Immunosuppressive Sesquiterpene Pyridine Alkaloids from Tripterygium wilfordii Hook. f.

Tripterygium wilfordii Hook. f. is a well-known traditional Chinese medicine used to treat autoimmune diseases. Sesquiterpene pyridine alkaloids (SPAs) are a major class of components found in this herb that have piqued the interest of researchers due to their complex and diverse structures as well as significant biological activities. In this study, ten new SPAs, wilfordatine A-J (1-10), were isolated from the roots of T. wilfordii, along with ten known analogues (11-20). Their structures were primarily elucidated by extensive 1D and 2D NMR spectroscopic analysis. To search for more immunosuppressive ingredients related to the clinical efficacy of T. wilfordii, the total alkaloids (TA) and compounds 4, 5, and 9-16 were tested for their inhibitory effects on nuclear factor-kappa B (NF-κB) pathway in Lipopolysaccharide (LPS) induced HEK293/NF-κB-Luc cells. Among them, TA, compounds 5, 11, and 16 showed potent immunosuppressive activity, with IC50 values of 7.25 µg/mL, 8.75 µM, 0.74 µM, and 15.66 µM, respectively, and no influence on the cell viability at a concentration of 100 µg/mL (TA) or 100 µM (5, 11, and 16). Accordingly, TA, 5, 11, and 16, especially 11, were identified as promising candidates for further investigation into their potential use as immunosuppressive agents.

[Spectral characteristics of sesquiterpene pyridine alkaloids from Tripterygium plants].

Sesquiterpene pyridine alkaloids are important components in Tripterygium plants, possessing a wide range of pharmacological activities, such as anti-inflammation immunosuppression, anti-tumor, anti-virus, and deinsectization, and are of great research value. They are composed of highly oxidized dihydro-ß-furansquiterpene and pyridine dicarboxylic acid through ester bonds. According to the structural characteristics of pyridine dicarboxylic acid fragments, they can be divided into various structural subtypes. Up to now, more than 110 sesquiterpene pyridine alkaloids have been isolated and identified from Tripterygium plants. This study reviewed the structural features and spectral(i.e., UV, IR, MS, and NMR) characteristics of sesquiterpene pyridine alkaloids and summarized the structural elucidation process in detail to provide references for their further research and development.

Comprehensive Evaluation of the Quality of Tripterygium Glycosides Tablets Based on Multi-Component Quantification Combined with an In Vitro Biological Assay.

Tripterygium glycosides tablets (TGTs) are widely used in clinical practice to treat rheumatoid arthritis and other autoimmune diseases, with significant beneficial effects but also high toxicity, necessitating rigorous quality evaluation and control. In current study, a rapid resolution liquid chromatography tandem electrospray ionization triple quadrupole mass spectrometry (RRLC-ESI-MS/MS) method was developed and validated for the quantitative analysis of 14 components of ten batches of TGTs produced by different manufacturers, including four diterpenoids, three triterpenoids, and seven sesquiterpene alkaloids. Meanwhile, the NO inhibition effects of these TGTs were evaluated in LPS-induced RAW264.7 cells for their downstream anti-inflammatory activities, as well as their cytotoxicity. The results indicate that the TGTs from different manufacturers showed poor quality consistency, as evidenced by large variations in chemical profiles and biological effects, which may increase the risks associated with clinical use. To improve the quality status of TGTs, it is crucial to identify indicator components whose characterization can accurately reflect the efficacy and toxicity of TGTs from which they were derived. Our study reveals that triptolide, triptoquinone B, celastrol, and demethylzelaysteral considerably contributed to the anti-inflammatory activity and/or cytotoxicity of TGTs, implying that they should be further investigated as candidate indicator components for TGT quality control.

A general procedure for establishing composite quality evaluation indices based on key quality attributes of traditional Chinese medicine.

