Optimisation of extraction procedure for black fungus polysaccharides and effect of the polysaccharides on blood lipid and myocardium antioxidant enzymes activities

Carbohydrate Polymers 84 (2011) 1061–1068 Contents lists available at ScienceDirect Carbohydrate Polymers journa l homepage: www.e lsev ier .com Optimi fu of the p car enzym Ma Jiang Department of i, 2014 a r t i c l Article history: Received 13 O Received in re 14 December 2 Accepted 22 D Available onlin Keywords: Black fungus p Antitumour ac Blood lipid High fat diet Cardiovascular diseases of bl samp owed centa l stru edly r l. Adm ioxid at bl s com 1. Introduction Cardiova ity and deat world. Ove onset and of CVD, resu metabolism 2005). A nu occurrence cholesterol cholesterol 2000). In lin are increas the prevent suggest tha min E, -c risk of card Kardinaal e gest that po well-known vegetables w disease (He ∗ Correspon E-mail add to their interesting biological activities, mushrooms have recently 0144-8617/$ – doi:10.1016/j.scular diseases (CVDs) are the leading causes of disabil- h in industrialized nations and much of the developing r the past three decades it has become clear that the progression of atherosclerosis, the pathological basis lt from a combination of abnormalities in lipoprotein , oxidative stress and chronic inflammation (Hansson, mber of risk factors have been associated with the of CVD including high blood concentrations of total (TC), triglycerides (TG) and homocysteine, low HDL- (HDL-C), hypertension, obesity and diabetes (Lusis, e with the oxidation hypothesis, dietary antioxidants ingly recognized as potentially important factors in ion of cardiovascular disease. Epidemiological studies t a high intake of dietary antioxidants such as vita- arotene and vitamin C is associated with a reduced iovascular disease (Gey, Brubacher, & Stahelin, 1987; t al., 1993; Rimm et al., 1993). Recent observations sug- tentially beneficial effects may not be limited to these antioxidants. High intake of flavonoids from tea and as also associated with a reduced risk of coronary heart rtog, Feskens, Hollman, Katan, & Kromhout, 1993).Due ding author. Tel.: +86 021 57412833; fax: +86 021 57412833. ress: qiaozyfxsh@yahoo.com.cn (Q. Zengyong). become an attractive source material for the development of phar- maceutical products (VanCott et al., 1996; Adebayo-Tayo et al., 2010; Mavundza et al., 2010). Many polysaccharides have been isolated from mushrooms, fungi, yeast, algae, lichens, and plants in recent years, and screened for biological activity (Murata, Shimamura, Tagami, Takatsuki, & Hamuro, 2002; Markova et al., 2003). Most polysaccharides derived from plants are relatively nontoxic and do not cause significant side effects. These could allow development of an effective natural anticancer with few side effects. The mushroom black fungus, belonging to heterobasidiae of basidiomycetes and also called Jew’s ear, wood ear, red ear, black tree fungus or ear fungus, is frequently consumed as a food and a traditional medicine in the far east. Its nutritional value and taste components have been investigated (Blinova et al., 2003; Vattem & Shetty, 2003), and a few studies have reported its biological activity and active substances. Lentinan, a polysaccharide from the Shi- itake mushroom (Lentinula edodes), has been demonstrated to have strong activity (Djordjevic et al., 2009; Feng et al., 2010; Vattem & Shetty, 2003). Ultrasonic-assisted extraction (UAE) is an expeditious, inexpen- sive and efficient alternative to traditional extraction techniques and, in some cases, even to supercritical fluid and microwave- assisted extraction, which has been demonstrated by application to both organic and inorganic analytes in a wide variety of samples (Jalbani et al., 2006). Therefore, ultrasonic treatment is widely used in the fractionation of