Yan-Bin Wu

Antioxidant activity of polysaccharide extracted from Ganoderma lucidum using response surface methodology.

Superfine grinding technology was applied for polysaccharide extraction from the fruiting bodies of Ganoderma lucidum, and response surface methodology (RSM) was used to optimize the effects of processing parameters on polysaccharide extraction yield. Results showed that the maximum yield of G. lucidum polysaccharides (GLP) was obtained at an optimum condition: extraction time 137 min, extraction temperature 66 ̊C, the ratio of water to material 35 mL/g, and the GLP extracting yield reached 2.44% under this condition. GLP were precipitated into three crude polysaccharides, viz. GLP40, GLP60 and GLP80. The basic characterization of polysaccharides was determined by using HPLC and FT-IR methods. GLP, GLP80, GLP60, and GLP40 were composed of Man, Rib, Glc, Gal and Fuc with the molar ratios of 1.27:0.36:22.89:1.61:0.33, 1.40:0.31:23.02:3.46:0.91, 0.96:0.34:25.76:2.47:0.46, and 2.81:1.42:23.83:1.61:0.33, respectively. The result of FT-IR suggested that the monosaccharide residue of the four polysaccharides was β-pyranoid ring. Moreover, the antioxidant activities of these four polysaccharides were evaluated. The results showed that GLP80 had the best reducing power, DPPH radical scavenging ability and oxygen radical scavenging ability followed by GLP, GLP60 and GLP40. Our results demonstrated that RSM might be a valuable technique for optimizing the efficient extraction of GLP, and G. lucidum could be considered as sources of natural antioxidants and preservatives of food industry. Moreover, polysaccharides, especially GLP80, extracted from the fruiting bodies of G. lucidum, exhibited promising antioxidant activities.

Chemical composition, antimicrobial activity against Staphylococcus aureus and a pro-apoptotic effect in SGC-7901 of the essential oil from Toona sinensis (A. Juss.) Roem. leaves

Abstract Ethnopharmacological relevance Leaves of Toona sinensis (A. Juss.) Roem. (TSL), a popular vegetable in China, have anti-inflammatory, antidoting, and worm-killing effects and are used in folk medicine for the treatment of enteritis, dysentery, carbuncles, boils, and especially abdominal tumors. Our aim was to investigate the in vitro antimicrobial activity against Staphylococcus aureus and anticancer property of the essential oil from TSL (TSL-EO), especially the pro-apoptotic effect in SGC-7901. Materials and methods TSL-EO obtained by hydrodistillation was analyzed by GC/MS and was tested in vitro against twenty clinically isolated strains of Staphylococcus aureus (SA 1–20), which were either methicillin-sensitive Staphylococcus aureus (MSSA) or methicillin-resistant Staphylococcus aureus (MRSA) and two standard strains viz. ATCC 25923 and ATCC 43300. The anticancer activity of TSL-EO was evaluated in vitro against HepG2, SGC7901, and HT29 through MTT assay. Moreover, the apoptosis-inducing activity of TSL-EO in SGC7901 cells was determined by Hoechst 33324 staining and flow cytometry methods. Also, the apoptosis-related proteins viz. Bax, Bcl-2 and caspase-3 were detected by western-blotting. Results GC–MS analysis showed that TSL-EO contained a high amount of sesquiterpenes (84.64%), including copaene (8.27%), β-caryophyllene (10.16%), caryophyllene (13.18%) and β-eudesmene (5.06%). TSL-EO inhibited the growth of both MSSA and MRSA, with the lowest MIC values of 0.125 and 1mg/ml, respectively. Treatment with TSL-EO for 24h could significantly suppress the viability of three different cancer cell lines (P<0.05). Furthermore, the apoptosis-inducing activity of TSL-EO in SGC7901 cells increased in a dose-dependent manner, potentially resulting from the up-regulated expression of Bax, caspase-3 and down-regulated expression of Bcl-2. Conclusions TSL-EO possessed antibacterial activity against Staphylococcus aureus and significant cytotoxicity against cancer cells and particularly prominent pro-apoptotic activity in SGC7901 cells. These bioactivities were probably due to the high content of sesquiterpenes. Our results suggested that TSL-EO possessed potential health benefits and could serve as a promising natural food addictive.