Licorice, a medicinal herb and food flavor ingredient, has been widely used in traditional Chinese medicine (TCM) for the past 4000 years. In this study, we propose a new quality evaluation approach for licorice quality control based on the key quality attributes commonly used in TCM. The high quality of TCM formulations is ensured by verifying the genuine origin and implementing good agricultural and collection practices for each medicinal herb. In our study, the genuine production area, the harvest season, and the number of growth years were considered the key quality attributes of TCM. To ensure the representativeness of our analysis, we obtained a total of 158 licorice sample batches that differed in the number of growth years, the location of the production areas, and the season for harvesting. Initially, the 158 sample batches were subjected to ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). A preliminary screen identified 11 licorice compounds related to the three key quality attributes of TCM . An analysis by ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-TQ-MS/MS) verified the presence of 34 compounds in all licorice samples. These 34 compounds included the 11 compounds related to the three key quality attributes of the samples, along with other bioactive components identified in previous studies. After using UHPLC-TQ-MS/MS to assess the signal peak intensities of the 34 compounds, we selected 17 licorice compounds to establish sample content evaluation indices, which were determined by high-performance liquid chromatography at four different wavelengths in all 158 licorice sample batches. Finally, the screen identified nine compounds that were closely associated with the quality attributes of licorice based on principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Our results suggested that liquiritin and eight other compounds could be used as quality control indicators of licorice, which provided a foundation to establish the TCM quality composite evaluation index (TCM QCEI). In summary, this research concept can serve as a reference for research on quality markers and the evaluation of TCM.

Accumulated Clinical Experiences from Successful Treatment of 1377 Severe and Critically Ill COVID-19 Cases.

In late December 2019, COVID-19 was firstly recognized in Wuhan, China and spread rapidly to all of the provinces of China. The West Campus of Wuhan Union Hospital, the designated hospital to admit and treat the severe and critically ill COVID-19 cases, has treated a large number of such patients with great success and obtained lots of valuable experiences based on the Chinese guideline (V7.0). To standardize and share the treatment procedures of severe and critically ill cases, Wuhan Union Hospital has established a working group and formulated an operational recommendation, including the monitoring, early warning indicators, and several treatment principles for severe and critically ill cases. The treatment experiences may provide some constructive suggestions for treating the severe and critically ill COVID-19 cases all over the world.

[Study on potential hepatotoxicity of rhein in Rhei Radix et Rhizoma based on liver metabolism].

The bilirubin metabolism mediated by the phase Ⅱ metabolizing enzyme UGT1A1 in the liver was evaluated to study the potential hepatotoxicity risk based on investigation on the inhibitory effect of rhein and its metabolites on the UGT1A1 enzyme in Rhei Radix et Rhizoma. Firstly, in vitro liver microsomes incubation was used to initiate the phase Ⅱ metabolic reaction to investigate the inhibitory effect of rheinon UGT1A1 enzyme. Secondly, the phase Ⅰ and phase Ⅱ metabolic reactions were initiated to investigate the hepatotoxicity risk of rhein metabolites. It was found that the rhein and its phase Ⅱ metabolites had no significant inhibitory effect on UGT1A1 enzyme, but its phase Ⅰ metabolites significantly reduced UGT1A1 enzyme activity. Based on the metabolites analysis, it is speculated that the rhein phase Ⅰ metabolite rheinhydroxylate and its tautomers have certain hepatotoxicity risks, while the toxicity risk induced by the prototype and phase Ⅱ metabolites of rheinglucoside, rheinglucuronic acid and rhein sulfate is small.

[Prediction of potential drug interactions of apigenin based on molecular docking and in vitro inhibition experiments].

The purpose of this study was to investigate the effect of apigenin on UGT1 A1 enzyme activity and to predict the potential drug-drug interaction of apigenin in clinical use. First,on the basis of previous experiments,the binding targets and binding strength of apigenin to UGT1 A1 enzyme were predicted by computer molecular docking method. Then the inhibitory effect of apigenin on UGT1 A1 enzyme was evaluated by in vitro human liver microsomal incubation system. Molecular docking results showed that apigenin was docked into the active region of UGT1 A1 enzyme protein F,consistent with the active region of bilirubin docking,with moderate affinity. Apigenin flavone mother nucleus mainly interacted with amino acid residues ILE343 and VAL345 to form hydrophobic binding Pi-Alkyl. At the same time,the hydroxyl group on the mother nucleus and the amino acid residue LYS346 formed an additional hydrogen bond,which increased the binding of the molecule to the protein. These results suggested that the flavonoid mother nucleus structure had a special structure binding to the enzyme protein UGT1 A1,and the introduction of hydroxyl groups into the mother nucleus can increase the binding ability. In vitro inhibition experiments showed that apigenin had a moderate inhibitory effect on UGT1 A1 enzyme in a way of competitive inhibition,which was consistent with the results of molecular docking. The results of two experiments showed that apigenin was the substrate of UGT1 A1 enzyme,which could inhibit the activity of UGT1 A1 enzyme competitively,and there was a risk of drug interaction between apigenin and UGT1 A1 enzyme substrate in clinical use.

[Research progress on chemical constituents and quality control of Tripterygium wilfordii preparations].

