Marsdenia tenacissima genome reveals calcium adaptation and tenacissoside biosynthesis.

Marsdenia tenacissima is a medicinal plant widely distributed in the calcium-rich karst regions of southwest China. However, the lack of a reference genome has hampered the implementation of molecular techniques in its breeding, pharmacology and domestication. We generated the chromosome-level genome assembly in Apocynaceae using combined SMRT sequencing and Hi-C. The genome length was 381.76 Mb, with 98.9% of it found on 11 chromosomes. The genome contained 222.63 Mb of repetitive sequences and 21 899 predicted gene models, with a contig N50 of 6.57 Mb. Phylogenetic analysis revealed that M. tenacissima diverged from Calotropis gigantea at least 13.43 million years ago. Comparative genomics showed that M. tenacissima underwent ancient shared whole-genome duplication. This event, together with tandem duplication, contributed to 70.71% of gene-family expansion. Both pseudogene analysis and selective pressure calculations suggested calcium-related adaptive evolution in the M. tenacissima genome. Calcium-induced differentially expressed genes (DEGs) were mainly enriched in cell-wall-related processes. Domains (e.g. Fasciclin and Amb_all) and cis-elements (e.g. MYB and MYC) frequently occurred in the coding and promoter regions of cell-wall DEGs, respectively, and the expression levels of these genes correlated significantly with those of calcium-signal-related transcription factors. Moreover, calcium addition increased tenacissoside I, G and H contents. The availability of this high-quality genome provides valuable genomic information for genetic breeding and molecular design, and lends insights into the calcium adaptation of M. tenacissima in karst areas.

Polyoxypregnane Glycosides from Root of Marsdenia tenacissima and Inhibited Nitric Oxide Levels in LPS Stimulated RAW 264.7 Cells.

Six new polyoxypregnane glycosides, marstenacisside F1−F3 (1−3), G1−G2 (4−5) and H1 (6), as well as 3-O-ß-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1→4)-ß-D-oleandropyranosyl-11α,12ß-di-O-benzoyl-tenacigenin B (7), were isolated from the roots of Marsdenia tenacissima. Their structures were established by an extensive interpretation of their 1D and 2D NMR and HRESIMS data. Compounds 1−7 were tenacigenin B derivatives with an oligosaccharide chain at C-3. This was the first time that compound 7 had been isolated from the title plant and its 1H and 13C NMR data were reported. Compounds 4 and 5 were the first examples of C21 steroid glycoside bearing unique ß-glucopyranosyl-(1→4)-ß-glucopyranose sugar moiety. All the isolated compounds were evaluated for anti-inflammatory activity by inhibiting nitric oxide (NO) production in the lipopolysaccharide-induced RAW 264.7 cells. The results showed that marstenacisside F1 and F2 exhibited significant NO inhibitory activity with an inhibition rate of 48.19 ± 4.14% and 70.33 ± 5.39%, respectively, at 40 µM, approximately equal to the positive control (L-NMMA, 68.03 ± 0.72%).

Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima.

The ethnobotanical plant Marsdenia tenacissima has been used for hundreds of years for Dai people in Yunnan Province, China. Previously, chemical investigations on this plant have revealed that pregnane glycosides were the main biological constituents. Nine new pregnane glycosides, marsdeosides A-I (1-9), were isolated from cultivated dried stems of the medicinal plant Marsdenia tenacissima in this study. The structures were analyzed by extensive spectroscopic analysis, including 1D, 2D NMR, HRESIMS, and IR spectroscopic analysis. The absolute configurations of the sugar moieties were identified by comparing the Rf values and specific optical rotations with those of the commercially available standard samples and the data reported in the literature. Marsdeosides A (1) featured an unusual 8,14-seco-pregnane skeleton. Compounds 1, 8, and 9 showed activity against nitric oxide production in lipopolysaccharide-activated macrophage RAW264.7, with IC50 values of 37.5, 38.8, and 42.8 µM (L-NMMA was used as a positive control, IC50 39.3 µM), respectively. This study puts the knowledge of the chemical profile of the botanical plant M. tenacissima one step forward and, thereby, promotes the sustainable utilization of the resources of traditional folk medicinal plants.

