Stephania tetrandra and Its Active Compound Coclaurine Sensitize NSCLC Cells to Cisplatin through EFHD2 Inhibition.

BACKGROUND: Adjuvant chemotherapy, particularly cisplatin, is recommended for non-small cell lung carcinoma (NSCLC) patients at high risk of recurrence. EF-hand domain-containing protein D2 (EFHD2) has been recently shown to increase cisplatin resistance and is significantly associated with recurrence in early-stage NSCLC patients. Natural products, commonly used as phytonutrients, are also recognized for their potential as pharmaceutical anticancer agents. RESULT: In this study, a range of Chinese herbs known for their antitumor or chemotherapy-enhancing properties were evaluated for their ability to inhibit EFHD2 expression in NSCLC cells. Among the herbs tested, Stephania tetrandra (S. tetrandra) exhibited the highest efficacy in inhibiting EFHD2 and sensitizing cells to cisplatin. Through LC-MS identification and functional assays, coclaurine was identified as a key molecule in S. tetrandra responsible for EFHD2 inhibition. Coclaurine not only downregulated EFHD2-related NOX4-ABCC1 signaling and enhanced cisplatin sensitivity, but also suppressed the stemness and metastatic properties of NSCLC cells. Mechanistically, coclaurine disrupted the interaction between the transcription factor FOXG1 and the EFHD2 promoter, leading to a reduction in EFHD2 transcription. Silencing FOXG1 further inhibited EFHD2 expression and sensitized NSCLC cells to cisplatin. CONCLUSIONS: S. tetrandra and its active compound coclaurine may serve as effective adjuvant therapies to improve cisplatin efficacy in the treatment of NSCLC.

Multi-omics analysis to reveal the synergistic mechanism underlying the multiple ingredients of Stephania tetrandra extract on rheumatoid arthritis through the PI3K/Akt signaling pathway.

Background: Stephania tetrandra has been used for treating rheumatic diseases for thousands of years in rural areas of China. Several studies have found that tetrandrine and fangchinoline can inactivate the PI3K/Akt signaling pathway by reducing the expression and phosphorylation of AKT. However, the mechanism underlying the therapeutic actions of S. tetrandra on RA is not well known. Methods: In this study, we determined the molecular mechanism of the therapeutic effects of the multiple ingredients of S. tetrandra extract (STE) on collagen-induced arthritic (CIA) rats by integrating pharmacometabolomics, proteomics, and PTMomics. Results: In the multi-omics joint analysis, first, the expression signatures of proteins, PTMs, metabolites, and STE ingredients were profiled in CIA rats PBMCs that underwent STE treatment. Bioinformatics analysis were subsequently probed that STE mainly regulated tryptophan metabolism, inflammatory response, and cell adhesion pathways in CIA rats. The interrelated pathways were further constructed, and the findings revealed that STE attenuated the inflammatory response and proliferation of PBMCs in CIA rats by mediating the key targets of the PI3K/Akt pathway, including Hint1, ACP1, FGR, HSP90@157W + dioxidation, and Prkca@220N + 845.4540 Da. The rheumatic functions of Hint1 and ACP1 were further confirmed by applying a transcriptomic data of RA patients who clinically received abatacept therapy. Furthermore, a cross-ome correlation analysis was performed and major in vivo ingredients of STE, including coclaurine-N-glucuronide, Me,coclaurine-O-glc, N-gluA-schefferine, corydamine, corypamine, tetrandrine, and fangchiniline, were found to act on these targerts to inactivate the PI3K/Akt pathway. Conclusion: These results elucidated the molecular mechanism by which the ingredients of STE mediate the expression of the key targets in the PI3K/Akt pathway, leading to anti-rheumatic functions. The findings of this study provided new insights into the synergistic effect of STE against arthritis in rats.

Development of Stephania tetrandra S. MOORE hairy root culture process for tetrandrine production.

