Bioactive prenylated phenolic compounds from the aerial parts of Glycyrrhiza uralensis.

In this work, a bioassay-guided fractionation strategy was used to isolate 26 phenolic compounds from the ethyl acetate partition of an ethanol extract of the aerial parts of Glycyrrhiza uralensis Fisch. ex DC. Among them, 8 prenylated phenolic compounds (glycyuralins Q-X) were described for the first time. The two enantiomers of glycyuralin Q were purified and their absolute configurations were established by ECD spectral calculations. (1″R, 2″S)-glycyuralin Q and (1″S, 2″R)-glycyuralin Q showed significant inhibitory activities against SARS-CoV-2 virus proteases 3CLpro with IC50 values of 1.5 ± 1.0 and 4.0 ± 0.3 µM, and PLpro with IC50 values of 2.4 ± 0.2 and 1.9 ± 0.1 µM, respectively. Four compounds showed potent cytotoxic activities against A549, Huh-7, and HepG2 human cancer cells with IC50 values ranging from 0.5 to 2.5 µM.

Analysis of curcuminoids and volatile components in 160 batches of turmeric samples in China by high-performance liquid chromatography and gas chromatography mass spectrometry.

Turmeric (Curcuma longa L.) is a popular herbal medicine worldwide. Curcuminoids and volatile constituents are its major bioactive components. To improve the quality control of turmeric, we determined the contents of three main curcuminoids in 160 batches of turmeric samples collected from five major production areas of China by HPLC, and analyzed the volatile components by GC/MS. The results indicated that samples with red cross sections (2.75 ±â€¯0.82 mg/g) contained significantly higher amounts of curcuminoids than samples with yellow sections (1.23 ±â€¯0.60 mg/g) (p < 0.001). This result was consistent with empirical standard of TCM pharmacists. The contents of curcuminoids in samples from Hainan (4.51±0.25%), Guizhou (3.17±0.41%), and Sichuan (2.25±0.54%) were relatively high and consistent. Moreover, the GC/MS profiles of turmeric may be affected by storage and processing. This study sets a good example for comprehensive quality control of herbal medicines.

AChE inhibitory alkaloids from Coptis chinensis.

Coptis chinensis is a widely used traditional Chinese herbal medicine. In this work, 6 new alkaloids (coptisine A-F, 1-6) and 26 known alkaloids (7-32) were isolated from the chloroform extract of the rhizomes of C. chinensis. Compounds 1-3 are α-carbonylated benzylisoquinolines, and 4-6 are berberidic acid type alkaloids. Their structures were elucidated on the basis of extensive NMR and MS analyses. Seven compounds (7, 20, 22, 23, 25, 26, 29) exhibited significant AChE inhibitory activities at 10 µM (inhibition rates >80%).

[UHPLC-HRMS metabonomics to study effect of sulfur-fumigated Ophiopogonis Radix on endogenous metabolites in rats].

The project was launched to analyze the effects of sulfur-fumigated Ophiopogonis Radix on endogenous metabolites in rats by metabonomics. The preparation method of sulfur-fumigated Ophiopogonis Radix in laboratory was established. Then the blood samples of SD rats in blank group,Ophiopogonis Radix extract group and sulfur-fumigated Ophiopogonis Radix extract group were investigated by UHPLC-Q-Exactive. The differential metabolites were screened and identified by PCA(principal component analysis),OPLSDA(orthogonal partial least squares discriminant analysis) and variable importance projection(VIP),and the metabolic pathways were analyzed. Finally,a total of 15 potential biomarkers were identified. Compared with the samples of Ophiopogonis Radix extract group,sulfur-fumigated Ophiopogonis Radix mainly affected the biosynthesis and metabolism of amino acids in normal rats. Its mechanism may be related to the biosynthesis of phenylalanine,tyrosine,tryptophan and aminoacyl-tRNA as well as the metabolism of phenylalanine and tryptophan. Based on UHPLC-HRMS metabonomics,this paper discussed the effects of sulfur-fumigated Ophiopogonis Radix on endogenous metabolites in rats,which provided an idea for the metabolic study of other sulfur-fumigated traditional Chinese medicines.

Rapid quantitation and identification of the chemical constituents in Danhong Injection by liquid chromatography coupled with orbitrap mass spectrometry.

