Modulation of in Vitro SARS-CoV-2 Infection by Stephania tetrandra and Its Alkaloid Constituents.

Botanical natural products have been widely consumed for their purported usefulness against COVID-19. Here, six botanical species from multiple sources and 173 isolated natural product compounds were screened for blockade of wild-type (WT) SARS-CoV-2 infection in human 293T epithelial cells overexpressing ACE-2 and TMPRSS2 protease (293TAT). Antiviral activity was demonstrated by an extract from Stephania tetrandra. Extract fractionation, liquid chromatography-mass spectrometry (LC-MS), antiviral assays, and computational analyses revealed that the alkaloid fraction and purified alkaloids tetrandrine, fangchinoline, and cepharanthine inhibited WT SARS-CoV-2 infection. The alkaloids and alkaloid fraction also inhibited the delta variant of concern but not WT SARS-CoV-2 in VeroAT cells. Membrane permeability assays demonstrate that the alkaloids are biologically available, although fangchinoline showed lower permeability than tetrandrine. At high concentrations, the extract, alkaloid fractions, and pure alkaloids induced phospholipidosis in 293TAT cells and less so in VeroAT cells. Gene expression profiling during virus infection suggested that alkaloid fraction and tetrandrine displayed similar effects on cellular gene expression and pathways, while fangchinoline showed distinct effects on cells. Our study demonstrates a multifaceted approach to systematically investigate the diverse activities conferred by complex botanical mixtures, their cell-context specificity, and their pleiotropic effects on biological systems.

Total alkaloids in Stephania tetrandra induce apoptosis by regulating BBC3 in human non-small cell lung cancer cells.

PURPOSE: This study investigated the effects of total alkaloids in Stephania tetrandra (TAS) and the main alkaloid components tetrandrine, fangchinoline and cepharanthine on the biological function of lung cancer cells and the mechanism underlying the synergistic antitumor effects of TAS and cisplatin. METHODS: RNA sequencing analysis was performed on TAS-treated H1299 cells. Differentially expressed genes were identified and analyzed, and the regulatory pathway was identified by gene set enrichment analysis. The mRNA and protein expression levels of the differentially expressed genes in cells were determined using quantitative reverse transcription-polymerase chain reaction and western blotting, respectively. Cell viability and wound healing assays evaluated the biological function of TAS and the main alkaloid components in non-small cell lung cancer (NSCLC) cells. Flow cytometry was used to determine the apoptosis rate in NSCLC cells. RESULTS: TAS inhibited the proliferation and migration of A549 and H1299 cells and increased the apoptosis rate in a time- and dose-dependent manner. When H1299 cells were treated with TAS (7.5 µg/ml), MGLL and BBC3 were identified as the possible differentially expressed genes. Pathways associated with cisplatin resistance were screened to investigate the effect of TAS on the apoptosis of NSCLC cells. TAS may regulate fatty acid metabolism and induce apoptosis through the upregulated expression of MGLL and BBC3. The combination of TAS at noncytotoxic concentrations (A549: 1.0 µg/ml; H1299: 3.0 µg/ml) and cisplatin significantly inhibited the viability of A549 and H1299 cells. CONCLUSION: TAS and the main alkaloid components exert anticancer activity in NSCLC by regulating tumor cell proliferation and apoptosis. Therefore, TAS and the main alkaloid components have the potential to be used as multi-targeted drugs for lung cancer treatment.

An integrated strategy for characterization of chemical constituents in Stephania tetrandra using LC-QTOF-MS/MS and the target isolation of two new biflavonoids.

