Porous phenolic resin regulated by alcohol-based deep eutectic solvent for selective extraction and separation of tanshinones from Salvia miltiorrhiza.

Deep eutectic solvents (DES), regarded as promising green solvents, have gained attention due to their distinctive properties, particularly in analytical chemistry. While the use of DES in solvent extraction and separation has been extensively studied, its application in the synthesis of adsorbents has just begun. Phenolic resin, with its polyhydroxy structure and stable spherical morphology, could serve as an effective as adsorbents for enrichment of active ingredients in herbal medicine. Designing adsorbents with high selectivity and adsorption capacity presents a critical challenge in the enrichment of active ingredients in herbal medicine. In this study, alcohol-based DESs were employed as regulators of morphology and structure instead of organic solvents, facilitating the creation of polyhydroxy structure, adjustable pores and high specific surface areas. The resulting DES-regulated porous phenolic resin demonstrated enhanced extraction and separation capacity for active ingredients compared to conventional spherical phenolic resin owing to the alcohol-based DES offering more interaction modes with the analytes.

Targeted metabolomics reveals PFKFB3 as a key target for elemene-mediated inhibition of glycolysis in prostate cancer cells.

BACKGROUND: Elemene, an active anticancer extract derived from Curcuma wenyujin, has well-documented anticarcinogenic properties. Nevertheless, the role of elemene in prostate cancer (PCa) and its underlying molecular mechanism remain elusive. PURPOSE: This study focuses on investigating the anti-PCa effects of elemene and its underlying mechanisms. METHODS: Cell-based assays, including CCK-8, scratch, colony formation, cell cycle, and apoptosis experiments, to comprehensively assess the impact of elemene on PCa cells (LNCaP and PC3) in vitro. Additionally, we used a xenograft model with PC3 cells in nude mice to evaluate elemene in vivo efficacy. Targeted metabolomics analysis via HILIC-MS/MS was performed to investigate elemene potential target pathways, validated through molecular biology experiments, including western blotting and gene manipulation studies. RESULTS: In this study, we discovered that elemene has remarkable anti-PCa activity in both in vitro and in vivo settings, comparable to clinical chemotherapeutic drugs but with fewer side effects. Using our established targeted metabolomics approach, we demonstrated that ß-elemene, elemene's primary component, effectively inhibits glycolysis in PCa cells by downregulating 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression. Furthermore, we found that ß-elemene accomplishes this downregulation by upregulating p53 and FZR1. Knockdown and overexpression experiments conclusively confirmed the pivotal role of PFKFB3 in mediating ß-elemene's anti-PCa activity. CONCLUSION: This finding presents compelling evidence that elemene exerts its anti-PCa effect by suppressing glycolysis through the downregulation of PFKFB3. This study not only improves our understanding of elemene in PCa treatment but also provides valuable insights for developing more effective and safer therapies for PCa.

Screening of anti-cancer compounds from Vaccariae Semen by lung cancer A549 cell fishing and UHPLC-LTQ Orbitrap MS.

Vaccariae Semen, derived from the dried ripe seed of Vaccaria segetalis (Neck.) Garcke, has various therapeutic characteristics in traditional Chinese medicine (TCM), containing promoting blood circulation and unblocking meridians. It exhibits significant anti-cancer activity and is therapeutically utilized to treat and reduce chemotherapy adverse effects in cancer patients, notably those with lung cancer. However, the active ingredients responsible for its anti-lung cancer efficacy remain unknown. In this study, we used A549 cell fishing in conjunction with UHPLC-LTQ Orbitrap MS to screen for anti-lung cancer active components in Vaccariae Semen. The cell counting Kit-8 (CCK-8) assay revealed that the n-butanol extract substantially reduced A549 cell growth. Through the cell fishing assay, we found 14 A549 cell-binding compounds in the n-butanol extract, all of which were identified as triterpenoid saponins. The total saponins of Vaccariae Semen were subsequently purified using macroporous adsorption resin (MAR), and they showed a significant inhibitory effect on the proliferation of A549 lung cancer cells, as well as alterations in cell morphology, apoptosis, and fragmentation. In conclusion, saponins were discovered as the key active components responsible for the anti-lung cancer activity of Vaccariae Semen.