Antioxidant Activities of Extract and Fractions from Receptaculum Nelumbinis and Related Flavonol Glycosides

The antioxidant activities of ethanolic crude extract (ECE) and its four different solvent sub-fractions (namely, petroleum ether fraction (PEF), ethyl acetate fraction (EAF), n-butanol fraction (BF) and the aqueous fraction (AF) from the receptacles of Nelumbo nucifera Gaertn. (Receptaculum Nelumbinis) were investigated using two in vitro antioxidant assays. BF showed the highest total phenolic content (607.6 mg/g gallic acid equivalents), total flavonoid content (862.7 mg/g rutin equivalents) and total proanthocyanidin content (331.0 mg/g catechin equivalents), accompanied with the highest antioxidant activity compared to other fractions through 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assays. Five flavonol glycosides, namely hyperoside (1), isoquercitrin (2), quercetin-3-O-β-d-glucuronide (3), isorhamnetin-3-O-β-d-galactoside (4) and syringetin-3-O-β-d-glucoside (5) were isolated from the Receptaculum Nelumbinis. Compounds 2–5 were isolated for the first time from the Receptaculum Nelumbinis. The five isolated flavone glycosides, particularly compounds 1–3, demonstrated significant DPPH and ABTS radical scavenging activity, with IC50 values of 8.9 ± 0.2, 5.2 ± 0.2, 7.5 ± 0.1 for DPPH and 114.2 ± 1.7, 112.8 ± 0.8, 172.5 ± 0.7 μg/mL for ABTS, respectively. These results suggest that Receptaculum Nelumbinis has strong antioxidant potential and may be potentially used as a safe and inexpensive bioactive source of natural antioxidants.

Quantitative and Chemical Fingerprint Analysis for the Quality Evaluation of Receptaculum Nelumbinis by RP-HPLC Coupled with Hierarchical Clustering Analysis

A simple and reliable method of high-performance liquid chromatography with photodiode array detection (HPLC-DAD) was developed to evaluate the quality of Receptaculum Nelumbinis (dried receptacle of Nelumbo nucifera) through establishing chromatographic fingerprint and simultaneous determination of five flavonol glycosides, including hyperoside, isoquercitrin, quercetin-3-O-β-d-glucuronide, isorhamnetin-3-O-β-d-galactoside and syringetin-3-O-β-d-glucoside. In quantitative analysis, the five components showed good regression (R > 0.9998) within linear ranges, and their recoveries were in the range of 98.31%–100.32%. In the chromatographic fingerprint, twelve peaks were selected as the characteristic peaks to assess the similarities of different samples collected from different origins in China according to the State Food and Drug Administration (SFDA) requirements. Furthermore, hierarchical cluster analysis (HCA) was also applied to evaluate the variation of chemical components among different sources of Receptaculum Nelumbinis in China. This study indicated that the combination of quantitative and chromatographic fingerprint analysis can be readily utilized as a quality control method for Receptaculum Nelumbinis and its related traditional Chinese medicinal preparations.

Hepatoprotective effect of ganoderma triterpenoids against oxidative damage induced by tert-butyl hydroperoxide in human hepatic HepG2 cells

Abstract Context: Ganoderma triterpenoids (GTs) have been recognised as an important bioactive ingredient in Ganoderma Lucidum (Leyss. ex Fr.) Karst. (Polyporaceae), widely used for treating and preventing chronic hepatopathy of various etiologies. Objective: The objective of this study is to better understand the hepatoprotective effect of GTs and to enhance their use in food supplement pharmaceutical and medical industries. Materials and methods: HepG2 cells were pretreated in the presence or absence of GTs (50, 100 and 200 μg/ml) for 4 h, then exposed with 60 μmol/L of t-BHP for an additional 4 h. The cell viability was evaluated by MTT method. ALT, AST and LDH production in culture medium and intracellular MDA, GSH and SOD levels were determined. Moreover, the total triterpenoid content and chemical constituents in GTs were detected by ultraviolet spectrophotometry and HPLC/Q-TOF-MS, respectively. Results: GTs (50, 100 and 200 μg/ml) significantly increased the relative cell viability by 4.66, 7.78 and 13.46%, respectively, and reduced the level of ALT by 11.44%, 33.41% and 51.24%, AST by 10.05%, 15.63% and 33.64%, and LDH by 16.03%, 23.4% and 24.07% in culture medium, respectively. GTs could also remarkably decrease the level of MDA and increase the content of GSH and SOD in HepG2 cells. Furthermore, the total triterpenoid content in GTs was 438 mg GAAEs/g GTs. And 16 triterpenoids in GTs were identified or tentatively characterised. Discussion and conclusion: Our results showed that GTs had potent cytoprotective effect against oxidative damage induced by t-BHP in HepG2 cells, thus suggesting their potential use as liver protectant.

