microwave assisted extraction (UMAE) of polysaccharides from Inonotus obliquus and evaluation of its anti-tumor activities

International Journal of Biological Macromolecules 46 (2010) 429–435

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Optimization of ultrasonic/microwave assisted extraction (UMAE) of polysaccharides from Inonotus obliquus and evaluation of its anti-tumor activities Yiyong Chen a,b , Xiaohong Gu a , Sheng-quan Huang c,d , Jinwei Li a , Xin Wang e , Jian Tang a,∗ a

State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, China c College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China d Infinitus(China) Co., Ltd., Jiangmen 529156, China e Wuxi Scientific Research and Designing Institute of the State Administration of Grain, Wuxi 214122, China b

a r t i c l e

i n f o

Article history: Received 31 December 2009 Received in revised form 28 January 2010 Accepted 2 February 2010 Available online 10 February 2010 Keywords: Inonotus obliquus Ultrasonic/microwave assisted extraction (UMAE) Polysaccharides Anti-tumor

a b s t r a c t Recently, the use of ultrasonic and microwave has attracted considerable interest as an alternative approach to the traditional extraction methods. In this paper, in order to maximize the yield and purity of polysaccharides from Inonotus obliquus, response surface methodology (RSM) was employed to optimize the ultrasonic/microwave assisted extraction (UMAE) conditions. The results indicated that the optimal conditions for UMAE were 90 W microwave power, 50 W ultrasonic power together with 40 kHz ultrasonic frequency, solid/water ratio was 1:20 (W/V) and the extracting time was 19 min, respectively. Under the optimal conditions, the yield and purity of polysaccharides were 3.25% and 73.16%, respectively, which are above that of traditional hot water extraction and close to the predicted value (3.07% and 72.54%, respectively). These results confirmed that ultrasonic/microwave assisted extraction (UMAE) of polysaccharides had great potential and efficiency compared with traditional hot water extraction. At the same time, the anti-tumor activities of the polysaccharides from I. obliquus with UMAE were evaluated. The results suggested that polysaccharides from I. obliquus exhibited obvious anti-tumor activities. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Inonotus obliquus is a white rot fungus that belongs to the family Hymenochaetaceae of Basidiomycetes. This fungus is usually found as a sterile conk (sclerotia) called ‘Chaga’ on Betula species in nature [1,2]. It grows on living trunks of the mature birch, and is mainly found atlatitudes of 45◦ N–50◦ N. Traditionally, ‘Chaga’ has been used as a folk remedy for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes since the 16th century in Russia, Poland and most of the Baltic countries [2–4]. A decoction of fungal sclerotia did not show toxic effects and has been used in treatment of cancersand digestive system diseases [1,5]. At the same time, it is widely consumed in Russia and Korea as a tea or concentrate for its health-benefiting functions [6]. In recent years, many polyphenolic compounds, triterpenoids, and steroids, such as lanosterol, inotodiol, trametenolic acids, and ergosterol peroxide from Inonotus sclerotia [1], have shown various biological activities, including hypoglycemic, hepato-protective [7].

∗ Corresponding author. Tel.: +86 510 85329016; fax: +86 510 85919837. E-mail addresses: [email protected], [email protected] (J. Tang). 0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.02.003

Polysaccharides extracted used in the food industry and in medicine for a long time, have attracted much attention in recent years due to their biological activities [8], including anti-tumor [9], immunostimulation [9,10], anti-oxidation [11,12]. However, there is little information about anti-tumor activities of polysaccharides from I. obliquus. Extraction of polysaccharides is an important processing for its application or further research & development, which has prompted numerous research papers on the extraction technology of polysaccharides from plentiful of plants or fungus in recent years. Response surface methodology (RSM) was applied to optimize the hot water extraction process of crude polysaccharides from boat-fruited sterculia seeds [13] and wild edible BaChu mushroom [14]. Ultrasonic technology was employed to extract polysaccharides from jujube [15] and from longan fruit pericarp [16] and the optimal extracted condition was obtained by response surface methodology. They found the DPPH radical scavenging activity of polysaccharides could be improved by application of ultrasonic treatment. In general, hot-water treatment has been used for classical extraction of polysaccharides. However, it should be noted that hot water extraction of polysaccharides is associated with long extraction time and high temperature [15]. It is necessary to find