The Protective Effect of Marsdenia tenacissima against Cisplatin-Induced Nephrotoxicity Mediated by Inhibiting Oxidative Stress, Inflammation, and Apoptosis.

Cisplatin (Cis) is considered to be one of the most effective drugs for killing cancer cells and remains a first-line chemotherapeutic agent. However, Cis's multiple toxicities (especially nephrotoxicity) have limited its clinical use. Marsdenia tenacissima (Roxb.) Wight et Arn. (MT), a traditional Chinese medicine (TCM) employed extensively in China, not only enhances the antitumor effect in combination with Cis, but is also used for its detoxifying effect, as it reduces the toxic side effects of chemotherapy drugs. The aim of this study was to explore the therapeutic effect of MT on Cis-induced nephrotoxicity, along with its underlying mechanisms. In this study, liquid-mass spectrometry was performed to identify the complex composition of the extracts of MT. In addition, we measured the renal function, antioxidant enzymes, and inflammatory cytokines in mice with Cis-induced nephrotoxicity and conducted renal histology evaluations to assess renal injury. The expressions of the proteins related to antioxidant, anti-inflammatory, and apoptotic markers in renal tissues was detected by Western blotting (WB). MT treatment improved the renal function, decreased the mRNA expression of the inflammatory factors, and increased the antioxidant enzyme activity in mice. A better renal histology was observed after MT treatment. Further, MT inhibited the expression of the phospho-NFκB p65 protein/NFκB p65 protein (p-p65)/p65, phospho-inhibitor of nuclear factor kappa B kinase beta subunit/inhibitor of nuclear factor kappa B kinase beta subunit (p-IKKß/IKKß), Bcl-2-associated X (Bax), and Cleaved Caspase 3/Caspase 3 proteins, while the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Recombinant NADH Dehydrogenase, Quinone 1 (NQO1), and B-cell lymphoma-2 (Bcl-2) was increased. The present study showed that MT ameliorated renal injury, which mainly occurs through the regulation of the Nrf2 pathway, the NF-κB pathway, and the suppression of renal tissue apoptosis. It also suggests that MT can be used as an adjuvant to mitigate the nephrotoxicity of Cis chemotherapy.

[Mechanism of Marsdenia tenacissima against ovarian cancer based on network pharmacology and experimental verification].

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-12ß-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.

Laticifer ontogenesis and the chemical constituents of Marsdenia zehntneri (Apocynaceae) latex in a semiarid environment.

MAIN CONCLUSION: Anastomosed laticifers with intrusive growth produce latex containing methyl comate and betulin with economic and ecological value in arid environments. Climatic factors influence laticifer development in the apical meristem and vascular cambium. Latex is a complex emulsion with high medicinal as well as ecological value related to plant survival. Marsdenia zehntneri is a shrubby plant that grows on limestone outcrops in the semiarid regions of Brazil. We sought to characterize the ontogenesis of the laticifers of this species and to relate that process to climatic seasonality and phenology through anatomical, ultrastructural, and micro-morphometric evaluations of the apical meristem and vascular cambium. The histochemistry of the secretory structure was investigated and the chemical composition of the latex was analyzed. Phenological assessments were performed by monitoring phenological events for 1 year. The laticifers network of M. zehntneri permeates the entire primary and secondary body of the plant, providing a wide distribution system of defensive compounds. Its laticifers, of a distinct mixed type (anastomosed, with intrusive growth), are numerous and voluminous in the apical meristem but scarce and minute in the secondary phloem. Latex secretion involves the participation of oleoplasts, polysomes, and dictyosomes. Methyl 2,3-dihydroxy-ursan-23-oate, methyl 3-hydroxy-ursan-23-oate, and betulin are encountered in high proportions in the latex and have ecological and medicinal functions. The development of primary laticifers is related to the resumption of apical meristem activity with increasing day length at the end of the austral winter. The development of secondary laticifers is related to high summer temperatures and rainfall that favor vascular cambium activity. The wide distribution of laticifers, their seasonal pattern of secretion, and their latex composition contribute to the adaptation of M. zehntneri to its natural environment.

Distributions of eight bioactive components in rat tissues administered Marsdenia tenacissima extract orally detected through UPLC-MS/MS.

