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.

Investigating the Underlying Mechanisms of Ardisia japonica Extract's Anti-Blood-Stasis Effect via Metabolomics and Network Pharmacology.

OBJECTIVE: Our study aims to assess Ardisia japonica (AJ)'s anti-blood-stasis effect and its underlying action mechanisms. METHODS: The primary components of AJ were determined using liquid chromatography-mass spectrometry (LC-MS). The blood stasis model was used to investigate the anti-blood-stasis effect of AJ extract. The underlying mechanisms of AJ against blood stasis were investigated via network pharmacology, molecular docking, and plasma non-targeted metabolomics. RESULTS: In total, 94 compounds were identified from an aqueous extract of AJ, including terpenoids, phenylpropanoids, alkaloids, and fatty acyl compounds. In rats with blood stasis, AJ reduced the area of stasis, decreased the inflammatory reaction in the liver and lungs of rats, lowered the plasma viscosity, increased the index of erythrocyte deformability, and decreased the index of erythrocyte aggregation, suggesting that AJ has an anti-blood-stasis effect. Different metabolites were identified via plasma untargeted metabolomics, and it was found that AJ exerts its anti-blood-stasis effect by reducing inflammatory responses through the cysteine and methionine metabolism, linolenic acid metabolism, and sphingolipid metabolism. For the effect of AJ on blood stasis syndrome, the main active ingredients predicted via network pharmacology include sinensetin, galanin, isorhamnetin, kaempferol, wogonin, quercetin, and bergenin, and their targets were TP53, HSP90AA1, VEGFA, AKT1, EGFR, and PIK3CA that were mainly enriched in the PI3K/AKT and MAPK signaling pathways, which modulate the inflammatory response. Molecular docking was also performed, and the binding energies of these seven compounds to six proteins were less than -5, indicating that the chemical components bind to the target proteins. CONCLUSIONS: This study suggests AJ effectively prevents blood stasis by reducing inflammation.

Co-Fermentation of Marsdenia tenacissima with Ganoderma lucidum and Anti-Lung Cancer Effect of the Fermentation Products.

Marsdenia tenacissima (Roxb.) Wight et Arn. (MT), as a traditional Chinese and Dai herbal medicine, has anti-inflammatory, antibacterial, and antitumor properties. However, most of its main active substances are aglycones, such as tenacigenin A and tenacigenin B. As the bioavailability of MT is low and its medicinal active components are challenging to synthesize, it is primarily studied by biotransformation. This study aims to produce biotransformation products rich in pungent saponins by using MT as a fermentation medium for Ganoderma lucidum (G. lucidum). Through the preliminary screening of three medicinal fungi, it was found that G. lucidum and Ophiocordyceps sinensis (O. sinensis) can generally grow in the medium for MT; hence, the efficacy of the fermentation of the two types of fungi was screened using a mouse model of lung cancer. Finally, the co-fermentation of G. lucidum and MT was selected for further investigation. Non-target metabolomics analysis was performed on the products of MT with G. lucidum co-fermentation. We identified 12 specific saponins of MT from the fermentation products, and obtained a monomeric compound, tenacigenin A, from fermentation products. Most of the tenacigenin showed a significant upward trend, through tenacigenin A and tenacigenin B levels. The results showed that the efficacy of MT improved after fermentation by G. lucidum. Furthermore, the biotransformation of C21 steroidal glycosides in MT was the central reaction in this fermentation process. In summary, this study established a systematic and comprehensive co-fermentation system and pharmacodynamic evaluation method for MT, which not only enhanced the full utilization of effective active substances in MT but also provided a methodological reference for the development of other ethnic drugs.

Optimization of Cordyceps sinensis fermentation Marsdenia tenacissima process and the differences of metabolites before and after fermentation.