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a novel method for extracting polysaccharides economically that could avoid the disadvantage of hot water extraction. Ultrasound and microwave radiation could accelerate the extracting process and this may improve extraction of bioactive compound [17–19]. The main advantage of applying microwave approaches is the time saving it achieves, while its disadvantage is inhomogeneous heating [19]. The possible benefits of ultrasound in extraction are mass transfer intensification, cell disruption, improved penetration and capillary effects [20]. Consequently, combining ultrasonic with microwave is a complementary technique and may show their several advantages. There are few reports regarding optimization of ultrasonic/ microwave assisted extraction (UMAE) of polysaccharides, especially polysaccharides from I. obliquus. The first purpose of the present study is to optimize the ultrasonic/microwave assisted extraction (UMAE) conditions and compare with traditional hot water extraction. Moreover, there is little information about antitumor activities of polysaccharides from I. obliquus. Consequently, the second purpose is to evaluate the anti-tumor activities of polysaccharides from I. obliquus with UMAE and to broaden its application in medicine field. 2. Materials and methods 2.1. Materials and equipments Commercially available dried I. obliquus samples were purchased from Ya Buli Co. Ltd. (Hei Long Jiang., China). They were ground in a high disintegrator (Micron Co. Ltd., China) to pass through 40 mesh screen and kept at 4 ◦ C in refrigerator during experiments. An UMAE apparatus (CW-2000, Shanghai Xintuo Microwave Instrument Co. Ltd., China) with maximal microwave power of 800 W at a frequency of 2450 MHz and an ultrasonic transducer with a fixed power of 50 W at a frequency of 40 kHz was used to extract polysaccharides. The schematic diagram of UMAE apparatus is shown in Fig. 1. The traditional hot water extraction procedure was carried out in the water bath (SHAB/SHA-D, Wei er Experiment Equipment Company, Suzhou, China). FT-IR spectrometer (Nicolet Nexus, Thermo Electron Co. Ltd., USA)

Fig. 1. Schematic diagram of UMAE apparatus.

Table 1 Independent variables and their levels in the response surface design. Independent variables

Symbol

Extraction time (min) Solid/water ratio (g/ml) Microwave power (W)

