Food Sci. Biotechnol. 19(6): 1463-1469 (2010) DOI 10.1007/s10068-010-0209-5
RESEARCH ARTICLE
Antioxidant and Immunomodulatory Activities of Polysaccharides from Moxa (Artemisia argyi) Leaf Min-Bo Lan, Yan-Hong Zhang, Ying Zheng, Hui-Hui Yuan, Hong-Li Zhao, and Feng Gao
Received: 18 May 2010 / Revised: 13 August 2010 / Accepted: 14 September 2010 / Published Online: 31 December 2010 © KoSFoST and Springer 2010
Abstract The purpose of the current study was to characterize the polysaccharides isolated from the moxa (Artemisia argyi) leaf and investigate their bioactivities. The molecular weights of the purified polysaccharides were at a range from 6,000 to 10,000 Da and consisted mostly of carbohydrates (>90%), mainly the monosaccharide of xylose, arabinose, mannose, and glucose. The isolated polysaccharides were further fractionated to form 4 subfractions and all of the subfractions exhibited high antioxidant capability on both hydroxide (IC50 ranging from 8 to 18 µg/mL) and superoxide anion radicals (IC50 ranging from 10 to 30 µg/mL). The moxa leaf polysaccharides were also capable of enhancing the Concanavallin A (ConA)-induced T cell proliferation, but not the lipopolysaccharides (LPS)-induced B cell proliferation in the in vitro immunological tests. Furthermore, the polysaccharides also strongly facilitate ConA-induced secretion of interferon-γ (IFN-γ) and interleukin-2 (IL-2) in a dose-dependent manner. The results suggested that the moxa leaf polysaccharides may have potential applications as antioxidants and immune enhancers. Keywords: moxa leaf, polysaccharides, isolation, antioxidation, immunomodulation Min-Bo Lan, Yan-Hong Zhang, Ying Zheng, Hui-Hui Yuan ( ), Hong-Li Zhao Shanghai Key Laboratory of Functional Materials Chemistry, and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237, China Tel: +86-21-64252055; Fax: +86-21-64250624 E-mail:
[email protected] Min-Bo Lan Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, China Feng Gao Department of Pharmaceutics, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Introduction Polysaccharides, a kind of carbohydrate polymers consisting of multiple units of monosaccharides, represent a structurally diverse class of macromolecules that are widely distributed in nature. In the last few decades, polysaccharides have brought much attention in the field of both biochemistry and medicine. The biological activities of polysaccharides have been revealed including antiviral, antitumor, immunostimulating, antiinflammatory, anticomplementary, anticoagulant, hypoglycemic, and antitussive effects (1). Furthermore, researchers have found that polysaccharides isolated from traditional medicines could profoundly affect the immune responses both in vivo and in vitro and have the potential of being immunomodulators (2). In fact, polysaccharides such as lentinan, schizophyllan, and krestin have already been used in clinical cancer therapies (3). Moxa (Artemisia argyi) leaf is one of the most popular plants in Chinese traditional medicine and frequently used for diseases treatment such as eczema, inflammation, hemostasis, menstruation-related symptoms, and tuberculosis. Recent study on moxa leaf found that both its alcohol and water extracts exhibited antitumor effects. Extensive studies on the components of the plant have led to the identification of many components, such as monoterpenes, sesquiterpenes, triterpenes, and avones from the dry leaves (4-7). Nevertheless, a few of them in literature has focused on the antioxidant and immunostimulatory activities of the polysaccharides isolated from moxa leaf. In the present study, crude moxa leaf polysaccharides (AYC) was extracted using hot water extraction, ethanol precipitation, and freeze-drying. The extract was then fractionated through diethyl aminoethyl (DEAE)-Sepharose Fast Flow column and 4 subfractions of the polysaccharides were obtained. The antioxidant and immunomodulatory effect of the polysaccharides were investigated.
