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Enzymatic water extraction of taxifolin from wood sawdust of Larix gmelini (Rupr.) Rupr. and evaluation of its antioxidant activity
Food Chemistry 126 (2011) 1178–1185
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Enzymatic water extraction of taxifolin from wood sawdust of Larix gmelini (Rupr.) Rupr. and evaluation of its antioxidant activity Ying Wang a,b,1, Yuangang Zu a,b,1, Jingjing Long a,b, Yujie Fu a,b,⇑, Shuangming Li a,b, Dongyang Zhang a,b, Ji Li a,b, Michael Wink c, Thomas Efferth d a
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, China Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany d Department of Pharmaceutical Biology, Institute of Pharmacy, University of Mainz, 55128 Mainz, Germany b c
a r t i c l e
i n f o
Article history: Received 6 May 2010 Received in revised form 12 October 2010 Accepted 26 November 2010 Available online 3 December 2010 Keywords: Enzymatic water extraction Taxfolin Antioxidant Larix gmelini (Rupr.) Rupr. wood sawdust Central composite design Scanning electron microscopy
a b s t r a c t An enzyme incubation–water extraction (EI–WE) method was developed and optimised for the extraction of the natural antioxidant taxifolin and of the total flavonoids from wood sawdust of Larix gmelini (Rupr.) Rupr. A factorial design and a central composite design approach were used for method optimisation. Optimal conditions were 0.5 mg/ml cellulase and 0.5 mg/ml pectinase, a pH of 5.0, a temperature of 32 °C and 18 h incubation time. The flavonoids and taxifolin were extracted in hot water at 50 °C for 30 min, with a solid to liquid ratio of 1:20. Under optimised conditions, the yields of taxifolin and total flavonoids increased from 1.06 ± 0.08 to 1.35 ± 0.04 mg/g and 4.13 ± 0.17 to 4.96 ± 0.29 mg/g, respectively. DPPH and BHT assays revealed that the EI–WE samples had 1.8- and 1.68-fold higher antioxidant activities than the controls. SEM results revealed the structural disruption of wood sawdust with enzyme incubation. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Taxifolin (dihydroquercetin, 2-(3,4-dihydroxyphenyl)-2,3-dihydro-3,5,7-trihydroxy-4H-benzopyran-4-one) is a dihydroflavonol. It can eliminate free radicals in the body, improve the impermeability of capillary vessels and recover their elasticity effectively. Taxifolin has a distinguished better antioxidant activity (Audron, Regina, Jonas, & Narimantas, 2000; Bong-Sik et al., 2000) compared to other antioxidants, and can remarkably prolong the shelflife of lard, plant oils, powdered milk and candy. It is not embryotoxic and does not lead to malformations, hypersusceptibility or mutations. An antiviral effect of taxifolin has also been reported (Evangelos, Jean-Marc, Jacques, & Robert, 1987; Shu-Chen, Yih-Shou, & Jung-Yaw, 1992). Furthermore, taxifolin can also restrain the synthesis of fat in human liver (Andre et al., 2000) and activate phosphodiesterase (Kuppusamy & Das, 1992). Taxifolin derivatives may be useful as sweeteners without calories. In general, taxifolin can be used as a natural antioxidant additive in food industry. Abbreviations: EI–WE, enzyme incubation–water extraction; FD, factorial design; CCD, central composite design; SEM, scanning electron microscopy; RSM, response surface methodology. ⇑ Corresponding author. Tel.: +86 451 82190535; fax: +86 451 82102082. E-mail addresses: [email protected], [email protected] (Y. Fu). 1 First two authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.11.155
Larches (genus Larix) are widespread conifers of the cool northern hemisphere and comprise 10 species. They are one of the three dominant conifers in northeast China. They usually grow in cool temperature zones and are the most cold-resistant species of conifers. In China, the genus Larix mainly includes Larix gmelini (Rupr.) Rupr., L. principisrupprechtii, L. kaempferi, L. sibirica and L. olgensi. Among these, L. gmelini (Rupr.) Rupr. is best known for its wide distribution and extensive use. It occupies nearly 55% of Great Khingan and Lesser Khingan (Sun, Zhang, Han, & Wang, 2007). With physical properties, such as rigidness, corrosion resistance and straight grain, L. gmelini (Rupr.) Rupr. has been widely used for building and furniture (Xin & Li, 2009). As a result, large quantities of wood sawdust are produced every year as a side product. Therefore, the exploitation of L. gmelini (Rupr.) Rupr. as a conifer resource has become a main task. It has been reported that L. gmelini (Rupr.) Rupr. contains valuable bioactive secondary metabolites, such as arabinogalactan (AG) and flavonoids, including taxifolin, oligomeric proanthocyanidins (OPC) and others (Huang & Fang, 2005). Extraction of these bioactive components from wood sawdust could be an economically interesting utilisation of this conifer resource. Enzyme-assisted extraction can be used as an alternative to the release of secondary metabolites from biological materials. It possesses the advantages of environmental compatibility, high
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efficiency, and easy operation processing (Zu et al., 2009). Hydrolytic enzymes, including cellulase, pectinase and beta-glucosidase, are commonly used for the extraction of secondary metabolites (Mark, Wilbur, Karel, & Randall, 2007; Young et al., 2005). They interact with cell walls, break down their structural integrity and in consequence enhance the release of flavonoid aglycones. However, such an enzyme-assisted extraction of taxifolin from the L. gmelini (Rupr.) Rupr. wood sawdust has not yet been reported. Most studies on the extraction of taxifolin and flavonoid have used ethanol as a solvent at ambient temperatures. However, consuming large volumes of organic solvents for extraction represents a considerable shortcoming (Chattip, Wanchai, & Artiwan, 2008; Huang, Xue, Niu, Jia, & Wang, 2009). Therefore, the development of an environmentally friendly method to extract taxifolin and total flavonoids from wood sawdust of L. gmelini (Rupr.) Rupr. is a major challenge. The aim of this investigation was to obtain an economically and environmentally friendly method for the extraction of taxifolin and total flavonoids from L. gmelini (Rupr.) Rupr. The influence of hydrolytic enzymes on the yields of taxifolin was optimised by factorial design (FD) and central composite design (CCD). The antioxidant activity of the extracts and taxifolin was evaluated by DPPH and BHT assays. Furthermore, the ultra-structural changes of plant materials were analysed by scanning electron microscopy (SEM). The present study offers an alternative method for the highly effective utilisation of a side product of conifer utilisation. 2. Materials and methods 2.1. Plant materials Wood sawdust of L. gmelini (Rupr.) Rupr. was obtained from the Xinming furniture factory in Harbin. 2.2. Chemicals and reagents Taxifolin was obtained from Shanghai Ronghe Bio-Technology Co., Ltd. (Shanghai, China). Cellulase (EC 1.1.1.27, P1000 U/mg), beta-glucosidase (EC 3.2.1.21, P47 U/mg) from Shanghai WeeBeyond Scientific and Trade Co., Ltd., and pectinase (EC 3.2.1.15, 1.41 U/mg) from Fluka Chemical Co. Methanol of chromatographic grade were obtained from J&K Chemical Ltd. (China). Double-distilled water was used in all experiments. All solvents for HPLC were filtered through a membrane filter (0.45 lm pore size) and degassed under ultrasonic condition before use. The total flavonoids yield of the samples was determined by a modified colorimetric method previously described by Ordonez, Gomez, Vattuone, and Isla (2006) using rutin (Sigma, St. Louis, MO, USA) as a standard. Aliquots (1 ml) of appropriately diluted extracts or standard solutions were mixed with 0.3 ml 5% NaNO2 solution. After 6 min, 0.3 ml 10%AlCl3 solution were added, and the mixture was allowed to stand for another 6 min. Then, 4 ml 4% NaOH were added. The reaction solution was well mixed, kept for 15 min, and the absorbance was determined at 510 nm. Total flavonoids yield was calculated using a standard curve. 2.3. Apparatus and chromatographic conditions HPLC analysis was performed with a Jasco LC system (Jasco Company, Japan) equipped with a Jasco PU-1580 intelligent HPLC pump and coupled to Jasco UV-1575 UV/VIS Detector as well as Millennium 32 system software. Chromatographic separation was performed on a HIQ sil C18V reversed-phase column (4.6 mm £ 250 mm, KYA TECH Corporation, Japan) packed with 5 lm diameter particles. The mobile phase was methanol–water–