X1 X2 X3

Coded factor level −1

0

1

15 1:15 55

20 1:20 85

25 1:25 115

was used for infrared spectrum analysis. Jurkat cell and Daudi cell were presented by Academy of Military Medical Science (Beijing, China), Medium RPMI1640, dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Life Technologies, Inc., USA). 5-Fluorouracil (5-Fu) was purchased from Jiangsu Heng Rui Pharmaceutic Co. (Lianyungang, China). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin and streptomycin were purchased from Sigma Chemicals Co., USA. All reagents were of analytical grade. 2.2. Extraction procedure 2.2.1. Pre-treatment of I. obliquus Ground I. obliquus samples were refluxed twice with ethanol (volume fraction 80%) at 70 ◦ C in a water bath for 3 h to remove some colored materials, oligosaccharides and some small molecule materials. The ethanolic mixture extract was centrifuged (3000 × g, 20 min). After removing supernatant, precipitation was vacuumdried at 60 ◦ C for 12 h, pre-treated I. obliquus powder was obtained. 2.2.2. Ultrasonic/microwave assisted extraction The pre-treated I. obliquus powder (5 g) was weighed accurately and then transfered into the flask, 100 ml water was added into the flask. Then the flask was transfered into the chamber of the apparatus connected with condensing tubes. Finally, the door of chamber was closed and the program of different extraction time and microwave power was set. When extraction of polysaccharides was accomplished, the flask was removed from apparatus. The treated mixture was cooled to room temperature using ice water and then centrifuged (5000 × g, 15 min). The supernatant was concentrated in a rotary evaporator under reduced pressure at 85 ◦ C, and then precipitated by the addition of ethanol (12 h, 4 ◦ C) to a final concentration of 85%(v/v). The precipitates were collected by centrifugation (5000 × g, 15 min), washed with cold 80% ethanol, and finally lyophilized to obtain polysaccharides from I. obliquus. All experiments were performed at least in duplicate. 2.2.3. Experimental design of RSM Response surface methodology (RSM) was used to explore the effect of independent variables on the response within the range of investigation, which is a collection of statistical techniques for designing experiments, building models, evaluating the effects of factors and searching optimum conditions of factors for desirable responses [21–23]. A central composite design (CCD) with three independent variables (X1 , extraction time; X2 , the ratio of water to solid; X3 , power of microwave in UMAE) at three levels (Table 1) was performed to optimize the extracting conditions and investigate effects of above independent variables on the yield and purity of polysaccharides. Yield and purity of the extracted polysaccharides were the dependent variables. The complete design consisted of 15 experimental points including 12 factorial points, 2 axial points and 3 centre points and the experiment was carried out in a random order. Three replicates (treatment 13–15) at the centre of the design were used to allow for estimation of a pure error sum of squares.

Y. Chen et al. / International Journal of Biological Macromolecules 46 (2010) 429–435

2.2.4. Statistical analysis Multiple regressions to fit the following quadratic polynomial model were expressed below: yk = bk0 +

3  i=1

bk xi +

3  i=1

bkii xi2 +

3 

bkij xi xj

(1)

i
where yk is the response function; bk0 is the centre point of the system; bki , bkii and bkij represent the coefficients of the linear, quadratic and interactive terms, respectively; xi and xi xj represent the linear and interactive terms of the coded independent variables, respectively. The Statistical Analysis System (SAS, version 8.0) was used to analyze the experimental data. ANOVA procedure was used to analyze variance and the data obtained in anti-tumor activity experiment. The significances of all terms in the polynomial were considered statistically different when P < 0.05. Significance of any differences between groups was evaluated using Duncan’s multiple range test. 2.2.5. Traditional hot water extraction (THWE) A water bath (SHA-B/SHA-D, Wei er Experiment Equipment Company, Suzhou, China) was used to extract polysaccharides from I. obliquus with traditional hot water extraction at the optimum extraction condition: extraction temperature of 100 ◦ C, extraction time of 240 min, and solid/water ratio of 1:20 based on the preliminary three-factor and three level designed orthogonal optimal experiment. 2.2.6. Analytical methods The sugar content was determined using phenol-sulfuric acid colorimetric method [24]. The yield of crude polysaccharides was calculated based on the weight of the lyophilized crude polysaccharidess to the total weight of the I. obliquus powder used. The purity of polysaccharides extracted was determined by soluble non-cellulose polysaccharides to the weight of crude polysaccharides. 2.3. Infrared spectrum analysis of polysaccharides Fourier-transform infrared spectra were recorded from extracted polysaccharides powder (1 mg) in KBr pellets on a Nicolet Nexus FT-IR spectrometer in the range of 4000–400 cm−1 . 2.4. Evaluation of anti-tumor activities of polysaccharides 2.4.1. Cell lines and culture Human T lymphadenoma jurkat cell and human B lymphadenoma daudi cell were used in the tumor therapeutical study and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/L) at 37 ◦ C in a humidified atmosphere with 5% CO2 . The culture was passaged every 2 or 3 days. Finally, cell concentration was 2 × 106 cells/ml. 2.4.2. Assay of inhibition of tumor cell proliferation in vitro The inhibition effects of jurkat and daudi cells (2 × 106 cells/ml) proliferation were determined in vitro using the colorimetric MTT assay [25]. Briefly, cells were incubated in 96-well plates containing 100 ␮l of the culture medium at 37 ◦ C in a humidified atmosphere with 5% CO2 for 12 h. Polysaccharides from I. obliquus with UMAE were added into each well, the dosages of polysaccharides from I. obliquus extracted were 0.7, 2.3, 7, 70, 200 ␮g/ml, respectively. The volume of every dosage of polysaccharides solution was 0.5 ml. Every dosage was set three parallel wells, while the negative controls were treated with the medium only. The cells in each well