plant materials (Riera et al., see front matter © 2011 Elsevier Ltd. All rights reserved. carbpol.2010.12.068sation of extraction procedure for black olysaccharides on blood lipid and myo es activities wei, Qiao Zengyong ∗, Xiang Xia Cardiovascular Medicine, Fengxian Branch of Shanghai 6th People’s Hospital, Shangha e i n f o ctober 2010 vised form 010 ecember 2010 e 30 December 2010 olysaccharides tivities a b s t r a c t Optimal conditions for the extraction for ultrasonic power, ratio of water to Gas chromatography (GC) analysis sh mannose and ribose. Their molar per NMR analysis showed typical chemica high fat diet feeding for 29 days mark and enhanced lipid peroxidation leve enhanced myocardium and blood ant high fat mice. Our results indicated th against cardiovascular diseases and it/ locate /carbpol ngus polysaccharides and effect dium antioxidant 00, PR China ack fungus polysaccharides were 350 W, 5, 35 min and 90 ◦C, le, extraction time and extraction temperature, respectively. that black fungus polysaccharides contained glucose, xylose, ges were 6.8%, 34.2%, 50.7% and 8.9%, respectively. FT-IR and cture of black fungus polysaccharides. In animal experiment, educed myocardium and blood antioxidant enzyme activities inistration of black fungus polysaccharides had significantly ant enzyme activities and reduced lipid peroxidation level in ack fungus polysaccharides could be beneficial for protection plications. © 2011 Elsevier Ltd. All rights reserved. 1062 M. Jiangwei et al. / Carbohydrate Polymers 84 (2011) 1061–1068 2010) and well established in the processing of plant materials, particularly, for extracting low molecular substances (Banjoo & Nelson, 2005; KaZˇys & Svilainis, 1997; Salisova, Toma, & Mason, 1997). In this w utilized for by a respon still design charides as oxidative in to ascribe a interventio 2. Materia 2.1. Plant m Black fu China. Thes China. The pharmacolo was deposi ground in a 2.2. Prepara Black fu powder (60 sonic clean 40 kHz), us various dur immersing selected tem was collect 2.3. Box–Be Accordin tion tempe extraction on the resp (Martendal tested in a As shown i designated +1, 0, −1 fo test variabl xi = (Xi − X X where xi is actual value independen of an indep 2.4. Analys The poly 2 M trifluor residue was and the rem ing alditol a Japan) on a ization det was increas 8 ◦C/min to Table 1 Experimental design and response values. RUN X1 X2 X3 X4 Y1 1 − ◦ − 1 1 0 0 0 0 − − 1 1 0 0 0 0 − − 1 1 0 0 0 0 0 0 0 -IR s IR wa of b MR sp ples h a geab ze–d for a perfo ed w softw imals and dietary treatment rty kunming mice weighing 16 ± 1 g were housed in stain- el cages in a room with controlled lighting (12-h light:dark constant temperature (24 ◦C) and relative humidity (60%). imals were randomly divided into four groups of 10 each d a different diet for 4 weeks, as follows: one group fed containing 1% cholesterol and 0.5% cholic acid, i.e. high terol diet (HCD) and the other group fed the same diet mented with black fungus polysaccharides (0.6% and 1.2%). r mice fed with basic diet and served as control. Diets p water were freely available. The animals were weighed . We followed the general guidelines on the use of living ani- scientific investigations (Council of European Communities, tioxidant enzyme measurements day 29, the mice were fasted overnight, killed and blood and amples were collected. Then, it was centrifuged at 3000 × g in at 4 ◦C to obtain the serum for the measurement of TG, TC, and LDL-c levels, according to the commercial instructionsork, an ultrasonically assisted extraction technique was the extraction of polysaccharides from black fungus se surface methodology design. The present study was ed to investigate the efficacy of black fungus polysac- sources of water-soluble antioxidants on myocardium jury in cholesterol-fed mice. This study will allow us