acetic acid (44:56:0.4, v/v/v). The eluents were monitored at 290 nm UV wavelength. The injection volume and the flow rate of the mobile phase were 10 ll and 1 ml/min, respectively. The column was maintained at 35 °C. 2.4. Enzyme incubation–water extraction process Cellulase, pectinase and beta-glucosidase were dispersed in 100 ml deionised water to obtain solutions of defined concentrations (0.25–2 mg/ml). Powder of 2 g sawdust was added to the enzymatic solution, which was then adjusted to defined pH-values (2.5–7.0) with 0.1 M HCl solution and shaken on a flat-bed orbital shaker 0–72 h at defined temperatures (20–50 °C). After the enzymatic treatment the samples were filtered. The residues were collected into conical flasks, and distilled water was added. Then, the conical flasks were put into an ultrasonic bath (Kunshan Ultrasonic Instrument Co., Ltd., China). The extraction was performed for 30 min at 50 °C and 100% power. The residues were then filtered, and the entire process was repeated twice. The filtrates collected were concentrated in vacuo (50 °C) in a rotary evaporator. The syrup was reconstituted with HPLC mobile phase. The sample solutions were then centrifuged at 12,000 rpm before being analysed by HPLC. All experiments were performed in triplicate. 2.5. Central composite design (CCD) Response surface methodology (RSM) (Deniz & Ismail, 2007) was applied to identify the optimum levels of the three variables: enzyme concentration (mg/ml), pH, and incubation temperature (°C) and two responses, the yields of taxifolin and total flavonoids. The ranges of concentration (A), pH (B), temperature (C) and the central points were selected based on results of a factorial design. The experiments were designed according to the central composite design (CCD) using a 23 full factorial design. The general equation of the second degree polynomial equation is
Y ¼ b0 þ
k X i¼1
Bi X i þ
k X
Bii X 2 þ
i¼1
k X
Bij X i X j
ð1Þ
i>j
where Y represents the measured response variables, b0 is defined as the constant, Bi, Bii and Bij are the linear, quadratic and interactive coefficients of the model, respectively. Xi and X j are the levels of the independent variables. The test variables were transformed to range between 1 and 1 for the evaluation of the factors. In all cases, the calculations were carried out with the help of the statistical package Statgraphics Plus for Windows V6.0. 2.6. 1,1-Diphenyl-2-picrylhydrazyl assay (DPPH assay) The antioxidant activity of the crude extracts with or without enzyme treatment and pure taxifolin was studied using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay as reported by Amarowicz, Pegg, Moghaddam, Barl, and Weil (2004). An aliquot of the sample (100 ll) at various concentrations or water as negative control was mixed with ethanol (1.4 ml) and then added to 4‰ DPPH (1 ml, Sigma–Aldrich) in ethanol. The mixture was vigorously shaken followed by incubation for 70 min. The absorbance was read at 517 nm (UNICO, Shanghai, China). Ascorbic acid (Sigma–Aldrich), a stable antioxidant, was used as positive control. The radical-scavenging activities of the samples, expressed as percent inhibition of DPPH, were calculated according to the formula:
Scavenging Effectð%Þ ¼
Absorbance sample 100% 1 Absorbance control
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where absorbancesample and absorbancecontrol are the absorbance values of the tested sample and of the blank samples determined after 70 min, respectively. The DPPH scavenging activity is presented by its IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution. 2.7. Butylated hydroxytoluene assay (BHT assay) The antioxidant activity of the samples was determined using the b-carotene–linoleic acid test (Burtun, 1989; Sies & Stahl, 1995). Approximately 10 mg of b-carotene (type I synthetic, Sigma–Aldrich) was dissolved in chloroform (10 ml). The carotene–chloroform solution (0.2 ml) was pipetted into a boiling flask containing linoleic acid (20 mg, Sigma–Aldrich) and 200 mg TweenÒ 40 (Sigma–Aldrich). Chloroform was removed using a rotary evaporator at 40 °C for 5 min, and distilled water (50 ml)
was added to the residue slowly with vigorous agitation, to form an emulsion. A portion of the emulsion (5 ml) was added to a tube containing the sample solution (0.2 ml) and the absorbance was immediately measured at 470 nm against a blank, consisting of an emulsion without b-carotene. The tubes were placed in a water bath at 50 °C and the oxidation of the emulsion was monitored over a 60 min period. Control samples contained 200 ll of water instead. Butylated hydroxytoluene (BHT, Sigma–Aldrich), a stable antioxidant, was used as synthetic reference. The antioxidant activity was expressed as inhibition percentage with reference to the control after a 60 min incubation using the following equation:
AA ¼ 100ðDRC DRS Þ=DRC where AA = antioxidant activity; DRC = degradation rate of the control = [ln(a/b)/60]; DRS = degradation rate in presence of the
Fig. 1. Effect of (a) enzyme type; (b) enzyme concentration; (c) incubation time; (d) pH; and (e) incubation temperature on the yields of taxifolin and total flavonoids (n = 3), p < 0.05. aDW: dry weight.
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sample = [ln(a/b)/60]; a = absorbance at time 0; b = absorbance at 60 min. 2.8. Scanning electron microscopy Samples with or without EI–WE treatment were scanned by SEM (Hitachi, San Jose, CA, USA). The materials were collected and air-dried after extraction of taxifolin and total flavonoid. They were fixed on aluminium stubs with adhesive tape and covered by gold using a sputter coater (JEOL JEC-1200). All samples were examined under high vacuum conditions at a voltage of 15.0 kV (50 lm, 500 magnification). 2.9. Statistical analysis All results were subjected to statistical analyses. Mean values of all data were obtained from triplicate experiments. Differences were considered to be statistically significant at p < 0.05. 3. Results and discussion 3.1. Optimisation of taxifolin and total flavonoids extraction using FD 3.1.1. Selection of enzyme type Cellulase catalyses the breakdown of cellulose into glucose, cellubiose and glucose polymers. Pectinase has the ability to disintegrate pectic compounds and pectin. Beta-glucosidase breaks the
beta-1,4 glucosidic linkages in glucosides. In the present investigation, the effect of enzyme combinations on taxifolin and total flavonoids extraction yields was evaluated, under the conditions of 1 mg/ml total enzyme concentration, pH 7.0, temperature 40 °C, and incubation time 24 h. Fig. 1a shows the yields of taxifolin and total flavonoids after incubation with enzymes. Generally, a similar variation tendency for the yields of taxifolin and total flavonoids were found in the EI–WE samples. The taxifolin yield was increased with the enzymatic treatment (p < 0.05) as expected. The groups treated with cellulase alone or with pectinase and cellulase (p & c) revealed higher yields as compared to other groups. Under these conditions, the yields of taxifolin were 1.26 ± 0.02 and 1.35 ± 0.05 mg/g, which were 1.47-fold and 1.57-fold higher compared to control, respectively. It is well documented in the literature that pectinase and cellulase have synergistic effects (Elias, Foda, & Attia, 1984; Grohman & Baldwin, 1992; Marshall, Graumlich, Braddock, & Messersmith, 1985). They hydrolyse the cellulose and pectin of cell walls, which leads to an enhanced release of taxifolin. The variation of the total flavonoids yield expressed as mg rutin per g of extract was similar to that of taxifolin (p < 0.05). The total flavonoids yields in cellulase alone and p & c groups were also better compared to other treatments. The yields were 4.204 ± 0.24 and 4.202 ± 0.41 mg/g (1.26-fold and 1.25-fold compared to control), respectively. Acceptable yields were achieved in the p & c group for taxifolin; also, the yields of total flavonoids treated with cellulase alone did not show significant improvements over the p & c group. Thus,
Table 1 Central composite experimental design and response values obtained under different extraction conditions. No.