Tetrandrine, a bioactive active compound mainly found in the roots of Stephania tetrandra, exhibits various pharmacological properties. In vitro hairy root (HR) culture may serve as a promising solution for the extraction of tetrandrine, overcoming the limitations of natural cultivation. The present study describes the consistent production of tetrandrine from S. tetrandra hairy roots induced by different strains of Agrobacterium rhizogenes. Cultivation in woody plant medium (WPM) resulted in the highest HR biomass (0.056 g/petri-dish) and tetrandrine content (7.28 mg/L) as compared to other media. The maximum HR biomass (6.95 g dw/L) and tetrandrine production (68.69 mg/L) were obtained in the fifth week of cultivation. The presence of ammonium nitrate (800 mg/L), calcium nitrate (1156 mg/L), sucrose (20 g/L) and casein (2 g/L) enhanced the tetrandrine production. Moreover, the fed-batch cultivation demonstrated that the NH4NO3 (1200 mg/L) was an important growth limiting factor that yielded the highest tetrandrine amount (119.59 mg/L). The cultivation of hairy roots in a mist trickling bioreactor for eight weeks was less (26.24 mg/L) than in the flask. Despite a lower tetrandrine yield observed in bioreactors compared to flask cultures, refining the growth medium and fine-tuning bioreactor operations hold promise for boosting tetrandrine yield.

[Discovery, isolation and structural identification of alkaloid glycosides in six traditional Chinese medicine such as Coptis chinensis].

Alkaloids are important active ingredients occurring in many traditional Chinese medicines, and alkaloid glycosides are one of their existence forms. The introduction of saccharide units improves the water solubility of alkaloid glycosides thus presenting better biological activity.Because of the low content in plants, alkaloid glycosides have been not comprehensively studied. In this study, ultrahigh performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-QTOF-MS/MS) was employed to identify and analyze the alkaloid glycosides in Coptis chinensis, Phellodendron chinense, Menispermum dauricum, Sinomenium acutum, Tinospora sagittata and Stephania tetrandra. The results showed that except Tinospora sagittata, the other five herbal medicines contained alkaloid glycosides. Furthermore, the alkaloid glycosides in each herbal medicine were identified based on UV absorption spectra, quasimolecular ion peaks in MS, fragment ions information in the MS/MS, and previous literature reports. A total of 42 alkaloid glycosides were identified. More alkaloid glycosides were identified in C. chinensis and Menispermum dauricum, and eleven in C. chinensis were potential new compounds. Furthermore, the alkaloid glycosides in the water extract of C. chinensis were coarsely se-parated by macroporous adsorption resin, purified by column chromatography with D151 cation exchange resin, ODS and MCI, combined with semi-preparative high performance liquid chromatography. Two new alkaloid glycosides were obtained, and their structures were identified by mass spectrometry and NMR data as(S)-7-hydroxy-1-(p-hydroxybenzyl)-2,2-N,N-dimethyl-1,2,3,4-tetrahydroisoquinoline-6-O-ß-D-glucopyranoside and(S)-N-methyltetrahydropalmatubine-9-O-ß-D-glucopyranoside, respectively. This study is of great significance for enriching the information about the chemical composition and the in-depth development of C. chinensis. Meanwhile, it can provide a reference for rapid identification and isolation of alkaloid glycosides from other Chinese herbal medicines.

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.

Metabolomic Analysis of Stephania tetrandra-Astragalus membranaceus Herbal Pair-Improving Nephrotic Syndrome Identifies Activation of IL-13/STAT6 Signaling Pathway.

The Stephania tetrandra−Astragalus membranaceus herbal pair (FH) is a classic herbal pair widely used in the treatment of nephrotic syndrome (NS). The effects of Stephania tetrandra (FJ) and Astragalus membranaceus (HQ) on NS have been reported, but the mechanism of their combination on the improvement of NS are still unclear. The NS model was established by injecting adriamycin into the tail vein. FH intervention reduced the levels of serum triglyceride, total cholesterol, interleukin-6 (IL-6), blood urea nitrogen (BUN), urinary protein, and the gene expression levels of aquaporin 2 (AQP2) and arginine vasopressin (AVP) in NS rats. In addition, FH improved kidney injury in NS rats by inhibiting the expression of interleukin 13 (IL-13), phospho-signal transducers, and activators of transcription 6 (p-STAT6), Bax, cleaved-caspase3, while promoting the expression of Bcl-2. By comprehensive comparison of multiple indexes, the effects of FH on lipid metabolism, glomerular filtration rate, and inflammation were superior to that of FJ and HQ. Metabonomic studies showed that, compared with FJ and HQ, FH intervention significantly regulated tricarboxylic acid (TCA) cycle, cysteine and methionine metabolism, and alanine, aspartic acid and glutamic acid metabolism. Pearson correlation analysis showed that succinic acid and L-aspartic acid were negatively correlated with urinary protein, cystatin C (Cys C) and BUN (p < 0.05). In summary, FH could reduce renal injury and improve NS through inhibiting the IL-13/STAT6 signal pathway, regulating endogenous metabolic pathways, such as TCA cycle, and inhibiting the expression of AQP2 and AVP genes. This study provides a comprehensive strategy to reveal the mechanism of FH on the treatment of NS, and also provides a reasonable way to clarify the compatibility of traditional Chinese medicine.