In this paper, we explored the interaction of factors which influenced the Cordyceps sinensis fermentation Marsdenia tenacissima (Roxb.) Wight et Arn, a Dai (a national minority of China) medicine, and the optimal fermentation conditions. The differences of C. sinensis metabolites in normal state (CN) and products of two-way liquid fermentation of C. sinensis and Marsdenia tenacissima (CM) and Marsdenia tenacissima (MT). The interactive effect of factors was analyzed and the best conditions are obtained through the box-behnken design (BBD) in response surface methodology (RSM). All metabolites were determined by ultra high performance liquid chromatography quadrupole time of flight mass spectrometer (UHPLC-Q-TOF-MS), analyzed and identified by metabonomics technology. Results showed that the optimum fermentation conditions were the concentration of raw medicinal materials is 160 g/L, the fermentation time is 6 days, the inoculation volume is 9.5%, the rotating speed is 170 rpm. 197 metabolites were identified in both positive ion and negative ion. 119 metabolites were significantly different between CN and CM. 43 metabolites were significantly different between CM and MT. Differential metabolic pathways were enriched. In conclusion, this paper optimizes the bidirectional fermentation process of M. tenacissima and C. sinensis through response surface methodology, and analyzes the changes of components from the level of metabonomics, so as to provide reference for exploring medicinal fungi fermentation of traditional Chinese medicine.

A Combined Phytochemistry and Network Pharmacology Approach to Reveal Potential Anti-NSCLC Effective Substances and Mechanisms in Marsdenia tenacissima (Roxb.) Moon (Stem).

Marsdeniae tenacissimae Caulis is a traditional Chinese medicine, named Tongguanteng (TGT), that is often used for the adjuvant treatment of cancer. In our previous study, we reported that an ethyl acetate extract of TGT had inhibitory effects against adenocarcinoma A549 cells growth. To identify the components of TGT with anti-tumor activity and to elucidate their underlying mechanisms of action, we developed a technique for isolating compounds, which was then followed by cytotoxicity screening, network pharmacology analysis, and cellular and molecular experiments. We isolated a total of 19 compounds from a TGT ethyl acetate extract. Two novel steroidal saponins were assessed using an ultra-performance liquid chromatography-photodiode array coupled with quadrupole time-of-flight mass (UPLC-ESI-Q/TOF-MS). Then, we screened these constituents for anti-cancer activity against non-small cell lung cancer (NSCLC) in vitro and obtained six target compounds. Furthermore, a compound-target-pathway network of these six bioactive ingredients was constructed to elucidate the potential pathways that controlled anticancer effects. Approximately 205 putative targets that were associated with TGT, as well as 270 putative targets that were related to NSCLC, were obtained from online databases and target prediction software. Protein-protein interaction networks for drugs as well as disease putative targets were generated, and 18 candidate targets were detected based on topological features. In addition, pathway enrichment analysis was performed to identify related pathways, including PI3K/AKT, VEGF, and EGFR tyrosine kinase inhibitor resistance, which are all related to metabolic processes and intrinsic apoptotic pathways involving reactive oxygen species (ROS). Then, various cellular experiments were conducted to validate drug-target mechanisms that had been predicted using network pharmacology analysis. The experimental results showed the four C21 steroidal saponins could upregulate Bax and downregulate Bcl-2 expression, thereby changing the mitochondrial membrane potential, producing ROS, and releasing cytochrome C, which finally activated caspase-3, caspase-9, and caspase-8, all of which induced apoptosis in A549 cells. In addition, these components also downregulated the expression of MMP-2 and MMP-9 proteins, further weakening their degradation of extracellular matrix components and type IV collagen, and inhibiting the migration and invasion of A549 cells. Our study elucidated the chemical composition and underlying anti-tumor mechanism of TGT, which may be utilized in the treatment of lung cancer.