X1
X2
X3
X1 Concentration (mg/ml)
X2 pH
X3 Temperature (°C)
Taxifolin yield (mg/g)
Total flavonoids yield (mg/g)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 1 1 1 1 1 1 1 0 0 0 0 1.68 1.68 0 0 0
1 1 1 1 1 1 1 1 0 0 1.68 1.68 0 0 0 0 0
1 1 1 1 1 1 1 1 1.68 1.68 0 0 0 0 0 0 0
0.25 0.75 0.25 0.75 0.25 0.75 0.25 0.75 0.5 0.5 0.5 0.5 0.08 0.92 0.5 0.5 0.5
4.2 4.2 5.8 5.8 4.2 4.2 5.8 5.8 5.0 5.0 3.7 6.4 5.0 5.0 5.0 5.0 5.0
25 25 25 25 35 35 35 35 21.6 38.4 30 30 30 30 30 30 30
1.281 1.203 1.216 1.261 1.260 1.341 1.277 1.233 1.236 1.380 1.247 1.242 1.274 1.227 1.431 1.411 1.406
4.361 4.687 4.738 4.159 4.381 4.482 4.870 4.147 4.841 5.123 4.515 4.897 4.610 4.246 4.876 4.987 5.012
Table 2 Significance of regression coefficient for taxifolin and total flavonoids yield. Variables
Model X1 (enzyme concentration) X2 (pH) X3 (temperature) X1X1 X2X2 X3X3 X1X2 X1X3 X2X3 Lack of fit R2 Adjusted R2
Eq. (2)
Eq. (3)
Mean square
F-value
p-value
Mean square
t-value
p-value
7.85 103 8.98 105 2.52 104 8.59 103 0.28 0.43 0.17 3.23 104 1.76 103 2.22 103 1.67 103
6.31 0.072 0.20 6.91 22.51 34.23 13.78 0.26 1.42 1.79 9.46 0.8903 0.7491
0.120 0.7959 0.6662 0.0340 0.0021 0.0006 0.0075 0.6262 0.2727 0.2232 0.0983
0.15 0.16 3.10 102 0.12 0.62 0.21 1.70 102 0.37 1.70 102 1.20 102 0.043
4.53 5.04 0.95 0.38 19.31 6.52 0.53 11.64 0.53 0.36 8.18 0.8535 0.6652
0.0295 0.0596 0.3622 0.5563 0.0032 0.0380 0.4921 0.0113 0.4903 0.5663 0.1125
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the p & c group was further considered to be optimum for the simultaneous extraction of taxifolin and total flavonoids. 3.1.2. Effect of enzyme concentration The effect of different enzyme concentrations on the yields of taxifolin and total flavonoids was evaluated (Fig. 1b) under the conditions of pectinase and cellulase combination, pH 7.0, temperature 40 °C, and incubation time 24 h. Acceptable yields were achieved at 0.5 mg/ml for both taxifolin and total flavonoids. Higher concentrations than 0.5 mg/ml did not increase yields. We conclude that a combination of 0.5 mg/ml cellulase and 0.5 mg/ml pectinase is suitable for the extraction of taxifolin and total flavonoids from L. gmelini (Rupr.) Rupr. Thus, 0.5 mg/ml was selected as an optimal enzyme concentration for the subsequent steps. 3.1.3. Effect of incubation time Under the conditions of 0.5 mg/ml pectinase and 0.5 mg/ml cellulase combination, pH 7.0 and temperature 40 °C, the effect of incubation time on taxifolin and total flavonoids extraction yields
is shown in Fig. 1c. It was apparent that extending the extraction period increased the taxifolin and total flavonoids yields (p < 0.05). The yields of taxifolin reached a peak (1.29 ± 0.04 mg/ g) at 12 h, and 18 h for total flavonoids (5.24 ± 0.15 mg/g). As a result, 18 h was considered optimum for enzyme incubation. 3.1.4. Effect of pH value of enzyme solution The pH of the enzyme solution considerably influences the activity of the enzyme. Fig. 1d shows the taxifolin and total flavonoids yields in enzyme solutions of different pH-values under the conditions of 0.5 mg/ml pectinase and 0.5 mg/ml cellulase combination, temperature 40 °C and incubation time 24 h. The taxifolin yields at pH 5.0 and 6.5 were higher than for other pH values. The enzymes work optimally at these two pH-values and disintegrated cell walls more completely. Unlike taxifolin, the highest yield of total flavonoids was achieved only at pH 5.0. The reason may be that flavonoids represent a chemically diverse group of compounds, and the retention behaviour of each compound might be different in plant cells.
Fig. 2. Response surface plot for maximal extraction yields of taxifolin as a function of (a) enzyme concentration and pH; (b) enzyme concentration and temperature; and (c) pH and temperature.
Y. Wang et al. / Food Chemistry 126 (2011) 1178–1185
3.1.5. Effect of temperature on enzyme activity Fig. 1e presents the influence of increased temperatures on taxifolin and total flavonoids yields under the conditions of 0.5 mg/ml pectinase and 0.5 mg/ml cellulase combination, incubation time of 24 h and pH 7.0. Rising temperatures caused a slight increase of taxifolin and total flavonoids yields. The best yields were obtained at 30 °C. Higher temperatures rather decreased than increased the amount of taxifolin and total flavonoids. It is likely that enzymes are heat sensitive and are denatured at higher temperatures. A higher yield of total flavonoids at 50 °C is probably due to the better solubility of flavonoids in hot water. The date of this section was shown in Table S1, and Duncan’s Multiple Range Test was used to describe the significant difference between pairs of treatments. 3.2. Optimisation of taxifolin and total flavonoids extraction using CCD 3.2.1. Fitting the models To optimise maximal yields of taxifolin and total flavonoids using the EI–WE method, the response surface methodology was applied. Enzyme concentration (X1), pH (X2) and incubation temperature (X3) were used for the optimised experiment conducted by a 23 full factorial central composite design. All the experimental data obtained from a 17-run-experiment and the predicted data from the response surface analysis model established are shown in Table 1. The yields of taxifolin (Y1) and total flavonoids (Y2) are functions of these variables. By applying multiple regression analysis to the experimental data, the following second order polynomial equations were found to represent the extraction yields adequately.
