Rhizoma Menispermi Research Articles

Daurisoline attenuates H2O2-induced chondrocyte autophagy by activating the PI3K/Akt/mTOR signaling pathway

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degeneration and intra-articular inflammation. Daurisoline (DAS) is an isoquinoline alkaloid isolated from Rhizoma Menispermi, whose antitumor and anti-inflammatory pharmacological effects have been demonstrated, but the effects of DAS on OA have rarely been researched. In this study, we aimed to explore the potential role of DAS in OA and its partial mechanism.Materials and methodsThe cytotoxicity of H2O2 and DAS toward chondrocytes was detected by the Cell Counting Kit-8 assay. Safranin O staining was used to detect chondrocyte phenotype changes. Cell apoptosis was measured by both flow cytometry and quantitative analysis of the protein levels of the apoptosis-related factors Bax, Bcl-2 and cleaved caspase-3 by western blot. Western blotting and immunofluorescence were used to assess the expression of the autophagy-related proteins LC3, Beclin-1 and p62. In addition, key signal pathway targets and matrix-degrading indicators were measured by western blot.ResultsOur results indicated that H2O2 induced human chondrocyte apoptosis and activated autophagy in a dose-dependent manner. DAS treatment dose-dependently reversed the expression of apoptosis-related proteins (Bax, Bcl-2 and cleaved caspase3) and the apoptosis rate induced by H2O2. Western blot and immunofluorescence analyses showed that DAS decreased the H2O2-induced upregulation of the autophagy marker Beclin-1 and the LC3 II/LC3 I ratio and upregulated the p62 protein level. Mechanistically, DAS inhibited autophagy through the activation of the classical PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis. In addition, DAS alleviated the H2O2-induced degradation of type II collagen and the high expression of matrix metalloproteinase 3 (MMP3) and MMP13.ConclusionOur research demonstrated that DAS alleviated chondrocyte autophagy caused by H2O2 through activation of the PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis and matrix degradation. In conclusion, these findings suggest that DAS may serve as a promising therapeutic strategy for OA.

Daurisoline inhibits hepatocellular carcinoma progression by restraining autophagy and promoting cispaltin-induced cell death

Hepatocellular carcinoma (HCC) is a common malignancy with high cancer-associated mortality. Suppressing autophagy has been reported to promote the efficiency of chemotherapy in HCC. Daurisoline (DAS) is a constituent of Rhizoma Menispermi, and functions as a potential autophagy inhibitor to perform different cellular events. In the present study, we found that DAS treatment up-regulated autophagosomes in HCC cells, accompanied with the increases of LC3-II and p62, demonstrating the disturbance of autophagic flux. Then, by the colocalization analysis, we identified that DAS did not repress the fusion of autophagosomes and lysosomes in HCC cells. However, Lysotracker and acridine orange (OA) staining showed that DAS could suppress lysosomal acidification, as evidenced by the decreased red fluorescence. Consistently, significant decreases in mature form of cathepsin B and cathepsin D were detected in DAS-treated HCC cells. Furthermore, DAS treatment markedly promoted the anti-cancer effects of cisplatin (cDDP) on HCC cells, as revealed by the dramatically reduced cell viability and proliferation, whereas the enhanced apoptosis. Moreover, the nude mice xenograft models with HCC confirmed that compared with cDDP alone group, DAS combined with cDDP significantly reduced tumor progression in vivo. Taken together, these findings elucidated that DAS could restrain autophagic flux, potentiating the chemosensitivity of HCC cells to cDDP treatment.

Direct targeting of HSP90 with daurisoline destabilizes β-catenin to suppress lung cancer tumorigenesis.

Lung cancer is the most frequent cancer worldwide with a poor prognosis. Identification of novel cancer targets and useful therapeutic strategies without toxicity are urgently needed. In this study, we screened natural products for anticancer bioactivity in a library consisting of 429 small molecules. We demonstrated for the first time that daurisoline, a constituent of Rhizoma Menispermi, repressed lung cancer cell proliferation by inducing cell cycle arrest at the G1 phase. Furthermore, daurisoline was found not only to suppress the growth of lung tumor xenografts in animals without obvious side effects, but also to inhibit cell migration and invasion. Mechanistically, quantitative proteomics and bioinformatics analyses, Western blotting and qRT-PCR confirmed that daurisoline exerted its anticancer effects by inhibiting the expression levels of β-catenin and its downstream targets c-myc and cyclin D1. Furthermore, our data from Drug Affinity Responsive Target Stability (DARTS), isothermal titration calorimetry (ITC) and a series of functional assays demonstrated that daurisoline could target HSP90 directly and disrupt its interaction with β-catenin, therefore increasing the ubiquitin-mediated proteasomal degradation of β-catenin. This study reveals that daurisoline could be a promising therapeutic strategy for the treatment of lung cancer.

