Optimised extraction of Erythronium sibiricum bulb polysaccharides and evaluation of their bioactivities

International Journal of Biological Macromolecules 82 (2016) 898–904

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

Optimised extraction of Erythronium sibiricum bulb polysaccharides and evaluation of their bioactivities Chunli Chen a , Rena Kasimu a,∗ , Xiangyun Xie a , Yanling Zheng b , Wenhuan Ding c a b c

Pharmacy College of Xinjiang Medical University, Urumqi, 830011, China College of Medical Engineering and Technology, Xinjiang Medical University, Urumqi, 830011, China Central Laboratory of Xinjiang Medical University, Urumqi, 830011, China

a r t i c l e

i n f o

Article history: Received 2 September 2015 Received in revised form 12 October 2015 Accepted 19 October 2015 Available online 23 October 2015 Keywords: Erythronium sibiricum Polysaccharide extraction Biological activities

a b s t r a c t In this study, the extraction of Erythronium sibiricum bulb polysaccharides (ESBP) through hot water decoction was optimised using response surface methodology (RSM) and a three-level, four-factor Box–Behnken design. The optimum extraction conditions were as follows: extraction time of 4.28 h, extraction temperature of 90 ◦ C, ratio of liquid to raw material of 37 mL/g and extraction cycle number of three. The experimental yield (37.25% ± 0.17%) agreed with the predicted value of the RSM model (37.465%). Preliminary ESBP characterisation was conducted through physicochemical analysis. Biological activity test results showed that ESBP exhibited antioxidant activities and excellent anti-inflammatory and analgesic activities, indicating its potential as an anti-inflammatory and analgesic agent. © 2015 Published by Elsevier B.V.

1. Introduction Erythronium sibiricum (Fisch. et Mey.) Kryl. of the Liliaceae family is mainly distributed in Tian Shan Mountains, Altai and Fuhai regions in Xinjiang, China, as well as in Siberia and Central Asia [1,2]. E. sibiricum bulb has been used as a traditional herbal medicine for centuries by the Kazak ethnic population in Xinjiang. The Kazaks believe that E. sibiricum bulb, when boiled with millet or goat milk, can relieve waist and knee soreness and improve physical symptoms of weakness. However, little is known about the bioactive components of the bulb. Our previous study revealed that E. sibiricum bulb contains amino acids, sugar, polysaccharides and their glycosides, organic acids, phenols, tannins, alkaloids, steroids, triterpene and volatile compounds [3]. Medicinal herbal polysaccharides have attracted increasing attention in recent years because of their potential biological and pharmacological activities, including immunomodulatory, antiinflammatory, anti-tumour, cardiovascular, antifungal, anti-viral, anti-complementary, anti-diabetic, antioxidant and free radical scavenging properties [4–7]. Thus far, no study has analysed the polysaccharides in E. sibiricum bulb.

∗ Corresponding author. Tel.: +86 991 4362473; fax: +86 991 4362473. E-mail address: [email protected] (R. Kasimu). http://dx.doi.org/10.1016/j.ijbiomac.2015.10.058 0141-8130/© 2015 Published by Elsevier B.V.

The present study aimed to optimise the extraction of polysaccharides from E. sibiricum bulb by using response surface methodology (RSM). A three-level, four-factor Box–Behnken design (BBD) was employed to study the effects of extraction time, extraction temperature, ratio of liquid to raw material and extraction cycle number on the extraction yield of E. sibiricum bulb polysaccharides (ESBP). ESBP were characterised using ultraviolet (UV) spectrophotometry and high-performance liquid chromatography (HPLC). The antioxidant activities of ESBP were evaluated in vitro through the 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay, the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assay and the OH radical scavenging assay. The anti-inflammatory and analgesic activities of ESBP in mice were investigated using xylene-induced ear oedema test, cotton pellet-induced granuloma test and acetic acid-induced writhing response test.