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were incubated in culture medium with 20 ␮l MTT (500 mg/L) 48 h later. After incubation at 37 ◦ C for 4 h, the supernatant was aspirated, and 150 ␮l DMSO was added to each well. Absorbance at 570 nm was measured by a 96-well microplate ELISA reader (BioTeK Instruments, Winoski, VT). All determinations were done in triplicate. Inhibition ratio of tumor cell proliferation was calculated according to the formula below: Inhibition ratio (%) =



1−

OD OD0



× 100

(2)

where OD and OD0 were the absorbance of treated cells and untreated cells, respectively. 2.4.3. Assay of anti-tumor activities in vivo Balb/c-nu/nu nude mice (3–5 weeks old, weighing 20 ± 2 g, male-to-female ratio of 1:1) and jurkat tumor cells were purchased from the Animal Center of Academy of Military Medical Science (Beijing, China). At day 0, the nude mice were injected subcutaneously into the right groin with 0.2 ml jurkat tumor cells (2 × 106 cells/ml). Mice were randomly divided into four groups with 10 mice per group. Twenty-four hours after tumor implantation, the tumor-bearing mice were orally treated with normal saline (10 ml/kg d) as negative control, 5-Fluorouracil (5-Fu) dissolved in 0.9% aqueous NaCl (20 mg/kg d) as positive control and polysaccharides from I. obliquus at dosages of 50, 100 mg/kg everyday for 10 days. Then the mice were put to death, and the tumors were dissected and weighed. The inhibition ratio was calculated by the following formula: Inhibition ratio (%) =

CW − TW × 100 CW

(3)

where CW is the weight of untreated control and TW is the weight of treated tumor. This study was repeated three times. 3. Results and discussion 3.1. Statistical analysis and modeling of extraction of polysaccharides The fitted model for yield (Y1 ) and purity (Y2 ) to predict the relationships between the independent variables and the dependent variables can be expressed by: Y1 = 3.066667 + 0.04 × X1 + 0.03375 × X2 + 0.05375 × X3 − 0.013333 × X1 × X1 − 0.0725 × X1 × X2 − 0.1125 × X1 × X3 − 0.065833 × X2 × X2 − 0.01 × X2 × X3 − 0.090833 × X3 × X3

(4)

Y2 = 72.63333 + 2.275 × X1 + 1.3125 × X2 + 2.7375 × X3 − 2.766667 × X1 × X1 + 0.075 × X1 × X2 − 2.025 × X1 × X3 − 0.791667 × X2 × X2 + 1.2 × X2 × X3 − 3.391667 × X3 × X3 (5) The process variables and experimental data for yield and purity of the crude polysaccharides under different treatment conditions are presented in Table 2. The results of the analysis of variance, adequacy and fitness of the models are summarized in Table 3. The data indicated that the proposed regression model for yield and purity was adequate with satisfactory R2 value (determined coefficient). The R2 values for yield and purity were 0.9574 and 0.8749, respectively, which showed a close agreement between the experimental results and the theoretical values predicted by

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Table 2 Results of response surface analysis of the variation of the yield and the purity of polysaccharides from I. obliquus with time (X1 ), solid/water ratio (X2 ) and microwave power (X3 ). Number

X1

X2

X3

Yield (%)