Tripterygium wilfordii preparations,with various biological activities such as immunosuppressive,anti-inflammatory and anti-cancer effects,are widely used in the treatment of autoimmune diseases such as rheumatoid arthritis,lupus erythematosus,and nephrotic syndrome. They have definite therapeutic effect,but often cause serious adverse reactions and result in damages to liver,kidney,blood,reproduction,and other systems due to their complex compositions,great toxicity,and narrow margin between the toxic and therapeutic dosages. At present,T. wilfordii preparations produced by different manufacturers exhibit large variations in clinical efficacy and side effects in account of their different chemical compositions and quality fluctuation due to differences in raw materials and production process. However,the existing quality standards are controversial in terms of index components and content limit,which cannot be effectively used for the overall quality control of the preparations. In this paper,the research progress on chemical constituents,quality standard and quality control methods of four T. wilfordii preparations including Tripterygium Tablets,Tripterygium Zongtie Tablets,Tripterygium Shuangceng Tablets and Tripterygium Glycosides Tablets was reviewed,in order to provide ideas and reference for the quality improvement of this type of preparations.

[Study on hepatotoxicity of physcion based on liver metabolism in vitro].

To evaluate the hepatotoxicity risks of physcion on the basis of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1A1(UGT1A1 enzyme). The monomers were added into the rat liver microsomes to test the hepatotoxicity by using bilirubin as UGT1A1 enzyme substrate, with apparent inhibition constant K_i as the evaluation index. Liver microsome incubation in vitro was adopted to initiate phase Ⅱ metabolic reaction and investigate the inhibitory effect of physcion. Then the phase Ⅰ and Ⅱ metabolic reactions were initiated to investigate the comprehensive inhibition of metabolites and prototype components. The results showed that when only the phase Ⅱ reaction was initiated, physcion directly acted on the UGT1A1 enzyme in a prototype form, exhibited weak inhibition and the inhibition type was mixed inhibition; When the phase Ⅰ and Ⅱ reactions were initiated simultaneously, the inhibitory effects of physcion on UGT1A1 enzyme became strong and the inhibition type was mixed inhibition, suggesting that physcion had phase Ⅰ and Ⅱ metabolic processes, and the metabolites had strong inhibitory effect on UGT1A1 enzyme. This experiment preliminarily proved that the metabolites of physcion may be the main components to induce hepatotoxicity.

[Quality control of Guci tablets using UPLC-ELSD fingerprint analysis coupled with chemometrics].

Ultra-performance liquid chromatography-evaporative light scattering detection (UPLC-ELSD) fingerprint analysis method was established for quality control of Guci tablets. Chromatographic separation was performed on Waters Acquity UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) at 30 °C of column temperature. Acetonitrile-0.1% formic acid solution was adopted as mobile phase for gradient elution. The flow rate was set at 0.3 mL·min⁻¹, and the injection volume was 3 µL. Detection was carried out on an ELSD with a nitrogen pressure of 0.28 MPa, drift tube temperature of 60 °C, and gain of 400. A total of 39 batches of samples produced by six manufacturers were measured by using the above method and the data were analyzed by ChemPattern software. The peak present in more than 75% of the samples was defined as a common peak, and 30 common peaks were determined. Among them, 19 peaks were identified by rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) method, 16 of which were confirmed by reference substances. The similarity of the tested samples was 0.47-0.98, suggesting that the quality of the samples from different manufacturers varied greatly. Furthermore, principal component analysis (PCA) and hierarchical analysis (HCA) were performed to clarify the main different components in samples. The results indicated that there might be some feeding problems about Paeoniae Radix Alba, Notoginseng Radix et Rhizoma, and Clematidis Radix et Rhizoma in a few manufacturers. This study provided some evidences for the overall quality control of Guci tablets, as well as its quality standard improvements.

Effect of drying processes on prenylflavonoid content and antioxidant activity of Epimedium koreanum Nakai.

Epimedium koreanum Nakai is a famous Chinese herbal medicine for the treatment of impotence, osteoporosis, immune suppression and cardiovascular diseases. Drying is the most common and fundamental procedure in post-harvest processing of E. koreanum, which contributes to the variations of flavonoid content, especially prenylflavonoids, the bioactive components. In present study, effect of drying processes on flavonoid content and antioxidant activity were investigated. High performance liquid chromatography coupled with diode-array detection and electrospray ionization quadrupole time-of-flight tandem mass spectrometry methods were employed. Twenty seven compounds were identified and 11 of them, including eight prenylflavonoids and three other types of flavonoids, were further quantified. The antioxidant activity of E. koreanum was evaluated by the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging method. The results showed that content of the eight prenylflavonoids exhibited significant variations after different drying processes, especially icariin and baohuoside I. The variation tendency of antioxidant activity was positively correlated with the content of total flavonoid, afzelin and icariin.