2. Materials and methods 2.1. Materials and chemicals E. sibiricum bulbs were collected in Altai or Fuhai regions in Xinjiang Province (2013, 2014). Monosaccharide standards (mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose), m-hydroxydiphenyl, DPPH, ABTS and HPLC-grade acetonitrile were purchased from Sigma-Aldrich Co., USA. All other

C. Chen et al. / International Journal of Biological Macromolecules 82 (2016) 898–904 Table 1 Independent variables and their levels in BBD. Independent variable

A—Extraction time (h) B—Extraction temperature (◦ C) C—Ratio of liquid to raw material (mL/g) D—Extraction cycle number

2.5. Preliminary characterisation of ESBP Level −1

0

1

3 70 30 1

4 80 35 2

5 90 40 3

chemicals and solvents used were of analytical grade and produced in China unless otherwise specified. 2.2. Animals Male Kunming mice weighing 18–22 g were used in this study. The animals were obtained from the Experimental Animal Center of Xinjiang Medical University, Urumqi, China. They were maintained in an air-conditioned room and provided with rodent chow and water. Before the experiments, the animals were allowed to acclimatise to the environment for 3 days. All animal experiments were approved by the Animal Ethics Committee of Xinjiang Medical University and were in accordance with China Guidelines on Animals Care (No. A-20100920002). 2.3. Polysaccharide extraction Dried bulbs were ground in a blender to obtain a fine powder, which was then ultrasonically extracted three times with 95% ethanol at 40 ◦ C to defat and remove small-molecule materials. The pre-treated samples were subsequently separated from the organic solvent by centrifugation (3500 rpm for 15 min) and dried under reduced pressure (40 ◦ C for 24 h). Exactly 2 g of the pre-treated dried power was extracted with hot water under certain conditions (extraction time, extraction temperature, ratio of liquid to raw material and extraction cycle number). The extracted slurry was centrifuged at 5000 rpm for 15 min to collect the supernatant, which was then concentrated and precipitated with a fourfold volume of anhydrous ethanol for 24 h at 4 ◦ C. The resulting precipitate was collected via centrifugation at 5000 rpm for 15 min and then dried under reduced pressure at 50 ◦ C for 24 h to obtain ESBP. ESBP were weighed with a balance. The percentage yield (%) of polysaccharides was calculated as follows: Yield (%w/w) =

899

dried crude ESBP weight × 100. powder weight

2.4. Experimental design and statistical analysis On the basis of a single-factor test, extraction time (A), extraction temperature (B), ratio of liquid to raw material (C) and extraction cycle number (D) were selected as the independent variables. A three-level, four-factor BBD was applied to optimise statistically the extraction process. The range of independent variables and their levels are presented in Table 1. The complete design consisted of 29 experimental points, including 24 factorial points and 5 axial points. Each single-factor experiment was repeated thrice, and the average percentage yield (% yield) of polysaccharides was taken as the response value. The Design-Expert® software (Design-Expert 8.0, trial version, Stat-Ease Inc., Minneapolis, USA) was used for the experimental design, data analysis and quadratic model building.

UV spectra in the range of 200–700 cm−1 were recorded on a UV spectrometer (UV-2550, Shimadzu, Japan). The total sugar content was determined using the phenol–sulphuric acid method with glucose as the standard [8,9]. The uronic acid content was estimated using the m-hydroxydiphenyl method with galacturonic acid as the standard [10]. The monosaccharide content of ESBP was determined using HPLC as previously described [11,12] with slight modifications. ESBP (10 mg) was hydrolysed to monosaccharides in 2 mL of 2 M trifluoroacetic acid at 110 ◦ C for 4 h. After complete hydrolysis, the digested solution was neutralised with 2 M NaOH, and 500 ␮L of this solution was mixed with 500 ␮L of 0.5 M 1-phenyl-3-methyl5-pyrazolone solution in methanol and 500 ␮L of 0.3 M NaOH and reacted for 30 min in a 70 ◦ C water bath. After cooling to room temperature (20–25 ◦ C), the resulting mixture was neutralised with 500 ␮L of 0.3 M HCl and extracted with 2 mL of chloroform. The extraction process was repeated three times, and the aqueous layer was filtered through a 0.22 ␮m membrane for HPLC at 250 nm on an Agilent 1200 system equipped with a Diode Array Detector (Agilent, USA). A Hypersil-BDS C18 column (250 mm × 4.6 mm, 5 ␮m; Dalian Elite, China) was used in the analysis. Elution was carried out at a flow rate of 1.0 mL/min at 25 ◦ C. The mobile phase was a mixture of 0.02 M ammonium acetate buffer (pH 5.5) and acetonitrile (78:22). The injection volume was 10 ␮L. Mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose were used as the monosaccharide standards. 2.6. In vitro antioxidant activity 2.6.1. Assay of DPPH radical scavenging activity The DPPH radical scavenging activity was determined as previously described [13,14] with some modifications. A 2.0 mL volume of different concentrations was added to 2.0 mL of 0.2 mM DPPH in ethanol. After 60 min of incubation in the dark at room temperature, the absorbance (A) was measured at 517 nm. Measurements were performed in triplicate. Ascorbic acid was used as a positive control. The scavenging activity was calculated as follows: Scavenging activity (%) = 1 −