LC-MS has been a widely used analytical technique for identification of natural compounds. However, sophisticated and laborious data analysis is required to identify chemical components, especially new compounds, from a large LC-MS dataset. The aim of this study is to develop an integrated data-mining strategy that combines molecular networking (MN), in-house polygonal mass defect filtering (MDF), and diagnostic fragment ion filtering (DFIF) to identify phytochemicals in Stephania tetrandra based on LC-MS data. S. tetrandra samples were prepared by matrix solid-phase dispersion extraction methods and then raw MS spectra were acquired using LC-QTOF-MS/MS. MN and in-house polygonal MDF classified the compounds roughly. Modified DFIF were then used in succession to place each spectrum into a specific class. Finally, the exact structures were deduced by fragmentation pathways and related botanical biogenesis, with the help of the narrowed classification from MN and MDF. The total workflow was a combination of data filtering and identification methods for rapid characterization of known compounds (dereplication) and discovery of new compounds. Consequently, 144 compounds were identified or tentatively identified in the aerial parts and roots of S. tetrandra, including 11 potentially new compounds and 63 compounds first identified in this species. Among 144 compounds, 61 were from the aerial parts exclusively, 8 were from the roots exclusively, and 75 were found in both parts. Furthermore, two new biflavonoids were isolated with the guide of LC-MS analysis and structurally elucidated by spectroscopic methods. In conclusion, the proposed data-mining strategy based on LC-MS can be used to profile chemical constituents with high efficiency and guide the isolation of new compounds from medicinal plants. The comparison of the components of the aerial parts and roots of S. tetrandra would be helpful for the rational utilization of the medicinal plant.

Stephtetrandrine A-D, bisbenzylisoquinoline alkaloids from Stephania tetrandra.

Four undescribed bisbenzylisoquinoline alkaloids, designated as Stephtetrandrine A-D, were isolated from the roots of Stephania tetrandra. Their structures were elucidated by IR, HRESIMS, ECD spectra, 1 D and 2 D NMR spectra and comparison with the literature data. Additional five known compounds (limacine, tetrandrine, N-trans-Feruloyltyramine, 2'-N-chloromethyltetrandrine, 2,2'-N-N-dichloromethyltetrandrine) were also isolated. N-trans-Feruloyltyramine was isolated from Stephania tetrandra for the first time. The isolated compounds were tested for monoamine oxidase, acetylcholinesterase, phosphoinositide 3-kinase α and human hepatoma cell HepG2 inhibitory activities. Stephtetrandrine C showed obvious inhibitory effect on human hepatoma HepG2, with IC50 value of 16.2 µM. Limacine and 2'-N-chloromethyltetrandrine showed moderate monoamine oxidase inhibitory effect with the IC50 values of 37.7 and 29.2 µM, respectively.

Effective extraction of bioactive alkaloids from the roots of Stephania tetrandra by deep eutectic solvents-based ultrasound-assisted extraction.

The efficient and green extraction of bioactive ingredients from natural plants play a vital role in their corresponding drug effects and subsequent studies. Recently, deep eutectic solvents (DESs) have been considered promising new green solvents for efficiently and selectively extracting substances from varied plants. In this work, an environment-friendly DESs-based ultrasonic-assisted extraction (DESs-UAE) procedure was developed for highly efficient and non-polluting extraction of alkaloids from the roots of Stephania tetrandra (ST). A total of fifteen different combinations of DESs, compared with traditional organic solvents (methanol and 95% ethanol) and water, were evaluated for extraction of bioactive alkaloids (FAN and TET) from ST, and the results revealed that DESs system made up of choline chloride and ethylene glycol with mole ratio of 1:2 exhibited the optimal extraction efficiency for alkaloids. Additionally, a four-factor and three-level Box-Behnken design (BBD), a particular pattern of response surface methodology (RSM), was used to optimize extraction conditions. RSM results indicated that the maximum extraction yields of FAN, TET, and TA were attained 7.23, 13.36, 20.59 mg/g, respectively, within extraction temperature of 52 °C, extraction time of 82 min, DES water content of 23% (v/v), and liquid-solid ratio of 23 mL/g. The measured results were consistent with the predicted values. Notably, the optimized DES extraction efficiency of TA, according to the experimental data analysis, is 2.2, 3.3 and 4.1 times higher than methanol, 95% ethanol and water, respectively. Meanwhile, based on 3D response surface plots, interactive effects plots and contour maps, the effects of the aforementioned four essential factors on the extraction yield and their interactions on the response were visualized. The results revealed that the mutual interactions between extraction temperature and liquid-solid ratio exhibited positive effects on all responses, while extraction time and water content in DES posed a negative effect. Therefore, these results suggest that DESs, as a class of novel green solvents, with the potential to substitute organic solvent and water, can be widely and effectively applied to extract bioactive compounds from natural plants.