Elemene induces cell apoptosis via inhibiting glutathione synthesis in lung adenocarcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Curcuma wenyujin Y.H. Chen & C. Ling, also known as Wen-E-Zhu, has been used for cancer treatment since ancient times, with roots dating back to the Song Dynasty. Elemene (EE), a sesquiterpene extract with potent anticancer properties, is extracted from Wen-E-Zhu, with ß-elemene (BE) being its main active compound, along with trace amounts of ß-caryophyllene (BC), γ-elemene and δ-elemene isomers. EE has demonstrated broad-spectrum anti-cancer effects and is commonly used in clinical treatments for various types of malignant cancers, including lung cancer. Studies have shown that EE can arrest the cell cycle, inhibit cancer cell proliferation, and induce apoptosis and autophagy. However, the exact mechanism of its anti-lung cancer activity remains unclear and requires further research and investigation. AIM OF THE STUDY: In this study, the possible mechanism of EE and its main active components, BE and BC, against lung adenocarcinoma was investigated by using A549 and PC9 cell lines. MATERIALS AND METHODS: The subcutaneous tumor model of nude mice was constructed to evaluate the efficacy of EE in vivo, then the in vitro half-inhibitory concentration (IC50) of EE and its main active components, BE and BC, on A549 and PC9 cells at different concentrations were determined by CCK-8. Flow cytometry was used to detect the apoptosis and cycle of A549 and PC9 cells treated with different concentrations of BE and BC for 24 h. Non-targeted metabolomics analysis was performed on A549 cells to explore potential target pathways, which were subsequently verified through kit detection and western blot analysis. RESULTS: Injection of EE in A549 tumor-bearing mice effectively suppressed cancer growth in vivo. The IC50 of EE and its main active components, BE and BC, was around 60 µg/mL. Flow cytometry analysis showed that BE and BC blocked the G2/M and S phases of lung adenocarcinoma cells and induced apoptosis, leading to a significant reduction in mitochondrial membrane potential (MMP). Results from non-targeted metabolomics analysis indicated that the glutathione metabolism pathway in A549 cells was altered after treatment with the active components. Kit detection revealed a decrease in glutathione (GSH) levels and an increase in the levels of oxidized glutathione (GSSG) and reactive oxygen (ROS). Supplementation of GSH reduced the inhibitory activity of the active components on lung cancer and also decreased the ROS content of cells. Analysis of glutathione synthesis-related proteins showed a decrease in the expression of glutaminase, cystine/glutamate reverse transporter (SLC7A11), and glutathione synthase (GS), while the expression of glutamate cysteine ligase modified subunit (GCLM) was increased. In the apoptosis-related pathway, Bax protein and cleaved caspase-9/caspase-9 ratio were up-regulated and Bcl-2 protein was down-regulated. CONCLUSIONS: EE, BE, and BC showed significant inhibitory effects on the growth of lung adenocarcinoma cells, and the mechanism of action was linked to the glutathione system. By down-regulating the expression of proteins related to GSH synthesis, EE and its main active components BE and BC disrupted the cellular redox system and thereby promoted cell apoptosis.

Chemical profiling of Fritillariae thunbergii Miq prepared by different processing methods reveals two new quality markers: Zhebeininoside and imperialine-3-ß-D-glucoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae thunbergii Miq (FTM)exhibit versatile biological activities including the significant antitussive and expectorant activities. As a herbal medicine, the therapeutic effects of FTM may be expressed by multi-components which have complicated integration effects on multi-targets. With the time going, the different processing methods of FTM has been changed a lot. Thus，the study described the effect of processing methods to FTM and its quality. MATERIAL AND METHOD: Studies were undertaken by using UHPLC-LTQ Orbitrap MS and pharmacodynamic models. All reagents were involved of analytical grade. While a HPLC-ELSD's method has been developed and validated, a certified Quality System is conformed to ICH requirements. The experimental animals followed the animal welfare guidelines. AIM OF THE STUDY: We aimed to found the differences after the different processing methods of FTM, and to demonstrate the changes could be selected as quality control indicators, and established a method for simultaneous determination of these for quality control. RESULTS: we have previously found two new steroidal alkaloids: zhebeininoside and imperialine-3-ß-D-glucoside from the different processing methods of FTM, which is the difference between the different processing methods of FTM, mainly on the steroidal alkaloids. The activity analysis of zhebeininoside, imperialine-3-ß-D-glucoside, verticine and verticinone showed that the mouse model of cough expectorant has antitussive effect. The positive drug selected was dextromethorphan syrup. The positive group showed biological activity, but the blank group showed nothing. The model group showed illness which means that the model was effective. There are two ways of the mechanism of action of the expectorant action which can make sputum thin, reduce its viscosity, and be easy to cough up, or can accelerate the movement of mucous cilia in the respiratory tract and promote the discharge of sputum. In our study, the content of phenol red was significantly reduced in the administration group. CONCLUSIONS: To sum up, our results suggest that zhebeininoside and other three components cloud be selected as quality control indicators, and a method for simultaneous determination of zhebeininoside and other three components was established for quality control.