Marsdenia tenacissima (Roxb.) Wight et Arn. (M. tenacissima) is considered an anticancer medicine in traditional Chinese medicine, which is extensively used in clinical application since it has great therapeutic effects. Currently, although a number of articles have examined M. tenacissima in terms of its pharmacology and quality control, few have investigated the in vivo mechanism of M. tenacissima active ingredients. Previously, we have studied the pharmacokinetics of eight active ingredients after oral administration of M. tenacissima extracts in rat plasma. This study constructed a new scientific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach to simultaneously quantify the contents of tenacissosides B, G, H and I, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid and caffeic acid in rats orally administered M. tenacissima extract. The proposed approach was successfully used for investigating the distributions of those eight analytes in rat tissues, with digoxin being used as an internal control. The Eclipse Plus C18 RRHD column was used for determination at a column temperature of 30°C. The mobile phase system consisted of acetonitrile and water (supplemented with 0.1% formic acid) under optimal gradient elution conditions. Afterwards, this approach was validated according to the requirements for the analysis of biological samples developed by the US Food and Drug Administration, including precision, accuracy, stability and matrix effects. Based on tissue distribution analysis, those eight analytes showed rapid distribution within all the tested tissues. With regard to organic acid distribution, it followed the order stomach > liver > kidney > small intestine > lung > spleen > heart, whereas the four steroids followed the order stomach > lung > spleen > small intestine > liver > kidney > heart. The present study lays the theoretical foundation for the use and development of M. tenacissima in clinical practice.

Simultaneous determination of eight bioactive components in Marsdenia tenacissima extract in rat plasma by LC-MS/MS and its application in a pharmacokinetic study.

As a traditional Chinese medicine, Marsdenia tenacissima (Roxb.) Wight et Arn. plays an indispensable role in clinical practice owing to its specific efficacy in treating malignant tumors, leukocythemia, cystitis and asthma. This study aimed to establish a novel and scientific LC-MS/MS approach to simultaneously determine tenacissoside B, H, G and I, caffeic acid, cryptochlorogenic acid, chlorogenic acid and neochlorogenic acid from M. tenacissima extract within the rat plasma samples. Digoxin was used as the internal reference. All determinations were carried out using the Eclipse Plus C18 column, and water (containing 0.1% formic acid) was used as the mobile phase A, while acetonitrile was the mobile phase B for gradient elution. The UPLC methods were validated, including calibration curves, accuracy, precision, stability and recovery of the total eight analytes, in accordance with the requirements for biopharmaceutical analysis. Moreover, the proposed approach was also used in comprehensive pharmacokinetic research on those eight analytes in rats following M. tenacissima extract gavage. According to the pharmacokinetic parameters, tenacissoside B, I, H and G are the long-acting and primary bioactive constituents in M. tenacissima extract, with long mean residence times and high concentrations. Our findings shed light on the absorption mechanism and provide significant information for the clinical application of M. tenacissima.

Screening potential antagonists of epidermal growth factor receptor from Marsdenia tenacissima via cell membrane chromatography model assisted by HPLC-ESI-IT-TOF-MS.

Marsdenia tenacissima, or Tongguanteng in Chinese, is a traditional Chinese herb and has a broad application in clinical practice for its pharmacological effects of treating asthma, pneumonia, tonsillitis, pharyngitis tumors, etc. However, few studies have reported the screening of the active components of this medicine for tumor therapy. In this work, a two-dimensional analytical system was developed to screen antagonists of epidermal growth factor receptor (EGFR) from M. tenacissima. A fraction was retained on the EGFR cell membrane chromatography (CMC) column, separated and identified as tenacissoside G (TG), tenacissoside H (TH) and tenacissoside I (TI) by two-dimensional HPLC-IT-TOF-MS. Molecular docking and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay were carried out to assess the activity of TS (including TG, TH and TI). Molecular docking results showed that the binding mode of TS on EGFR is similar to that of gefitinib. The MTT assay demonstrated that gefitinib and TS (especially TI) could inhibit the growth of EGFR highly expressed cell lines in a dose-dependent manner in the range of 5-50 µmol/L. In conclusion, the two-dimensional EGFR/CMC-HPLC-IT-TOF-MS system could be a useful approach in drug discovery from traditional Chinese medicines for searching for potential antitumor candidates.