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The Model F-value of 4.53 implies the model is significant. There is only a 2.95% chance that a ‘‘Model F-value’’ this large could occur due to noise. The ‘‘Lack of Fit F-value’’ of 8.18 implies the Lack of Fit is not significant relative to the pure error. There is 11.25% chance that a ‘‘Lack of Fit F-value’’ this large could occur due to noise. A maximal value of total flavonoids (5.06 mg/g) was observed at 0.31 mg/ml complex enzyme, pH 5.72 and 35.0 °C. 3.2.2. Optimisation of taxifolin extraction with the EI–WE method 3D surface plots were employed to illustrate the interactions between enzyme concentrations, pH and temperature on the extraction yields of taxifolin and total flavonoids. The response surfaces based on these variables are shown with one variable kept at optimum level and varying the other two within the experimental range. In general, the response surfaces indicated a complex interaction between the variables. All these three variables had a quadratic effect on the extraction yield of taxifolin (Fig. 2a–c). As the concentrations of enzymes, and in addition the pH and temperature increased, the response increased to a maximal value and then decreased. These three variables acted synergistically. Slightly higher temperatures, intermediate pH values and enzymes concentration of 0.5 mg/ml gave maximal taxifolin yields, as explained above in the FD experiments. The interactive effects of enzymes concentration, pH and temperature on the yield of total flavonoids were similar to taxifolin (data not shown). 3.3. Validation of the method In order to validate the adequacy of the model equations [Eqs.(2) and (3)], the extraction was carried out under optimal con-
Y 1 ¼ 3:233 þ 0:590X 1 þ 1:096X 2 þ 0:114X 3 0:798X 21 0:096X 22 0:0016X 23 0:032X 1 X 2 þ 0:012X 1 X 3 0:0042X 2 X 3 ð2Þ Y 2 ¼ 4:763 þ 9:827X 1 þ 2:442X 2 þ 0:070X 3 3:753X 21 0:213X 22 0:0015X 23 1:081X 1 X 2 þ 0:037X 1 X 3 0:0095X 2 X 3 ð3Þ The significance of each coefficient was determined using the F test and p-value. The corresponding variables were more significant, if the absolute F-value became larger and the p-value smaller. For taxifolin, the coefficients calculated from the regression model [Eqs. (2) and (3)] are listed in Table 2. They contain one constant, three linear, three quadratic, and three interaction terms. For [Eq. (2)], the results showed that the linear and quadratic term of X3 were very important, followed by X1, X2, and X3 (p < 0.05). None of the other terms (X1, X2, X1X2, X1X3, X2X3) are significant (p > 0.05), so the model should be reduced. The reduced equation was
Y 1 ¼ 3:233 þ 0:114X 3 0:798X 21 0:096X 22 0:0016X 23
ð4Þ
The Model F-value of 6.31 implies the model is significant. There is only a 1.20% chance that a ‘‘Model F-value’’ this large could occur due to noise. The ‘‘Lack of Fit F-value’’ of 9.46 implies there is a 9.83% chance that a ‘‘Lack of Fit F-value’’ this large could occur due to noise. A maximum value of taxifolin (1.42 mg/g) was observed at 0.51 mg/ml enzyme concentration, pH 4.93 and 31.73 °C. For Eq. (3), the results showed that only the interaction terms of X1X2, X12 and X22 are significant (p < 0.05). So the model was reduced as that
Y 2 ¼ 4:763 3:753X 21 0:213X 22 0:0015X 23 1:081X 1 X 2
ð5Þ
Fig. 3. (a) DPPH scavenging capacity of extracts and taxifolin and (b) inhibition of linoleic acid oxidation. Values are means ± SD (n = 3), p < 0.05.
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Fig. 4. Scanning electron microscopy images of L. gmelini (Rupr.) Rupr. wood sawdust: (a) untreated and (b) EI–WE.
ditions. Enzyme incubation conditions were: incubation with 0.5 mg/ml cellulase and 0.5 mg/ml pectinase at pH 5.0, and 32 °C for 18 h. Hot water (20:1) was used to extract taxifolin and total flavonoids (30 min). Under optimal conditions, taxifolin and total flavonoids yields were 1.35 ± 0.04 and 4.96 ± 0.29 mg/g (n = 3), respectively. The p-values of taxifolin and total flavonoids yields were 0.037 and 0.042 (p < 0.05), respectively, which were obtained by a one-sided t-test. 3.4. Antioxidant activities 3.4.1. 1,1-Diphenyl-2-picrylhydrazyl assay (DPPH assay) In the present investigation, the DPPH assay was used to evaluate the radical-scavenging activity of L. gmelini (Rupr.) Rupr. extracts with and without enzyme treatment, as well as of pure taxifolin. Their scavenging potential was compared to ascorbic acid (Fig. 3a). The EI–WE samples contained higher amounts of taxifolin and total flavonoids than the controls. With increasing extraction yields, the DPPH radical scavenging effect increased. Taxifolin exhibited the highest radical-scavenging activity. One gram taxifolin, equal to 21.83 mmol ascorbic acid, exerted a fourfold higher radical-scavenging activity (1 g ascorbic acid is 5.68 mmol). These results suggest that taxifolin is one of the major components contributing to the total antioxidant capacity of L. gmelini (Rupr.) Rupr. 3.4.2. Butylated hydroxytoluene assay (BHT assay) The antioxidant activity of the samples with or without EI–WE treatment and pure taxifolin, as well as the positive control BHT is presented in Fig. 3b. These samples prevented b-carotene bleaching by neutralising the free radicals. The weakest free radical-scavenging activity was exhibited by the samples without EI–WE treatment. The EI–WE samples showed better radical-scavenging activity (equal to 99 lmol BHT/g dry weight). It is apparent that b-carotene was protected from oxidation by the larch antioxidants in a dose dependent fashion. Taxifolin exhibited the best antioxidant activity in this assay, i.e. 1 g taxifolin was equal to 32.44 mmol of BHT (6.15-fold higher antioxidant activity). The order of antioxidant activity was taxifolin > BHT > EI–WE > control. 3.5. Scanning electron microscopy Fig. 4 shows the scanning electron micrographs of wood sawdust without (a) and after enzyme treatment (b). The morphological structures represent imperforate tracheary cells of the xylem, a rigid structure for the transport of water and mineral salts. As expected, the wood structures were partly destroyed by EI–WE treatment; the cell walls became thinner and disorganised (Fig. 4b). The
hydrolysis of cell walls apparently leads to the release of taxifolin and other secondary metabolites. 4. Conclusions An enzyme water extraction (EI–WE) method was used for the extraction of taxifolin and total flavonoids from L. gmelini (Rupr.) Rupr. for the first time. This method was evaluated using a factorial design (FD) and a central composite design (CCD). The enzyme type, concentration, incubation time, pH and temperature were studied and optimised. The results showed that all these factors were important for the extraction process. Optimal conditions were 0.5 mg/ml cellulase and 0.5 mg/ml pectinase, pH 5.0, and 32 °C. The water extraction was conducted at 50 °C, and the solid to liquid ratio was 1:20. Under these conditions, the yields of taxifolin and total flavonoids were 1.27-fold and 1.20-fold, respectively, higher than the controls. The antioxidant activities of EI–WE samples were higher than those of the controls as determined by the DPPH and BHT assays. Pure taxifolin exhibited the best radical scavenging ability, which was 3.84-fold higher than that of ascorbic acid and 7.15-fold higher than that of BHT. This indicates that taxifolin may be the main constituent responsible for the high antioxidant activity of the larch extracts. SEM results showed that EI– WE disintegrated the rigid wood material efficiently, increasing the release of secondary metabolites. The enzymatic water extraction procedure could be used for the economical large scale extraction of natural products without environmental pollution. Acknowledgements The authors gratefully acknowledge the financial supports by the Key Program for Science and Technology Development of Harbin (2009AA3BS083), National Natural Science Foundation of China (30770231), Heilongjiang Province Science Foundation for Excellent Youths (JC200704), Agricultural Science and Technology Achievements Transformation Fund Program (2009GB23600514), Key Project of Chinese Ministry of Education (108049), Project for Distinguished Teacher Abroad, Chinese Ministry of Education (MS2010DBLY031), Technology Innovation Project of Northeast Forestry University (GRAM 09), Fundamental Research Funds for the Central Universities (DL09EA04) and the Special Fund of Forestry Industrial Research for Public Welfare of China (201004040). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2010.11.155.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Enzymatic water extraction of taxifolin from wood sawdust of Larix gmelini (Rupr.) Rupr. and evaluation of its antioxidant activity Ying Wang a,b,1, Yuangang Zu a,b,1, Jingjing Long a,b, Yujie Fu a,b,⇑, Shuangming Li a,b, Dongyang Zhang a,b, Ji Li a,b, Michael Wink c, Thomas Efferth d a