[Research on revision of origins to Chinese medicinal materials in 2020 edition of Chinese pharmacopoeia].

The origins of 9 species of the Chinese medicinal materials in the 2015 edition of the Chinese pharmacopoeia(ChP) has revised in the 2020 edition of ChP. The revision is based on the investigation and textual research on the problems found after screening the original plants, animals or minerals of all the Chinese medicinal materials in the 2015 edition. Among them the Chinese names of Alismatis Rhizoma, Cassiae Semen, Coicis Semen, Corydalis Bungeanae Herba and Echinopsis Radix all do not match to the Latin scientific names, and also do not match the name of the actual medicinal origins. In addition, Alismatis Rhizoma has the omission of original plant. There is confusion about the Chinese name and the family name of the original insect of Cera Chinensis. The original mineral of Gypsum Fibrosum has the wrong group names. Alumstone and melanterite, the original mineral of Alumen and Melanteritum respectively, of which the group names are missing. To solve these problems, field survey and literature research were conducted on the medicinal materials and their origins. The source of these problems are explored. The correct origins and the Chinese names or Latin names are all determined according to the research results to the situation, in which the Chinese and Latin names of the original plants of the medicinal materials do not match. The correct family name and group name are obtained through textual research by taxonomy if the names are confused or mis-sing. The scientific evidence and correct results of revision in the 2020 edition of ChP are determined at last.

Bioactivity-guided discovery of quality control markers in rhizomes of Curcuma wenyujin based on spectrum-effect relationship against human lung cancer cells.

BACKGROUND: Due to the diversity of the ingredients, the complexity of the mechanism of action, the uncertainty of the effective ingredients, coupled with the multiple species and multiple growing areas, the quality control (QC) of Traditional Chinese Medicines (TCMs) is challenging. Discovering and identifying effective compounds from the complex extracts of TCMs and then establishing a scientific QC method is the key to the holistic QC of TCMs. PURPOSE: To develop an anti-lung-cancer-guided spectrum-effect relationship approach for the discovery of QC markers of the rhizome of Curcuma wenyujin (WEZ) and establish a bioactive compounds-based holistic QC method. METHODS: The chemical profiling of the volatile oil (WVO) from 42 batches of WEZ collected from different growing areas was performed by GC-MS. The anti-lung cancer activity of different WVO samples was determined by CCK-8 assay against human lung cancer cells (A549). The apoptosis and cell cycle analysis under different concentrations of WVO were detected by flow cytometry. SIMCA-P software was used to perform multivariate statistical analysis on the chemical composition of different WVO samples and to find the different components. Active compounds were screened using a PLSR model of the spectrum-effect relationship. Bioactive compounds-based fingerprint and quantification of the leading bioactive compounds were developed by GC-MS and GC-FID, respectively. RESULTS: Seventy-eight compounds were detected in WVO and 54 were successfully identified. The multivariate statistical analysis uncovered that WVO components and the anti-A549 activity of WVO at the concentration of 60 nl/ml differ greatly according to the origin of the plant. The WVO at the concentration of 60 nl/ml (IC50) increased A549 cells apoptosis significantly with late and early apoptosis of 15.61% and 7.80%, and the number of cells in the G2/M phase were also increased significantly under this concentration. The spectrum-effect relationship analysis revealed that 44 compounds were positively correlated with their activities, and the result was verified by A549 cell viability assay. Sixteen positively correlated compounds were further selected as QC markers according to their relative amount > 0.5% and anticancer activity. Finally, the 16 QC markers-based GC-MS fingerprint was established to holistically control the quality of WEZ, and a GC-FID method was developed for the quantification of leading bioactive compounds, ß-elemene and ß-caryophyllene. CONCLUSION: Based on an anti-lung-cancer-guided spectrum-effect relationship approach, the bioactive compounds-based holistic QC method was successfully developed for WEZ, which could provide a valuable reference for the QC of TCMs.