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Materials and Methods Materials and regents Moxa (Artemisia argyi) leaf cultivated in Anhui Province was purchased from Chinese medicine shop of Yanghetang (Shanghai, China). Calf serum was obtained from HyClone (China). Concanavallin A (ConA), lipopolysaccharides (LPS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) were purchased from Sigma-Aldrich (USA). Interferon-γ (IFNγ), interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), and enzyme-linked immunosorbent assay (ELISA) kits were purchased from Senxiong Biological Technical Ltd. (China). RPMI 1640 medium was obtained from Gibco (USA). H2O2 (30%), ferrous sulfate (FeSO4 ·7H2O), chloride ferric (FeCl3 ·6H2O), cupric sulfate (CuSO4 · 10H2O), ascorbic acid, sodium tetraborate (Na2B4O7 · 10H2O), 1,10-phenanthroline, sulfanilic, and pyrogallol were purchased from Shanghai Chemical Reagent Ltd. (China). All solvents and reagents obtained from commercial source were analytical reagent (AR) grade and used without further purification. Extraction and purification of the polysaccharides from moxa leaf Dried leaves of moxa leaf (2 kg) was extracted with hot water (10 L) for 1 hr. The extract was collected and the residue was extracted further with an equal amount of hot water. The obtained solution was concentrated under reduced pressure to a proper volume and precipitated by addition of 4 fold volume of 95%(v/v) ethanol. The precipitate collected by centrifugation was resuspended in distilled water and centrifuged at 3,000×g for 20 min. The supernatant was dialyzed (cut-off value of 5,000 Da) with running water for 48 hr. Finally, 60 g of aqueous soluble crude polysaccharides (AYC) was obtained by lyophilization. AYC was fractionated with different concentrations of NaCl in distilled water (0.1, 0.2, 0.5, and 1.0 M) using pretreated DEAE-Sepharose Fast Flow column. The eluates were combined as separate subfractions according to the phenol-sulfuric acid method (8). Five subfractions were obtained and named as AY00, AY01, AY02, AY05, and AY10. The subfractions were dialyzed against distilled water for 48 hr and then lyophilized. Total sugar and molecular weight measurements Total sugar content of the fractions was determined by the phenol-sulfuric acid method using glucose as the standard (8). Molecular weights of the purified polysaccharides were carried out on a Waters high performance liquid chromatography (HPLC, USA) system equipped with combined columns of TSK-GEL G5000PWXL and G4000PWXL (7.5×300 mm, 10 µm). The mobile phase was 0.2 M NaCl and the flow rate was 0.4 mL/min.
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Preliminary calibration of the column was performed with standard dextrans (Mw: 5, 10.5, 50, 270, 410, 670, and 1,400 kDa, purchased from Fluka, Switzerland). Multichrom with gel permeation chromatography (GPC) software designed for polysaccharides was employed to acquire and analyze the molecular weight data. Polysaccharides composition measurement The analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled monosaccharides was carried out on a Waters 517 HPLC system equipped with a Lichrospher NH2 (Merck) column (4.6× 250 mm, 5 µm). A carbohydrate standard mixture (10 mg/ mL), including rhamnose, xylose, arabinose, mannose, glucose, and galactose was used for monosaccharide composition identification. The hydrolyzed polysaccharides were prepared as follows (9,10): The samples were hydrolyzed with 3 M trifluoroacetic acid (TFA) in boiling water bath under nitrogen atmosphere for 4 hr. The reaction mixture was centrifuged at 1,000×g for 5 min and the supernatant was diluted with distilled water. The hydrolyzed polysaccharides and the carbohydrate standard mixture were injected into the HPLC system respectively. The mobile phase was acetonitrile-water (78:22) at a flow rate of 1.0 mL/min. Antioxidant activities The chemiluminescence (CL) method was applied to evaluate the antioxidant activities of the polysaccharides (11). The integrated area of the curve expressed the relative luminescent intensity. Antioxidant activity of the test samples was expressed as the percentage of inhibition based on the luminescent intensity of a test sample relative to that of the control group and calculated from the following equation: CLc – CLs - × 100 I(%) = ---------------------CLc – CL0
where, CLC is the relative luminescent intensity of the control group, CL0 is the relative luminescent intensity of the background group, and CLS is the relative luminescent intensity of the experimental group. Hydroxide radical scavenging activity assay The scavenging capability of the moxa leaf polysaccharides on hydroxide radical was measured in a CuSO4-Phen-VcH2O2 CL system (12). The sample solution (50 µL), CuSO4 solution (1.0 mmol/L, 50 µL), 1,10-phenanthroline solution (1 mmol/L, 50 µL), borate buffer (pH 9.18, 780 µL), and ascorbate solution (1 mmol/L, 20 µL) were placed in the instrument in a 2-mL glass tube. The reaction was initiated immediately after the injection of the H2O2 (0.15%, 20 µL) solution with a Hamilton syringe, and the kinetic curves were obtained at 1 sec intervals over a period of 100 sec.