Exploration of Habitat-Related Chemical Markers for Stephania tetrandra Applying Multiple Chromatographic and Chemometric Analysis.

Geo-authentic herbs refer to medicinal materials produced in a specific region with superior quality. Stephania tetrandra S. Moore (S. tetrandra) is cultivated in many provinces of China, including Anhui, Zhejiang, Fujian, Jiangxi, Hunan, Guangxi, Guangdong, Hainan, and Taiwan, among which Jiangxi is the geo-authentic origin. To explore habitat-related chemical markers of herbal medicine, an integrated chromatographic technique including gas chromatography-mass spectrometry (GC-MS), ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) combined with chemometric analysis was established. The established methods manifested that they were clearly divided into two groups according to non-authentic origins and geo-authentic origins, suggesting that the metabolites were closely related to their producing areas. A total of 70 volatile compounds and 50 non-volatile compounds were identified in S. tetrandra. Meanwhile, tetrandrine, fangchinoline, isocorydine, magnocurarine, magnoflorine, boldine, and higenamine as chemical markers were accurately quantified and suggested importance in grouping non-authentic origins and geo-authentic origins samples. The discriminatory analysis also indicated well prediction performance with an accuracy of 80%. The results showed that the multiple chromatographic and chemometric analysis technique could be used as an effective approach for discovering the chemical markers of herbal medicine to fulfill the evaluation of overall chemical consistency among samples from different producing areas.

Tetrandrine Suppresses Human Brain Glioblastoma GBM 8401/luc2 Cell-Xenografted Subcutaneous Tumors in Nude Mice In Vivo.

Tetrandrine (TET), a bisbenzylisoquinoline (BBI) alkaloid, is isolated from the plant Stephania tetrandra S. Moore and has a wide range of biological activity, including anticancer properties in vitro and in vivo. At first, we established a luciferase-expressing stable clone that was named GBM 8401/luc2 cells. Herein, the primary results indicated that TET reduced the total cell viability and induced cell apoptosis in GBM 8401/luc2 human glioblastoma cells. However, there is no available information showing that TET suppresses glioblastoma cells in vivo. Thus, we investigated the effects and mechanisms of TET on a GBM 8401/luc2 cell-generated tumor in vivo. After the tumor volume reached 100-120 mm3 in subcutaneously xenografted nude mice, all of the mice were randomly divided into three groups: Group I was treated with phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 25 mg/kg of TET, and Group III with 50 mg/kg of TET. All mice were given the oral treatment of PBS or TET by gavage for 21 days, and the body weight and tumor volumes were recorded every 5 days. After treatment, individual tumors, kidneys, livers, and spleens were isolated from each group. The results showed that TET did not affect the body weights, but it significantly decreased the tumor volumes. The TET treatment at 50 mg/kg had a two-fold decrease in tumor volumes than that at 25 mg/kg when compared to the control. TET decreased the total photon flux, and treatment with TET at 50 mg/kg had a lower total photon flux than that at 25 mg/kg, as measured by a Xenogen IVIS imaging system. Moreover, the higher TET treatment had lower tumor volumes and weights than those of the lower dose. The apoptosis-associated protein expression in the tumor section was examined by immunohistochemical analysis, and the results showed that TET treatment reduced the levels of c-FLIP, MCL-1, and XIAP but increased the signals of cleaved-caspase-3, -8, and -9. Furthermore, the hematoxylin and eosin (H & E) staining of kidney, liver, and spleen tissues showed no significant difference between the TET-treated and control groups. Overall, these observations demonstrated that TET suppressed subcutaneous tumor growth in a nude-mice model via the induction of cell apoptosis.