Quality evaluation of the leaves of Magnolia officinalis var. biloba using high-performance liquid chromatography fingerprint analysis of phenolic compounds.

The high-performance liquid chromatography fingerprint method is a simple and reliable technique to evaluate the quality of leaves of Magnolia officinalis Rehd.et Wils. var. biloba Rehd.et Wils. We used the following bioactive phenolic constituents as reference compounds: rutin, afzelin, hyperoside, isoquercitrin, quercetin-3-O-α-l-rhamnoside, honokiol and magnolol. The conditions of an Agilent 1200 HPLC were: YMC-Pack-ODS-AQ column (250 × 4.6 mm id S-5 μm, 12 nm), mobile phase acetonitrile and 0.2% phosphoric acid in a gradient elute mode, flow rate 1.0 mL/min, detection wavelength 280 nm and column temperature 30°C. The analytical method was validated in terms of linearity, stability, repeatability, precision and recovery tests. While performing fingerprint analysis, we identified 11 peaks as characteristic peaks and assessed the similarities of 17 samples collected from different geological regions of China. The peak areas were used to evaluate the variation in the chemical composition of the tested samples. For this purpose, we performed hierarchical cluster analysis of the peak areas. Our results indicate that simultaneous determination of multiple ingredients could be done through chromatographic fingerprint analysis. Therefore, this high-performance liquid chromatography fingerprint method was readily utilized to evaluate the quality of leaves of M. officinalis var.biloba, which are used in several traditional herbal preparations.

Pharmacokinetics and metabolism study of isoboldine, a major bioactive component from Radix Linderae in male rats by UPLC–MS/MS

ETHNOPHARMACOLOGICAL RELEVANCE Isoboldine is one of the major bioactive constituents in the total alkaloids from Radix Linderae (TARL) which could effectively alleviate inflammation and joints destruction in mouse collagen-induced arthritis. To better understand its pharmacological activities, we need to determine its pharmacokinetic and metabolic profiles. MATERIALS AND METHODS In this study, a sensitive and simple UPLC-MS/MS method was developed and validated for determination of isoboldine in rat plasma. Isoboldine in plasma was recovered by liquid-liquid extraction using 1 mL of methyl tert-butyl ether. Chromatographic separation was performed on a C18 column at 45°C, with a gradient elution consisting of acetonitrile and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. The detection was performed on an electrospray triple-quadrupole MS/MS by positive ion multiple-reaction monitoring mode. This newly developed method was successfully applied to a pharmacokinetic study after oral and intravenous dosing in rats. For metabolites identification, isoboldine was orally administered to rats and the metabolite in plasma, bile, urine and feces were characterized by the established UPLC-MS/MS method. RESULTS Good linearity (r(2)>0.9956) was achieved in a concentration range of 4.8-2400 ng/mL with a lower limit of quantification of 4.8 ng/mL for isoboldine. The intra- and inter-day precisions of the assay were 1.7-5.1% and 2.2-4.4% relative standard deviation with an accuracy of 91.3-102.3%. A total of five phase II metabolites in rat plasma, bile, urine and feces were characterized by comparing retention time in UPLC, and by molecular mass and fragmentation pattern of the metabolites by mass spectrometry with those of isoboldine. CONCLUSION isoboldine has extremely low oral bioavailability due to the strong first-pass effect by the rats, and glucuronidation and sulfonation were involved in metabolic pathways of isoboldine in rats. These results have paved the way for further clarifying therapeutic ingredients and provided new knowledge regarding pharmacokinetic features of this category of isoquinoline alkaloids.

Fatty acids, essential oils, and squalene in the spore lipids of Ganoderma lucidum by GC-MS and GC-FID