Mechanism of Marsdenia tenacissima extract promoting apoptosis of lung cancer by regulating Ca2+/CaM/CaMK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima is a traditional Chinese medicine that has been used since the Ming dynasty. It is famous for its apoptotic effects on lung cancer. However, limited information is available about the underlying mechanisms. AIM OF THE STUDY: We aimed to determine the mechanisms by which different organic M. tenacissima extracts induce apoptosis in lung cancer cells. MATERIALS AND METHODS: PubMed and CNKI databases were screened for M. tenacissima components that may targets lung cancer; 223 components were selected for drug-like and pharmacokinetic analysis. Moreover, the inhibitory effect of different extracts of M. tenacissima on tumor cells was illustrated using CCK-8 and apoptosis assays, and intracellular [Ca2+] was measured. The in vivo effects were examined by body weight, tumor pathology, and TUNEL staining analysis in a Lewis lung carcinoma mouse model. In vivo levels of the Ca2+ signaling-related proteins Calmodulin, CaMKⅡ, p-CaMKⅡ, MEK1/2, p-MEK1/2, ERK, and p-ERK were measured by Western blot. RESULTS: Petroleum ether and ethyl acetate extracts of M. tenacissima have stronger inhibitory effects than other extracts on lung cancer cells, with IC50 values of 0.35 ± 0.04 mg/ml and 0.29 ± 0.02 mg/ml for LLC cells and 0.56 ± 0.05 mg/ml and 0.85 ± 0.04 mg/ml for A549 cells, respectively. Compared with the normal control group, A549 and LLC cells of treatment groups exhibited morphological changes typical of apoptosis, and the apoptosis rate was significantly higher, as determined by flow cytometry. Furthermore, the i[Ca2+] changed accordingly, causing a decrease in vivo in Calmodulin, CaMKⅡ, p-CaMKⅡ, p-MEK1/2 and p-ERK levels. CONCLUSIONS: M. tenacissima induces apoptosis, both in vitro and in vivo. Hydrophobic extracts are most effective; they increase i[Ca2+], decrease intracellular Calmodulin, CaMKⅡ, p-CaMKⅡ, p-MEK1/2 and p-ERK levels, and activate the apoptotic cascade.

Neuroprotective Effects of Ginsenosides against Cerebral Ischemia.

Ginseng has been used worldwide as traditional medicine for thousands of years, and ginsenosides have been proved to be the main active components for their various pharmacological activities. Based on their structures, ginsenosides can be divided into ginseng diol-type A and ginseng triol-type B with different pharmacological effects. In this study, six ginsenosides, namely ginsenoside Rb1, Rh2, Rg3, Rg5 as diol-type ginseng saponins, and Rg1 and Re as triol-type ginseng saponins, which were reported to be effective for ischemia-reperfusion (I/R) treatment, were chosen to compare their protective effects on cerebral I/R injury, and their mechanisms were studied by in vitro and in vivo experiments. It was found that all ginsenosides could reduce reactive oxygen species (ROS), inhibit apoptosis and increase mitochondrial membrane potential in cobalt chloride-induced (CoCl2-induced) PC12 cells injury model, and they could reduce cerebral infarction volume, brain neurological dysfunction of I/R rats in vivo. The results of immunohistochemistry and western blot showed that the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), silencing information regulator (SIRT1) and nuclear transcription factor P65 (NF-κB) in hippocampal CA1 region of some ginsenoside groups were also reduced. In general, the effect on cerebral ischemia of Rb1 and Rg3 was significantly improved compared with the control group, and was the strongest among all the ginsenosides. The effect on SIRT1 activation of ginsenoside Rb1 and the inhibition effect of TLR4/MyD88 protein expression of ginsenoside Rb1 and Rg3 were significantly stronger than that of other groups. The results indicated that ginsenoside Rg1, Rb1, Rh2, Rg3, Rg5 and Re were effective in protecting the brain against ischemic injury, and ginsenoside Rb1 and Rg3 have the strongest therapeutic activities in all the tested ginsenosides. Their neuroprotective mechanism is associated with TLR4/MyD88 and SIRT1 activation signaling pathways, and they can reduce cerebral ischemic injury by inhibiting NF-κB transcriptional activity and the expression of proinflammatory cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

Sotetsuflavone inhibits proliferation and induces apoptosis of A549 cells through ROS-mediated mitochondrial-dependent pathway.