Complete chloroplast genome of Stephania tetrandra (Menispermaceae) from Zhejiang Province: insights into molecular structures, comparative genome analysis, mutational hotspots and phylogenetic relationships.

BACKGROUND: The Stephania tetrandra S. Moore (S. tetrandra) is a medicinal plant belonging to the family Menispermaceae that has high medicinal value and is well worth doing further exploration. The wild resources of S. tetrandra were widely distributed in tropical and subtropical regions of China, generating potential genetic diversity and unique population structures. The geographical origin of S. tetrandra is an important factor influencing its quality and price in the market. In addition, the species relationship within Stephania genus still remains uncertain due to high morphological similarity and low support values of molecular analysis approach. The complete chloroplast (cp) genome data has become a promising strategy to determine geographical origin and understand species evolution for closely related plant species. Herein, we sequenced the complete cp genome of S. tetrandra from Zhejiang Province and conducted a comparative analysis within Stephania plants to reveal the structural variations, informative markers and phylogenetic relationship of Stephania species. RESULTS: The cp genome of S. tetrandra voucher ZJ was 157,725 bp, consisting of a large single copy region (89,468 bp), a small single copy region (19,685 bp) and a pair of inverted repeat regions (24,286 bp each). A total of 134 genes were identified in the cp genome of S. tetrandra, including 87 protein-coding genes, 8 rRNA genes, 37 tRNA genes and 2 pseudogene copies (ycf1 and rps19). The gene order and GC content were highly consistent in the Stephania species according to the comparative analysis results, with the highest RSCU value in arginine (1.79) and lowest RSCU value in serine of S. tetrandra, respectively. A total of 90 SSRs have been identified in the cp genome of S. tetrandra, where repeats that consisting of A or T bases were much higher than that of G or C bases. In addition, 92 potential RNA editing sites were identified in 25 protein-coding genes, with the most predicted RNA editing sites in ndhB gene. The variations on length and expansion extent to the junction of ycf1 gene were observed between S. tetrandra vouchers from different regions, indicating potential markers for further geographical origin discrimination. Moreover, the values of transition to transversion ratio (Ts/Tv) in the Stephania species were significantly higher than 1 using Pericampylus glaucus as reference. Comparative analysis of the Stephania cp genomes revealed 5 highly variable regions, including 3 intergenic regions (trnH-psbA, trnD-trnY, trnP) and two protein coding genes (rps16 and ndhA). The identified mutational hotspots of Stephania plants exhibited multiple SNP sites and Gaps, as well as different Ka/Ks ratio values. In addition, five pairs of specific primers targeting the divergence regions were accordingly designed, which could be utilized as potential molecular markers for species identification, population genetic and phylogenetic analysis in Stephania species. Phylogenetic tree analysis based on the conserved chloroplast protein coding genes indicated a sister relationship between S. tetrandra and the monophyletic group of S. japonica and S. kwangsiensis with high support values, suggesting a close genetic relationship within Stephania plants. However, two S. tetrandra vouches from different regions failed to cluster into one clade, confirming the occurrences of genetic diversities and requiring further investigation for geographical tracing strategy. CONCLUSIONS: Overall, we provided comprehensive and detailed information on the complete chloroplast genome and identified nucleotide diversity hotspots of Stephania species. The obtained genetic resource of S. tetrandra from Zhejiang Province would facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of Stephania plants.

Mechanism of Tetrandrine Against Endometrial Cancer Based on Network Pharmacology.