Identification of Confusable Herbal Medicines by Mapping of Partial Degradation Products from Herbal Medicine Polysaccharides.

Partial degradation products (PDPs) of herbal medicine (HM) polysaccharides with precolumn derivatization using 1-phenyl-3-methy-5-pyrazolone (PMP) were mapped by high performance liquid chromatography (HPLC), and three groups of confusable HMs were differentiated using the PDP fingerprints assisted with cluster analysis (CA). Three variables of HPLC mobile phase, i.e. acetonitrile proportion, buffer concentration and pH value were optimized with PDP of β-cyclodextrin. Radix Glehniae and Radix Adenophorae; Radix Sophorae Tokinensis and Rhizoma Menispermi; Radix Achyranthis Bidentatae and Radix Cyathulae were successfully distinguished by the method, respectively. The results involving mass spectrometry analysis showed that these PDPs primarily included oligosaccharides and a few monosaccharides. The method can be used as an effective approach for the identification and quality control of HMs, and can also facilitate the in-depth study of biological activity and further development of HM polysaccharides to some extent.

Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells to camptothecin-induced toxicity.

Autophagy is a cellular bulk degradation pathway implicated in various diseases. Inhibition of autophagy has been regarded as a new therapeutic strategy for cancer treatment, especially in combination with chemotherapy. In our study, we identified two natural compounds, dauricine (DAC) and daurisoline (DAS), as two potent autophagy blockers through a high-content screening. DAC and DAS are alkaloids isolated from traditional Chinese medicine Rhizoma Menispermi. We systematically examined the effects of DAC and DAS on autophagy function in HeLa cells and found that DAC and DAS induced massive formation of autophagic vacuoles and lipidation of LC3. The accumulation of autophagic vacuoles and LC3 lipidation are due to blockage of autophagosome maturation as evidenced by interrupted colocalization of autophagsosome and lysosome, increased GFP-LC3/RFP-LC3 ratio and accumulation of autophagic substrate p62. Moreover, DAC and DAS impaired lysosomal function, as indicated by reduced lysosomal protease activity and increased lysosomal pH values. Importantly, we showed that DAC and DAS strongly inhibited the lysosome V-type ATPase activity. For the therapeutic potential, we found that DAC and DAS blocked the campothecin (CPT)-induced protective autophagy in HeLa cells, and dramatically sensitized the multiple cancer cells to CPT-induced cell death. In conclusion, our result shows that DAC and DAS are autophagy inhibitors which inhibit the lysosomal degradation of auophagic vacuoles, and sensitize the CPT-induced cancer cell death. The study implies the therapeutic potential of DAC and DAS in the treatment of cancers in combination of chemotherapy by inhibiting autophagy.

A sensitive and selective UPLC–MS/MS method for simultaneous determination of 10 alkaloids from Rhizoma Menispermi in rat plasma and its application to a pharmacokinetic study

A sensitive and selective liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous quantitation of 10 alkaloids (dauricine, daurisoline, N-desmethyldauricine, dauricicoline, dauriporphinoline, bianfugecine, dauricoside, stepholidine, acutumine and acutumidine) from Rhizoma Menispermi in rat plasma. After addition of internal standard (verapamil), plasma samples were pretreated by a single-step protein precipitation with acetonitrile. Chromatographic separation was performed on a Waters BEH C18 column with gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.3mL/min. The analytes were detected without interference in the multiple reaction monitoring (MRM) mode with positive electrospray ionization. The validated method exhibited good linearity over a wide concentration range (r≥0.9914), and the lower limits of quantification were 0.01–5.0ng/mL for all the analytes. The intra-day and inter-day precisions (RSD) at three different levels were both less than 13.4% and the accuracies (RE) ranged from −12.8% to 13.5%. The mean extraction recoveries of analytes and IS from rat plasma were all more than 77%. The validated method was successfully applied to a comparative pharmacokinetic study of 10 alkaloids in rat plasma after oral administration of Rhizoma Menispermi extract.

Rapid determination of eight oxoisoaporphine alkaloids in Rhizoma Menispermi by the optimal homogenate extraction followed by UPLC-MS/MS.

The objective of this study was to develop a rapid and reliable homogenate extraction (HGE) and ultra high-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method for simultaneous analysis of eight bioactive oxoisoaporphine alkaloids (including two new alkaloids) in Rhizoma Menispermi. HGE was optimized by response surface methodology (RSM) to obtain the maximum extraction efficiency of eight alkaloids. Separation was achieved on a Waters ACQUITY UPLC® BEH C18 column (50 × 2.1 mm(2), 1.7 μm) using gradient elution with a mobile phase consisting of acetonitrile and 0.2% formic acid in water. Quantification was performed with multiple reaction monitoring (MRM) mode using positive ESI as an interface. This is the first report of the simultaneous analysis of eight oxoisoaporphine alkaloids in Rhizoma Menispermi using a UPLC-MS/MS method; this analysis afforded good linearity, precision, and accuracy. Then, the method was successfully applied to determine the alkaloids in Rhizoma Menispermi from different sources.