Spores of Ganoderma lucidum contain a large amount of bioactive components such as polysaccharides and lipids and have a higher bioactivity than the fruit bodies [1, 2]. It was reported that 24.82% (w/w) yield of spore oil was obtained from sporoderm-broken spores of G. lucidum by supercritical CO2 fluid extraction (SFE-CO2) [3]. But if the crude spores were not superfine-pulverized, the yield of spore oil was only 3.96% (w/w) even under the optimal condition of SFE-CO2 [4]. This result was evidenced that sporoderm-broken was a necessary technical step before SFE extraction that enables the endo-substances such as lipids to be easily extracted. Table 1 shows the results of analysis of 15 kinds of fatty acids in the spore lipids by GC-MS. The main components were palmitic acid (16:0, 16.12%), stearic acid (18:0, 4.97%), oleic acid (18:1, 67.11%), and linoleic acid (18:2, 9.63%). The totaled relative contents of unsaturated fatty acids were 77.84% (mass fraction), including 16:1, 18:1, 18:2, and 20:1. Among them, 9,12-octadecadienoic acid and (Z)-9-octadecenoic acid (total up to 76.74%) were the major compounds in the spore lipids of G. lucidum. The GC-MS results also showed that the chemical compositions of essential oils (from separator II) were different from the fatty acids (from separator I). At least 39 compounds were analyzed; among them 11 compounds were identified (see Table 2), including terpenoids (such as -pinene, D-limonene), aromatic aldehydes (such as nonanal, (E)-2-heptenal, (E)-2-decenal, (E,E)-2,4-decadienal), and several alkanes (see Table 2). In addition, squalene in the spore lipids was isolated together with fatty acids from separator I and was separately determined by GC-FID with a standard substance (tR/min 23.062 and 22.863) purchased from Sigma-Aldrich. The content of squalene was 0.43 mg/g. It is known that squalene exists in codliver oil, olive oil, and other major oil products and can improve the in vivo activity of superoxide dismutase, enhance immunity and improve sexual function, and has anti-aging, anti-cancer, and other physiological functions [5]. According to early reports, there were 20 kinds of fatty acids (C14–C25) from spore oils of G. lucidum obtained by GC-MS/LC-MS, and the major compounds were oleic acid (18:1, 63.28%), linoleic acid (18:2, 6.59%), and palmitic acid (C16:0, 20.73%) [6]. In our preliminary study, nine kinds of known fatty acids were determined by GC, and their main constituents were also oleic acid (18:1, 57.5%), linoleic acid (18:2, 13.4%), and palmitic acid (16:0, 19.6%) [7]. There are four types of components (lipids, terpenes, aromatics, and heterocyclic) in spore essential oil [8]. Moreover, some other reports have indicated that spore oils or lipids extracted from Ganoderma lucidum have antioxidant activity [4], and show positive antitumor effects in vitro and in vivo [9, 10]. The aim of this study was to use different operational conditions of SFE-CO2 from those reported previously for extracts of spore lipids and essential oils [3]. The conditions were 30 MPa of extract pressure, 40 C extract temperature, 25 L/h of CO2 flux, and 2.0 h of extract time. The spore lipids were collected from separator I (at 8 MPa and 45 C) with an average yield of 24.16% (w/w), and the essential oils were collected from separator II (at 5.0–5.5 MPa, 32 C) with an average yield of 0.64% (w/w). The chemical components of the spore lipids and essential oils were analyzed by GC and GC-MS, respectively.