BACKGROUND: Endometrial cancer (EC) is one of the most common gynaecological malignancies, and its incidence has been rising over the past decade. Tetrandrine, a bisbenzylisoquinoline alkaloid, has been isolated from a vine used in traditional Chinese medicine, Stephania tetrandra. However, the key mechanism of tetrandrine in EC is still unclear. PURPOSE: This research was designed to predict the molecular mechanisms of tetrandrine against EC based on network pharmacology and to further verify these predictions by in vitro experiments. METHODS: The potential therapeutic targets of tetrandrine against EC were predicted by using public databases. Afterwards, the protein-protein interaction (PPI) network of the common targets was constructed, and the key gene targets were obtained. Biological function and pathway enrichment analyses were performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Furthermore, molecular docking and in vitro experiments were carried out to verify the predictions. The cell counting kit­8 (CCK­8) assay, Hoechst 33258 staining, flow cytometry analysis, qRT-PCR, Western blot analysis and an immunofluorescence assay were performed. RESULTS: Our findings identified 111 potential therapeutic targets of tetrandrine against EC. We obtained 7 key gene targets from the PPI network analysis. Furthermore, GO enrichment analysis indicated that these targets were mainly associated with metabolic processes, responses to stimulus, and biological regulation. The KEGG pathway analysis showed that the common targets were mainly distributed in the PI3K/Akt signalling pathway. A potential interaction of tetrandrine with Akt1 was revealed by molecular docking. In addition, in vitro experiments showed that tetrandrine significantly inhibited cell proliferation and induced apoptosis in Ishikawa and HEC-1-B cells in dose- and time-dependent manners. The results also revealed that tetrandrine can downregulate the expression of Bcl-2 and upregulate the expression of Bax at the mRNA level. The mRNA levels of Akt were not significantly different in the various tetrandrine (0, 10 and 20µM) groups. However, Western blot analysis demonstrated that the protein expression ratios of p-Akt/Akt decreased at the protein level. The results were further confirmed by immunofluorescence assays. CONCLUSION: Based on bioinformatic analysis and experimental verification, our findings demonstrated that tetrandrine exerted tumour-suppressive effects on EC by regulating the PI3K/Akt signalling pathway.

An overview on the chemistry, pharmacology and anticancer properties of tetrandrine and fangchinoline (alkaloids) from Stephania tetrandra roots.

Tetrandrine (TET) and fangchinoline (FAN) are dominant bisbenzylisoquinoline (BBIQ) alkaloids from the roots of Stephania tetrandra of the family Menispermaceae. BBIQ alkaloids comprise two benzylisoquinoline units linked by oxygen bridges. The molecular structures of TET and FAN are exactly the same, except that TET has a methoxy (-OCH3) group, while FAN has a hydroxyl (-OH) group at C7. In this overview, the current knowledge on the chemistry, pharmacology and anticancer properties of TET and FAN have been updated. The focus is on colon and breast cancer cells, because they are most susceptible to TET and FAN, respectively. Against colon cancer cells, TET inhibits cell proliferation and tumor growth by inducing apoptosis and G1 cell cycle arrest, and suppresses adhesion, migration and invasion of cells. Against breast cancer cells, FAN inhibits cell proliferation by inducing apoptosis, G1-phase cell cycle arrest and inhibits cell migration. The processes involve various molecular mechanisms and signaling pathways. Some insights on the ability of TET and FAN to reverse multi-drug resistance in cancer cells and suggestions for future research are provided.

An overview on the chemistry, pharmacology and anticancer properties of tetrandrine and fangchinoline (alkaloids) from Stephania tetrandra roots / 中西医结合学报

Tetrandrine (TET) and fangchinoline (FAN) are dominant bisbenzylisoquinoline (BBIQ) alkaloids from the roots of Stephania tetrandra of the family Menispermaceae. BBIQ alkaloids comprise two benzylisoquinoline units linked by oxygen bridges. The molecular structures of TET and FAN are exactly the same, except that TET has a methoxy (-OCH

The Cholinesterase Inhibitory Properties of Stephaniae Tetrandrae Radix.