Ai − Aj A0

× 100

where Ai is the absorbance of the reaction solution, Aj is the absorbance of the mixture of 2 mL of sample and 2 mL of ethanol, and A0 is the absorbance of the solution of 2 mL of DPPH and 2 mL of water. 2.6.2. ABTS radical scavenging activity assay ABTS radical scavenging activity was measured as previously described [15,16]. A 2.0 mL sample of different concentrations was added to 2.0 mL of 7 mmol/L ABTS+ •. After 60 min of reaction, the absorbance of the resulting solution was measured at 729.5 nm. Ascorbic acid served as a positive control. The ABTS radical scavenging activity was expressed as follows: ABTS radical scavenging activity (%) = 1 −

Ai − Aj A0

× 100

where Ai is the absorbance of the reaction solution, Aj is the absorbance of the mixture of 2 mL of sample and 2 mL of water, and A0 is the absorbance of the solution of 2 mL of ABTS+ • and 2 mL of water. 2.6.3. Hydroxyl radical scavenging activity assay Hydroxyl radical scavenging activity was determined as previously described [17,18]. A 1 mL sample was mixed with 1 mL

900

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Table 2 BBD with the observed response for extraction yield. Run

A: time (h)

B: temperature (◦ C)

C: ratio (mL/g)

D: number

Yield (%)

1 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 28 29

4 3 5 4 3 4 5 4 4 4 5 4 4 4 3 5 3 4 4 5 4 4 3 4 3 5 4 4 4

80 70 80 80 90 70 90 90 70 70 70 90 80 80 80 80 80 80 80 80 80 80 80 70 80 80 90 80 90

40 35 35 35 35 35 35 40 35 40 35 30 35 30 40 35 35 40 35 40 30 35 30 30 35 30 35 35 35

1 2 3 2 2 1 2 2 3 2 2 2 2 1 2 1 1 3 2 2 3 2 2 2 3 2 1 2 3

23.34 16.12 26.7 26.88 30.42 16.58 34.24 35.22 18.32 16.34 18.56 30.22 29.28 22.14 21.54 24.78 21.68 28.5 29.34 25.68 28.14 29.22 21.86 17.48 25.38 24.6 30.12 28.86 36.70

of 1,10-phenanthroline ethanol solution (0.75 mM), 1 mL of FeSO4 (0.75 mM), 1 mL of H2 O2 (0.01%) and 2 mL of phosphate-buffered saline (pH 7.4). The mixture was warmed to 37 ◦ C and used as the sample solution. After 60 min, the absorbance was measured at 512.0 nm. The blank group contained the same solutions as the sample group, except that deionised water was used instead of the sample. The control group contained the same solution as the blank group, except that deionised water was used instead of H2 O2 . The hydroxyl radical scavenging activity was calculated as follows: Hydroxyl radical scavenging activity (%) =

Asample − Ablank Acontrol − Ablank

× 100.

2.7. Anti-inflammatory and analgesic tests 2.7.1. Animal treatment protocols Mice were randomly divided into five groups of 10 animals each. The mice in the negative control group were treated with saline (10 mL/kg, b.w. i.g.), and the mice in the positive control groups for anti-inflammatory and analgesic tests received indomethacin (10 mg/kg, b.w. i.g.) and aspirin (100 mg/kg, b.w. i.g.). The treatment groups were administered with ESBP at 0.6, 1.2 and 2.4 g/kg through gastric infusion. The dosages of ESBP were estimated on the basis of the recommended oral dosage for folk use in humans.

2.7.2. Xylene-induced ear oedema in mice This experiment was performed as previously described [19,20]. Three groups of mice received ESBP, one group received indomethacin and one group received saline for 7 consecutive days before the experiment. Thirty minutes after the last oral treatment, 50 ␮L of xylene was administered on the anterior and posterior surfaces of the right ear lobe. The left ear was considered as a control. Mice were sacrificed by cervical dislocation 30 min after xylene application. Both ears were removed and

weighed. The inhibition of oedema in percentage was calculated as follows:

%Inhibition =

Difference in ear weightcontrol −Difference in ear weightdrug Difference in ear weightcontrol

× 100.