Oral and injectable Marsdenia tenacissima extract (MTE) as adjuvant therapy to chemotherapy for gastric cancer: a systematic review.

BACKGROUND: Marsdenia tenacissima extract (MTE) is a phytochemical widely used as complementary therapy in cancer care. This systematic review was conducted to investigate the anticancer and detoxification effects of MTE, as an adjuvant therapy to chemotherapy, for treating gastric cancer. METHODS: Ten databases were searched to identify randomized controlled trials (RCTs) comparing oral or injectable MTE plus chemotherapy versus chemotherapy alone for treating gastric cancer up to May 1, 2019. In meta-analyses, proportional odds ratios (PORs) with 95% confidence intervals (CIs) were pooled for the ordinal outcomes using the generalized linear model, and risk ratios (RRs) with 95% CIs were pooled for dichotomous outcomes using the Mantel-Haenszel method. RESULTS: Seventeen RCTs with 1329 individuals were included, with a moderate to high risk of selection and performance bias. Compared to chemotherapy alone, MTE adjuvant therapy significantly improved the response to anticancer treatment (POR 2.01, 95% CI 1.60-2.53) and patients' performance status (POR 3.15, 95% CI 2.22-4.48) and reduce the incidences of chemotherapy-induced leukopenia (RR 0.66, 95% CI 0.56-0.78), thrombocytopenia (RR 0.64, 95% CI 0.48-0.86), anemia (RR 0.89, 95% CI 0.72-1.10), nausea/vomiting (RR 0.79, 95% CI 0.69-0.91), hepatic injury (RR 0.77, 95% CI 0.61-0.96), and peripheral neurotoxicity (RR 0.77, 95% CI 0.59-1.01). However, MTE did not significantly alleviate anemia, diarrhea, constipation, kidney injury, and oral mucosal lesions after chemotherapy. Incidence of nausea/vomiting was lower in patients receiving oral MTE than those receiving injectable MTE (RR 0.47 vs. 0.82, interaction P = 0.04). Heterogeneity was generally low among these outcomes. Three out of five RCTs that reported survival data supported the effects of MTE for prolonging progression-free and/or overall survival. No studies reported safety outcomes of MTE. CONCLUSIONS: The current evidence with limitations of risk of selection and performance bias suggests that MTE, as an adjuvant therapy to chemotherapy, is effective for inhibiting cancer growth and reducing incidences of multiple chemotherapy side effects. Oral MTE may be a better choice. Uncertainty remains regarding the effects of MTE on survival endpoints and the subgroup differences between acute and chronic use of MTE and between different chemotherapy regimens.

Marsdenia Tenacissima Extract Inhibits Proliferation and Promotes Apoptosis in Human Ovarian Cancer Cells.

BACKGROUND Marsdenia tenacissima extract (MTE) is a traditional Chinese medicine that can be effectively used against various cancers. However, to the best of our knowledge, its role in ovarian cancer is not known. This study investigated the effects of MTE on human ovarian cancer SKOV3 cells. MATERIAL AND METHODS The viability and cell cycle of SKOV3 cells were assessed using the cell counting kit-8 (CCK-8) and propidium Iodide (PI) staining kit, respectively. Cell apoptosis and mitochondrial membrane potential (MMP) were detected by flow cytometry. The expression levels of proliferation-related and apoptosis-related factors were tested by quantitative real-time PCR (qRT-PCR) and Western blot assays, respectively. RESULTS We found that MTE markedly reduced the viability of SKOV3 cells in dose-dependent and time-dependent manners. MTE induced cell cycle arrest by downregulating the levels of cyclin D1and cyclin B1. MTE (10, 20, and 40 mg/mL) markedly increased apoptosis rates (2.77±0.6%, 4.95±0.97%, and 12.16±0.69%, respectively), and enhanced the loss of MMP. MTE obviously downregulated the expression of B cell lymphoma-2 (Bcl-2) and upregulated the expression levels of fibroblast-associated (Fas), Fas ligand (FasL), cleaved cysteinyl aspartate-specific proteinas-3 (caspase-3), and Bcl-2-associated X protein (Bax) compared to the control group. In addition, the expressions of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated protein kinase B (p-AKT), and phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) were decreased by MTE. CONCLUSIONS MTE inhibited proliferation and induced apoptosis of SKOV3 cells. The depression of the PI3K/AKT/mTOR pathway may augment the protective effect of MTE. Thus, MTE might be expected to be a new drug for curing ovarian cancer.