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.

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.

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.

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.

First Report of Sclerotium rolfsii Causing Southern Blight on Stephania tetrandra in China.

Stephania tetrandra S. Moore is a perennial liana and is widely cultivated in southern China for traditional Chinese medicine as a diuretic, anti-inflammatory, and antirheumatic treatment (Jiang et al. 2020). In August 2021, it was observed that a severe stem rot disease affected St. tetrandra cultivated in Anfu, Jiangxi province, China (114°27'26" E, 27°22'46" N). The disease symptoms included constriction and rot at the base of the stem, and covered with a layer of white mycelia. The plants above-ground finally wilted and dried with a disease incidence ranging from 8% to 16%. Lots of dried plants formed withered patches of field. Sections (1.0~2.0 cm) from browning stem tissues were surface-disinfected with 75% ethanol for 15 s, followed by 60 s in 4% NaClO, rinsed twice in sterile water, dried on sterilized filter paper, placed on potato dextrose agar (PDA), and incubated at 26°C in the dark for 3 days. A white rhizomorphic fungal mycelium, that is similar to the mycelium of strain FJSR0 on the surface of an infected plant in the field, was isolated from the cultured tissues with 67% frequency. When incubated on PDA, white and fluffy mycelia with even margins and a slight halo formed. Mycelia-produced clamp connections were observed. Colonies grew quickly and covered the dish (diameter: 9 cm) in 5 or 6 days. After that, sclerotia were initially white, then turned yellow, and chestnut brown at maturity. Spherical and subspherical sclerotia were observed after 8 days, with each plate containing 448 to 634 sclerotia (0.8 to 1.4 mm diameter; mean = 0.94 mm; n = 50). On the basis of morphology, the pathogen was similar to Sclerotium rolfsii Sacc. [teleomorph: Athelia rolfsii (Curzi) Tu & Kimbrough] (Sun et al. 2020; Ling et al. 2021). For molecular confirmation, the internal transcribed spacer (ITS) region with approximately 680 bp was amplified from strains FJRS0 and FJRS1 using primers ITS1/ITS4 (White et al. 1990). Two distinct types (different in one SNP and one 1-bp InDel) of ITS sequences were obtained from each isolate, and all isolates contain the two types (FJSR0: ON972516, ON972517; FJSR1: ON972520, ON972518). BLAST analysis of each type found that the hits, with identities >99%, are A. rolfsii except for two Sc. delphinii sequences (GU567775.1 and MK073010.1). Phylogenetic analysis placed strains FJSR0 and FJSR1 in the same clade as Sc. rolfsii but away from Sc. delphinii based on the previous method (Sun et al. 2021). Both morphological and molecular characteristics confirmed that the strains were Sc. rolfsii. For pathogenicity tests, PDA plugs (8 mm in diameter) covered with 5-day-old fungal mycelium were inoculated at the stem bases of three healthy St. tetrandra seedings and incubated at 26℃ and relative humidity of 80%. On the fifth day, inoculated plants were wilting. The infected stem bases turned brown to black and constricted as previously observed in the field. Some leaves, infected by the mycelium expanded from the PDA plugs, developed an orange and irregular spot. Sclerotia were observed 20 days post inoculation. In contrast, the leaves and stems of non-inoculated control plants remained symptomless. Pathogenicity tests were repeated three times. The fungus was reisolated consistently from each symptomatic tissue, thus completing Koch's postulates. Although Sc. rolfsii has been previously reported to cause a southern blight symptoms on vegetables, ornamentals, grass, and medicinal and leguminous crops (Sun et al. 2020; Ling et al. 2021), this is the first report of Sc. rolfsii causing similar symptoms of southern blight on St. tetrandra in China.

Transcriptome Analysis of Stephania tetrandra and Characterization of Norcoclaurine-6-O-Methyltransferase Involved in Benzylisoquinoline Alkaloid Biosynthesis.