2.7.3. Cotton pellet-induced granuloma The writhing test was carried out as previously described [21,22]. After shaving the fur under ether anaesthesia, 25 mg of sterile cotton pellets were inserted into the right groin region of each rat. Three groups of mice received ESBP, one group received indomethacin and one group received saline orally for 7 consecutive days from the day of cotton pellet implantation. On the 7th day, the animals were sacrificed by cervical dislocation. The cotton pellets were surgically removed and then dried at 60 ◦ C for 24 h, after which the weights of granuloma were determined.

%Inhibition =

Weight of granulomacontrol −Weight of granulomadrug Weight of granulomacontrol

× 100.

2.7.4. Acetic acid-induced writhing response in mice This test was conducted as previously described [23,24]. Three groups of mice received ESBP, one group received aspirin and one group received saline for 7 days. One hour after the last administration, the mice were intraperitoneally injected with acetic acid (0.7%, 10 mL/kg b.w.). The number of stretching or writhing was counted for 15 min after a latency period of 5 min. The analgesic activity in percentage was calculated as follows:

%Inhibition =

number of writhingcontrol −number of writhingdrug number of writhingcontrol

× 100.

C. Chen et al. / International Journal of Biological Macromolecules 82 (2016) 898–904 1.5

300

5

250

1.2

PMP

200

0.9

mAU

Absorbance

901

A B

150

1

0.6 100

0.3

0.0 200

4

2

7

50

6

3

0

300

400

500

600

700

0

5

10

wavelength(nm) Fig. 1. UV scanning spectrum of ESBP.

2.7.5. Statistical analysis Statistical analysis was performed using SPSS (version 16.0). Data were expressed as mean ± S.D. One-way ANOVA was carried out to compare the results of the control and test drug groups. The difference between means was considered significant when P < 0.05 and highly significant when P < 0.01. 3. Results and discussion 3.1. Extraction optimisation in BBD assay The response values at different experimental combinations are presented in Table 2. Results showed that the polysaccharide yields ranged from 16.12% to 36.70%. Multiple regression analysis of the experimental data revealed that the predicted response yield can be calculated using the following second-order polynomial equation:

15

20

25

30

time(min) Fig. 2. HPLC analysis of monosaccharide composition in ESBP. The standards were separated under the conditions as described in (A), and derivatives of hydrolysate were separated as described in (B). (1 mannose, 2 rhamnose, 3 glucuronic acid, 4 galacturonic acid, 5 glucose, 6 galactose and 7 arabinose).

3.2. Chemical properties of ESBP ESBP was preliminarily characterised via physicochemical analysis. The total sugar and uronic acid contents of ESBP were 58.1% and 5.31%, respectively. The UV scanning spectrum of the ESBP solution is shown in Fig. 1. No apparent UV absorption peak was observed at 260–280 nm, indicating that the protein content of crude polysaccharide was negligible. The results of monosaccharide composition analysis are shown in Fig. 2. No other chromatographic peaks aside from glucose were observed. The HPLC analysis shows that glucose is the predominant monosaccharide in ESBP.

Yield = +28.72 + 1.46 ∗ A + 7.79 ∗ B + 0.51 ∗ C + 2.09 ∗ D + 0.35 ∗ A ∗ B + 0.35 ∗ A ∗ C − 0.45 ∗ A ∗ D + 1.53 ∗ B ∗ C + 1.21 ∗ B ∗ D − 0.21 ∗ C ∗ D − 2.69 ∗ A2 − 1.60 ∗ B2 − 2.25 ∗ C 2 − 1.34 ∗ D2 . The significance of the model was evaluated using ANOVA. ANOVA results in Table 3 showed that the predicted model was highly significant (F = 68.35, P < 0.0001). The F-value and P-value of lack-of-fit (F = 0.81, P = 0.6429) implied that the lack-of-fit was not significant relative to the pure error. The value of R2 (R2 = 0.9856) suggested that the total variation of 99.28% was attributed to the independent variable and that only approximately 0.72% of the total variation cannot be explained by the model. The value of adj-R2 (R2 = 0.9712) indicated a good agreement between the experimental and predicted values. As shown in Table 3, the linear coefficients (A, B and D), quadratic term coefficients (A2 , B2 , C2 and D2 ) and cross product coefficients (BC and BD) were significant (P < 0.05). The other term coefficients were not significant (P > 0.05). The optimum conditions for the response were evaluated using Design-Expert as follows: extraction time of 4.28 h, extraction temperature of 90 ◦ C, ratio of liquid to raw material of 37 mL/g and extraction cycle number of three. The predicted maximum polysaccharide yield was 37.465%. Considering the operability in the actual experiment, we modified the optimal extraction time from 4.28 h to 4.3 h and retained the other conditions. Under the modified optimal conditions, a mean yield of 37.25% ± 0.17% was obtained from the laboratory experiment. This result indicated that the regression model was accurate and adequate for ESBP extraction.