Purity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

−1 −1 1 1 0 0 0 0 −1 1 −1 1 0 0 0

−1 1 −1 1 −1 −1 1 1 0 0 0 0 0 0 0

0 0 0 0 −1 1 −1 1 −1 −1 1 1 0 0 0

2.87 3.04 3.08 2.96 2.78 2.93 2.91 3.02 2.76 3.08 3.07 2.94 3.03 3.08 3.09

66.4 65.6 72.4 71.9 63.4 67.6 66.9 75.9 60.8 67.8 69.2 68.1 73.2 72.9 71.8

the polynomial model. The P-values are used as a tool to check the significance of each coefficient. The smaller the value of P, the more significant is the corresponding coefficient. From the model of yield, linear terms of extraction time (X1 ) and microwave power (X3 ), quadratic terms of solid/water ratio (X22 ) and microwave power (X32 ) were significant (P < 0.05). Among the interaction terms, X1 with X2 and X1 with X3 were significant (P < 0.05). From the model of purity, linear terms of X1 , X3 and quadratic terms of X32 were significant (P < 0.05). From the two models tested, all the linear terms except X2 exhibited no obvious significant effect. From the above analysis, it was found that the independent variables of the extraction time and microwave power were the most important factors which had more effects on yield and purity of crude polysaccharides. The full model filled Eqs. (4) and (5) were made threedimensional and contour plots to predict the relationships between the independent variables and the dependent variables. The influence of UMAE variables on the yield and purify of polysaccharides is presented in Fig. 2. As can be seen from Fig. 2, it was observed that the yield of polysaccharides increased gradually with the increase of extraction time and microwave power. The similar result that the changing of microwave power had more effects on lycopene extraction than the others was observed in the study of ultrasound/microwave assisted extraction (UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomatoes [19]. When solid/water ratio was constant (1:20), the purity increased substantially with the increase of extraction time and microwave power. Solid/water ratio had no obvious influence on purity of polysaccharides.

Table 3 Regression coefficient and analysis of the model for two response variables. Coefficient

Yield (%)

Purity (%)

b0 b1 b2 b3 b11 b12 b13 b22 b23 b33 R2

3.0667 0.04a 0.03375 0.05375a −0.013333 −0.0725a −0.1125b −0.065833a −0.01 −0.090833b 0.9574

72.6333 2.275a 1.3125 2.7375a −2.766667 0.075 −2.025 −0.791667 1.2 −3.391667a 0.8749

Note: ‘a’ means P < 0.05; ‘b’ means P < 0.01.

3.2. Optimization of UMAE conditions Optimization of the extraction procedure was based on higher extraction yield and purity of crude polysaccharides. The effects of extraction conditions on the yield and purity of polysaccharides were optimized and analyzed. It was concluded that the yield and purity changed obviouslly with extraction time and microwave power. So solid/water ratio was held at 1:20 for optimizing the other two parameters. The overlaying plot attained is shown in Fig. 3. The optimal conditions of UMAE were given by the RSM optimization approach as following: microwave power was 90 W; extracting time was 19 min; solid/water ratio was 1:20. 3.3. Validation of the models The suitability of the model equations for predicting optimum response values was tested under the conditions: extracting time 19 min, microwave power 89 W, solid/water ratio, 1:20. Under these conditions, the experiment was performed three times. Mean values of the yield and purity of polysaccharides from I. obliquus were expected to be 3.25% and 73.16%, respectively, which was close to the predicted value (3.07% and 72.54%, respectively). The results of analysis indicated that the yield and purity of experimental values were in good agreement with the predicted ones and also suggested that the models of Eqs. (4) and (5) are satisfactory and accurate for the extraction process. 3.4. Comparison of traditional hot water extraction and UMAE Compared with traditional hot water extraction (THWE), the application of ultrasonic/microwave assisted extraction (UMAE) affected positively the yield and purity of polysaccharides extracted. The result was seen in Table 4. Under the optimal conditions of UMAE, the yield of polysaccharides increased from 2.12% to 3.25% and the purity increased from 64.03% to 73.16% at substantially shorter extraction time (19 min), which represented an increase of about 53.3% to THWE in yield and an increase of 14.3% to THWE in purity, respectively. The extraction time shortened greatly compared with THWE. This is because ultrasound and microwave radiation could accelerate the extracting process and may improve extraction of bioactive compounds [16,18]. Simultaneously, the possible benefits of ultrasound in extraction are mass transfer intensification, cell disruption, improved penetration and capillary effects [20]. It was confirmed that UMAE should be an appropriate and effective extraction technique for polysaccharides from I. obliquus because of the maximum extraction values. In order to prove the effect of THWE and UMAE on functional groups of polysaccharides, IR spectra of I. obliquus polysaccharides extracted with two methods were done shown in Fig. 4. As can be seen from Fig. 4, there was a broad and strong absorption peak of polysaccharides extracted by two different methods at 3412.28 cm−1 , which attributed to hydroxyl –OH groups. A weak –CH stretching peak near 2934 cm−1 was displayed. A strong absorption peak at 1613.28 cm−1 attributed to carboxyl group in uronic acid. A peak at 1387.38 cm−1 was an indication of the presence of carboxyl group. Based on the above analysis, the functional Table 4 Comparison of THWE and UMAE in extraction time, yield and purity of polysaccharides from I. obliquus. Methods

