-2-methylbutyryl-12?-O-acetyl-tenacigenin B (Ⅱ),tenacissoside H (Ⅲ),and marsdenoside A (Ⅳ),respectively.Conclusion Compound Ⅰ is a new C21 steroid compound,named tenacissoside O.

HPLC fingerprint analysis of Marsdenia tenacissima / 中成药

AIM:To establish the method of fingerprint analysis of Marsdenia tenacissima by HPLC-UV. METHODS: Analysis was performed on an Alltech C_(18)(4.6 mm?250 mm,5 ?m) column with the acetonitrile-(0.1%) acetic acid gradient.Fingerprint was finished in 55 min. The flow rate was 1.0 mL/min.The monitoring wavelength was at 328 nm. The column temperature was 30 ℃. RESULTS: 15 peaks were separated on HPLC fingerprint in Marsdenia tenacissima analyzed for chlorogenic acid,caffeic acid and part of derivatives. CONCLUSION: The method is reliable,accurate and can be used as a quality control method for Marsdenia tenacissima.