Identification and characterization of the structure-activity relationships involved in UGT1A1 inhibition by anthraquinone and dianthrone constituents of Polygonum multiflorum.

The adverse effects of Polygonum (P.) multiflorum, including abnormal bilirubin metabolism, are a serious public health issue. As uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) is the only enzyme responsible for bilirubin metabolism, we investigated the inhibitory effect of a P. multiflorum extract and 10 anthraquinone and dianthrone compounds on UGT1A1 in rat liver microsomes in vitro. The P. multiflorum extract exhibited the strongest inhibitory effect on UGT1A1 activity (inhibition constant [Ki] = 0.3257 µM, 1422 µg of material/mL), followed by cis-emodin dianthrones (Ki = 0.8630 µM), trans-emodin dianthrones (Ki = 1.083 µM), emodin-8-O-glc (Ki = 3.425 µM), and polygonumnolide C2 (Ki = 4.291 µM). Analysis of the structure-activity relationships of these compounds suggested that the spatial orientation of the molecules and the presence of particular functional groups affect UGT1A1 inhibition. A mechanistic analysis showed that all the tested compounds docked into two of the nine active sites of UGT1A1 and suggested that hydrophobic interactions and hydrogen bonds are important for the affinity of the tested compounds for UGT1A1; moreover, their interaction energies were generally in agreement with the Ki values. These findings provide insight into adverse reactions to P. multiflorum and identify the pharmacophores involved in inhibition of UGT1A1.

A comparative study on bioactive constituents in different parts of Lonicera japonica determined by HPLC-ESI-MS(n).

Lonicera japonica Thunb. is a well-known traditional herbal medicine in most East Asian countries. In China, the flower bud and stem of this plant are used for various clinical therapies, while the leaf is not officially recognized as an active part. Due to the similarities in their chemical constituents but great differences in their commercial values, the flower bud has been found to be adulterated with leaf and/or stem during the production of formulations by some drug manufactures. In order to identify adulteration in products and enable quality control, it is necessary to chemically discriminate these three parts of L. japonica. In the current study, an HPLC-ESI-MS(n) method was developed and validated for the quantitative analysis of 18 bioactive compounds: 7 organic acids, 6 iridoids, and 5 flavonoids, in batches of flower bud, stem, and leaf samples. Subsequently, chemometric analyses, such as one-way analysis of variance, principal component analysis, and hierarchical clustering analysis, were performed based on the quantitative data. The results indicated that there were remarkable differences in the distribution of the investigated compounds among the three parts of L. japonica, and that they could be straightforwardly and reliably distinguished according to their chemical profiles. Twelve compounds were selected as potential differential metabolites, which would be useful for quality control of L. japonica. As the content of caffeic acid was low in the flower bud but much higher in the stem and leaf, it could be used as a chemical marker to identify adulteration.

Simultaneous determination of five triterpenoid saponins in different parts of Lonicera macranthoides by RRLC-MS/MS method.

A rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS) method was developed and validated for the determination of five major saponins (macranthoidin B, macranthoidin A, dipsacoside B, akebiasaponin D, and dipsacoside A) in the flower bud, stem, and leaf parts of Lonicera macranthoides. Chromatographic separation was performed on a ZORBAX SB-C18 column (2.1 x 50 mm, 1.8 µm). Acetonitrile and 0.1% aqueous formic acid were adopted as mobile phase. Detection was carried out on a triple quadrupole mass spectrometer in the negative ion mode using an electrospray source. Multiple reaction monitoring (MRM) mode was employed. The established method showed good linearity (r2 ≥ 0.9994) for all the analytes within the test ranges and the recoveries were 95.19-103.28%. Desirable intra-day and inter-day precision as well as repeatability were obtained with relative standard deviations (RSDs) less than 5%. The method was simple, sensitive, accurate and performed well in application to the sample determination within a short analysis time of 15 min. The saponin profiles of different parts of Lonicera macranthoides were obtained based on the quantitative data, showing that the flower bud contained much higher level of saponins than the stem and leaf by several orders of magnitude, and that the quantity ratios varied remarkable between these three part. The conclusions might provide scientific evidences for the reasonable application of Lonicera macranthoides, and the proposed RRLC-MS/MS method might be useful for the quality control of this medicinal plant.

[Simultaneous determination of ten bioactive constituents in Lonicerae Flos by HPLC-MS-MS].