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Polysaccharides from Moxa Leaf
Superoxide anion radical scavenging activity assay For investigation of the superoxide anion radical scavenging abilities of the crude and fractionated polysaccharides, the autoxidation of pyrogallol method was used (13). A mixture of 10 µL of sample solution and 940 µL of aqueous luminol-sodium carbonate solution (1 mmol/L, pH=10.2) was placed in the instrument in a 2-mL glass tube. The reaction was initiated by adding 50 µL of HCl with a Hamilton syringe. For the control group, 10 µL of double distilled water was added to the 940 µL of aqueous luminol-sodium carbonate solution followed by addition of 50 µL of HCl with a Hamilton syringe. The CL intensity and reaction time were recorded simultaneously and the integral CL from 0 to 10 sec was recorded. Animal cell isolation and culture Spleen cells and peritoneal macrophages were obtained from the Kunming mice at the age of 10 weeks old (Animal Experimental Center of China Pharmaceutical University). The mice were sacrificed by cervical dislocation and the spleens were removed aseptically. Spleen cells of the mice were obtained by gently teasing the organ in RPMI 1640 medium under aseptic condition. The cell suspension was centrifuged at 2,000×g for 10 min at room temperature. The cells were cultured for propagation in RPMI 1640 medium after the red blood cells were removed by erythrocyte lysate. Final density of the spleen cells was 107 cells/mL determined by trypan blue exclusion. Peritoneal macrophages were obtained from the mice after scarification and centrifuged at 1,000×g for 5 min. The cells were cultured in RPMI 1640 medium after 3 wash with Hank’s solution. Final density of the precipitate is 106 cells/mL determined by trypan blue exclusion. The cells were allowed to adhere to a 96-well culture plate in a 5% CO2 incubator at 37oC for 2 hr. Then the cultures were washed twice with Hank’s solution to remove unattached cells and fed with 1 mL of fresh RPMI 1640 containing 10% fatal bovine serum (FBS). In vitro splenocyte proliferation test Prepared spleen cell suspension (100 µL) was plated in a 96-well culture plate in the absence and presence of ConA (5.0 µg/mL) or LPS (10.0 µg/mL). The crude and purified polysaccharides were added to each cell suspension at concentrations of 1, 10, and 100 µg/mL, respectively. ConA alone (5.0 µg/mL) or LPS alone (10.0 µg/mL) was used as positive control. After incubated in a 5% CO2 incubator at 37oC for 44 hr, the culture was treated with 20 µL of MTT for an additional 4 hr. After centrifugation at 1,000×g, the supernatant was aspirated from the wells and an aliquot of 100 µL of dimethylsulfoxide (DMSO) was added to the plate to dissolve the resulted formazan in the living cells. Cell viability was determined at 570 nm using an automatic
ELISA plate reader and the results were expressed as stimulation index (S.I.), the ratio of the optical density (OD) values between treated and blank groups. IFN-γ and IL-2 assays in vitro Prepared spleen cell suspension (100 µL) was incubated with various concentrations of polysaccharides in the presence of ConA (5.0 µg/mL) at 37oC for 48 hr. After incubation, the supernatants were collected for determination of IL-2 and IFN-α. ConA alone group (5.0 µg/mL) was used for positive control. IL-2 and IFN-γ concentration were measured using an ELISIA kit according to manufacturer's protocol. In vitro TNF-α secretion Adhered macrophages (1 mL) was incubated with various concentrations of polysaccharides in the presence of LPS (10.0 µg/mL) at 37oC. The cell supernatant was collected after incubation for 48 hr. LPS alone (10.0 µg/mL) was used for positive control. TNF-α concentration was measured using an ELISIA kit according to the manufacturer’s protocol. Statistical analysis Data were analyzed using the SPSS 14.0 software. One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was used to compare the parameters among the groups and the level of pAY01>AY05>AY00 (Table 1). Total sugar contents of the 4 purified polysaccharides were all higher than 90%. Table 1 presents the components of the 4 polysaccharides. Xylose, arabinose, mannose, and glucose were found in all of the polysaccharides, whereas rhamnose was found only in AY10. Antioxidant activities A large number of studies have shown that polysaccharides possess immunomodulating, antitumor, antiradiation, antiinflammatory, and antiaging effects, and these effects are mostly related to their antioxidant capabilities (14). As shown in Fig. 2, AYC and the 4 subfractions of the polysaccharides exhibited scavenging effect on the hydroxide radical and superoxide anion
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Fig. 1. Chromatographic fractionation of moxa leaf polysaccharides separated by DEAE-sepharose F. F. chromatography under stepwise elution with NaCl solutions at 0.1, 0.2, 0.5, and 1.0 M.