3.3. Antioxidant activity The antioxidant activities of ESBP were measured in vitro by using the DPPH, ABTS and OH radical scavenging assays. The DPPH radical scavenging assay has been widely used to evaluate the free radical scavenging activities of various samples [25]. The scavenging effects of ESBP and ascorbic acid on the DPPH radical were evaluated, and the results are presented in Fig. 3a. ESBP showed a certain DPPH scavenging activity which increased with increasing concentration up to 0.672 mg/mL, after which the scavenging activity slowly increased. The capacity of ESBP to scavenge the DPPH radical was lower than that of ascorbic acid. ABTS radical cation decolourisation is used to assess the antioxidant activity of a water-soluble substance [26,27]. The scavenging rate was 4.81% at 0.168 mg/mL, which increased to 34.06% at 1.68 mg/mL in a concentration-dependent manner (Fig. 3b). These data indicate that ESBP exhibits a certain ABTS radical scavenging activity. Hydroxyl radicals can react with almost all biomacromolecules functioning in living cells and induce severe damage to the adjacent biomolecules [28]. Thus, hydroxyl radical removal is beneficial to the body. The scavenging activities of ESBP and ascorbic acid

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C. Chen et al. / International Journal of Biological Macromolecules 82 (2016) 898–904

Table 3 ANOVA for the response surface quadratic model. Source

Sum of squares

df

Mean square

F value

P-value Prob > F

Model A—time B—temperature C—ratio D—number AB AC AD BC BD CD A2 B2 C2 D2 Residual Lack of fit Pure error Cor total R-squared Adj R-squared

900.73 25.7 728.83 3.18 52.5 0.48 0.49 0.79 9.42 5.86 0.18 46.95 16.51 32.93 11.61 13.18 8.82 4.35 913.9 0.9856 0.9712

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 10 4 28

64.34 25.7 728.83 3.18 52.5 0.48 0.49 0.79 9.42 5.86 0.18 46.95 16.51 32.93 11.61 0.94 0.88 1.09

68.35 27.3 774.33 3.38 55.78 0.51 0.52 0.84 10.01 6.22 0.19 49.89 17.54 34.98 12.34

<0.0001 0.0001 < 0.0001 0.0872 < 0.0001 0.4886 0.4825 0.3745 0.0069 0.0257 0.6717 < 0.0001 0.0009 < 0.0001 0.0034

0.81

0.6429

Table 4 Effect of ESBP on xylene-induced ear oedema in mice. Group Control (saline, 10 mL/kg) Indomethacin ESBP

*

Dose (g/kg)

Difference in ear weight (mg)

0.01 0.60 1.20 2.40

36.92 18.002 30.78 25.60 20.52

± ± ± ± ±

8.47 4.02** 8.42 7.64* 9.62**

Percentage of inhibition (%) 51.24 16.63 30.66 44.47

p < 0.05 and ** p < 0.01 when compared with control.

Table 5 Effects of ESBP on cotton pellet-induced granuloma in mice. Group Control (saline, 10 mL/kg) Indomethacin ESBP

*

Dose (g/kg)

Weight of granuloma (mg)

0.01 0.60 1.20 2.40

69.48 31.81 52.38 43.41 39.92

± ± ± ± ±

9.41 8.45** 10.66** 12.79** 12.24**

Percentage of inhibition (%) 54.21 24.60 37.55 42.59

p < 0.05 and ** p < 0.01 when compared with control.

Table 6 Effect of ESBP on acetic acid-induced writhing response in mice. Group Control (saline, 10 mL/kg) Aspirin ESBP

*

Dose (g/kg)

Number of writhing

Percentage of inhibition (%)

0.10 0.60 1.20 2.40

50.30 ± 10.264 27.90 ± 23.154** 39.90 ± 15.800 30.30 ± 15.642** 25.10 ± 13.119**

44.53 20.68 39.76 50.10

p < 0.05 and ** p < 0.01 when compared with control.