BACKGROUND: Sotetsuflavone is isolated from Cycas revoluta Thunb., which has biological activity against tumors. However, the anti-proliferative effects of sotetsuflavone on A549 cells and its mechanism are not fully elucidated. METHODS: This study investigated the mechanisms of growth inhibition, cell cycle arrest and apoptosis in non-small cell lung cancer A549 cells induced by sotetsuflavone and evaluated whether sotetsuflavone can be safely utilized by humans as therapeutic agent. RESULTS: We found that sotetsuflavone had significant antiproliferative activity against A549 cells. At the same time, the reactive oxygen species (ROS) content increased while the mitochondrial membrane potential and the ratio of Bcl-2/Bax decreased. Cleaved caspase-3, cleaved caspase-9, cytochrome C and Bax expression increased, and Cyclin D1, CDK4, cleaved caspase-8 and Bcl-2 expression decreased. Interestingly, we demonstrated that sotetsuflavone could effectively inhibit the G0/G1 cycle progression, and then induce the endogenous apoptosis pathway. Our results show that sotetsuflavone could inhibit the growth of A549 cells by up-regulating intracellular ROS levels and causing the mitochondrial membrane potential to collapse, inducing G0/G1 phase arrest and endogenous apoptosis. CONCLUSIONS: In short, we confirm that sotetsuflavone had an inhibitory effect on A549 cells and discovered that it causes apoptosis of A549 lung cancer cells. Sotetsuflavone may be used as a novel candidate for anti-tumor therapy in patients with lung cancer.

[Transformation pathways in methanol of echinacoside, a principal effective ingredient of Cistanches Herba].

Echinacoside (ECH) is one of the active ingredients in Cistanche Herba and the principal effective component of Memoregain© as well. Moreover, a new agent namely Naoqing Zhiming tablet, derived from ECH has been licensed for clinical trials. However, the knowledge regarding the stability of is limited, till now, initiating a significant barrier for its further development along with the clinical trials. Herein, we aim to in depth characterize the transformation pattern of ECH in methanol. When ECH was stored in methanol, two primary products (P1 and P2) could be observed in HPLC chromatogram. A home-made automated fraction collector was configured via employing two 2-phase/6-port electronic valves to prepare P1 and P2. Following ¹H-NMR and LC-MS/MS assays, P1 and P2 were unambiguously identified as acteoside and cistanoside A, respectively. Moreover, the existences of cis-ECH, cis-acteoside, and cis-cistanoside A were claimed after careful analysis of the ¹H-NMR spectra of ECH, P1 and P2. Above all, the primary transformation pathways of ECH in methanol included methylation as well as hydrolysis, and mild transformation could also be initiated by cis/trans- configuration transferring for the caffeoyl group. The findings obtained in current study are envisioned to provide useful insight for the further development of ECH and the impurity detection of Naoqing Zhiming tablet. Moreover, the automated fraction collector configured in current study is able to serve as a versatile tool for the collection of signals-of-interest within phytochemical evaluations and impurity isolation.

Simultaneous determination of components with wide polarity and content ranges in Cistanche tubulosa using serially coupled reverse phase-hydrophilic interaction chromatography-tandem mass spectrometry.