Stephaniae tetrandrae radix (STR) is a commonly used traditional Chinese medicine in alleviating edema by inducing diuresis. In the clinic, STR extracts or its components are widely used in the treatment of edema, dysuria, and rheumatism for the regulation of water metabolism. Furthermore, STR has been used in treating emotional problems for years by combining with other Chinese herbs. However, the material basis and mechanism of STR on the nervous system have not been revealed. Here, the main components of STR extracts with different extracting solvents were identified, including three major alkaloids, i.e., cyclanoline, fangchinoline, and tetrandrine. The cholinesterase inhibitory activity of STR extracts and its alkaloids was determined using the Ellman assay. Both cyclanoline and fangchinoline showed acetylcholinesterase (AChE) inhibitory activity, demonstrating noncompetitive enzyme inhibition. In contrast, tetrandrine did not show enzymatic inhibition. The synergism of STR alkaloids with huperzine A or donepezil was calculated by the median-effect principle. The drug combination of fangchinoline-huperzine A or donepezil synergistically inhibited AChE, having a combination index (CI) < 1 at Fa = 0.5. Furthermore, the molecular docking results showed that fangchinoline bound with AChE residues in the peripheral anionic site, and cyclanoline bound with AChE residues in the peripheral anionic site, anionic site, and catalytic site. In parallel, cyclanoline bound with butyrylcholinesterase (BChE) residues in the anionic site, catalytic site, and aromatic site. The results support that fangchinoline and cyclanoline, alkaloids derived from STR, could account for the anti-AChE function of STR. Thus, STR extract or its alkaloids may potentially be developed as a therapeutic strategy for Alzheimer's patients.

Comparative Transcriptome Analysis Reveals Candidate Genes Involved in Isoquinoline Alkaloid Biosynthesis in Stephania tetrandra.

The roots of Stephania tetrandra are used as a traditional Chinese medicine. Isoquinoline alkaloids are considered to be the most important and effective components in this herb, but little is known about the molecular mechanism underlying their biosynthesis. In this context, this study aimed to reveal candidate genes related to isoquinoline alkaloid biosynthesis in S. tetrandra. Determination of tetrandrine and fangchinoline in the roots and leaves of S. tetrandra by HPLC showed that the roots had much higher contents of the two isoquinoline alkaloids than the leaves. Thus, a comparative transcriptome analysis of the two tissues was performed to uncover candidate genes involved in isoquinoline alkaloid biosynthesis. A total of 71 674 unigenes was obtained and 31 994 of these were assigned putative functions based on BLAST searches against 6 annotation databases. Among the 79 isoquinoline alkaloid-related unigenes, 51 were differentially expressed, with 42 and 9 genes upregulated and downregulated, respectively, when the roots were compared with the leaves. The upregulated differentially expressed genes were consistent with isoquinoline alkaloid accumulation in roots and thus were deemed key candidate genes for isoquinoline alkaloid biosynthesis in the roots. Moreover, the expression profiles of 10 isoquinoline alkaloid-related differentially expressed genes between roots and leaves were validated by quantitative real-time polymerase chain reaction, which indicated that our transcriptome and gene expression profiles were reliable. This study not only provides a valuable genomic resource for S. tetrandra but also proposes candidate genes involved in isoquinoline alkaloid biosynthesis and transcription factors related to the regulation of isoquinoline alkaloid biosynthesis. The results lay a foundation for further studies on isoquinoline alkaloid biosynthesis in this medicinal plant.

