Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum.

Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs' biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, and the enzyme reaction products were detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) methods. The results indicated that both XsGAO1 and XsGAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum.

Chemical Constituents of Pyrrosia calvata.

A novel flavanone glycoside, 3',5',5,7-tetrahydroxy-6-C-ß-D-glucopyranosyl-flavanone (1), along with 16 known compounds, (R/S)-eriodictyol-8-C-ß-D-glucopyranoside (2), quercetin-3-O-α-L-rhamnosyl (1''' --> 3''')-ß-D-glucopyranoside (3), hemipholin (4), 4ß-carboxymethyl-(-)-epicatechin methyl ester (5), kaempferol (6), quercetin (7), mangiferin (8), chlorogenic acid (9), 1,5-O-dicaffeoylquinic acid (10), 3,5-O-dicaffeoylquinic acid (11), 3-O-caffeoylquinic acid methyl ester (12), 1-O-caffeoyl glycoside (13), 4-O-ß-D-glucopyranosyl-caffeic acid (14), 3'-O-methyleplcatechin-7-O-ß-D-glucopyranoside (15), hop-22(29)-en-30-ol (16) and diploptene (17), were isolated from the whole plant of Pyrrosia calvata (Backer) Ching. Among them, compounds 2, 3, 4, 10, 11, 13 and 14 were isolated from the family Polypodiaceae for the first time, and compound 5 has not been recorded previously from the genus Pyrrosia.

Cause and control of Radix Ophiopogonis browning during storage.

In the storage of Radix Ophiopogonis, browning often happens to cause potential risk with regard to safety. Previously few reports investigate the browning of Radix Ophiopogonis. In this research, the causes and mechanisms of the browning of Radix Ophiopogonis were preliminarily elucidated. Content determination by high-performance liquid chromatography (HPLC) and spectrophotometry, enzyme activity determination by colorimetry, and morphological observation by electron microscopy were performed in the present study. Uniform design and three-dimensional response surfaces were applied to investigate the relationship between browning and storage factors. The cortex cell wall of browned Radix Ophiopogonis was ruptured. Compared with the normal Radix Ophiopogonis, cellulase and polyphenol oxidase enzymes were activated, the levels of 5-hydroxymethylfurfural (5-HMF), total sugars, and reducing sugars were increased, while the levels of polysaccharides and methylophiopogonanone A were decreased in browned Radix Ophiopogonis. The relationship between the storage factors and degree of browning (Y) could be described by following correlation equation: Y = - 0.625 4 + 0.020 84 × X3 + 0.001 514 × X1 × X2 - 0.000 964 4 × X2 × X3. Accompanied with browning under storage conditions, the chemical composition of Radix Ophiopogonis was altered. Following the activation of cellulase, the rupture of the cortex cell wall and the outflow of cell substances flowed out, which caused the Radix Ophiopogonis tissue to become soft and sticky. The main causes of the browning were the production of 5-HMF, the activation of polyphenol oxidase, Maillard reactions and enzymatic browning. Browning could be effectively prevented when the air relative humidity (HR), temperature, and moisture content were under 25% RH, 12 °C and 18%, respectively.

Post-harvest induced production of salvianolic acids and significant promotion of antioxidant properties in roots of Salvia miltiorrhiza (Danshen).

Danshen, the dried roots of Salvia miltiorrhiza, is an extremely valued Traditional Chinese Medicine. Previously, we have demonstrated that salvianolic acid B (SaB), the important bioactive ingredient in this herb, was a post-harvest product. Here, we further reported that all salvianolic acids (SAs) in the roots were post-harvest products of the drying process. In addition, the results of various radical scavenging activity assays, including lipid peroxidation (1), DPPH (2), hydroxyl (3) and superoxide (4), were significantly increased along with the accumulation of total salvianolic acids in the process. The contents of chemical targets and antioxidant activities both reached the highest value under thermal treatment at 130 °C for 80 min. In this dehydration period, contents of SaB, and sum of nine SAs increased from 0.01% to 5.51%, and 0.20% to 6.61%; and IC50 of antioxidant activity decreased from 4.85 to 2.69 (1); 7.75 to 0.43 (2); 2.57 to 1.13 (3) and 17.25 to 1.10 mg/mL. These results further supported the hypothesis that the newly harvested plant roots were still physiologically active and the secondary metabolites might be produced due to dehydration stress after harvest. Our findings supplied an important and useful theoretical basis for promoting the quality of Danshen and other medicinal plant materials.

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 IKKß/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.

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'-ß-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 Ehrlich's 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.

[Chemical constitunents of seeds of Oroxylum indicum].

OBJECTIVE: To study the chemical constituents in the seeds of Oroxylum indicum. METHOD: Twenty compounds were isolated and purified by silica gel, and Sephadex LH-20 column chromatography, and their structures were determined by spectroscopic analysis including NMR and MS. RESULT: Twenty compounds were isolated and identified as oroxin A (1), oroxin B (2), chrysin (3), baicalein (4), quercetin (5), apigenin (6), kaempferol (7), quercetin-3-O-ara-binopyranoside (8), lupeol C9), lup-20 (29)-ene-2alpha,3beta-diol (10), pinosylvin (11), dihydropinosylvin (12), cholest-5-ene-3, 7-diol (13), rengyol (14), isorengyol (15), zarzissine (16), (E) -pinosylvin-3-O-beta-D-glucopyranoside (17), adenosine (18), sitosterol (19) and daucosterol (20). CONCLUSION: Compounds 11-13 and 15-18 were obtained from the genus Oroxylum for the first time, and except compound 18, the remaining 6 compounds were obtained from the family Bignoniaceae for the first time.

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-[ß-D-xylopyranosyl-(1→6)-ß-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.

A new lignan with anti-HBV activity from the roots of Bombax ceiba.

A new lignan bombasinol A (1), together with three known compounds was obtained from the ethanol (95%) extract of roots of Bombax ceiba L. through its being subjected to silica gel and Sephadex LH-20 chromatography. Their structures were elucidated as 4-(4-(3,5-dimethoxyphenyl)hexahydrofuro[3,4-c]furan-1-yl)-2-methoxy-phenol (1), 5,6-dihydroxymatairesinol (2), (+)-pinoresinol (3) and matairesinol (4) on the basis of spectroscopic methods, including 1-D and 2-D NMR (HSQC and HMBC) experiments and by comparison of the data with those previously reported literatures. All these compounds were the first reported from Bombacaceae. The anti-Hepatitis B Virus (HBV) activity of all compounds isolated from B. ceiba in the research was evaluated. From the results of the HBV assay, these tested compounds showed inhibitory activity against HepG2 2.2.15 cell lines. Compounds 1-4 showed relative differences in their abilities to inhibit HBsAg secretion, with IC50 values of 118.3, 123.7, 118.9 and 218.2 mM, respectively.

New cytotoxic bis-labdanic diterpenoids from Alpinia calcarata.

Two novel bis-labdanic diterpenoids named calcaratarin D and calcaratarin E were isolated from the rhizomes of Alpinia calcarata. Their structures were elucidated as a pair of stereoisomers on the basis of the spectral evidence. 2D NMR techniques including (1)H- (1)H COSY, HMQC, HMBC, NOESY spectra and HRFABMS were extensively applied to establish the structures. The two bis-labdanic diterpenoids showed cytotoxic activity against human KB cells in vitro.