Stephania tetrandra (S. Moore) is a source of traditional Chinese medicine that is widely used to treat rheumatism, rheumatoid arthritis, edema, and hypertension. Benzylisoquinoline alkaloids (BIAs) are the main bioactive compounds. However, the current understanding of the biosynthesis of BIAs in S. tetrandra is poor. Metabolite and transcriptomic analyses of the stem, leaf, xylem, and epidermis of S. tetrandra were performed to identify candidate genes associated with BIAs biosynthesis. According to the metabolite analysis, the majority of the BIAs accumulated in the root, especially in the epidermis. Transcriptome sequencing revealed a total of 113,338 unigenes that were generated by de novo assembly. Among them, 79,638 unigenes were successfully annotated, and 42 candidate structural genes associated with 15 steps of BIA biosynthesis identified. Additionally, a new (S)-norcoclaurine-6-O-methyltransferase (6OMT) gene was identified in S. tetrandra, named St6OMT2. Recombinant St6OMT2 catalyzed (S)-norcoclaurine methylation to form (S)-coclaurine in vitro. Maximum activity of St6OMT2 was determined at 30°C and pH 6.0 in NaAc-HAc buffer. Its half-life at 50°C was 22 min with the Km and kcat of 28.2 µM and 1.5 s-1, respectively. Our results provide crucial transcriptome information for S. tetrandra, shedding light on the understanding of BIAs biosynthesis and further gene functional characterization.

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.

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.

A critical review: traditional uses, phytochemistry, pharmacology and toxicology of Stephania tetrandra S. Moore (Fen Fang Ji).

ABSTRACT: Stephania tetrandra S. Moore (S. tetrandra) is distributed widely in tropical and subtropical regions of Asia and Africa. The root of this plant is known in Chinese as "Fen Fang Ji". It is commonly used in traditional Chinese medicine to treat arthralgia caused by rheumatism, wet beriberi, dysuria, eczema and inflamed sores. Although promising reports have been published on the various chemical constituents and activities of S. tetrandra, no review comprehensively summarizes its traditional uses, phytochemistry, pharmacology and toxicology. Therefore, the review aims to provide a critical and comprehensive evaluation of the traditional use, phytochemistry, pharmacological properties, pharmacokinetics and toxicology of S. tetrandra in China, and meaningful guidelines for future investigations.

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.

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.

The complete chloroplast genome of Stephania tetrandra (Menispermaceae).

The complete chloroplast genome of Stephania tetrandra was sequenced and assembled for the first time. The chloroplast genome is 159,974 bp in length, containing a large single-copy (LSC) region of 90,539 bp and a small single-copy region (SSC) of 20,735 bp, separated by a pair of inverted repeats (IRs) of 24,350 bp. The genome contains 113 unique genes, including 79 protein-coding genes (PCGs), 30 tRNA genes, and four rRNA genes. Among them, 15 genes have one intron each and three genes contain two introns. The overall GC content is 37.8%, while the corresponding values of LSC, SSC, and IR regions are 35.8, 32.4, and 43.7%, respectively. Phylogenetic analysis showed that S. tetrandra is more closely related to the clade of two species within Stephania, providing new insight into the evolution of Menispermaceae.

Otoprotective Effects of Stephania tetrandra S. Moore Herb Isolate against Acoustic Trauma.

Noise is the most common occupational and environmental hazard, and noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit. Although therapeutics that target the free-radical pathway have shown promise, none of these compounds is currently approved against NIHL by the United States Food and Drug Administration. The present study has demonstrated that tetrandrine (TET), a traditional Chinese medicinal alkaloid and the main chemical isolate of the Stephania tetrandra S. Moore herb, significantly attenuated NIHL in CBA/CaJ mice. TET is known to exert antihypertensive and antiarrhythmic effects through the blocking of calcium channels. Whole-cell patch-clamp recording from adult spiral ganglion neurons showed that TET blocked the transient Ca2+ current in a dose-dependent manner and the half-blocking concentration was 0.6 + 0.1 µM. Consistent with previous findings that modulations of calcium-based signaling pathways have both prophylactic and therapeutic effects against neural trauma, NIHL was significantly diminished by TET administration. Importantly, TET has a long-lasting protective effect after noise exposure (48 weeks) in comparison to 2 weeks after noise exposure. The otoprotective effects of TET were achieved mainly by preventing outer hair cell damage and synapse loss between inner hair cells and spiral ganglion neurons. Thus, our data indicate that TET has great potential in the prevention and treatment of NIHL.