Danhong Injection (DHI) is a Chinese medicine patent drug to treat cardiovascular diseases. It is derived from the herbal medicines Dan-shen and Hong-hua. The bioactive compounds of DHI are polar phenolic acids and flavonoid glycosides. Thus far, the contents of major compounds in DHI are not well understood, and the identification of minor compounds lacks rapid methods. In this work, quantitative and qualitative analyses of DHI compounds were performed using ultra-high performance liquid chromatography coupled with orbitrap mass spectrometry (UHPLC/orbitrap-MS). DHI was separated on an Acquity HSS T3 column (1.8 µm, 100 mm × 2.1 mm) and eluted with acetonitrile-water (containing 0.1% formic acid) to determine the contents of 12 compounds within 6 min. The method was fully validated according to the ICH guidance. To identify the minor compounds, an ion statistics-based strategy was used to dig for 4 filtering ions and 6 diagnostic ions from 22 reference standards. A total of 117 compounds, including 76 phenolic acids, 20 flavonoids, and 21 other compounds were tentatively identified. The poor stability of salvianolic acid A upon storage was also discussed.

Danshen: a phytochemical and pharmacological overview.

Danshen, the dried root or rhizome of Salvia miltiorrhiza Bge., is a traditional and folk medicine in Asian countries, especially in China and Japan. In this review, we summarized the recent researches of Danshen in traditional uses and preparations, chemical constituents, pharmacological activities and side effects. A total of 201 compounds from Danshen have been reported, including lipophilic diterpenoids, water-soluble phenolic acids, and other constituents, which have showed various pharmacological activities, such as anti-inflammation, anti-oxidation, anti-tumor, anti-atherogenesis, and anti-diabetes. This article intends to provide novel insight information for further development of Danshen, which could be of great value to its improvement of utilization.

[Identification of metabolites of Danshensu in vivo in rats].

To identify the metabolites of Danshensu in plasma and urine in rats by using UHPLC-LTQ-Orbitrap method. After oral gavage of Danshensu CMC-Na suspension in SD rats, urine and plasma samples were collected and processed by solid phase extraction. ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) was utilized, with 0.1% formic acid (A)-acetonitrile (B) solution as the mobile phase for gradient elution. Negative electrospray ion mode based data-acquisition method was established to collect the mass spectrometry data of biological samples. As a result, Danshensu and 21 Danshensu Ⅰ phase and Ⅱ phase metabolites were finally identified according to the accurate mass measurements, mass fragmentation behaviors and comparing with the reference standards. The main metabolic pathways included dehydration, methylation, glucuronide conjugation, sulfate conjugation and their composite reactions. Consequently, our study expounded metabolites of Danshensu in rats based on UHPLC-LTQ-Orbitrap method and provided a reference for further researches on therapeutic material basis and mechanism of Danshensu.

[Metabolites of tanshinone â and tanshinone â ¡A in vivo in rats].

An efficient method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap (UHPLC-LTQ-Orbitrap) mass spectrometer was established to elucidate the in vivo metabolites of tanshinone Ⅰ and tanshinone ⅡA in rats. Urine and plasma samples were collected after oral gavage. After processing biological sample by solid phase extraction, Waters ACQUITY HPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) was used with 0.1% formic acid (A) - acetonitrile (B) solution as the mobile phase for gradient elution. The plasma, urine and the blank samples were then analyzed by ESI-LTQ-Orbitrap equipped with an ESI ion source under positive ion mode. On the basis of the accurate mass measurements, multiple mass spectra and comparison of data with published literature, a total of 26 metabolites were tentatively identified and characterized in the rat samples. Among them, 7 metabolites were derived from tanshinone Ⅰ through metabolic pathways of glucuronide conjugation, hydroxylation, reduction reaction, demethylation reaction, methylation, sulfate conjugation and their composite reactions. Nineteen metabolites were derived from tanshinone ⅡA through metabolic pathways of hydroxylation, reduction reaction, methylation, sulfate conjugation, glucuronidation, glucosylation and their complicated reactions. The results showed that the metabolism of tanshinone Ⅰ and tanshinone ⅡA in rats could be comprehensively clarified by using UHPLC-LTQ-Orbitrap mass spectrometer, providing material basis for the further research in terms of pharmacodynamics, toxicology, and secondary development of Chinese medicine.

Rapid screening and identification of sesquiterpene lactones in Kudiezi injection based on high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry.