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, China Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany d Department of Pharmaceutical Biology, Institute of Pharmacy, University of Mainz, 55128 Mainz, Germany b c
a r t i c l e
i n f o
Article history: Received 6 May 2010 Received in revised form 12 October 2010 Accepted 26 November 2010 Available online 3 December 2010 Keywords: Enzymatic water extraction Taxfolin Antioxidant Larix gmelini (Rupr.) Rupr. wood sawdust Central composite design Scanning electron microscopy
a b s t r a c t An enzyme incubation–water extraction (EI–WE) method was developed and optimised for the extraction of the natural antioxidant taxifolin and of the total flavonoids from wood sawdust of Larix gmelini (Rupr.) Rupr. A factorial design and a central composite design approach were used for method optimisation. Optimal conditions were 0.5 mg/ml cellulase and 0.5 mg/ml pectinase, a pH of 5.0, a temperature of 32 °C and 18 h incubation time. The flavonoids and taxifolin were extracted in hot water at 50 °C for 30 min, with a solid to liquid ratio of 1:20. Under optimised conditions, the yields of taxifolin and total flavonoids increased from 1.06 ± 0.08 to 1.35 ± 0.04 mg/g and 4.13 ± 0.17 to 4.96 ± 0.29 mg/g, respectively. DPPH and BHT assays revealed that the EI–WE samples had 1.8- and 1.68-fold higher antioxidant activities than the controls. SEM results revealed the structural disruption of wood sawdust with enzyme incubation. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Taxifolin (dihydroquercetin, 2-(3,4-dihydroxyphenyl)-2,3-dihydro-3,5,7-trihydroxy-4H-benzopyran-4-one) is a dihydroflavonol. It can eliminate free radicals in the body, improve the impermeability of capillary vessels and recover their elasticity effectively. Taxifolin has a distinguished better antioxidant activity (Audron, Regina, Jonas, & Narimantas, 2000; Bong-Sik et al., 2000) compared to other antioxidants, and can remarkably prolong the shelflife of lard, plant oils, powdered milk and candy. It is not embryotoxic and does not lead to malformations, hypersusceptibility or mutations. An antiviral effect of taxifolin has also been reported (Evangelos, Jean-Marc, Jacques, & Robert, 1987; Shu-Chen, Yih-Shou, & Jung-Yaw, 1992). Furthermore, taxifolin can also restrain the synthesis of fat in human liver (Andre et al., 2000) and activate phosphodiesterase (Kuppusamy & Das, 1992). Taxifolin derivatives may be useful as sweeteners without calories. In general, taxifolin can be used as a natural antioxidant additive in food industry. Abbreviations: EI–WE, enzyme incubation–water extraction; FD, factorial design; CCD, central composite design; SEM, scanning electron microscopy; RSM, response surface methodology. ⇑ Corresponding author. Tel.: +86 451 82190535; fax: +86 451 82102082. E-mail addresses: [email protected], [email protected] (Y. Fu). 1 First two authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.11.155
Larches (genus Larix) are widespread conifers of the cool northern hemisphere and comprise 10 species. They are one of the three dominant conifers in northeast China. They usually grow in cool temperature zones and are the most cold-resistant species of conifers. In China, the genus Larix mainly includes Larix gmelini (Rupr.) Rupr., L. principisrupprechtii, L. kaempferi, L. sibirica and L. olgensi. Among these, L. gmelini (Rupr.) Rupr. is best known for its wide distribution and extensive use. It occupies nearly 55% of Great Khingan and Lesser Khingan (Sun, Zhang, Han, & Wang, 2007). With physical properties, such as rigidness, corrosion resistance and straight grain, L. gmelini (Rupr.) Rupr. has been widely used for building and furniture (Xin & Li, 2009). As a result, large quantities of wood sawdust are produced every year as a side product. Therefore, the exploitation of L. gmelini (Rupr.) Rupr. as a conifer resource has become a main task. It has been reported that L. gmelini (Rupr.) Rupr. contains valuable bioactive secondary metabolites, such as arabinogalactan (AG) and flavonoids, including taxifolin, oligomeric proanthocyanidins (OPC) and others (Huang & Fang, 2005). Extraction of these bioactive components from wood sawdust could be an economically interesting utilisation of this conifer resource. Enzyme-assisted extraction can be used as an alternative to the release of secondary metabolites from biological materials. It possesses the advantages of environmental compatibility, high
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efficiency, and easy operation processing (Zu et al., 2009). Hydrolytic enzymes, including cellulase, pectinase and beta-glucosidase, are commonly used for the extraction of secondary metabolites (Mark, Wilbur, Karel, & Randall, 2007; Young et al., 2005). They interact with cell walls, break down their structural integrity and in consequence enhance the release of flavonoid aglycones. However, such an enzyme-assisted extraction of taxifolin from the L. gmelini (Rupr.) Rupr. wood sawdust has not yet been reported. Most studies on the extraction of taxifolin and flavonoid have used ethanol as a solvent at ambient temperatures. However, consuming large volumes of organic solvents for extraction represents a considerable shortcoming (Chattip, Wanchai, & Artiwan, 2008; Huang, Xue, Niu, Jia, & Wang, 2009). Therefore, the development of an environmentally friendly method to extract taxifolin and total flavonoids from wood sawdust of L. gmelini (Rupr.) Rupr. is a major challenge. The aim of this investigation was to obtain an economically and environmentally friendly method for the extraction of taxifolin and total flavonoids from L. gmelini (Rupr.) Rupr. The influence of hydrolytic enzymes on the yields of taxifolin was optimised by factorial design (FD) and central composite design (CCD). The antioxidant activity of the extracts and taxifolin was evaluated by DPPH and BHT assays. Furthermore, the ultra-structural changes of plant materials were analysed by scanning electron microscopy (SEM). The present study offers an alternative method for the highly effective utilisation of a side product of conifer utilisation. 2. Materials and methods 2.1. Plant materials Wood sawdust of L. gmelini (Rupr.) Rupr. was obtained from the Xinming furniture factory in Harbin. 2.2. Chemicals and reagents Taxifolin was obtained from Shanghai Ronghe Bio-Technology Co., Ltd. (Shanghai, China). Cellulase (EC 1.1.1.27, P1000 U/mg), beta-glucosidase (EC 3.2.1.21, P47 U/mg) from Shanghai WeeBeyond Scientific and Trade Co., Ltd., and pectinase (EC 3.2.1.15, 1.41 U/mg) from Fluka Chemical Co. Methanol of chromatographic grade were obtained from J&K Chemical Ltd. (China). Double-distilled water was used in all experiments. All solvents for HPLC were filtered through a membrane filter (0.45 lm pore size) and degassed under ultrasonic condition before use. The total flavonoids yield of the samples was determined by a modified colorimetric method previously described by Ordonez, Gomez, Vattuone, and Isla (2006) using rutin (Sigma, St. Louis, MO, USA) as a standard. Aliquots (1 ml) of appropriately diluted extracts or standard solutions were mixed with 0.3 ml 5% NaNO2 solution. After 6 min, 0.3 ml 10%AlCl3 solution were added, and the mixture was allowed to stand for another 6 min. Then, 4 ml 4% NaOH were added. The reaction solution was well mixed, kept for 15 min, and the absorbance was determined at 510 nm. Total flavonoids yield was calculated using a standard curve. 2.3. Apparatus and chromatographic conditions HPLC analysis was performed with a Jasco LC system (Jasco Company, Japan) equipped with a Jasco PU-1580 intelligent HPLC pump and coupled to Jasco UV-1575 UV/VIS Detector as well as Millennium 32 system software. Chromatographic separation was performed on a HIQ sil C18V reversed-phase column (4.6 mm £ 250 mm, KYA TECH Corporation, Japan) packed with 5 lm diameter particles. The mobile phase was methanol–water–