A combination index and glycoproteomics-based approach revealed synergistic anticancer effects of curcuminoids of turmeric against prostate cancer PC3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal medicines (HMs) often exert integration effects, including synergistic, additive and antagonistic effects, in such ways that they act on multiple targets and multiple pathways on account of their multiple components. Turmeric, made from the rhizome of Curcuma longa L., is a well-known HM prescribed in the polyherbal formulas for cancer treatment in traditional Chinese medicines (TCMs). However, neither the multiple anticancer compounds of turmeric nor the integration effects of these components are fully known. AIM OF THE STUDY: This work aims to develop a systematic approach to reveal the integration effect mechanisms of multiple anticancer compounds in turmeric against prostate cancer PC3 cells. MATERIALS AND METHODS: Combination index and omics technologies were applied to profile the integration effect mechanisms of bioactive compounds in proportions naturally found in turmeric. PC3 cell line (a prostate cancer cell line) fishing and high resolution mass spectrometry were employed to screen and identify the anticancer compounds from turmeric. The combinations which contain different cell-bound compounds in natural proportions were prepared for further evaluation of anti-cancer activity by using cell viability assays, and assessment of cell apoptosis and cell cycle analysis. Combination index analysis was applied to study the integration effects of the anticancer compounds in their natural proportions. Finally, quantitative glycoproteomics/proteomics and Western blot were implemented to reveal the potential synergistic effect mechanisms of the anticancer compounds based on their natural proportions in turmeric. RESULTS: Three curcuminoids (curcumin, CUR; demethoxycurcumin, DMC; bisdemethoxycurcumin, BDMC) in turmeric were discovered and shown to possess significant synergistic anticancer activities. Combination index analysis revealed an additive effect of CUR combined with DMC or BDMC and a slight synergistic effect of DMC combined with BDMC in natural proportions in turmeric, while a combination of all three curcuminoids (CUR, DMC and BDMC) at a ratio of 1:1:1 yielded superior synergistic effects. Interestingly, the presence of BDMC and DMC are essential for synergistic effect. Glycoproteomics and proteomics demonstrated that different curcuminoids regulate various protein pathways, such as ribosome, glycolysis/gluconeogenesis, biosynthesis of amino acids, and combination of CUR + DMC + BDMC showed the most powerful effects on down-regulation of protein expression. CONCLUSIONS: Our analytical approach provides a systematic understanding of the holistic activity and integration effects of the anti-cancer compounds in turmeric and three curcuminoids of turmeric showed a synergistic effect on PC3 cells.

Discovery and validation of peptide biomarkers for discrimination of Dendrobium species by label-free proteomics and chemometrics.

The stems of Dendrobium officinale, a well-known and expensive food material and herbal medicine in Asia, has recently suffered adulterants and counterfeits by using lower-price confusing Dendrobium species such as D. devonianum or D. transparens in the herbal market. However, robust methods that could authenticate D. officinale from its confusing species effectively are still lacking, especially for the dried samples. This study committed to discover specific peptides biomarkers for the authentication of D. officinale from the other two Dendrobium species using label-free proteomics by nanoLC LTQ Orbitrap mass spectrometry. Multivariate statistical analysis was applied to visualize the difference between the three Dendrobium species. As a result, 29 peptides among a total of 343 measurable peptides were selected to be potential biomarkers for the classification of these Dendrobium species. The validation of the representative peptide biomarkers was carried out by the synthesized peptides and 3 peptide biomarkers were found significant for the authentication of D. officinale. Further analysis showed that peptide ALGLELDLSER may also be a biomarker for the discrimination of the D. officinale originated from different geographical regions.

Chemical markers' knockout coupled with UHPLC-HRMS-based metabolomics reveals anti-cancer integration effects of the curcuminoids of turmeric (Curcuma longa L.) on lung cancer cell line.