on hydroxyl radicals are shown in Fig. 3c. The scavenging activity of ESBP on hydroxyl radicals increased with increasing concentration from 0.168 mg/mL to 1.68 mg/mL. These data suggest that ESBP exhibits a certain scavenging effect on hydroxyl radicals. 3.4. Anti-inflammatory and analgesic activities Ear oedema induced by xylene may involve inflammatory mediators, such as histamine, serotonin, bradykinin and prostaglandins, which promote vasodilatation and increase vascular permeability [29]. The xylene-induced ear oedema model was used to determine the anti-acute inflammatory activity of ESBP [30]. The statistical data (Table 4) obtained in this study showed that ESBP

dose-dependently decreased mouse ear oedema compared with the control group. Ear oedema was significantly reduced by ESBP at 1.2 (P < 0.05) and 2.4 g/kg (P < 0.01) but not by ESBP at 0.6 g/kg, with inhibition rates of 30.66% and 44.47%, respectively. However, the inhibitory effect of 2.4 g/kg ESBP was weaker than that of indomethacin at 10 mg/kg (51.24%). Cotton pellet-induced granuloma formation is common in an established chronic inflammatory reaction and can serve as a sub-chronic inflammatory test model [31]. This method has been extensively used to assess the transudative, exudative and proliferative components of sub-chronic inflammation [32]. As shown in Table 5, ESBP at all tested doses significantly inhibited (P < 0.01) granuloma formation in a dose-dependent manner; the strongest

C. Chen et al. / International Journal of Biological Macromolecules 82 (2016) 898–904

with the control (39.76% and 50.10%, respectively). The inhibitory effect of ESBP at 2.4 g/kg was stronger than those of the positive control 0.1 g/kg aspirin (44.53%). Similar to aspirin, ESBP possibly exerts its analgesic activity by blocking the release of PGs and other cytokines which excite pain nerve endings.

A 100

Scavenging activity(%)

80

4. Conclusion

ESBP Ascorbic acid

60

40

20

0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

Concentration(mg/mL)

B 100

80

Scavenging activity(%)

903

ESBP Ascorbic acid

60

40

20

0 0.0

0.4

0.8

1.2

1.6

2.0

Polysaccharides were extracted from the bulb of E. sibiricum through hot water decoction, and BBD was used to optimise the extraction conditions. ANOVA showed that the linear coefficients (A, B and D), quadratic term coefficients (A2 , B2 , C2 and D2 ) and cross product coefficients (BC and BD) were significant (P < 0.05), and a quadratic model was established. The optimal experiment yield of 37.25% ± 0.17% was obtained under the following optimum conditions: extraction time of 4.3 h, extraction temperature of 90 ◦ C, ratio of liquid to raw material of 37 mL/g and extraction cycle number of three. The aforementioned experimental yield of ESBP closely agreed with the predicted value (37.465%). The chemical compositions were also analysed. ESBP contained 58.10% total sugar and 5.31% uronic acid, with almost no protein. The HPLC analysis showed that ESBP mainly consists of glucose. The antioxidant activities of ESBP were also evaluated using the DPPH, ABTS and OH radical scavenging assays in vitro. ESBP exhibited certain effects on radical scavenging. ESBP exerted significant anti-inflammatory and analgesic effects as determined through cylene-induced ear oedema test, cotton pellet-induced granuloma and acetic acidinduced writhing response test in mice. Compared with the control, ESBP at 1.2 and 2.4 g/kg exhibited significant inhibitory effects in all three models. Hence, ESBP should be investigated as a potential functional food ingredient and anti-inflammatory and analgesic product. Further research should focus on the purification, structural characterisation and bioactivities of ESBP.

Concentration(mg/mL)

Acknowledgement

C

This study was supported by the Science and Technology Projects of Xinjiang Uygur Autonomous Region of China (No. 201130105-2).

100

Scavenging activity(%)

80

References ESBP Ascorbic acid

60

40

20

0 0.0

0.4

0.8

1.2

1.6

2.0

Concentration(mg/mL) Fig. 3. Effect of ESBP on DPPH (a), ABTS (b) and hydroxyl radicals (c).

inhibitory effect was obtained at the highest ESBP dose of 2.4 g/kg (42.59%). However, the inhibitory effect of indomethacin (54.21%) was stronger than that of ESBP at 2.4 g/kg. Writhing response in mice is induced by acetic acid, which causes pain by liberating prostaglandins (PGs) and other cytokines which excite pain nerve endings [33]. The acetic acid-induced writhing test model is a classical peripheral analgesic animal model which is widely used to screen analgesic drugs [34]. The statistical data obtained in this study are listed in Table 6. ESBP at 1.2 and 2.4 g/kg significantly inhibited (P < 0.01) the writhes compared

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