acetic acid (44:56:0.4, v/v/v). The eluents were monitored at 290 nm UV wavelength. The injection volume and the flow rate of the mobile phase were 10 ll and 1 ml/min, respectively. The column was maintained at 35 °C. 2.4. Enzyme incubation–water extraction process Cellulase, pectinase and beta-glucosidase were dispersed in 100 ml deionised water to obtain solutions of defined concentrations (0.25–2 mg/ml). Powder of 2 g sawdust was added to the enzymatic solution, which was then adjusted to defined pH-values (2.5–7.0) with 0.1 M HCl solution and shaken on a flat-bed orbital shaker 0–72 h at defined temperatures (20–50 °C). After the enzymatic treatment the samples were filtered. The residues were collected into conical flasks, and distilled water was added. Then, the conical flasks were put into an ultrasonic bath (Kunshan Ultrasonic Instrument Co., Ltd., China). The extraction was performed for 30 min at 50 °C and 100% power. The residues were then filtered, and the entire process was repeated twice. The filtrates collected were concentrated in vacuo (50 °C) in a rotary evaporator. The syrup was reconstituted with HPLC mobile phase. The sample solutions were then centrifuged at 12,000 rpm before being analysed by HPLC. All experiments were performed in triplicate. 2.5. Central composite design (CCD) Response surface methodology (RSM) (Deniz & Ismail, 2007) was applied to identify the optimum levels of the three variables: enzyme concentration (mg/ml), pH, and incubation temperature (°C) and two responses, the yields of taxifolin and total flavonoids. The ranges of concentration (A), pH (B), temperature (C) and the central points were selected based on results of a factorial design. The experiments were designed according to the central composite design (CCD) using a 23 full factorial design. The general equation of the second degree polynomial equation is
Y ¼ b0 þ
k X i¼1
Bi X i þ
k X
Bii X 2 þ
i¼1
k X
Bij X i X j
ð1Þ
i>j
where Y represents the measured response variables, b0 is defined as the constant, Bi, Bii and Bij are the linear, quadratic and interactive coefficients of the model, respectively. Xi and X j are the levels of the independent variables. The test variables were transformed to range between 1 and 1 for the evaluation of the factors. In all cases, the calculations were carried out with the help of the statistical package Statgraphics Plus for Windows V6.0. 2.6. 1,1-Diphenyl-2-picrylhydrazyl assay (DPPH assay) The antioxidant activity of the crude extracts with or without enzyme treatment and pure taxifolin was studied using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay as reported by Amarowicz, Pegg, Moghaddam, Barl, and Weil (2004). An aliquot of the sample (100 ll) at various concentrations or water as negative control was mixed with ethanol (1.4 ml) and then added to 4‰ DPPH (1 ml, Sigma–Aldrich) in ethanol. The mixture was vigorously shaken followed by incubation for 70 min. The absorbance was read at 517 nm (UNICO, Shanghai, China). Ascorbic acid (Sigma–Aldrich), a stable antioxidant, was used as positive control. The radical-scavenging activities of the samples, expressed as percent inhibition of DPPH, were calculated according to the formula:
Scavenging Effectð%Þ ¼
Absorbance sample 100% 1 Absorbance control
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where absorbancesample and absorbancecontrol are the absorbance values of the tested sample and of the blank samples determined after 70 min, respectively. The DPPH scavenging activity is presented by its IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution. 2.7. Butylated hydroxytoluene assay (BHT assay) The antioxidant activity of the samples was determined using the b-carotene–linoleic acid test (Burtun, 1989; Sies & Stahl, 1995). Approximately 10 mg of b-carotene (type I synthetic, Sigma–Aldrich) was dissolved in chloroform (10 ml). The carotene–chloroform solution (0.2 ml) was pipetted into a boiling flask containing linoleic acid (20 mg, Sigma–Aldrich) and 200 mg TweenÒ 40 (Sigma–Aldrich). Chloroform was removed using a rotary evaporator at 40 °C for 5 min, and distilled water (50 ml)
was added to the residue slowly with vigorous agitation, to form an emulsion. A portion of the emulsion (5 ml) was added to a tube containing the sample solution (0.2 ml) and the absorbance was immediately measured at 470 nm against a blank, consisting of an emulsion without b-carotene. The tubes were placed in a water bath at 50 °C and the oxidation of the emulsion was monitored over a 60 min period. Control samples contained 200 ll of water instead. Butylated hydroxytoluene (BHT, Sigma–Aldrich), a stable antioxidant, was used as synthetic reference. The antioxidant activity was expressed as inhibition percentage with reference to the control after a 60 min incubation using the following equation:
AA ¼ 100ðDRC DRS Þ=DRC where AA = antioxidant activity; DRC = degradation rate of the control = [ln(a/b)/60]; DRS = degradation rate in presence of the
Fig. 1. Effect of (a) enzyme type; (b) enzyme concentration; (c) incubation time; (d) pH; and (e) incubation temperature on the yields of taxifolin and total flavonoids (n = 3), p < 0.05. aDW: dry weight.
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sample = [ln(a/b)/60]; a = absorbance at time 0; b = absorbance at 60 min. 2.8. Scanning electron microscopy Samples with or without EI–WE treatment were scanned by SEM (Hitachi, San Jose, CA, USA). The materials were collected and air-dried after extraction of taxifolin and total flavonoid. They were fixed on aluminium stubs with adhesive tape and covered by gold using a sputter coater (JEOL JEC-1200). All samples were examined under high vacuum conditions at a voltage of 15.0 kV (50 lm, 500 magnification). 2.9. Statistical analysis All results were subjected to statistical analyses. Mean values of all data were obtained from triplicate experiments. Differences were considered to be statistically significant at p < 0.05. 3. Results and discussion 3.1. Optimisation of taxifolin and total flavonoids extraction using FD 3.1.1. Selection of enzyme type Cellulase catalyses the breakdown of cellulose into glucose, cellubiose and glucose polymers. Pectinase has the ability to disintegrate pectic compounds and pectin. Beta-glucosidase breaks the
beta-1,4 glucosidic linkages in glucosides. In the present investigation, the effect of enzyme combinations on taxifolin and total flavonoids extraction yields was evaluated, under the conditions of 1 mg/ml total enzyme concentration, pH 7.0, temperature 40 °C, and incubation time 24 h. Fig. 1a shows the yields of taxifolin and total flavonoids after incubation with enzymes. Generally, a similar variation tendency for the yields of taxifolin and total flavonoids were found in the EI–WE samples. The taxifolin yield was increased with the enzymatic treatment (p < 0.05) as expected. The groups treated with cellulase alone or with pectinase and cellulase (p & c) revealed higher yields as compared to other groups. Under these conditions, the yields of taxifolin were 1.26 ± 0.02 and 1.35 ± 0.05 mg/g, which were 1.47-fold and 1.57-fold higher compared to control, respectively. It is well documented in the literature that pectinase and cellulase have synergistic effects (Elias, Foda, & Attia, 1984; Grohman & Baldwin, 1992; Marshall, Graumlich, Braddock, & Messersmith, 1985). They hydrolyse the cellulose and pectin of cell walls, which leads to an enhanced release of taxifolin. The variation of the total flavonoids yield expressed as mg rutin per g of extract was similar to that of taxifolin (p < 0.05). The total flavonoids yields in cellulase alone and p & c groups were also better compared to other treatments. The yields were 4.204 ± 0.24 and 4.202 ± 0.41 mg/g (1.26-fold and 1.25-fold compared to control), respectively. Acceptable yields were achieved in the p & c group for taxifolin; also, the yields of total flavonoids treated with cellulase alone did not show significant improvements over the p & c group. Thus,
Table 1 Central composite experimental design and response values obtained under different extraction conditions. No.