Polyoxypregnane Glycosides from the Roots of Marsdenia tenacissima and Their Anti-HIV Activities.

A continuous phytochemical study on the roots of Marsdenia tenacissima led to the isolation and identification of 13 new polyoxypregnane glycosides named marstenacissides B10-B17 (1, 2, 4, 7, 8, 11, 12, and 14) and marstenacissides A8-A12 (3, 9, 10, 13, and 15) in addition to two known polyoxypregnane glycosides marsdenosides M and L (5 and 6). Their structures were established by spectroscopic techniques and by comparison with the reported data in the literature. Moreover, the anti-HIV activities of these isolates and the previous isolated marstenacissides A1-A7 and B1-B9 were assessed, some of which exhibited slight or negligible effects against HIV-1.

Metabolic profiling of tenacigenin B, tenacissoside H and tenacissoside I using UHPLC-ESI-Orbitrap MS/MS.

Marsdenia tenacissima, which is widely used as an anticancer herb in traditional Chinese medicine, has been shown to possess anticancer activity. However, its metabolic profile is poorly investigated. Tenacigenin B is the major steroidal skeleton of C-21 steroids in M. tenacissima. Tenacissoside H and Tenacissoside I are detected at relatively high levels in M. tenacissima. Therefore, we studied their metabolic characteristics in human liver microsomes by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Fourteen metabolites were tentatively identified by accurate mass measurement and MS/MS fragmentation behavior. It was found that hydroxylation reactions were the major metabolic pathway of Tenacissoside H and Tenacissoside I in human liver microsomes, whereas the metabolic pathway of Tenacigenin B involved dehydrogenation reactions. This is the first time that the metabolic profile of C-21 steroids from M. tenacissima has been explored in human liver microsomes, which is of great significance for subsequent pharmacokinetic and interaction research. Biotransformation in vivo or in vitro may influence the structure of a compound and change its activity. Identification of their fragmentation behaviors and metabolites provides valuable and new information for further understanding the anti-tumor activity of M. tenacissima. Copyright © 2016 John Wiley & Sons, Ltd.

[Clinical value of Tongguanteng (Radix seu Herba Marsdeniae Tenacissimae) extract combined with chemotherapy in the treatment of advanced non-small cell lung cancer: a Meta-analysis].

OBJECTIVE: To investigate the clinical efficacy and safety of Tongguanteng (Radix seu Herba Marsdeniae Tenacissimae) extract combined with chemotherapy in the treatment of advanced non-small cell lung cancer (NCSLC) compared with chemotherapy alone. METHODS: Databases including Chinese National Knowledge Infrastructure, China Biology Medicine Disc, Wanfang, and MEDLINE were searched until April 1, 2014. Two assessors independently reviewed each trial. The primary outcome was the effective rate (ER) of Tongguanteng (Radix seu Herba Marsdeniae Tenacissimae) extract combined with chemotherapy. The secondary outcomes included quality of life improvement rate (QOLIR) and adverse reactions. Statistical calculations were performed by using Cochrane Collaboration Review Manager 5.2. RESULTS: A total of 888 patients from 15 studies, 13 randomized controlled trials (RCT) and two controlled clinical trials, were included. Compared with chemotherapy alone, Tongguanteng (Radix seu Herba Marsdeniae Tenacissimae) extract plus chemotherapy significantly improved ER [Risk ratio (RR) = 1.32, 95% CI, (1.14, 1.54)] (based on 15 studies) and QOLIR [RR = 2.04, 95% CI, (1.69, 2.47)] (based on 13 studies). Compared with chemotherapy alone, Tongguanteng (Radix seu Herba Marsdeniae Tenacissimae) extract plus chemotherapy significantly inhibited chemotherapy-induced white blood cell decline [RR = 0.79, 95% CI, (0.70, 0.90) (based on 10 studies), chemotherapy-induced platelet decline [RR = 0.77, 95% CI, (0.60, 0.98)] (based on 8 studies), and significantly alleviated nausea and vomiting (NV) [RR = 0.83, 95% CI, (0.71, 0.97)] (based on 7 studies). There was no significant difference in hemoglobin decline between the two therapies [RR = 0.88, 95% CI, (0.70, 1.09)] (based on 6 studies). CONCLUSION: This Meta-analysis suggests that Tongguanteng (Radix seu Herba Marsdeniae Tenacissimae) extract combined with chemotherapy may be more efficacious in the treatment of advanced NSCLC than chemotherapy alone. This effect includes enhancing ER and QOLIR, and weakening chemotherapy toxicity. However, large-scale RCTs are required to further investigate the short- and long-term effects of Tongguanteng (Radix seu Herba Marsdeniae Tenacissimae) extract.