ntiatherogenic effects to antioxidant properties of the n. ls and methods aterial ngus was purchased from a herb shop, Shanghai city, e black fungi originally grew in Shandong province, plant material was identified at the department of gy, Phd Hong where a voucher specimen 20100326 ted. The medicine was dried at room temperature and rotary mill and then sieved (60 mesh). tion of black fungus polysaccharides ngus polysaccharides (100 g) were ground into fine mesh). The extraction was performed using an ultra- er (SB-5200DTD, Xinzhi Biotech Co., Ningbo, China, ing selected ultrasonic power and temperature for ations. 10 g dry sample powders were extracted by in water at a selected ratio, then heating in water at perature for various periods of time. The supernatant ed for the determination of polysaccharides yield. hnken design g to the principle of Box–Behnken design, extrac- rature, extraction time, ratio of water to sample and number, which were identified to have strong effects onse in preliminary one-factor-at-a-time experiments , Budziak, & Carasek, 2007), were taken as the variables 27-run experiment to determine their optimum levels. n Table 1, the four factors chosen for this study were as X1, X2, X3, X4 and prescribed into three levels, coded r high, intermediate and low value, successively. Three es were coded according to the following equation (1): 0) i = 1, 2, 3 (1) the coded value of an independent variable; Xi is the of an independent variable; X0 is the actual value of an t variable at centre point; X is the step change value endent variable. is of carbohydrate composition saccharides sample (2 mg) was hydrolysed in 2 ml of oacetic acid (TFA) at 110 ◦C for 2 h. A small portion of the subjected to thin layer chromatography (TLC) analysis, aining portion was transformed into the correspond- cetates, which was analyzed by GC (Shimadzu, Kyoto, HP-5 chromosorb column and detected by a flame ion- ector (temperature 250 ◦C). The column temperature ed from 170 to 215 ◦C in a rate of 2 ◦C/min and then 250 ◦C (Dong, Yao, & Fang, 2003). 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2.5. FT FT- groups 2.6. N Sam throug exchan by free tubes were equipp Varian 2.7. An Thi less ste cycle), The an and fe a diet choles supple Anothe and ta weekly mals in 1986). 2.8. An On heart s for 15 m HDL-c1 (350 W) −1 (4) 0 (35 min) 0 (90 C) 15 1 1 (6) 0 0 17.2 (450 W) −1 0 0 17.5 1 0 0 18.3 (400 W) 0 (5) −1 (30 min) −1 (80 ◦C) 16 0 −1 1 (100 ◦C) 16.2 0 1 (40 min) −1 16.1 0 1 1 16 1 0 0 −1 16.2 1 0 0 1 16.3 0 0 −1 18 0 0 1 18 −1 −1 0 16.6 −1 1 0 16.7 1 −1 0 17.5 1 1 0 17.6 1 0 −1 0 14.8 1 0 1 0 16.8 0 −1 0 17.5 0 1 0 17.8 −1 0 −1 16.4 −1 0 1 16.3 1 0 −1 17.6 1 0 1 17.8 0 0 0 19.2 0 0 0 19.6 0 0 0 19.5 pectroscopy s analyzed using the KBr disc for detecting functional lack fungus polysaccharides. ectroscopy were dissolved in D2O (99.96% of atom), filtered 0.45-m syringe filter, and freeze–dried to remove le protons. After exchanging the samples three times rying from D2O, samples were transferred to Shigemi nalyses. One-dimensional (1D) 1H NMR experiments rmed on a Varian 500 MHz VXR-500 spectrometer ith 5-mm triple resonance tunable probe with standard are at 279, 298 and 313 K. M. Jiangwei et al. / Carbohydrate Polymers 84 (2011) 1061–1068 1063 for the automatic biochemical analyser (Biochemical analytic Cen- ter of Maigaoqiao Hospital, Nanjing, China). Lipid peroxidation was estimated by measuring thiobarbituric acid-reactive substances (TBARS) and expressed in terms of mal- ondialdehyde (MDA) content, according to the method of Draper and Hadley (1990). Reduced glutathione levels (GSH) were deter- mined by Ellman method (1959) modified by Jollow, Mitchell, Zamppaglione, and Gillette (1974). Superoxide dismutase activity was measured at 412 nm by the NADH oxidation procedure (Elstner, Youngman & Obwald, 1983). Glutathione peroxidase was determined by the method of Paglia and Valentine (1967) using cumene hydroperoxide as substrate. Catalase activity was determined by the method of Aebi (1974) by measuring the rate of decomposition of H2O2 at 240 nm. 2.9. Statistical analysis Results are expressed as means ± standard deviations (SD). Significant differences among the groups were determined by one- way ANOVA with Duncan’s multiple range test. Differences were considered significant if P < 0.05. 