X1
X2
X3
X1 Concentration (mg/ml)
X2 pH
X3 Temperature (°C)
Taxifolin yield (mg/g)
Total flavonoids yield (mg/g)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 1 1 1 1 1 1 1 0 0 0 0 1.68 1.68 0 0 0
1 1 1 1 1 1 1 1 0 0 1.68 1.68 0 0 0 0 0
1 1 1 1 1 1 1 1 1.68 1.68 0 0 0 0 0 0 0
0.25 0.75 0.25 0.75 0.25 0.75 0.25 0.75 0.5 0.5 0.5 0.5 0.08 0.92 0.5 0.5 0.5
4.2 4.2 5.8 5.8 4.2 4.2 5.8 5.8 5.0 5.0 3.7 6.4 5.0 5.0 5.0 5.0 5.0
25 25 25 25 35 35 35 35 21.6 38.4 30 30 30 30 30 30 30
1.281 1.203 1.216 1.261 1.260 1.341 1.277 1.233 1.236 1.380 1.247 1.242 1.274 1.227 1.431 1.411 1.406
4.361 4.687 4.738 4.159 4.381 4.482 4.870 4.147 4.841 5.123 4.515 4.897 4.610 4.246 4.876 4.987 5.012
Table 2 Significance of regression coefficient for taxifolin and total flavonoids yield. Variables
Model X1 (enzyme concentration) X2 (pH) X3 (temperature) X1X1 X2X2 X3X3 X1X2 X1X3 X2X3 Lack of fit R2 Adjusted R2
Eq. (2)
Eq. (3)
Mean square
F-value
p-value
Mean square
t-value
p-value
7.85 103 8.98 105 2.52 104 8.59 103 0.28 0.43 0.17 3.23 104 1.76 103 2.22 103 1.67 103
6.31 0.072 0.20 6.91 22.51 34.23 13.78 0.26 1.42 1.79 9.46 0.8903 0.7491
0.120 0.7959 0.6662 0.0340 0.0021 0.0006 0.0075 0.6262 0.2727 0.2232 0.0983
0.15 0.16 3.10 102 0.12 0.62 0.21 1.70 102 0.37 1.70 102 1.20 102 0.043
4.53 5.04 0.95 0.38 19.31 6.52 0.53 11.64 0.53 0.36 8.18 0.8535 0.6652
0.0295 0.0596 0.3622 0.5563 0.0032 0.0380 0.4921 0.0113 0.4903 0.5663 0.1125
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the p & c group was further considered to be optimum for the simultaneous extraction of taxifolin and total flavonoids. 3.1.2. Effect of enzyme concentration The effect of different enzyme concentrations on the yields of taxifolin and total flavonoids was evaluated (Fig. 1b) under the conditions of pectinase and cellulase combination, pH 7.0, temperature 40 °C, and incubation time 24 h. Acceptable yields were achieved at 0.5 mg/ml for both taxifolin and total flavonoids. Higher concentrations than 0.5 mg/ml did not increase yields. We conclude that a combination of 0.5 mg/ml cellulase and 0.5 mg/ml pectinase is suitable for the extraction of taxifolin and total flavonoids from L. gmelini (Rupr.) Rupr. Thus, 0.5 mg/ml was selected as an optimal enzyme concentration for the subsequent steps. 3.1.3. Effect of incubation time Under the conditions of 0.5 mg/ml pectinase and 0.5 mg/ml cellulase combination, pH 7.0 and temperature 40 °C, the effect of incubation time on taxifolin and total flavonoids extraction yields
is shown in Fig. 1c. It was apparent that extending the extraction period increased the taxifolin and total flavonoids yields (p < 0.05). The yields of taxifolin reached a peak (1.29 ± 0.04 mg/ g) at 12 h, and 18 h for total flavonoids (5.24 ± 0.15 mg/g). As a result, 18 h was considered optimum for enzyme incubation. 3.1.4. Effect of pH value of enzyme solution The pH of the enzyme solution considerably influences the activity of the enzyme. Fig. 1d shows the taxifolin and total flavonoids yields in enzyme solutions of different pH-values under the conditions of 0.5 mg/ml pectinase and 0.5 mg/ml cellulase combination, temperature 40 °C and incubation time 24 h. The taxifolin yields at pH 5.0 and 6.5 were higher than for other pH values. The enzymes work optimally at these two pH-values and disintegrated cell walls more completely. Unlike taxifolin, the highest yield of total flavonoids was achieved only at pH 5.0. The reason may be that flavonoids represent a chemically diverse group of compounds, and the retention behaviour of each compound might be different in plant cells.
Fig. 2. Response surface plot for maximal extraction yields of taxifolin as a function of (a) enzyme concentration and pH; (b) enzyme concentration and temperature; and (c) pH and temperature.
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3.1.5. Effect of temperature on enzyme activity Fig. 1e presents the influence of increased temperatures on taxifolin and total flavonoids yields under the conditions of 0.5 mg/ml pectinase and 0.5 mg/ml cellulase combination, incubation time of 24 h and pH 7.0. Rising temperatures caused a slight increase of taxifolin and total flavonoids yields. The best yields were obtained at 30 °C. Higher temperatures rather decreased than increased the amount of taxifolin and total flavonoids. It is likely that enzymes are heat sensitive and are denatured at higher temperatures. A higher yield of total flavonoids at 50 °C is probably due to the better solubility of flavonoids in hot water. The date of this section was shown in Table S1, and Duncan’s Multiple Range Test was used to describe the significant difference between pairs of treatments. 3.2. Optimisation of taxifolin and total flavonoids extraction using CCD 3.2.1. Fitting the models To optimise maximal yields of taxifolin and total flavonoids using the EI–WE method, the response surface methodology was applied. Enzyme concentration (X1), pH (X2) and incubation temperature (X3) were used for the optimised experiment conducted by a 23 full factorial central composite design. All the experimental data obtained from a 17-run-experiment and the predicted data from the response surface analysis model established are shown in Table 1. The yields of taxifolin (Y1) and total flavonoids (Y2) are functions of these variables. By applying multiple regression analysis to the experimental data, the following second order polynomial equations were found to represent the extraction yields adequately.