Extraction time (min)

Yield (%)

Purity (%)

THWE UMAE

240 19

2.12 3.25

64.03 73.16

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Fig. 2. Response surface (a) and contour (b) plots showing the effect of extraction time and microwave power (solid/water ratio 1:20) on yield (Y1 ) and purity (Y2 ) of crude polysaccharides.

groups of polysaccharides extracted by THWE and UMAE are fundamentally identical. 3.5. Evaluation of anti-tumor activities of polysaccharides 3.5.1. Inhibition of tumor cell proliferation in vitro Some polysaccharides, such as polysaccharides from Phellinus linteus [26] and Cordyceps sinensis [27], could directly inhibit the proliferation of cancer cell in vitro. Many polysaccharides have been found to induce apoptosis in cancer cells [28]. In this study, anti-

tumor acvitities in vitro of polysaccharides from I. obliquus against jurkat and daudi cells were investigated, the results were shown in Table 5. At the concentrations from 0.7 ␮g/ml to 200 ␮g/ml, the inhibition ability was in a dose-dependent manner. The inhibition ratio of polysaccharides from I. obliquus on the jurkat cells and daudi cells increased from 6.89% to 62.29% and from 20.23% to 66.42%, respectively. Polysaccharides from I. obliquus exhibited obvious anti-tumor activities against tumor cells and could directly inhibit the proliferation of tumor cells in vitro. These results were different from the other known anti-tumor native mushroom

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Table 5 Effect of polysaccharides from I. obliquus on jurkat cells and daudi cells proliferation in vitro. Group Control

IOP

Concentration (␮g/ml) 0 0.7 2.3 7 23 70 200

a

OD570 nm (X ± S)

1.233 1.148 1.038 0.931

± ± ± ±

a

b

Inhibition ratio (%)

OD570 nm (X ± S) ± ± ± ±

b

Inhibition ratio (%)

0.003 0.053 0.003a 0.016b,c

0 6.89 ± 0.27 15.82 ± 0.11 24.49 ± 0.83

1.087 0.867 0.630 0.523

0.021 0.006b 0.010b,c 0.058b,c

0 20.23 ± 0.66 42.04 ± 0.53 51.89 ± 0.70

0.666 ± 0.013b,c 0.523 ± 0.006b,d 0.465 ± 0.007b,d

45.99 ± 0.24 57.58 ± 0.25 62.29 ± 0.74

0.393 ± 0.006b,d 0.373 ± 0.006b,d 0.365 ± 0.007b,d

63.85 ± 0.37 65.69 ± 0.35 66.42 ± 0.61

Note: ‘c’ means jurkat cells; ‘d’ means daudi cells. Values are shown as means ± S.D. (n = 3). Significance was determined using Duncan’s multiple range test. a (P < 0.05) and b (P < 0.01) indicate a significant difference compared with negative control. c (P < 0.05) and d (P < 0.01) indicate a significant difference between the IOP groups.