3. Results and discussion 3.1. Effect of different extraction parameters on extraction yield of the polysaccharides As show for the extr extraction t tion yield o volume of w As shown i when temp extraction y temperatur Table 2 Analysis of variances in the regression model for optimisation of polysaccharide extraction from black fungus . Master model Predictive model Mean 17.12963 17.12963 R-Square 93.55% 89.01% Adj. R-square 86.03% 85.71% RMSE 0.454377 0.459619 CV 2.652578 2.683184 3.2. Optimisation of extraction process After the RSREG procedure, the regression equation was given as follows: Y1 = 19.43333 + 0.9 × X1 + 0.625 × X2 − 1.133333 × X1 × X1 − 0.995833 × X2 × X2 − 1.608333 × X3 × X3 − 1.445833 × X4 × X4 (2) The significance of each coefficient in Eq. (2) was determined using the Student’s t-test and p value as shown in Table 2. It was evi- dent that the linear coefficients (ultrasonic power, ratio of water to sample), and four quadratic coefficients (ultrasonic power, ratio of water to sample, extraction temperature and extraction time) were significant (p < 0.05), while all the cross product coefficients were insignificant (p > 0.5). These results suggest that ultrasonic power and ratio of water to sample were the most important fac- tors because it affected the polysaccharides extraction the most (p < 0.01). It is evident that the model was highly significant, as was evi- om t , F < 0 term coeffi tion ment Liu, M ed go of ex B D 10 10090807060500 extraction temperature (oC) ction yield of the polysaccharides.n in Fig. 1A, ultrasonic power of 400 W is favourable action of the polysaccharides. As shown in Fig. 1B, the ime of 35 min was enough to obtain maximum extrac- f the polysaccharides. As shown in Fig. 1C, 5 times ater was proper for extraction of this polysaccharides. n Fig. 1D, extraction yield did not markedly increased erature was between 90 ◦C and 100 ◦C. Therefore, high ield can be achieved with the increase of extraction e. dent fr model the de lation correla experi 2006; indicat values 500450400350300250200150100 4 6 8 10 12 14 16 18 20 22 24 ex tr ac tio n yi el d (% ) ultrasonic power (W) A C 5 6 8 10 12 14 16 18 20 22 24 ex tr ac tio n yi el d (% ) 876543210 2 4 6 8 10 12 14 16 18 20 22 24 ex tr ac tio n yi el d (% ) ratio of water to sample 4 10 12 14 16 18 20 22 24 ex tr ac tio n yi el d (% ) Fig. 1. Effect of different extraction parameters on extrahe model F-value and a very low probability value (P .0001). The goodness of the model could be checked by ination coefficient R2 (0.9355) and the multiple corre- cient R (0.8603). The closer the values of R (multiple coefficient) to 1, the better the correlation between the al and predicted values (Lin, Yang, Hsu, Hsu, & Chang, iao, Wen, & Sun, 2009). Here, the value of R (0.9355) od agreement between the experimental and predicted traction yield of polysaccharides. 45403530252015 extraction time (min) 1064 M. Jiangwei et al. / Carbohydrate Polymers 84 (2011) 1061–1068 5 16 43 .9 6 15 52 .4 5 15 cm-1 10 0 s poly 3.3. Chemic polysacchar The puri TFA into ind lylated for four mono ribose, wer standards. 