Target Spot on Menispermum dauricum Caused by Streptobotrys (≡Streptotinia) caulophylli, a New Disease in China.

Menispermum dauricum (moonseed) (family Menispermaceae), a perennial twining vine, is an ornamental plant traditionally used in Chinese medicine. M. dauricum is distributed mostly in northeastern, northern, and eastern China, Japan, Korea, and southern Siberia (4). Rhizoma menispermi is the dry root of M. dauricum, which has detoxifying and dehumidifying effects, and is mainly used for the treatment of sore throat, enteritis, diarrhea, and rheumatism. From June to September in 2012 and 2013, target spots were observed on moonseed plants, with an incidence above 30% in the medicinal herb garden of the Institute of Special Animal and Plant Sciences at the Chinese Academy of Agricultural Science (44°02' N, 126°05' E) in Jilin Province. Lesions on the leaves were roughly circular, forming concentric rings of alternating light and dark brown bands with yellow halos at the margins, and up to 50 mm in diameter. Lesions coalesced to a large area capable of destroying the leaf. Under humid conditions, the lesions enlarged rapidly. Occasionally, grayish-white mycelia appeared on the lesions. Subsequently, grayish-brown conidiophores arose, singly or in dense groups, up to 700 μm high, with large loose conidial heads. Side branches and branchlets were tightly twisted, brown and 7 to 11 μm in width. Conidiogenous cells were inflated at the apex of the branches and delimited by a septum. Conidia were globose to subglobose, pale brown, unicellular, minutely echinulate, and rounding to 7.8 to 16.9 μm in diameter. Four isolates were obtained from necrotic tissue from leaf spots and cultured on potato dextrose agar (PDA) at 25°C. Mycelia grown on PDA were sparse, whitish-gray, and produced small black sclerotia within 3 to 5 days. Sclerotia were round or oval to oblong and 0.3 to 1.7 × 0.8 to 1.7 mm. No conidiophores or conidia were produced on PDA. The similar species Streptobotrys streptothrix had smooth conidia and small sclerotia. So, all isolates were identified as S. caulophylli based on their morphological and cultural characteristics (2,3). The internal transcribed spacer (ITS) region was amplified by using the primers ITS4 and ITS5 (1). The ITS sequences (529 bp) were identical in these four isolates (Accession No. HG918042). Pathogenicity tests were performed on healthy 2-year-old moonseed plants. Ten leaves were inoculated with a 0.6-cm diameter mycelial plug from 3-day-old PDA cultures for each isolate, and the inoculation sites covered with moistened sterile absorbent cotton. Another 10 leaves were inoculated with sterile PDA plugs as controls. All plants in the experiments were covered with plastic bags and maintained in a greenhouse at 20 to 25°C for 24 h. After 3 days, dark brown spots were observed on all leaves inoculated with these isolates. After 7 days, the classical symptoms were evident, while control plants remained healthy. The re-isolated pathogen was identified as S. caulophylli based on morphological analysis. The pathogenicity test was repeated with similar results. Currently, the economic importance of this disease is limited, but it may become a more significant problem with the cultivation area of M. dauricum increasing. To our knowledge, this is the first report of S. caulophylli causing target spot on M. dauricum in China.

Pharmacology Research Progress of Phenolic Alkaloids from <i>Menisphermum dauricum</i>

Phenolic alkaloids from Menisphermum dauricum (PAMD) was a compound of multiple fat soluble alkaloid extracted from Rhizoma Menispermi. PAMD had wide pharmacological effects including of anti-arrhythmia, anti-MI, anti-thrombosis, anti-dementia, anti-cancer, anti-inflammatory, inhibiting respiratory depression, influencing on heart electrophysiology, ptotecting heart and nerve after cerebral ischemia, suppressing pain, lowering blood pressure, improving learning disorder and so on.

Identification of nuclear factor-κB inhibitors in the folk herb Rhizoma Menispermi via bioactivity-based ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis.