To meet the demands from plant physiologists and pharmacognosists, sustainable efforts are being devoted by the analytical chemists from all over the world to search an approach being capable of simultaneously monitoring primary along with secondary metabolites. The key technical bottlenecks currently lie at affording satisfactory chromatographic and spectrometric performances for both hydrophilic and hydrophobic substances that span a great content range. Herein, reverse phase liquid chromatography was directly coupled with hydrophilic interaction chromatography, namely RPLC-HILIC, to integrate their merits, whereas dilution pumps were employed to tackle the mismatching for the mobile phase between them. On the other side, inferior parameters rather than the optimal ones were applied for those abundant ingredients to advance the upper limits of quantitation, such as echinacoside (1250.0µg/mL), mannitol (100.0µg/mL), and acteoside (125µg/mL), in mass spectrometer domain. As a desert parasitic plant as well as tonic materials, Cistanche tubulosa (CT) has drawn widely interests from plant physiologists and pharmacognosists regarding its quantitative metabolome. Simultaneous determination of 23 abundant and minor ingredients covering most chemical families in CT, i.e. amino acid, nucleoside, organic acid, phenylethanoid glycoside, lignan, and iridoid, was attempted to understand the physiologic patterns as well as pharmacological values of the crude materials. Although the analytes spanned wide polarity (-3.326≤cLogP≤1.421) and content (more than 5 orders of magnitude) ranges, satisfactory chromatographic and spectrometric behaviors were gained for all analytes. Reliable quantitation was demonstrated via various method validation assays, such as recovery, linearity, sensitivity, and precision. The contents of 23 hydrophilic and hydrophobic substances were quantified in 20 batches of CT. Significant variations occurred for the contents of those components. Echinacoside (1.35-387.50mg/g) was usually observed as the most abundant component, whereas ferulic acid (<0.0043mg/g) always exhibited trace distributions. Above all, the integrated equipment setup could serve as a fit-for-purpose tool for in-depth quality evaluation of CT and more importantly, for comprehensively understanding the metabolome of plants.

[Establishment of induction and culture system for hairy roots of Psammosilene tunicoides].

OBJECTIVE: To establish a culture system for Psammosilene tunicoides hairy roots, and provide technological aid for the large-scale production of P. tunicoides material. METHOD: The young leaves and stem segments of sterile plantlets were infected with ACCC10060 strain, and subsequently a culture system suitable for hairy roots growth was further established. RESULT: When explants were co-cultured with ACCC10060 (A600 0.8) on B5 media containing 20 mg x L(-1) Acetosyringo (AS) for 48 h, the hairy roots could be successfully induced, and it could achieve a higher induction rate using young leaves as explants than that of stem segments. The transfected hairy roots possessed the ability of kanamycin resistance and growth on hormone-free media, and synthesis of opines. All above results demonstrated that the present hairy roots originated in the infection of P. tunicoides tissues by ACCC10060 strains. After 35 d culture in liquid hormone-free MS (1/2 strength), the biomass of hairy roots increased 14.11 times (fresh weight) and 8. 39 times (dry weight), respectively, and the content of total saponins in hairy roots reached to 0.857% (DW), by contrast, it's only 0.388% and 0.217% in callus and seedlings respectively. CONCLUSION: Establishment of hairy roots culture of P. tunicoides provided a foundation for industrial production of active components from P. tunicoides culture.

[Experimental study on effect of curcumin on inhibiting injury of free radical in pulmonary fibrosis rats].

OBJECTIVE: To study on the effect and mechanism of curcumin on inhibiting injury induced by free radical in pulmonary fibrosis. METHOD: One hundred and forty-four male SD rats were randomly divided into 6 groups (24 rats in each group). Rats in the model control group, positive medicine group, and high, moderate and low curcumin groups were injected with a single dose of bleomycin by trachea, and rats in sham-model control group with same volume normal saline. One day after the injection, curcumin solution of different dosages (200,100,50 mg x kg(-1) x d(-1)) was respectively given to rats in the high, moderate and low curcumin group by daily gastrogavage, while equal volume of normal saline was given to those in the sham-model control group and model control group, and an equal volume of prednisone (0.56 mg x kg(-1) x d(-1)) was saline was given to those in positive medicine control group. On the 7, 14, 28 days, the contents of GSH-Px, SOD, MDA and iNOS in pulmonary tissues of different groups were measured. RESULT: Curcumin can raise the content of SOD and GSH-Px and lessen the level of MDA and iNOS. CONCLUSION: Curcumin can regulate the level of free radical in the body of rats with pulmonary fibrosis and lessen the oxidative injury of pulmonary tissues caused by free radical, in the body of rats with pulmonary fibrosis. The mechanisms of curcumin on idiopathic pulmonary fibrosis lie in adjusting the level of free radical and inhibiting the injury of lung tissue induced by free radical.