Table 6 Anti-tumor effect of polysaccharides from I. obliquus on jurkat tumor-bearing nude mice in vivo. Group

Dose (mg/kg)

Tumor weight X ± S, (g)

Negative control 5-Fu IOP IOP

– 20 50 100

3.47 1.46 1.96 1.51

± ± ± ±

0.04 0.03a 0. 05b 0.02b,c

Inhibition ratio (%) – 57.93 ± 0.65 43.52 ± 0.72 57.48 ± 0.69

Values are shown as means ± S.D. of 10 mice. Significance was determined using Duncan’s multiple range test. a (P < 0.05) and b (P < 0.01) indicate a significant difference compared with negative control. c (P < 0.05) indicates a significant difference between the IOP groups.

polysaccharides such as schizophyllan and lentinan [29,30], all of which have no direct cytotoxicity to tumor cell lines in vitro.

Fig. 3. The contour plots of the yield (Y1 , %) and the purity (Y2 , %) of polysaccharides from I. obliquus were affected by extracted time and microwave power (solid/water ratio 1:20).

3.5.2. Anti-tumor activities of polysaccharides from I. obliquus in vivo The results of anti-tumor activities of polysaccharides from I. obliquus against jurkat tumor-bearing nude mice in vivo are summarized in Table 6. Obvious anti-tumor activities were observed in three samples. On day 10, the average tumor weight of negative control mice was 3.47 g, and the tumor weight of mice fed with polysaccharides from I. obliquus (50 mg/kg and 100 mg/kg, respectively) was 1.51–1.96 g. The tumor growth inhibition ratio was 43.52–57.48%. 5-Fu and high-dose polysaccharides from I. obliquus (100 mg/kg) exhibited higher inhibition ratio (P < 0.01). It was worth noting that polysaccharides from I. obliquus showed equivalently high activities even at much lower dose level (50 mg/kg) (P < 0.01). It implied that polysaccharides from I. obliquus had the higher anti-tumor activities as 5-Fu in vivo. At present, the proposed mechanisms by which polysaccharides exert anti-tumor effect include cancer-preventing, immunoenhancing and direct tumor inhibition. In this present study, polysaccharides from I. obliquus could directly inhibit the tumor cells proliferation. However, the anti-tumor mechanisms of polysaccharides from I. obliquus remain unclear, which are demanded to study further in the subsequent research. 4. Conclusions

Fig. 4. Contrast diagram of IR spectra of polysaccharides from I. obliquus extracted with two different methods. Note: ‘a’means THWE; ‘b’ means UMAE.

The response surface was used to optimize the extraction technology of polysaccharides from I. obliquus, which proved to be a useful method. The optimal conditions of UMAE were given as follows: microwave power was 90 W; extracting time was 19 min; solid/water ratio was 1:20. Compared with traditional hot extraction, UMAE of polysaccharides had great potential and efficiency, the extracting time was much shorter and the yield and purity of polysaccharides increased greatly. Futhermore, the functional groups of polysaccharides from I. obliquus extracted by traditional hot water extraction and UMAE are fundamentally identical.

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In the present study, the anti-tumor activities in vivo and in vitro of polysaccharides from I. obliquus were evaluated. The result showed that polysaccharides from I. obliquus had higher anti-tumor activities in vivo and in vitro and had a potential for clinical use in cancer prevention and treatment. Acknowledgement The authors are grateful to thank Dr. Renxi-Wang (Academy of Military Medical Science, Beijing, China) for his technical support and valuable advice. References [1] [2] [3] [4] [5] [6]

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