Turmeric (Curcuma longa L, Zingiberaceae) rhizomes exhibit versatile biological activities including the significant anti-cancer property. As an herbal medicine, the therapeutic effects of turmeric may be expressed by multi-components which have complicated integration effects on multi-targets. Therefore, having previously found three A549 cell-binding curcuminoids (curcumin, Cur; demethoxycurcumin, DMcur; bisdemethoxycurcumin, BMcur) from turmeric, studies were undertaken in this paper to determine the anti-cancer mechanism and integration effects of these curcuminoids by using chemical markers' knockout and UHPLC-LTQ Orbitrap MS-based metabolomics. Four curcuminoid-containing fractions including a mixture of 3 cell-binding curcuminoids (CE), and three individual curcuminoids with natural proportion in turmeric were prepared by chemical markers' knockout method. CE, Cur, DMcur and BMcur fractions showed significant anti-cancer activity on A549 cells. The activities of CE, Cur and BMcur fractions were comparative with the turmeric crude extract (TcE). In the metabolomics study, CE and three individual curcuminoid fractions changed the expression of 25 metabolites in A549 cells, which were involved in glycerophospholipid catabolism, sphingolipid metabolism and fatty acid metabolism, etc. Among them, glycerophospholipid catabolism was disordered greatly in CE group, while sphingolipid metabolism was suggested to be closely related to DMcur and BMcur activity. Furthermore, the metabolomics data showed that three curcuminoids existed synergistic and antagonistic actions and the use of multi-curcuminoids is more powerful than use of single curcuminoid on the metabolic alterations of A549 cells.

Comparison of three officinal species of Callicarpa based on a biochemome profiling strategy with UHPLC-IT-MS and chemometrics analysis.

Traditional Chinese medicine (TCM) materials with closely related species are frequently fungible in clinical use. Therefore, holistic comparison of the composition in bioactive compounds is essential to evaluate whether they are equivalent in efficacy. Taking three officinal species of Callicarpa as a case, we proposed and validated a standardized strategy for the discrimination of closely related TCM materials, which focused on the extraction, profiling and multivariate statistical analysis of their biochemome. Firstly, serial liquid-liquid extractions were utilized to prepare different batches of Callicarpa biochemome, and the preparation yields were utilized for the normalization of sampling quantity prior to UHPLC-IT-MS analysis. Secondly, 34 compounds, including 19 phenylethanoid glycosides, 10 flavonoids and 5 terpenoids, were identified based on an untargeted UHPLC-IT-MS method. Thirdly, method validation of linearity, precision and stability showed that the UHPLC-IT-MS system was qualified (R2>0.995, RSD<15%) for subsequent biochemome profiling. After PCA and PLS-DA analysis, 30 marker compounds were screened and demonstrated to be of good predictability using genetic algorithm optimized support vector machines. Finally, a heatmap visualization was employed for clarifying the distribution of marker compounds, which could be helpful to determine whether the three Callicarpa species are, in fact, equivalent substitutes. This study provides a standardized biochemome profiling strategy for systemic comparison analysis of closely related TCM materials, which shows promising perspectives in tracking the supply chain of pharmaceutical suppliers.

An in vitro approach for lipolysis measurement using high-resolution mass spectrometry and partial least squares based analysis.

The elevation of free fatty acids (FFAs) has been regarded as a universal metabolic signature of excessive adipocyte lipolysis. Nowadays, in vitro lipolysis assay is generally essential for drug screening prior to the animal study. Here, we present a novel in vitro approach for lipolysis measurement combining UHPLC-Orbitrap and partial least squares (PLS) based analysis. Firstly, the calibration matrix was constructed by serial proportions of mixed samples (blended with control and model samples). Then, lipidome profiling was performed by UHPLC-Orbitrap, and 403 variables were extracted and aligned as dataset. Owing to the high resolution of Orbitrap analyzer and open source lipid identification software, 28 FFAs were further screened and identified. Based on the relative intensity of the screened FFAs, PLS regression model was constructed for lipolysis measurement. After leave-one-out cross-validation, ten principal components have been designated to build the final PLS model with excellent performances (RMSECV, 0.0268; RMSEC, 0.0173; R2, 0.9977). In addition, the high predictive accuracy (R2 = 0.9907 and RMSEP = 0.0345) of the trained PLS model was also demonstrated using test samples. Finally, taking curcumin as a model compound, its antilipolytic effect on palmitic acid-induced lipolysis was successfully predicted as 31.78% by the proposed approach. Besides, supplementary evidences of curcumin induced modification in FFAs compositions as well as lipidome were given by PLS extended methods. Different from general biological assays, high resolution MS-based method provide more sophisticated information included in biological events. Thus, the novel biological evaluation model proposed here showed promising perspectives for drug evaluation or disease diagnosis.

[Screening of anti-lung cancer bioactive compounds from Curcuma longa by target cell extraction and UHPLC/LTQ Orbitrap MS].

A target cell extraction-chemical profiling method based on human alveolar adenocarcinoma cell line (A549 cells) and UHPLC/LTQ Orbitrap MS for screening the anti-lung cancer bioactive compounds from Curcuma longa has been developed in this paper. According to the hypothesis that when cells are incubated together with the extract of Curcuma longa, the potential bioactive compounds in the extract should selectively combine with the cells, then the cell-binding compounds could be separated and analyzed by LC-MS. The bioactive compounds in C. longa are lipophilic components. They intend to be absorbed on the inner wall of cell culture flask when they were incubated with A549 cells, which will produce interference in the blank solution. In this paper, by using cells digestion and multi-step centrifugation and transfer strategy, the interference problem has been solved. Finally, using the developed method, three cell-binding compounds were screened out and were identified as bisdemethoxycurcumin, demethoxycurcumin, and curcumin. These compounds are the main bioactive compounds with anti-lung cancer bioactivity in C. longa. The improved method developed in this paper could avoid the false positive results due to the absorption of lipophilic compounds on the inner wall of cell culture flask, which will to be an effective complementary method for current target cell extraction-chemical profiling technology.

[Fingerprint analysis of Fritillaria thunbergii using rapid resolution liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry].

A fingerprint method for quality assessment of Fritillaria thunbergii was developed by rapid resolution liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (RRLC-Q-TOF-MS). The separation was performed using Agilent Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 microm) by gradient elution with acetonitrile and 0.1% formic acid aqueous solution (containing 10 mmol x L(-1) ammonium formate) as the mobile phase. Q-TOF-MS was used to obtain the accurate mass for precursor and product ions. Under this chromatographic and MS condition, 12 batches of F. thunbergii and its adulterants (F. hupehensis and F. pallidiflora) were analyzed by RRLC-Q-TOF-MS. Fifteen steroidal alkaloids were identified from F. thunbergii, F. hupehensis and F. pallidiflora and nine were assigned as the common characteristic peaks for F. thunbergii. The RRLC-Q-TOF-MS fingerprint of F. thunbergii was different significantly with those of F. hupehensis and F. pallidiflora. The quality of 12 batches of F. thunbergii samples were finally evaluated by hierarchical clustering analysis (HCA) and principle component analysis (PCA). This convenient and high specific method could be used to identify and evaluate the quality of the F. thunbergii.

Mass spectrometry-based strategies for screening of bioactive natural products.

Natural products (NPs) are combinatorial chemical libraries with diversities in chemical structures and pharmacological activities. Screening active compounds is in many cases an important factor in drug discovery. It was not easy to screen out the bioactive compounds from complex extracts consisting of many NPs. Development of rapid, effective and accurate methods is in high demand. During last decades, mass spectrometry (MS)-based strategies, combining isolation, structures, and bioactivity in a single run, were programmed in the NPs screening. The current article reviews different assay formats and applications of MS-based methods for screening of active NPs. This review is divided into three sections based on methods classification. The first part introduces binding-based screening methods that directly assess the binding characteristics of a candidate molecule to its target. The second part describes function-based screening methods that monitor the functional output of a target-dependent biochemical reaction. The third part briefly discusses serum pharmacochemistry-based screening methods that analyze absorbed components and metabolites in plasma after oral administration of NPs extracts.

Characterization and identification of steroidal alkaloids in Fritillaria species using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI-QTOF-MS/MS) was performed to study the fragmentation behaviors of steroidal alkaloids from Fritillaria species, the antitussive and expectorant herbs widely used in traditional Chinese medicine. We propose, herein, a strategy that combining key diagnostic fragment ions and the relative abundances and amounts of major fragment ions (the ions exceeding 10% in abundance) to distinguish different sub-classes of Fritillaria alkaloids (FAs). It was found that hydrogen rearrangement and induction effects result in ring cleavage of the basic skeletons occurred in the MS/MS process and produced characteristic fragment ions, which are useful for structural elucidation. This method was finally used to investigate the primary steroidal alkaloids in the extracts of eight major Fritillaria species. As a result, 41 steroidal alkaloids (29 cevanine type, 1 jervine type, 6 veratramine type and 5 secosolanidine type alkaloids) were selectively identified in these Fritillaria species. Twenty-six compounds were unambiguously identified by comparing with the reference compounds and 15 compounds were tentatively identified or deduced according to their MS/MS data. Logical fragmentation pathways for different types of FAs have been proposed and are useful for the identification of these types of steroidal alkaloids in natural products especially when there are no reference compounds available.

Structural characterization and identification of oleanane-type triterpene saponins in Glycyrrhiza uralensis Fischer by rapid-resolution liquid chromatography coupled with time-of-flight mass spectrometry.

Oleanane-type triterpene saponins (OTS) are major active ingredients in Glycyrrhiza uralensis. In this work, a rapid-resolution liquid chromatography with time-of-flight mass spectrometry (RRLC/TOF-MS) method has been developed to characterize and identify OTS from G. uralensis. The major diagnostic ions and fragmentation pathways from thirteen OTS have been characterized for the first time. At a low fragmentor voltage of 120 V in positive ion mode, the precursor ion [M + H](+) or/and [M + Na](+) was obtained for accurate determination of molecular formula. When the fragmentor voltage was increased to 425 V, abundant characteristic fragment ions were observed for structural characterization. Neutral losses of sugar moieties, such as glucuronic acid (GlcA, 176 Da), glucose (Glc, 162 Da) and rhamnose (Rha, 146 Da), were commonly observed in the MS spectra for prediction of the sugar number and sequences. Other typical losses included AcOH (60 Da), CH(2)O (30 Da), 2 × H(2)O (2 × 18 Da) and HCOOH (46 Da) from [Aglycone + H-H(2)O](+) (named [B](+)), corresponding to the presence of a C(22)-acetyl group, C(24)-hydroxyl group, C(22)-hydroxyl group or C(30)-carboxyl group on the aglycone moiety, respectively. In particular, characteristic ring cleavages of the aglycone moieties on A- and B-rings were observed. Based on the fragmentation patterns of reference compounds, nineteen OTS have been identified in an extract of G. uralensis, thirteen of which were unambiguously identified and the other six were tentatively assigned.

Chemical markers' fishing and knockout for holistic activity and interaction evaluation of the components in herbal medicines.

A strategy based on chemical markers' fishing and knockout has been proposed for holistic activity and interaction evaluation of the bioactive components in herbal medicines (HMs). It was devised to screen bioactive-compound group that represents the efficacy of HM, estimate the bioactivity contribution of each component and elucidate the interactions of multi-components. This strategy was accomplished through the following steps: (1) screen out the chemical markers (target peaks) in a HM fingerprint using online two-dimensional turbulent flow chromatography/liquid chromatography-mass spectrometry technique, (2) fish target peaks and knockout any interested peak, and (3) evaluate the bioactivities of fishing and knockout portions. After comparison of the bioactivities of samples containing different target peaks, the efficacy of target-peak group, bioactivity contribution of each compound, and the interactions of multi-components are elucidated. Using Acetylcholinesterase (AChE) and Bulbs of Lycoris radiata (L. Herit.) Herb. (BLR) as the experimental materials, four target peaks were screened out as the AChE binders. By target peaks' fishing and knockout, combined with activity evaluation, we observed that the bioactivity of the four-peak mixture is similar with the global bioactivity of BLR extract, and there are significant suppressive actions among these four target peaks. These results indicate that this proposed strategy is a useful approach for holistic screening of bioactive-compound group and elucidation of the multi-component interactions in HM.

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine, verticinone and isoverticine in rat plasma.

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC-LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 microm particle size (Agilent Zorbax StableBond-C(18), 4.6 mm x 50 mm, 1.8 microm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic-lipophilic balanced copolymer (2.1 mm x 20 mm, 25 microm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC-MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R>0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.

Puqienine E: an angiotensin converting enzyme inhibitory steroidal alkaloid from Fritillaria puqiensis.

Eight steroidal alkaloids, puqienine A, puqienine B, puqienine C, puqienine D, puqienine E, puqietinone, puqiedine and peimisine were isolated from Fritillaria puqiensis G. D. Yu et. G. Y. Chen, and their anti-hypertensive effect were assessed in vitro based on the inhibition of the purified angiotensin converting enzyme (ACE) using high-performance liquid chromatography assay. The results showed that puqienine E, puqienine B and puqienine A exhibited better inhibitory activity against ACE than others, with inhibition ratios of 70.2+/-0.5%, 24.7+/-0.5% and 20.4+/-2.8%, respectively at the concentration of 200 microM. The 50% inhibiting concentration of puqienine E was determined to be 68 microM.