In this study, an HPLC-MS/MS method was developed and validated for simultaneous determination of six iridoids and four flavonoids in batches of Lonicerae Flos samples. Chromatographic separation was performed on a Shiseido Capcell Pak-C18 column (4.6 mm×250 mm, 5 µm). 0.1% Aqueous formic and acid (A) and acetonitrile (B) were adopted as mobile phase. Detection was carried out on a triple quadrupole mass spectrometer in the negative ion mode using an electrospray source. Multiple reaction monitoring (MRM) mode was employed. The developed method showed good linearity (R² ≥0.999 0) for all the analytes within the test ranges and the limits of quantification (LOQs) ranged from 7.4 to 31.0 µg•L⁻¹. The recoveries varied between 94.16% and 105.3%. The quantitative data indicated that the total content of iridoids (0.338%-1.440%) was much higher than that of flavonoids (0.015 4%-0.057 5%) in all samples. Moreover, it was found that there were significant differences in the content of six compounds among the samples from three different original plants, which might provide scientific evidences for the origin identification and quality control of Lonicerae Flos.

Microsporols A-C from the Plant Endophytic Fungus Pestalotiopsis microspore.

Three new ambuic acid derivatives, microsporols A-C (1-3) and the known compound ambuic acid (4), were isolated from the solid-substrate fermentation cultures of the plant endophytic fungus Pestalotiopsis microspora. Their structures were elucidated primarily by NMR experiments. The absolute configurations of the 6,7-diol moiety in 1 and 2 were assigned using the Snatzke's method, whereas that of 3 was deduced by circular dichroism (CD) exciton chirality method. Compounds 1, 3, and 4 showed moderate 5-lipoxygenase (5-LOX) inhibitory effects.

[Chemical constituents from roots of Illicium majus].

Ten compounds, including seven sesquiterpenes, two phenols and one phenylpropanoid, were isolated from the roots of Illicium majus by means of silica gel, ODS, Sephadex LH-20, and preparative HPLC. On analysis of MS and NMR spectroscopic data , their structures were established as cycloparviflorolide (1), cycloparvifloralone (2), tashironin (3), tashironin A (4), anislactone A(5), anislactone B (6), pseudomajucin (7), syringaldehyde (8), methyl-4-hydroxy-3, 5-dimethoxybenzoate (9), and (E)-3-methoxy-4,5-methylenedioxycinnamic alchol (10), respectively. Compounds 1-4 and 8-10 were first isolated from this plant. In the in vitro assays, at a concentration of 1.0 x 10(-5) mol x L(-1), compounds 5 and 6 were active against LPS induced NO production in microglia with a inhibition rate of 75.31% and 53.7%, respectively.

Five new compounds from Rhododendron mariae Hance.

Five new compounds (-)-(7R,8S,7'R,8'S)-4,9,4',9'-tetrahydroxy-3,3'-dimethoxy-7,7'-epoxylignan 9-O-ß-d-xylopyranoside (1), (-)-(7'R,8'S)-5'-methoxyl(dimeric coniferyl acetate) (2), (+)-(1R,2S)-1,2-bis(4-hydroxy-3-methoxyphenyl)-3-acetyl-1,3-propanediol (3), (-)-3-((2R,3R)-2-ethoxy-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)propan-1-ol (4), and (+)-3-((2S,3S)-2-ethoxy-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)propan-1-ol (5), together with 12 known compounds, were isolated from an ethanol extract of the dried stems of Rhododendron mariae Hance. Their structures were elucidated by NMR, HR-MS, CD, ORD experiments and chemical methods. Compounds 2, 3, 6, and 17 exhibited significant inhibitory effects on the nitric oxide production in lipopolysaccharide activated C57BL6/J mouse macrophages.

Five new fawcettimine-related alkaloids from Lycopodium japonicum Thunb.

Five new trace alkaloids with fawcettimine-related structures (1-5), i.e., 6-hydroxyl-6,7-dehydrolycoflexine (1), 6-hydroxyl-6,7-dehydro-8-deoxy-13-dehydroserratinine (2), together with three known ones (6-8), were isolated from the club moss Lycopodium japonicum Thunb. Their structures were elucidated by extensive NMR spectroscopic analysis, HRESIMS, CD and comparison with known ones. Compounds 1 and 2 are characterized by the enol moiety that is rarely reported in Lycopodium alkaloids, and Compound 5 is the second example of Lycopodium alkaloids with a C-16-C-4 linkage. A plausible biogenetic pathway for the isolates was proposed.