History of uses, phytochemistry, pharmacological activities, quality control and toxicity of the root of Stephania tetrandra S. Moore: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: the root of Stephania tetrandra S. Moore, known as Fangji in China (Chinese: ), is a traditional Chinese medicine (TCM) with a long history of use. Fangji is a type of medicine used to treat various diseases, including rheumatism, arthralgia, edema and beriberi, unfavorable urination, and eczema. AIM OF THIS REVIEW: There are many newly published reports on the history of uses, phytochemistry, pharmacological activity, quality control and toxicity of Fangji; however, no comprehensive systematic review exists. Therefore, the purpose of this review is to compile the latest and most comprehensive information on Fangji and provide a scientific basis for future research. MATERIALS AND METHODS: A systematic literature search was conducted using multiple electronic databases, including SciFinder, Web of Science, PubMed, Science Direct, ACS Publications, J-stage, SpringerLink, Thieme, Wiley, and CNKI. Information was also collected from journals and Chinese Pharmacopoeia. RESULT: Thus far, there were uses of Fangji against 20 different diseases/disorders, such as relieving edema and rheumatism pain, treating cough and asthma, treating enuresis, astringent urine and beriberi edema, purging blood and damp heat, and dispelling wind evil and dampness, etc. 48 compounds have been isolated from Fangji, belonging to alkaloids, flavonoids, and steroids, other compounds. The crude extracts and isolated compound of Fangji have shown a wide range of pharmacological activities, such as anti-tumor, anti-inflammatory, and neuroprotective activities, as well as role in reoxygenation, and antimicrobial effect, etc. Moreover, qualitative and quantitative analyses of quality control are reviewed, including qualitative analyses for the identification of compounds, as well as fingerprint and quantitative analyses by high performance liquid chromatography (HPLC) and capillary electrochromatography (CE). In the toxicity study, the hepatotoxicity, hepatorenal toxicity, nephrotoxicity, subacute and acute toxicities of the alcohol extract and water extract of Fangji, and tetrandrine were studied in-vitro and in-vivo experiments. CONCLUSION: In the history of uses, Fangji can be used to treat a variety of diseases, most of which are manifested in removing wind and dampness. In recent years, the phytochemistry of Fangji has rarely been reported. The pharmacological activities of Fangji mainly focus on the compounds, tetrandrine and fangchinoline, and there are a few reports on the pharmacological studies of other compounds in Fangji. Moreover, the quality control of Fangji lacks a standard fingerprint to distinguish Fangji from other easily-confused medicinal materials. In the toxicity study, there is no report on the mechanism of toxicity research. Therefore, further studies on such mechanisms are needed.

Characterization of O-methyltransferases involved in the biosynthesis of tetrandrine in Stephania tetrandra.

Tetrandrine is the most effective small molecule that has been found to inhibit the Ebola virus. It is a typical bisbenzylisoquinoline alkaloid and is the main active ingredient in Stephania tetrandra. Metabolic engineering and synthetic biology are potential methods for efficient and rapid acquisition of tetrandrine. S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting step involved in the biosynthesis of tetrandrine. In this study, we identify S-adenosyl-L-methionine: (S)-norcoclaurine-6-O-methyltransferase from S. tetrandra, which catalyzes the conversion of (S)-norcoclaurine to (S)-coclaurine. Four 6OMT-like genes were cloned from S. tetrandra. An in vitro enzyme assay showed that St6OMT1 could catalyze the conversion of (S)-norcoclaurine to produce (S)-coclaurine. St6OMT2 can catalyze the production of very few (S)-coclaurine molecules, accompanied by more by-products with m/z 300, compared to St6OMT1. The newly discovered 6OMTs will provide an optional genetic component for benzylisoquinoline alkaloid (BIA) synthetic biology research. This work will lay the foundation for the analysis of the biosynthetic pathway of tetrandrine in S. tetrandra.

[Research development on modern pharmacological effect of tetrandrine].

Han stephania, also known as Stephania tetrandra, expelling wind, relieve pain and inducing diuresis for removing edema, is a traditional Chinese medicine for treating rheumatic arthralgia. Alkaloids have an important pharmacodynamic basis in S. tetrandra, and tetrandrine is one kind content of bisbenzylisoquinoline alkaloids, which has many biological activities. These activities include anti-tumor in many ways, clinically inhibiting multiple inflammatory factors, preventing and treating liver fibrosis and renal fibrosis and many other kinds of fibrotic diseases, and in addition, tetrandrine could work synergistically with other drugs. In recent years, through in-depth research by scholars at home and abroad, it has been found that tetrandrine has a protective effect on the nervous system and ischemia-reperfusion injury. At the same time, as a calcium ion antagonist, tetrandrine could effectively block the deposition of calcium ions inside and outside the cell. In summary, the application prospect of tetrandrine in clinical practice is very extensive. In this paper, the pharmacological effects of tetrandrine and the possible mechanisms for these effects are summarized, and review its current research progress. It is hoped that the possible application direction of tetrandrine can be revealed more comprehensively, and provide better enlightenment and ideas for clinical application.