radical in dose dependent manner. These fractionated polysaccharides exhibited obviously higher antioxidant capability than that of the AYC, a phenomenon that has been reported in the previous studies on polysaccharides (15,16). The hydroxide radical scavenging capability of the 4 subfractions of the polysaccharides can be ranked in the order of AY05>AY10>AY00>AY01 (Fig. 2A). The IC50 on hydroxide radical activity of AY05 was 6.76 µg/mL. The scavenging capabilitiy against superoxide anion radical of the 4 fractionated polysaccharides was in the order of AY00>AY01>AY05>AY10 (Fig. 2B). All the 4 fractionated polysaccharides showed concentration-dependent scavenging activity against the superoxide anion radical and AY00 appeared to be most effective among the 4 with the IC50 of 10.48 µg/mL. The scavenging capability of the 4 subfractions of the polysaccharides against hydroxide radical and the superoxide anion were different. AY05 and AY10 showed higher scavenging capability on hydroxide radical but lower on superoxide anion radical comparing with the others, which could be due to different mechanisms involved in the scavenging activity against the radical species. Scavenging superoxide anion is totally depending on the number of active hydroxyl groups in the antioxidant molecules (17,18), whereas scavenging hydroxyl radical
Fig. 2. Antioxidant activities of the moxa leaf polysaccharides determined by chemiluminescence (CL) method. Hydroxyl radical scavenging activities (A) and superoxide anion radicalscavenging activities (B) of the moxa leaf polysaccharides. Scavenging activity of the polysaccharides was expressed as the percentage of inhibition (I, %) on the luminescent intensity by the moxa leaf polysaccharides at the various concentrations relative to the control group.
involves either suppressing the generation of the hydroxyl radical or trapping the generated hydroxyl radicals (19,20). In vitro splenocyte proliferation test Mitogen-stimulated lymphocytes proliferation can serve as an indicator for immunoactivation. More specifically, T lymphocyte activity can be evaluated by challenging the splenocytes with ConA whereas B lymphocyte activity can be assessed by
Table 1. Monosaccharide compositions and molecular weights (Mw) of the fractionated polysaccharides Sample AY00 AY01 AY05 AY10 1)
Monosaccharide content (mol. %)1) Xyl
Ara
Man
Glu
Rha
009.082) 13.00 11.20 18.18
13.38 15.73 13.91 14.91
24.86 43.09 21.03 37.83
52.68 28.18 53.86 25.88
3.20
Mw (Da)
Total sugar (wt %)
6,922 7,819 6,952 9,171
100.58 99.24 93.21 92.11
Xyl, xylose; Ara, arabinose; Man, mannose; Glu, glucose; Rha, rhamonse Monosaccharide content was measured by HPLC-ELSD and peak Mw was obtained by HPGPC; -, not detected
2)
Polysaccharides from Moxa Leaf
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ConA or LPS (Fig. 3). The moxa leaf polysaccharides showed dose-dependent stimulation (S.I.=1.8-3, p