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The Model F-value of 4.53 implies the model is significant. There is only a 2.95% chance that a ‘‘Model F-value’’ this large could occur due to noise. The ‘‘Lack of Fit F-value’’ of 8.18 implies the Lack of Fit is not significant relative to the pure error. There is 11.25% chance that a ‘‘Lack of Fit F-value’’ this large could occur due to noise. A maximal value of total flavonoids (5.06 mg/g) was observed at 0.31 mg/ml complex enzyme, pH 5.72 and 35.0 °C. 3.2.2. Optimisation of taxifolin extraction with the EI–WE method 3D surface plots were employed to illustrate the interactions between enzyme concentrations, pH and temperature on the extraction yields of taxifolin and total flavonoids. The response surfaces based on these variables are shown with one variable kept at optimum level and varying the other two within the experimental range. In general, the response surfaces indicated a complex interaction between the variables. All these three variables had a quadratic effect on the extraction yield of taxifolin (Fig. 2a–c). As the concentrations of enzymes, and in addition the pH and temperature increased, the response increased to a maximal value and then decreased. These three variables acted synergistically. Slightly higher temperatures, intermediate pH values and enzymes concentration of 0.5 mg/ml gave maximal taxifolin yields, as explained above in the FD experiments. The interactive effects of enzymes concentration, pH and temperature on the yield of total flavonoids were similar to taxifolin (data not shown). 3.3. Validation of the method In order to validate the adequacy of the model equations [Eqs.(2) and (3)], the extraction was carried out under optimal con-
Y 1 ¼ 3:233 þ 0:590X 1 þ 1:096X 2 þ 0:114X 3 0:798X 21 0:096X 22 0:0016X 23 0:032X 1 X 2 þ 0:012X 1 X 3 0:0042X 2 X 3 ð2Þ Y 2 ¼ 4:763 þ 9:827X 1 þ 2:442X 2 þ 0:070X 3 3:753X 21 0:213X 22 0:0015X 23 1:081X 1 X 2 þ 0:037X 1 X 3 0:0095X 2 X 3 ð3Þ The significance of each coefficient was determined using the F test and p-value. The corresponding variables were more significant, if the absolute F-value became larger and the p-value smaller. For taxifolin, the coefficients calculated from the regression model [Eqs. (2) and (3)] are listed in Table 2. They contain one constant, three linear, three quadratic, and three interaction terms. For [Eq. (2)], the results showed that the linear and quadratic term of X3 were very important, followed by X1, X2, and X3 (p < 0.05). None of the other terms (X1, X2, X1X2, X1X3, X2X3) are significant (p > 0.05), so the model should be reduced. The reduced equation was
Y 1 ¼ 3:233 þ 0:114X 3 0:798X 21 0:096X 22 0:0016X 23
ð4Þ
The Model F-value of 6.31 implies the model is significant. There is only a 1.20% chance that a ‘‘Model F-value’’ this large could occur due to noise. The ‘‘Lack of Fit F-value’’ of 9.46 implies there is a 9.83% chance that a ‘‘Lack of Fit F-value’’ this large could occur due to noise. A maximum value of taxifolin (1.42 mg/g) was observed at 0.51 mg/ml enzyme concentration, pH 4.93 and 31.73 °C. For Eq. (3), the results showed that only the interaction terms of X1X2, X12 and X22 are significant (p < 0.05). So the model was reduced as that
Y 2 ¼ 4:763 3:753X 21 0:213X 22 0:0015X 23 1:081X 1 X 2
ð5Þ
Fig. 3. (a) DPPH scavenging capacity of extracts and taxifolin and (b) inhibition of linoleic acid oxidation. Values are means ± SD (n = 3), p < 0.05.
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Fig. 4. Scanning electron microscopy images of L. gmelini (Rupr.) Rupr. wood sawdust: (a) untreated and (b) EI–WE.
ditions. Enzyme incubation conditions were: incubation with 0.5 mg/ml cellulase and 0.5 mg/ml pectinase at pH 5.0, and 32 °C for 18 h. Hot water (20:1) was used to extract taxifolin and total flavonoids (30 min). Under optimal conditions, taxifolin and total flavonoids yields were 1.35 ± 0.04 and 4.96 ± 0.29 mg/g (n = 3), respectively. The p-values of taxifolin and total flavonoids yields were 0.037 and 0.042 (p < 0.05), respectively, which were obtained by a one-sided t-test. 3.4. Antioxidant activities 3.4.1. 1,1-Diphenyl-2-picrylhydrazyl assay (DPPH assay) In the present investigation, the DPPH assay was used to evaluate the radical-scavenging activity of L. gmelini (Rupr.) Rupr. extracts with and without enzyme treatment, as well as of pure taxifolin. Their scavenging potential was compared to ascorbic acid (Fig. 3a). The EI–WE samples contained higher amounts of taxifolin and total flavonoids than the controls. With increasing extraction yields, the DPPH radical scavenging effect increased. Taxifolin exhibited the highest radical-scavenging activity. One gram taxifolin, equal to 21.83 mmol ascorbic acid, exerted a fourfold higher radical-scavenging activity (1 g ascorbic acid is 5.68 mmol). These results suggest that taxifolin is one of the major components contributing to the total antioxidant capacity of L. gmelini (Rupr.) Rupr. 3.4.2. Butylated hydroxytoluene assay (BHT assay) The antioxidant activity of the samples with or without EI–WE treatment and pure taxifolin, as well as the positive control BHT is presented in Fig. 3b. These samples prevented b-carotene bleaching by neutralising the free radicals. The weakest free radical-scavenging activity was exhibited by the samples without EI–WE treatment. The EI–WE samples showed better radical-scavenging activity (equal to 99 lmol BHT/g dry weight). It is apparent that b-carotene was protected from oxidation by the larch antioxidants in a dose dependent fashion. Taxifolin exhibited the best antioxidant activity in this assay, i.e. 1 g taxifolin was equal to 32.44 mmol of BHT (6.15-fold higher antioxidant activity). The order of antioxidant activity was taxifolin > BHT > EI–WE > control. 3.5. Scanning electron microscopy Fig. 4 shows the scanning electron micrographs of wood sawdust without (a) and after enzyme treatment (b). The morphological structures represent imperforate tracheary cells of the xylem, a rigid structure for the transport of water and mineral salts. As expected, the wood structures were partly destroyed by EI–WE treatment; the cell walls became thinner and disorganised (Fig. 4b). The
hydrolysis of cell walls apparently leads to the release of taxifolin and other secondary metabolites. 4. Conclusions An enzyme water extraction (EI–WE) method was used for the extraction of taxifolin and total flavonoids from L. gmelini (Rupr.) Rupr. for the first time. This method was evaluated using a factorial design (FD) and a central composite design (CCD). The enzyme type, concentration, incubation time, pH and temperature were studied and optimised. The results showed that all these factors were important for the extraction process. Optimal conditions were 0.5 mg/ml cellulase and 0.5 mg/ml pectinase, pH 5.0, and 32 °C. The water extraction was conducted at 50 °C, and the solid to liquid ratio was 1:20. Under these conditions, the yields of taxifolin and total flavonoids were 1.27-fold and 1.20-fold, respectively, higher than the controls. The antioxidant activities of EI–WE samples were higher than those of the controls as determined by the DPPH and BHT assays. Pure taxifolin exhibited the best radical scavenging ability, which was 3.84-fold higher than that of ascorbic acid and 7.15-fold higher than that of BHT. This indicates that taxifolin may be the main constituent responsible for the high antioxidant activity of the larch extracts. SEM results showed that EI– WE disintegrated the rigid wood material efficiently, increasing the release of secondary metabolites. The enzymatic water extraction procedure could be used for the economical large scale extraction of natural products without environmental pollution. Acknowledgements The authors gratefully acknowledge the financial supports by the Key Program for Science and Technology Development of Harbin (2009AA3BS083), National Natural Science Foundation of China (30770231), Heilongjiang Province Science Foundation for Excellent Youths (JC200704), Agricultural Science and Technology Achievements Transformation Fund Program (2009GB23600514), Key Project of Chinese Ministry of Education (108049), Project for Distinguished Teacher Abroad, Chinese Ministry of Education (MS2010DBLY031), Technology Innovation Project of Northeast Forestry University (GRAM 09), Fundamental Research Funds for the Central Universities (DL09EA04) and the Special Fund of Forestry Industrial Research for Public Welfare of China (201004040). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2010.11.155.
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