Antitumor Effect of C21 Steroidal Glycosides on Adenoid Cystic Carcinoma Cell Line SACC83.

BACKGROUND: To observe the anti-tumor effects and possible mechanism of C21 steroidal glycoside (17-ß-Marsdenia tenacissima glycoside B (17-ß-tenacigenin B)) and Xiaoaiping injection in the adenoid cystic carcinoma cell line SACC83. METHODS: We used the salivary adenoid cystic carcinoma cell line SACC83 for the study and used MTT, flow cytometry, and Western-blot (WB) to study the anti-tumor efficacy and mechanism of 17-ß-tenacigenin B and Xiaoaiping injection. Three groups were divided as follows: group 1: control group (without drug treatment), group 2: SACC83 treated with 17-ß-Marsdenia tenacissima glycoside B, group 3: SACC83 treated with Xiaoaiping injec- tion. RESULTS: The 17-ß-tenacigenin B had no significant anti-tumor effect while the Xiaoaiping injection had a dosage-dependent growth inhibition effect on the adenoid cystic carcinoma cell line SACC83. According to WB, after the administration of the 17-p-tenacigenin B, the expression of apoptosis-related protein was not very significant; however, after the administration of the Xiaoaiping injection, the expression of apoptosis inhibition protein bcl-2 decreased while the expression of apoptosis promotion protein bax increased. CONCLUSIONS: The 17-ß-tenacigenin B had no obvious anti-tumor effects while the Xiaoaiping injection had good anti-tumor effects. Even though the 17-ß-tenacigenin B is an important component of the Xiaoaiping injection, in the in vitro experiment, compared with the Xiaoaiping injection, the 17-ß-tenacigenin B showed no good anti-tumor effects. However, we need further studies on in vivo experiments of 17-ß-tenacigenin B and the anti-tumor effect of other C21 steroidal glycoside monomers in the Xiaoaiping injection.

Simultaneous quantification of two steroidal glycosides after oral gavage of Marsdenia tenacissima extract in rats using a LC-MS/MS method.

A specific, sensitive and accurate analytical LC-MS/MS assay was developed for the simultaneous determination of two steroidal glycosides, tenacissoside H and tenacissoside I, in rat plasma. An Agilent ZORBAX SB-C18 column was used with an isocratic mobile phase system composed of methanol-water-formic acid (70:30:0.1, v/v/v) at a flow rate of 0.3 mL/min. The analysis was performed on a positive ionization electrospray mass spectrometer via selected reaction monitoring mode scan. One-step protein precipitation with acetonitrile was chosen to extract the analytes from plasma. The lower limits of quantification were 0.9 ng/mL for tenacissoside H and tenacissoside I. The intra- and inter-day precisions were 2.03-11.56 and 3.76-11.62%, respectively, and the accuracies were <110.28% at all quality control levels. The validated method was applied to a pharmacokinetic study in rats after oral gavage of Marsdenia tenacissima extract.

Analysis of the transcriptome of Marsdenia tenacissima discovers putative polyoxypregnane glycoside biosynthetic genes and genetic markers.