Chemical Constituents of Bidens pilosa var. radiata

Bidens pilosa Linn. var. radiata Sch. Bip. (BP) belonging to the Asteraceae family is one variety of Bidens pilosa species originating from South America and transplanted to China [1]. From this species, groups of compounds with biological activity, mainly, polyacetylenes and flavonoids, have been isolated and identified [2]. However, the chemical composition of BP remains not fully understood. The plant material was dried at room temperature, powdered, and exhaustively extracted with 75% ethanol under reflux extraction. The extract was evaporated under vacuum to yield a syrupy residue. The residue was suspended in water, which was partitioned sequentially with petroleum ether, EtOAc, and n-BuOH to afford a petroleum ether fraction, an ethyl acetate fraction, a n-BuOH fraction, and the remainding water fraction after concentrating and drying. Using a series of chromatographic techniques, such as silica gel (mesh 200–300) and Sephadex LH-20 column chromatography, and PTLC, we isolated compounds 1–10 from the ethyl acetate fraction. Furthermore, the compounds were identified by their mass and NMR spectra, and all these data were in good agreement with the literature data. 3,3 -Dimethylquercetin (1). C17H14O7, yellow powder; mp, 210–212 C. ESI-MS m/z: 329 [M – H]–, 353 [M + Na]+. 1H NMR (400 MHz, DMSO-d6, , ppm, J/Hz): 3.79 (3H, s, 3-OCH3), 3.86 (3H, s, 3 -OCH3), 6.20 (1H, d, J = 2.0, H-6), 6.43 (1H, d, J = 2.1, H-8), 7.10 (1H, d, J = 8.3, H-5 ), 7.54 (1H, dd, J = 1.75, 8.4, H-6 ), 7.56 (1H, d, J = 2.2, H-2 ), 9.43 (1H, s, 4 -OH), 10.85 (1H, s, 7-OH), 12.67 (1H, s, 5-OH). 13C NMR (100 MHz, DMSO-d6, , ppm, J/Hz): 55.6 (3 -OCH3), 59.7 (3-OCH3), 93.6 (C-6), 98.6 (C-8), 104.2 (C-10), 111.9 (C-2 ), 114.9 (C-5 ), 120.3 (C-6 ), 122.2 (C-1 ), 138.0 (C-3), 146.3 (C-4 ), 150.2 (C-3 ), 155.2 (C-2), 156.3 (C-9), 161.2 (C-5), 164.2 (C-7), 177.9 (C-4) [3]. Diisopropyl Phthalate (2). C17H16O4, red crystals; identification of compound 2 was performed by comparison of the 1H NMR and 13C NMR data with those reported in [4]. 4-Hydroxy-6,4 -dimethoxychalcone (3). C14H18O4, yellow needles, identification of compound 3 was performed by comparison of the 1H NMR and 13C NMR data with those reported in [5]. Kaempferol (4). C15H10O6, yellow powder; mp 274–275 C. EI-MS m/z 286. 1H NMR (400 MHz, CD3OD, , ppm, J/Hz): 6.28 (1H, d, J = 2.2, H-6), 6.40 (1H, d, J = 2.2, H-8), 6.89 (2H, d, J = 8.7, H-3 and H-5 ), 8.07 (2H, d, J = 8.7, H-2 and H-6 ), 12.45 (1H, s, H-5). 13C NMR (100 MHz, CD3OD, , ppm, J/Hz): 94.8 (C-8), 99.6 (C-6), 104.1 (C-10), 115.0 (C-3 and C-5 ), 121.8 (C-1 ), 131.0 (C-2 and C-6 ), 137.5 (C-3), 148.4 (C-2), 158.6 (C-9), 160.7 (C-4 ), 162.8 (C-5), 165.5 (C-7), 177.7 (C-4) [6]. 4-O-(6 -O-p-Coumaroyl-D-glucopyranosyl)-p-coumaric Acid (5). C24H24O11, white powder. 1H NMR (400 MHz, DMSO-d6, , ppm, J/Hz): 4.32 (1H, dd, J = 7.2, 9.3, H-2 ), 4.35–4.36 (3H, m, H-3 , H-4 and H-5 ), 4.66 (1H, d, J = 7.2, H-1 ), 4.83 (1H, dd, J = 6.6, 11.8, H-6 ), 4.98 (1H, dd, J = 3.2, 11.8, H-6 ), 6.36 (2H, d, J = 15.8, H-8 ), 6.83 (2H, d, J = 15.8, H-8), 7.09 (2H, d, J = 8.6, H-5 and H-5 ), 7.44 (2H, d, J = 8.7, H-3 and H-3 ), 7.53 (2H, d, J = 8.7, H-2 and H-2 ), 7.53 (2H, d, J = 8.6, H-6 and H-6 ), 7.59 (2H, d, J = 15.8, H-7 ), 7.63 (2H, d, J = 15.8, H-7). 13C NMR (100 MHz, DMSO-d6, , ppm): 64.9 (C-6 ), 75.1 (C-2 ), 72.2 (C-4 ), 75.9 (C-5 ), 78.2 (C-3 ), 102.0 (C-1 ), 115.3 (C-8 ), 117.3 (C-5 and C-5 ), 118.0 (C-3 and C-3 ), 118.4 (C-8), 127.4 (C-1 ), 130.3 (C-1), 131.0 (C-6 and C-6 ), 131.5 (C-2 and C-2 ), 145.9 (C-7 ), 147.1 (C-7), 160.8 (C-4 ), 161.7 (C-4), 169.2 (C-9 and C-9 ) [7].

Supercritical fluid CO2 extraction, simultaneous determination of total sterols in the spore lipids of Ganoderma lucidum by GC-MS/SIM methods

0009-3130/12/4804-0657 2012 Springer Science+Business Media, Inc. 1) Institute of Edible and Medicinal Fungi, Fujian Academy of Agricultural Sciences, 10 Qianheng Road, 350014, Fuzhou, P. R. China, fax: +86 591 87572341, e-mail: efungi@hotmail.com; 2) Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, 1 Huatuo Road, 350108, Fuzhou, P. R. China, fax: 86 591 22861611, e-mail: jinzhongfj@126.com. Published in Khimiya Prirodnykh Soedinenii, No. 4, July–August, 2012, p. 591. Original article submitted May 3, 2011. Chemistry of Natural Compounds, Vol. 48, No. 4, September, 2012 [Russian original No. 4, July–August, 2012]