Jin-Zhong 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.

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]

Anticancer and anti-angiogenic activities of extract from Actinidia eriantha Benth root

ETHNOPHARMACOLOGICAL RELEVANCE The roots of Actinidia eriantha Benth (AER) are commonly used traditional folk medicine for the treatment of gastric carcinoma, nasopharyngeal carcinoma, and breast carcinoma. Besides, the anti-proliferative and immunomodulatory effects of AER polysaccharides on tumor-bearing mice have been reported previously. AIM OF THE STUDY This work was carried out to investigate the anticancer and anti-angiogenic activities of AER. MATERIALS AND METHODS The growth inhibitory effects of ethanol extracts from the leaves (EEL), stems (EES) and roots (EER) of A. eriantha on human gastric carcinoma SGC7901 cells, human nasopharyngeal carcinoma CNE2 cells, human breast carcinoma MCF7 cells and human umbilical vein endothelial cells (HUVECs) were evaluated by MTT assay. The ethyl acetate fraction from EER (EA-EER) was further investigated for the anticancer activity against SGC7901 cells and the anti-angiogenic activity in HUVECs in vitro. The apoptosis in SGC7901 cells and HUVECs was confirmed by DAPI nuclear staining and flow cytometry analysis, the effect on cellular DNA fragmentation was detected in SGC7901 cells. And the cell cycle-arresting activity in HUVECs was determined by flow cytometry. Moreover, the inhibitory effect of EA-EER on cell migration in HUVECs was observed by both wound-healing and Transwell migration assays. RT-PCR and Western-blotting were performed to determine the mRNA and protein expression levels, respectively, including Bax, Bcl-2 and caspase-3 in SGC7901 cells, as well as VEGF-A and VEGFR-2 in HUVECs. Furthermore, the in vivo anti-angiogenic activity of EA-EER was evaluated by using chick embryo chorioallantoic membrane (CAM) assay. Ultimately, the chemical components in EA-EER were isolated and purified by repeated column chromatography followed by structure characterization using 1H and 13C nuclear magnetic resonance and mass spectroscopy. RESULTS Compared with EEL and EES, EER displayed the strongest growth inhibitory effect on SGC7901 cells, CNE2 cells and HUVECs. Among the EER fractions, EA-EER exhibited the most potent growth inhibitory activity against SGC7901 cells, CNE2 cells and HUVECs. Moreover, EA-EER induced obvious apoptosis in SGC7901 and HUVECs, and significantly inhibited the proliferation of HUVECs via blockade of cell cycle G1 to S progression. Furthermore, EA-EER suppressed the expression of Bcl-2 and improved the expression Bax and caspase-3 in SGC7901 cells. EA-EER not only inhibited migration of HUVECs, but also down-regulated the expression of VEGF-A and VEGFR-2 in HUVECs. In vivo, EA-EER exposure reduced the formation of blood vessels in chick embryos. A bio-guided isolation of EA-EER led to the isolation of three compounds for the first time, namely (6R, 7E, 9S)-6, 9-hydroxy-megastigman-4, 7-dien-3-one-9-O-β-D-glucopyranoside, Oleanolic acid-23-O-β-D-glucopyranoside, 3β, 23, 24-trihydroxyl-12-oleanen-28-oic acid. CONCLUSION The present research demonstrated that the significant anticancer and anti-angiogenic effects of AER, providing the supportive evidence for its traditional use in the treatment for cancer. It was suggested that AER could be use as a potential source of cancer therapeutic drug.

Comparison of Chemical Composition of Spore Lipids Extracted from Two Species of Ganoderma

Ganoderma, commonly referred to as “Lingzhi” in Chinese, “Reizhi” in Japanese, and “Youngzhi” in Korean, is a family of medicinal mushrooms employed to maintain human vitality and to promote longevity in traditional Chinese medicine for over 2000 years [1]. To date, Ganoderma has been used as remedy for the treatment of various diseases such as migraine and headache, hypertension, arthritis, hepatitis, cardiovascular problems, and cancer. It has been shown to contain a variety of bioactive ingredients including trierpenes, polysaccharides, nucleosides, steroids, fatty acids, alkaloids, proteins, peptides, amino acids, and inorganic elements [2]. Recently, Ganoderma spores gained considerable attention owning to their possible higher bioactivity [3]. However, spores have extremely hard and resilient bilayer walls, thus making them difficult to break open, and their bioactive lipid is stored between the inner and outer walls and is difficult to extract [4]. The difficulty above could be overcome by using superfine grinding technology through breaking the sporoderm and removing plant cellulose barriers [5]. Furthermore, it was reported that alcohol extracts from sporoderm-broken spores were more cytotoxic against tumor cells than that from sporoderm-nonbroken spores [6]. Super CO2 fluid extraction (SFE), a well-known extract technology, was established to extract liposoluble materials, which presented some advantages such as being nontoxic, high-efficiently, and less-deteriorating compared with the conventional methods viz. hydrodistillation, steam distillation, or solvent extraction [7]. In our current work, spores from Ganoderma duropora Lloyd (G. duropora) and Ganoderma lucidum (Leyss.ex Fr.) Karst. (G. lucidum) were superfine pulverized prior to extraction by SFE. The chemical composition of DSL (G. duropora spores lipids) and LSL (G. lucidum spores lipids) were then determined and compared by GC-MS. The results showed that spore lipids from G. duropora and G. lucidum obtained by SFE gave an average yield of 14.14% and 20.66% on a dry weight basis, respectively. As given in Table 1, GC-MS analyses revealed the presence of 22 and 10 compounds whose total relative contents were 74.04% and 88.58% in DSL and LSL, respectively. Aliphatic compounds were the main constituents in these two spore lipids, except for naphthalene and ergosterol belonging to aromatics and steroids, respectively. The chromatographic profile showed that both contained eight compounds and high levels of n-hexadecanoic acid and oleic acid (9.52%, 61.19% in DSL and 7.99%, 80.01% in LSL, as shown in Table 1). Nevertheless, 1-nonadecene (0.02%) and 2,3-dihydroxypropyl elaidate (0.07%) were detected in LSL but not in DSL. According to the above-mentioned results, it was concluded that the components from two spore lipids resembled each other. From this point of view, G. duropora in high production amounts might be developed as an alternative to Ganoderma.