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine due to bioactive constituents of polyoxypregnane glycosides, such as tenacissosides, marsdenosides and tenacigenosides. Genomic information regarding this plant is very limited, and rare information is available about the biosynthesis of polyoxypregnane glycosides. To facilitate the basic understanding about the polyoxypregnane glycoside biosynthetic pathways, de novo assembling was performed to generate a total of 73,336 contigs and 65,796 unigenes, which represent the first transcriptome of this species. These included 27 unigenes that were involved in steroid biosynthesis and could be related to pregnane backbone biosynthesis. The expression patterns of six unigenes involved in polyoxypregnane biosynthesis were analyzed in leaf and stem tissues by quantitative real time PCR (qRT-PCR) to explore their putative function. Furthermore, a total of 15,295 simple sequence repeats (SSRs) were identified from 11,911 unigenes, of which di-nucleotide motifs were the most abundant.

[Effects of seed priming on physiology of seed germination and seeding growth of Marsdenia tenacissima under NaCl stress].

To offer the reference and method for salt damage in the cultivation of Marsdenia tenacissima, the seeds of M. tenacissima collected from Maguan city ( Yunnan province) were taken as the test materials to study the effects of different priming materials on improving germination and growth under high-level salt stress condition. Four different treatments, which were GA3, KNO3-KH2PO4, PEG-6000, NaCl, combined with ANOVA were applied to test the performance of germination energy, germination percentage, germination index, MDA, SOD, and CAT. The results showed that the seed germination was obviously inhibited under salt stress and the soaked seeds with different priming materials could alleviate the damage of salt stress. Under these treatments, the activities of SOD, CAT the content of soluble protein significantly increased. While the content of MDA significantly decreased. The maximum index was obtained when treated with 1.20% KNO3-KH2PO4, the germination percentage increased from 52.67% to 87.33% and the activity of SOD increased from 138.01 to 219.44 respectively. Comparing with the treatment of 1.20% KNO3-KH2PO4, the germination percentage of treating with 300 mg x L(-1) GA3 increased from 52.67% to 80.67%, while the activity of SOD increased from 138.01 to 444.61.

Polyoxypregnane steroids from the stems of Marsdenia tenacissima.

A new polyoxypregnane aglycone, tenacigenin D (1), and seven new C21 steroid glycosides, tenacissimosides D-J (2-8), were isolated from the stems of Marsdenia tenacissima. Their structures were determined by interpretation of their 1D and 2D NMR and other spectroscopic data, as well as by comparison with published values for related known compounds. Compound 1 was found to circumvent P-glycoprotein (P-gp)-mediated multidrug resistance through an inhibitory effect on P-gp with a similar potency to verapamil. In addition, compound 1 potentiated the activity of erlotinib and gefitinib in epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI)-resistant non-small-cell lung cancer cells.

In vitro and in vivo antitumor activities of tenacissoside C from Marsdenia tenacissima.

Tenacissoside C, a natural bioactive compound of C21 steroidal saponins, was isolated and purified from air-dried stems of Marsdenia tenacissima. In the present study, the MTT assay showed that tenacissoside C exhibited obvious cytotoxicity in K562 cells with IC50 values of 31.4, 22.2, and 15.1 µM for 24, 48, and 72 h, respectively. Flow cytometry analysis indicated that the antiproliferative activity induced by tenacissoside C might be executed via G0/G1 cell cycle arrest and proapoptosis in K562 cells. Western blotting analysis elucidated that: A) Tenacissoside C induced K562 cell cycle (G0/G1) arrest via downregulating cycline D1 protein expression; and B) Tenacissoside C induced K562 cell apoptosis via the mitochondrial pathway by downregulating Bcl-2 and Bcl-xL protein expression, upregulating Bax and Bak protein expression, and activating caspase-9 and caspase-3. In vivo, significant tumor growth inhibition activity of tenacissoside C was observed in K562 cell-bearing nude mice, accompanied by a significant antiangiogenic effect in vivo against K562 cells (a marked decrease in MVD) and associated with enhanced apoptotic cell death (TUNEL staining assay in vivo), both in dose-dependent manners. The treatment with tenacissoside C did not significantly affect body mass and macroscopic examination of the organs in this mouse tumor model.