Sesquiterpene lactones are considered as the major active compounds in Kudiezi injection in virtue of their special structures and activities. Herein, an analytical method was developed for rapid screening and identification of sesquiterpene lactones in Kudiezi injection using high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap) in negative ion mode. First, two sesquiterpene lactone reference standards were analyzed to obtain their characteristic ESI-MS/MS fragmentation patterns. Second, based on extracted ion chromatography (EIC) data-mining method and characteristic fragmentation pathways analysis, sesquiterpene lactones in Kudiezi injection were rapidly screened and identified. Finally, an important parameter Clog P was adopted to discriminate the isomers of sesquiterpene lactones. As a result, 50 sesquiterpene lactones were characterized, including 9 sesquiterpene lactone aglycones, 39 sesquiterpene lactone glycosides, and 2 amino acid-sesquiterpene lactone conjugates. Among them, 13 compounds were tentatively identified as new compounds. The results demonstrated that the established method would be a rapid, effective analytical tool for screening and identification of sesquiterpene lactones in the complex system of natural medicines.

[HUVECs extraction and UHPLC LTQ Orbitrap analysis for screening vascular endothelium protective components in Kudiezi injection].

An effective method has been employed as a tool for screening active components in Kudiezi injection by using cell chromatography and sensitive UHPLC-HR-MSn method. The potential bioactive components in Kudiezi injection could be selectively bound to the HUVECs target cells first. After cell target desensitization and inactivation, the chemical constituents with cell target affinity were identified by LC-MS, so as to screen the possible active components in Kudiezi injection. Based on the accurate mass measurements and the retention time, in total, 9 compounds were tentatively identified and characterized, including 4 sesquiterpene lactones, 3 phenolic acids and 2 flavonoids. HUVECs biospecific extraction coupled with UHPLC-LTQ-Orbitrap analysis could provide a rapid and efficient method for the identification of potential bioactive components in Kudiezi injection, and provide the reference for further research on its effective materials basis.

Analysis of Non-Volatile Chemical Constituents of Menthae Haplocalycis Herba by Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry.

Menthae Haplocalycis herba, one kind of Chinese edible herbs, has been widely utilized for the clinical use in China for thousands of years. Over the last decades, studies on chemical constituents of Menthae Haplocalycis herba have been widely performed. However, less attention has been paid to non-volatile components which are also responsible for its medical efficacy than the volatile constituents. Therefore, a rapid and sensitive method was developed for the comprehensive identification of the non-volatile constituents in Menthae Haplocalycis herba using ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap). Separation was performed with Acquity UPLC® BEH C18 column (2.1 mm × 100 mm, 1.7 µm) with 0.2% formic acid aqueous solution and acetonitrile as the mobile phase under gradient conditions. Based on the accurate mass measurement (<5 ppm), MS/MS fragmentation patterns and different chromatographic behaviors, a total of 64 compounds were unambiguously or tentatively characterized, including 30 flavonoids, 20 phenolic acids, 12 terpenoids and two phenylpropanoids. Finally, target isolation of three compounds named Acacetin, Rosmarinic acid and Clemastanin A (first isolated from Menthae Haplocalycis herba) were performed based on the obtained results, which further confirmed the deduction of fragmentation patterns and identified the compounds profile in Menthae Haplocalycis herba. Our research firstly systematically elucidated the non-volatile components of Menthae Haplocalycis herba, which laid the foundation for further pharmacological and metabolic studies. Meanwhile, our established method was useful and efficient to screen and identify targeted constituents from traditional Chinese medicine extracts.

[Rapid characterization of chemical constituents in capillary wormwood extract based on UHPLC-LTQ-Orbitrap].

A rapid and sensitive UHPLC-HR-MSn method was developed for the identification of chemical constituents in capillary wormwood extract. ACQUITY UHPLC HSS T3 chromatography column (2.1 mm×100 mm, 1.7 µm) was used with 0.1% formic acid-acetonitrile solution as the mobile phase in gradient elution. The extract was detected by ESI-LTQ-Orbitrap equipped with an ESI ion source in a negative mode. Based on the accurate mass measurements, retention time, mass fragmentation patterns and literature reports, a total of 50 compounds including 21 flavonoids, 22 phenolic acids, 6 coumarins and 1 other compound were tentatively screened and characterized. These results are helpful for the comprehensive quality control, better comprehension of the metabolism and further study of pharmacodynamic substance from capillary wormwood extract.