8.9%, respec Fig. 2 sh to the varia located at The range quencies (1 glucose and ciated with is associate the presen rides. The sign 3.64 ppm (C and are sho charides ha by - and  The sign of black fun C-6 of -d- 56 ppm cou on the data the resonan anomeric ca (Xylp), resp hibit in hig dicin med32 59 .2 3 29 26 .5 2000250030003500 Wavenumber 50 60 70 80 90 Tr an sm itt an ce [ % ] Fig. 2. FT-IR spectroscopy of black fungu al composition and structure of black fungus ides fied black fungus polysaccharides were hydrolysed by ividual monosaccharides that were further trimethylsi- 3.4. In injury Me tant regas chromatography analysis. The results showed that saccharides, including glucose, xylose, mannose and e identified after comparison with the monosaccharide Their molar percentages were 6.8%, 34.2%, 50.7% and tively. ows that the most important wavenumbers related bility of black fungus polysaccharides were the bands 3259, 2926, 1643, 1552, 1409, 1243, and 991 cm−1. (1243–1409 cm−1) is O–H-group vibrations. The fre- 243–991 cm−1) were polysaccharides with mannose, xylose, constituents. The band at 2926 cm−1 is asso- the vibrations of C–H bond. The band at 991 cm−1 d with the presence of -pyran ring, which indicated ce of  glucosidic bond in black fungus polysaccha- als of 1H NMR were 5.05 (-C-1), 4.73 ppm (-C-1), -5), 3.52 ppm (C-4), 3.56 ppm (C-3), and 3.43 ppm (C-2) wn in Fig. 3A. On the basis of these results, the polysac- s been determined to be a novel biomolecule combined -linkages. als identified at 95, 75, 73 and 63 ppm in the 13C spectra gus polysaccharides could be assigned to C-1, C-4 and mannose (Fig. 3B). The signals identified at 74, 57 and ld be assigned to C-1, C-5 and C-6 of-d-glucose. Based available in the literature, it was possible to identify that ces in the region of 75–95 ppm were attributed to the rbon atoms of glucopyranose (Glcp) and xylopyranose ectively. for thousa Philippouss et al., 199 effects has rooms inclu immuno-po Among the strated to b agents are Auricularia thermore, t pharmacolo could supp modulate im 2009; Shua Imaizumi, 2 In addition rides treatm Oyedemi et MDA an are present MDA level were mark diet was fed decreased M blood. Results a LDL-c level14 09 .4 8 12 43 .8 1 99 1. 33 100000 saccharides. ion of black fungus polysaccharides against oxidative h fat mice al mushroom extracts have been considered as impor- ies for the prevention and treatment of many diseases nds of years especially in the Orient (Israilides & is, 2003; Kidd, 2000; Wasser & Weis, 1999; Matsuo 6; Djordjevic et al., 2009). A plethora of medicinal been demonstrated for many traditionally used mush- ding antibacterial, antiviral, antifungal, antitumour and tentiating activities (Hobbs, 2003; Ooio & Liu, 1999). various bioactive components which have been demon- e most effective as antitumour and immunomodulatory polysaccharides and polysaccharopeptides. A lot of polytricha were consumed every year in the East. Fur- hese edible fungi are also well known for its multiple gical effects. It has been reported that A. polytricha ress platelet aggregating (Hokama & Hokama, 1981), mune function (Sheu, Chien, & Chien, 2004; Hu et al., i et al., 2010), exhibit antinociceptive (Koyama, Akiba, & 002) and antioxidative effect (Mau, Chao, & Wu, 2001). , previous study showed that black fungus polysaccha- ent can reduced blood lipid level (Han & Xu, 2007; al., 2009). d GSH levels of BFP-treated and untreated high fat mice ed in Figs. 4 and 5. Compared with normal control, and increased GSH level in myocardium and blood edly increased and decreased 29 days after high fat . Black fungus polysaccharides treatment significantly DA level and increased GSH level in myocardium and re given in Fig. 6. As seen from the table, blood TC, TG, s in untreated model control mice were significantly M. Jiangwei et al. / Carbohydrate Polymers 84 (2011) 1061–1068 1065 Fig. 3. NMR spectroscopy of black fungus polysaccharides. 1066 M. Jiangwei et al. / Carbohydrate Polymers 84 (2011) 1061–1068 NC MC BFP1 BFP2 0 5 10 15 20 25 ** ## ## B NC MC BFP1 BFP2 0 5 10 15 20 25