Elsevier

Food Chemistry

Volume 126, Issue 3, 1 June 2011, Pages 1178-1185
Food Chemistry

Enzymatic water extraction of taxifolin from wood sawdust of Larix gmelini (Rupr.) Rupr. and evaluation of its antioxidant activity

https://doi.org/10.1016/j.foodchem.2010.11.155Get rights and content

Abstract

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.

Research highlights

Organic solvent free. ► Combined enzyme system was used for the extraction of taxifolin and total flavonoids in Larix gmelini (Rupr.) Rupr. sawdust. ► Taxifolin extracted by EI–WE method can be used as food additive for its excellent antioxidant activity. ► CCD (central composite design) was used for the optimisation of taxifolin and total flavonoids extraction condition. ► Realise material recycle, for L. gmelini (Rupr.) Rupr. sawdust is generally deserted as castoff.

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 et al., 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 et al., 1987, Shu-Chen et al., 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.

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 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 et al., 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 et al., 2008, Huang et al., 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.

Section snippets

Plant materials

Wood sawdust of L. gmelini (Rupr.) Rupr. was obtained from the Xinming furniture factory in Harbin.

Chemicals and reagents

Taxifolin was obtained from Shanghai Ronghe Bio-Technology Co., Ltd. (Shanghai, China). Cellulase (EC 1.1.1.27, ⩾1000 U/mg), beta-glucosidase (EC 3.2.1.21, ⩾47 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

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

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

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

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