The genus Liriope: Phytochemistry and pharmacology.

Liriope (Liliaceae) species have been used as folk medicines in Asian countries since ancient times. From Liriope plants (8 species), a total of 132 compounds (except polysaccharides) have been isolated and identified, including steroidal saponins, flavonoids, phenols, and eudesmane sesquiterpenoids. The crude extracts or monomeric compounds from this genus have been shown to exhibit anti-tumor, anti-diabetic, anti-inflammatory, and neuroprotective activities. The present review summarizes the results on phytochemical and biological studies on Liriope plants. The chemotaxonomy of this genus is also discussed.

UHPLC-LTQ-Orbitrap-based metabolomics coupled with metabolomics pathway analysis method for exploring the protection mechanism of Kudiezi injection in a rat anti-ischemic cerebral reperfusion damage model.

Kudiezi injection has been used extensively in the treatment of cerebrovascular and cardiovascular diseases. However, its therapeutic effects and underlying mechanism of action are not fully understood. The aim of the present study was to clarify the protective mechanisms of Kudiezi injection on cerebral ischemic injury, using metabolomics methods. Middle cerebral artery occlusion (MCAO) was introduced in rats to build the cerebral ischemic damage. UHPLC-LTQ-Orbitrap-based analytical method was established for analysis of the metabolites. The raw mass data of all samples were normalized with Sieve 2.2 software and then introduced to orthogonal partial least squares discriminant analysis (OPLS-DA) model. Finally, 23 metabolites in plasma (15 were tentatively identified) were chosen as potential biomarkers, according to accurate mass measurements (< 5 ppm), MS/MS fragmentation patterns, and diagnostic product ions. Furthermore, on the basis of metabolic pathway analysis via metabolomics pathway analysis (MetPA), we first discovered that the protection mechanism in anti-ischemic cerebral reperfusion damage of Kudiezi injection was possibly related to the biosynthesis of phenylalanine, tyrosine, and tryptophan. The present study provided a useful approach for exploring the mechanism of ischemic stroke and evaluating the efficacy of Kudiezi injection or other traditional medicines.

[Rapid characterization of chemical constituents and rats metabolites of Kudiezi injection by UHPLC-LTQ-Orbitrap].

A rapid and sensitive UHPLC-HR-MSn method was used for the identification of Kudiezi injection and its main metabolites in rat plasma. After the tail intravenous injection of Kudiezi, ACQUITY UHPLC BEH C18 (2.1 mm×100 mm, 1.7 µm) was used, with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. Kudiezi injection and plasma were detected by ESI-LTQ-Orbitrap equipped with an ESI ion source in a negative mode. Based on the accurate mass measurements, the retention time and the mass fragmentation patterns, a total of 53 compounds were tentatively identified and characterized. Furthermore, metabolites in rat plasma after the intravenous administration of Kudiezi injection were also analyzed. A total of 38 compounds were identified, including 27 prototypes and 11 metabolites through metabolic pathways of methylation, glucuronide conjugation, sulfate conjugation and hydrolysis. As a result, UHPLC-LTQ-Orbitrap technique was applied to comprehensively expound Kudiezi injection's chemical components and constituents migrating to rat plasma, and provide scientific basis for further studies on Kudiezi injection's in vivo metabolic process and effective material base.

[Influence of different drying methods on steroidal saponins and homoisoflavonoids in Ophiopogon japonicus].

To study the influence of three different drying methods (including 50 ℃-drying, 80 ℃-drying and －70 ℃-freeze-drying methods) on steroidal saponins and homoisoflavonoids in Ophiopogon japonicus,a HPLC-DAD-ELSD-MSn method was investigated to screen and identify the differential components. Through comparing the HPLC chromatograms with that of fresh O. japonicus, 50 ℃-drying medicine was similar with fresh medicine whereas the other two drying methods had great influence on the components of O. japonicus. In this study, 36 differential components were screened, among which 24 constituents(13 homoisoflavonoids and 11 steroidal saponins) were identified via HPLC-LTQ-Orbitrap MS.As a result, it was revealed that different drying methods had significant influences on the components of steroidal saponins and homoisoflavonoids. Among them, 50 ℃-drying method was the most suitable drying approach when the stability of components, cost and practicability were considered.