BackgroundRhizoma Menispermi (RM) is the dried root of Menispermum dauricum DC, which is traditionally used to treat swelling and pain for sore throat, enteritis and rheumatic arthralgia in the clinic, but its bioactive compounds remain unclear.MethodsIn this study, RM extract was administered orally to ICR mice followed by challenging with an intratracheal Pseudomonas aeruginosa suspension. Then mortality, histological features of lung, and inflammatory cytokines were evaluated. RM treatment significantly ameliorated Pseudomonas aeruginosa-induced acute lung inflammation and reduced levels of inflammatory mediators. To screen for potential anti-inflammatory constituents of the RM extract, a simple and rapid method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) coupled with a luciferase reporter assay system to detect nuclear factor-κB (NF-κB) activity was established.ResultsUsing this system, seven potential NF-κB inhibitors were detected, including sinomenine, norsinoacutin, N-norsinoacutin-β-D-glucopyranoside, 6-O-methyl-laudanosoline-13-O-glucopyranoside, magnoflorine, laurifloline and dauricinoline. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which norsinoacutin, 6-O-methyl-laudanosoline-13-O-glucopyranoside laurifloline, dauricinoline and N-norsinoacutin-β-D-glucopyranoside were revealed as new NF-κB inhibitors.ConclusionThis method of UPLC-Q/TOF coupled with the luciferase reporter assay system was initially applied to the study of RM and was demonstrated to represent a simple, rapid and practical approach to screen for anti-inflammatory compounds. This study provided useful results for further investigation on the anti-inflammatory mechanism of RM.

Molecular Identification of Chinese Materia Medica and Its Adulterants Using ITS2 and <i>psbA-trnH</i> Barcodes: A Case Study on Rhizoma Menispermi

Rhizoma Menispermi, derived from the rhizoma of Menispermum dauricum DC., is one of the most popular Chinese medicines. However Rhizoma Menispermi is often illegally mixed with other species in the herbal market, including Aristolochia mollissimae Hance, which is toxic to the kidneys and potentially carcinogenic. The use of DNA barcoding to authenticate herbs has improved the management and safety of traditional medicines. In this paper, 49 samples belonging to five species, including 34 samples of M. dauricum, from different locations and herb markets in China were collected and identified using DNA barcoding. The sequences of all 34 samples of Rhizoma Menispermi are highly consistent, with only one site variation in internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA and no variations in the psbA-trnH region. The intra-specific genetic distance is much smaller than inter-specific one. Phylogenetic analysis shows that both sequences allow the successful identification of all species. Nearest distance and BLAST1 methods for the ITS2 and psbA-trnH regions indicate 100% identification efficiency. Our research shows that DNA barcoding can effectively distinguish Rhizoma Menispermi from its adulterants from both commercial and original samples, which provides a new and reliable way to monitor commercial herbs and to manage the modern medicine market.

Optimization of the micellar electrokinetic capillary chromatographic determination of dauricine and daurisoline in Rhizoma Menispermi and its herbal medicine using experimental design and radial basis function neural network

Orthogonal design has been applied to the optimization of separation and determination of dauricine and daurisoline in Rhizoma Menispermi and its herbal medicine by micellar electrokinetic capillary chromatography. Operational variables, such as the voltage, micelle concentration, buffer concentration and pH were optimized. Their different effects on peak resolution were studied by the experimental design method. Optimized separation conditions were obtained and successfully applied to the separation and determination of dauricine and daurisoline in real samples. The proposed method allows alkaloids in real samples to be determined within 15 min using a buffer system composed of 25 mM HAc, 25 mM NaAc and 2% polyoxyethylene sorbitan monolaurate (Tween-20) (pH 4.5). In addition, a radial basis function neural network with a “4-18-1” structure was developed based on the experimental results of orthogonal design and uniform design, and applied to the prediction of peak resolution of dauricine and daurisoline under the optimum separation conditions given by orthogonal design. The predicted results were in good agreement with the experimental values, indicating that radial basis function neural network may be a potential method for the selection of separation conditions in capillary electrophoresis.

An experimental study on four kinds of Chinese herbal medicines containing alkaloids using fluorescence microscope and microspectrometer

In order to find a useful method for uniting the identification and quality evaluation of Chinese herbal medicine, the techniques of fluorescence microscopy and microspectrometer were firstly applied to authenticate four kinds of herbal medicines, Caulis Sinomenii, Rhizoma Coptidis, Radix Sophorae Tonkinensis and Rhizoma Menispermi, as well as to measure the distribution of alkaloids in their cross-sections. The results showed that the fluorescence microscopic characteristics and the fluorescence emission spectra of the same tissues from the four kinds of Chinese herbal medicine were different, for example, the cortex of Radix Sophorae Tonkinensis emitted blue fluorescence instead of the yellow of Rhizoma Coptidis observed with emission filter of long-pass 397 nm, which could be effective to identify them. Furthermore, alkaloids, as active components of the above herbal medicines, were distributed in each part of herbal tissue but their fluorescence intensities in different parts of tissue were different. The results indicated that fluorescence microscope combined with microspectrometer could be a helpful method for identification and quality evaluation of Chinese herbal medicine.