Bioactive bisbenzylisoquinoline alkaloids from the roots of Stephania tetrandra.

Ten new bisbenzylisoquinoline alkaloids (1-10) and eight known analogues (11-18) were obtained from the roots of Stephania tetrandra. The structures of these compounds were determined by spectroscopic methods, single-crystal X-ray diffraction, electronic circular dichroism analyses, and chemical method. Compounds 1, 15, and 16 showed the better anti-inflammatory activities with IC50 values of 15.26 ± 2.99, 6.12 ± 0.25, and 5.92 ± 1.89 µM, respectively. Compound 18 possessed cytotoxic activities against MCF-7, HCT-116, and HepG2 cell lines with IC50 values of 2.81 ± 0.06, 3.66 ± 0.26, and 2.85 ± 0.15 µM, respectively.

Aporphine and phenanthrene alkaloids with antioxidant activity from the roots of Stephania tetrandra.

Five new alkaloids (1-5), including three new aporphine alkaloids and two new phenanthrene alkaloids, together with 10 known compounds (6-15) were obtained from the roots of Stephania tetrandra. Their structures were elucidated by spectroscopic methods, single-crystal X-ray diffraction, and electronic circular dichroism analyses. Compounds 7-10, and 13 showed antioxidant activities with malondialdehyde (MDA) inhibitory rates of 62.50 ± 1.91 to 98.44 ± 0.34% at the concentration of 10 µM.

Comprehensive profiling of Stephania tetrandra (Fangji) by stepwise DFI and NL-dependent structure annotation algorithm-based UHPLC-Q-TOF-MS and direct authentication by LMJ-HRMS.

Stephania tetrandra S. Moore, a widely used traditional antirheumatic herbal medicine (HM), is a rich source of isoquinoline alkaloids. With the exception of the two recognized isoquinolines, viz. tetrandrine and fangchinoline, the other isoquinoline alkaloids present in S. tetrandra have not been clearly clarified. In addition, due to their similar names and morphological similarities, S. tetrandra is often mistakenly substituted and adulterated with the nephrotoxic Aristolochia fangchi. In this study, ultra-high-performance liquid chromatography-triple time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was initially employed to comprehensively profile the isoquinolines from S. tetrandra. To overcome the complexities arising due to the similar mass behaviors of the isoquinolines, a stepwise diagnostic fragment ion (DFI) and neutral loss (NL)-dependent structure annotation algorithm was proposed, and this accelerated the identification of 393 isoquinolines distributed over twenty classes. Consequently, liquid microjunction surface sampling-high-resolution mass spectrometry (LMJ-HRMS) was deployed in an attempt to directly authenticate S. tetrandra by the chemical profiling of its crude slice. By matching the 393 isoquinolines, the 87 peaks detected by LMJ-HRMS were assigned to 270 isoquinolines, including the recognized tetrandrine and fangchinoline. The absence of aristolochic acid-related mass signals confirmed the authentication of S. tetrandra. In summary, LMJ-HRMS can be considered a direct, nondestructive, high-throughput, and environment-friendly analytical method for the authentication of HMs. Moreover, the stepwise DFI- and NL-dependent structure annotation algorithm-based UHPLC-Q-TOF-MS method allowed high-coverage detection and high-quality data processing of the inherent structural similarity and complexity of isoquinolines or other phytochemical compounds.

Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling.

OBJECTIVE: Podocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca2+ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra, with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6. METHODS: Immortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0-800 µM) and tetrandrine (0-40 µM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 µM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca2+ concentration were measured. RESULTS: Maximum safe concentrations of 100 µM valsartan and 10 µM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca2+ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 µM valsartan and 10 µM tetrandrine for 48 h significantly inhibited intracellular Ca2+ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6. CONCLUSION: Elevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca2+ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage.