Xiao-Ying Qin

Characterization and determination of the major constituents in Belamcandae Rhizoma by HPLC–DAD–ESI-MSn

Belamcandae Rhizoma, derived from the rhizome of Belamcanda chinensis (L.) DC., has been used as traditional Chinese medicine for the treatment of coughing and pharyngitis. However, there have been few studies dealing with the systematic analysis of the bioactive constituents in Belamcandae Rhizoma. In this work, high performance liquid chromatography-diode array detection-electrospray ionization multiple-stage mass spectrometry (HPLC-DAD-ESI-MS(n)) combined with liquid chromatography-time of flight-mass spectrometry (HPLC-TOF/MS) was established for profiling and characterization of multi-constituent in Belamcandae Rhizoma. The ESI-MS(n) fragmentation behaviors of the authentic references were proposed for aiding the structural identification of components in the extract. Thirty-five flavonoids, including 30 isoflavones and five xanthones, were identified or tentatively identified by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data. Twelve of the identified compounds (neomangiferin, mangiferin, tectoridin, iristectorin B, iristectorin A, iridin, tectorigenin, iristectorigenin A, irigenin, irisflorentin, irilone and dichtomitin) were determined by HPLC-DAD using a C(18) column. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in herbal medicine. This work is expected to provide comprehensive information for the quality evaluation of Belamcandae Rhizoma, which would be a valuable reference for the further study and development of this herb and related medicinal products.

Four new eudesmane-type sesquiterpenes from the basal leaves of Salvia plebeia R. Br.

Four new eudesmane-type sesquiterpenes, named plebeiolide A-C (1-3) and plebeiafuran (4), together with a known eudesmanolide (5), were isolated from the basal leaves of Salvia plebeia R. Br. Their structures were elucidated by extensive spectroscopic analysis including 1D and 2D NMR, HR-ESI-MS spectra. The absolute configuration of compound 1 was confirmed by single-crystal X-ray analysis and CD spectra. The inhibitory activity of isolated compounds toward NO production in lipopolysaccharide-induced RAW264.7 cells was evaluated and compounds 3 and 4 showed moderate inhibitory activity. In addition, the sesquiterpene lactones in Lamiaceae plants may possess some chemosystematic implications at intergeneric and intrageneric levels.

Tectorigenin Attenuates Palmitate-Induced Endothelial Insulin Resistance via Targeting ROS-Associated Inflammation and IRS-1 Pathway

Tectorigenin is a plant isoflavonoid originally isolated from the dried flower of Pueraria thomsonii Benth. Although its anti-inflammatory and anti-hyperglycosemia effects have been well documented, the effect of tectorigenin on endothelial dysfunction insulin resistance involved has not yet been reported. Herein, this study aims to investigate the action of tectorigenin on amelioration of insulin resistance in the endothelium. Palmitic acid (PA) was chosen as a stimulant to induce ROS production in endothelial cells and successfully established insulin resistance evidenced by the specific impairment of insulin PI3K signaling. Tectorigenin effectively inhibited the ability of PA to induce the production of reactive oxygen species and collapse of mitochondrial membrane potential. Moreover, tectorigenin presented strong inhibition effect on ROS-associated inflammation, as TNF-α and IL-6 production in endothelial cells was greatly reduced with suppression of IKKβ/NF-κB phosphorylation and JNK activation. Tectorigenin also can inhibit inflammation-stimulated IRS-1 serine phosphorylation and restore the impaired insulin PI3K signaling, leading to a decreased NO production. These results demonstrated its positive regulation of insulin action in the endothelium. Meanwhile, tectorigenin down-regulated endothelin-1 and vascular cell adhesion molecule-1 overexpression, and restored the loss of insulin-mediated vasodilation in rat aorta. These findings suggested that tectorigenin could inhibit ROS-associated inflammation and ameliorated endothelial dysfunction implicated in insulin resistance through regulating IRS-1 function. Tectorigenin might have potential to be applied for the management of cardiovascular diseases involved in diabetes and insulin resistance.

Analysis of catalpol derivatives by characteristic neutral losses using liquid chromatography combined with electrospray ionization multistage and time-of-flight mass spectrometry.

Iridoid glycosides are found in some families such as Scrophulariaceae, Labiatae, Rubiaceae and Gentianaceae. They have been of particular interest due to their important biochemical and physiological effects, including anti-inflammatory properties, anti-microbial or anti-viral effects, detoxification, diuretic functions, and resolving phlegm effects, etc. Veronica linariifolia Pall. ex Link ssp. dilatata (Nakai et Kitagawa) Hong is a member of the family Scrophulariaceae. Phytochemical studies and our previous studies show that iridoid glycosides are its main components, with catalpol being the predominant derivative. However, a systematic study of the mass spectrometry of catalpol derivatives and the fragmentation pathways under electrospray ionization (ESI) conditions has not been conducted yet, due to the complexity of the compounds, the easy degradation of iridoid glucosides and lack of authentic standards. Liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) has become a powerful technique, because of the increased power of resolution, full-scan capabilities, and accurate mass measurements, for the sensitive analysis of the multiple active components found in herbal medicines. It provides the elemental compositions of unknown constituents with high accuracy (routinely within 5 ppm) in complex matrices. In comparison, liquid chromatography/electrospray ionization multistage tandem mass spectrometry (LC/ESI-MS) has been widely used to provide highly sensitive and abundant information on the molecular mass and structural features on-line. The structural elucidation of the unknown constituents with the same parent nucleus using the LC/ ESI-MS platform is based on their structural similarities. Using the product ion scan mass spectrum of the authentic standards as a substructural template, detailed structural information of the compounds with similar structures can be rapidly characterized by extracting protonated or deprotonated molecules, subsequent or simultaneous product ions and neutral losses. Utilizing the combination of LC/TOF-MS and LC/ESIMS to gather accurate mass measurements and complementary structural information, we apply the powerful approach for the identification and structural elucidation of the iridoid glycosides, especially the catalpol derivatives in V. linariifolia. Characteristic neutral losses and product ions of the catalpol derivatives were concluded, and appropriate fragmentation pathways were readily proposed based on definite composition of the product ions. Entire plants of V. linariifolia were collected in Wanrong City, Shan Xi Province in China and identified by Prof. Minjian Qin. High-performance liquid chromatography (HPLC)-grade methanol was purchased from Merk (Darmstadt, Germany). Standards were isolated and purified from V. linariifolia in our laboratory, including verproside, catalpol, geniposidic acid and aucubin. Their structures (Fig. 1) were identified by spectroscopic methods (UV, IR, MS, H-NMR and C-NMR). The TOF-MS was performed using an Agilent orthogonal TOF mass spectrometer equipped with an ESI source (Agilent Corp., CA, USA), and was performed using full scan mode. The mass range was set at m/z 50–1000 in negative mode. ESI-MS analysis was performed on a diode-array detector (DAD) and a LC/ MSD trap SL mass spectrometer (Agilent Technologies, USA). The wavelength range was set as 210–600 nm and monitored at 255 nm. The ESI source conditions were as follows: drying gas (N2) flow rate, 9.0 L min ; drying gas temperature, 3508C; nebulizer, 35 psi; capillary, 3800 V; scan range, 50–1000 u in positive and negative ion mode; The capillary exit voltage was set at 150 V for positive ion mode and 150 V for negative ion mode. All the operations, acquisition and analysis of data were controlled by Chemstation software (Agilent Technologies, USA). ESI mass spectra in both negative and positive modes were examined in this study. The iridoid glycosides were not very stable in positive mode; they are prone to yield fragment ions rather than protonated molecules, and the MS and MS fragment ions produced in negative ion mode gave better results in our study. Hence, the MS data were mainly obtained in negative mode in the following experiments. The base peak chromatogram and typical HPLC chromatograms of the extracts obtained by HPLC/ESI-MS are presented in Fig. 2. More than 25 peaks were separated and detected according to the spectrum of each peak in the base peak chromatogram. Among them, 21 major constituents including 12 iridoids, 4 flavonoids, 4 phenolic acids and 1 lignan were identified (see Supporting Information). Structures of iridoids are shown in Fig. 1. The accurate mass measurements (<5 ppm), retention times (tR), formulas, experimental and theoretical masses and errors (in ppm) of iridoid compounds 1–12 (marked as C1–C12) are listed in Table 1. The UV and ESI-MS data of C1–C12 are listed in Table 2. Compound 1–4, 6–7 and 10–12 were characterized in this plant for the first time. Most of the iridoids occur in V. linariifolia in the glycoside forms, whose 7 and 8 positions are linked through an aglycone oxygen bridge with different benzene substituent groups at the 6 position (Fig. 3). The most common sugar residues are hexoses (glucose) linked to aglycone through glycosidic bonds at the O-1 position. Additionally, the fragmentation patterns indicate that the glycosyl residues of iridoids in Veronica are mostly glucose, which was employed to elucidate other iridoids in V. linariifolia according to the standards isolated. There are a few reports I: 10.1002/rcm.4676

Application of an efficient strategy for discovery and purification of bioactive compounds from Chinese herbal medicines, a case study on the Puerariae thomsonii Flos

In this study, an efficient strategy based on bioassay-guided fractionation, high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q/TOF-MS) and high-speed counter-current chromatography (HSCCC) was established to screen and purify bioactive compounds from Chinese herbal medicines (CHMs). This screening system was efficient and successfully applied to reveal anti-prostate cancer candidates from Puerariae thomsonii Flos. As a result, an active fraction with strong in vitro anti-prostate cancer activity was obtained, and the main compounds in the fraction were purified by HSCCC, giving 82 mg of tectoridin, 36 mg of tectorigenin-7-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside and 64 mg of tectorigenin. Among them, tectorigenin, possessing the highest anti-prostate cancer activity with IC₅₀ value of 0.08 μM, has priority to be lead compound. The results of this work demonstrated that the developed method was efficient and could be employed for the rapid screening, identification and purification of active components from CHMs.

New isoflavones with cytotoxic activity from the rhizomes of Iris germanica L.

Two new compounds, 5-methoxy-3′,4′-dihydroxy-6,7-methylenedioxy-4H-1-benzo-pyran-4-one(iriskashmirianin A) (1) and 5,3′-dihydroxy-3-(4′-β-d-glucopyranosyl)-6,7-methylenedioxy-4H-1-benzo-pyran-4-one (germanaism H) (2), along with eight known compounds (3–10), were isolated from the rhizomes of Iris germanica L. The cytotoxicities of these compounds were tested using Ehrlichs ascites carcinoma (EAC) cancer cell line by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazoli-umbromide (MTT) and ATP assays. The results showed that these compounds possessed antiproliferative effects on EAC cell line. Among them, compound 1 possessed the best cytotoxic activity with IC50 ± SD of 20.9 ± 2.7 and 4.3 ± 0.9 μM for MTT and ATP assay methods, respectively.

New flavonoids with cytotoxicity from the roots of Flemingia latifolia.

Flemingia latifolia is a folk medicine in China, which is used for treating rheumatism, arthropathy, chronic nephritis and menopausal syndrome. The phytochemicals of the plant have seldom been studied so far. In present study, three new compounds, a flavanone quinone (flemingiquinone A) (1), a prenylated dihydroflavonoid (khonklonginol I) (2) and an isoflavonoid (flemilatifolin B) (3) were isolated from the roots of F. latifolia. Their structures were established by (1)H and (13)C NMR spectra and 2D NMR experiments, including COSY, HMQC, HMBC and ROESY. Meanwhile, the compounds were evaluated for cytotoxicity against two human cancer cell lines, SMMC-7721 and A-549. The results showed that compounds 1 and 2 possessed moderate antiproliferative effects on SMMC-7721 and A-549 cell lines.

Alkaloids from the Rhizomes of Iris germanica

The genus Iris (Iridaceae) comprises over 300 species, which is distributed mainly in the northern hemisphere. Iris germanica L., a perennial herb, is cultivated as an ornamental and aromatic plant in many areas of the world. The rhizomes of I. germanica are traditionally used in Pakistan for dropsy and gall bladder diseases, and as a diuretic, antispasmodic, stimulant, and aperient [1]. Previous phytochemical investigations of the Iris plants have revealed a variety of compounds, including flavones, isoflavones and their glycosides, benzoquinones, triterpenoids, and stilbene glycosides [2–6]. In this study, nine alkaloid compounds were reported from the plant. The rhizomes of Iris germanica L. were purchased from Qianxiang Town, Dongyang City, Zhejiang Province of China, in August 2010. The plant was identied by Prof. Minjian Qin, and a voucher specimen (No. Xie 2010001) was deposited in the Herbarium of Medicinal Plants of China Pharmaceutical University. Air-dried rhizomes of I. germanica (5 kg) were extracted with 95% ethanol by heat reflux extraction. The extract was filtered, combined, and concentrated in vacuum to obtain a residue (ca. 300 g), which was isolated by silica gel column chromatography with a gradient elution system of CHCl3–MeOH (100:0–0:100) to obtain 18 fractions (F1–F18). Fraction F14 (CHCl3–MeOH, 9:1, 4:1) was separated on a silica gel column with a gradient of CHCl3–MeOH (50:1–1:1) and Sephadex LH-20 (CHCl3–MeOH, 1:1) to give four compounds: 5 (9 mg), 7 (11 mg), 8 (8 mg), and 9 (12 mg). Fraction F18 (CHCl3–MeOH, 1:1) was separated by column chromatography over RP-18 with a gradient of MeOH–H2O (4:6, 5:5, 7:3, 8:2) and Sephadex LH-20 (CHCl3–MeOH, 1:1) to yield five compounds: 1 (20 mg), 2 (10 mg), 3 (9 mg), 4 (9 mg), and 6 (16 mg). All compounds were isolated for the first time from the family of Iridaceae. By a comparison of their NMR and MS data with those reported in the literatures, the structures were identified as: 1,2,3,4-tetrahydro-carboline-3-carboxylic acid (1) [7], S-(–)-methyl-1,2,3,4-tetrahydro-9H-pyrido [3,4-b]indole-3-carboxylate (2) [8], (1S,3R)-methyl 1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (3) [9], (1R,3R)-methyl 1-methyl2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (4) [10], 4-(9H-carbolin-1-yl)-4-oxobut-2-enoic acid methyl ester (5) [11], 2-(furan-2-yl)-5-(2,3,4-trihydroxybutyl)-1,4-diazine (6) [12], 3-D-ribofuranosyluracil (7) [13], 6-hydroxymethyl3-pyridinol (8) [14], and 2-amino-1H-imidazo-[4,5-b]pyrazine (9) [15]. 1,2,3,4-Tetrahydro-carboline-3-carboxylic Acid (1). C12H12N2O2. White powder. 1H NMR (500 MHz, DMSO-d6, , ppm, J/Hz): 7.44 (1H, d, J = 7.8, H-5), 7.32 (1H, d, J = 8.1, H-8), 7.07 (1H, t, J = 7.4, 7.2, H-7), 6.98 (1H, t, J = 7.4, 7.3, H-6), 4.20 (1H, d, J = 15.3, H-1a), 4.15 (1H, d, J = 15.4, H-1b), 3.59 (1H, dd, J = 5.1, 10.1, H-3), 3.10 (1H, dd, J = 4.6, 16.0, H-4a), 2.81 (1H, dd, J = 5.1, 16.0, H-4b). 13C NMR (125 MHz, CD3OD, , ppm): 169.3 (C=O), 136.2 (C-8a), 127.9 (C-9a), 126.2 (C-4b), 121.2 (C-7), 118.7 (C-6), 117.7 (C-5), 111.1(C-8), 106.6 (C-4a), 79.1 (C-3), 56.6 (C-1), 22.9 (C-4). ESI-MS m/z 217 [M + H]+. S-(–)-Methyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylate (2). C13H14N2O2. White powder. 1H NMR (500 MHz, CD3OD, , ppm, J/Hz): 7.39 (1H, d, J = 7.7, H-5), 7.26 (1H, d, J = 7.9, H-8), 7.04 (1H, t, J = 7.6, 7.2, H-7), 6.97 (1H, t, J = 7.7, 7.1, H-6), 4.12 (1H, d, J = 15.8, H-1a), 4.03 (1H, d, J = 15.3, H-1b), 3.81 (1H, dd, J = 9.3, 4.7, H-3), 3.78 (3H, s, OCH3), 3.10 (1H, dd, J = 14.6, 4.7, H-4a), 2.88 (1H, dd, J = 9.2, 15.9, H-4b). 13C NMR (125 MHz, CD3OD, , ppm): 174.9 (C=O), 138.0 (C-8a), 132.9 (C-9a), 128.4 (C-4b), 122.1 (C-7), 119.8 (C-6), 118.3 (C-5), 111.9 (C-8), 107.1 (C-4a), 56.6 (OCH3), 54.5 (C-3), 42.6 (C-1), 25.9 (C-4). ESI-MS m/z 231 [M + H] +.