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1 s2.0 S0141022910002103 Main
1 s2.0 S0141022910002103 Main
a r t i c l e i n f o a b s t r a c t
Article history: We report a novel enzyme-involved approach to improve the extraction of flavonoids from Ginkgo biloba,
Received 31 August 2010 in which the enzyme is employed not only for cell wall degradation, but also for increasing the solubility
Accepted 28 September 2010 of target compounds in the ethanol–water extractant. Penicillium decumbens cellulase, a commercial cell
wall-degrading enzyme with high transglycosylation activity, was found to offer far better performance
Keywords: in the extraction than Trichoderma reesei cellulase and Aspergillus niger pectinase under the presence of
Enzyme-assisted extraction
maltose as the glycosyl donor. TLC, HPLC and MS analysis indicated that P. decumbens cellulase could
Penicillium decumbens cellulase
transglycosylate flavonol aglycones into more polar glucosides, the higher solubility of which led to
Transglycosylation
Flavonoid
improved extraction. The influence of glycosyl donor, pH, solvent and temperature on the enzymatic
Ginkgo biloba transglycosylation was investigated. For three predominant flavonoids in G. biloba, the transglycosyla-
Solubility tion showed similar optimal conditions, which were therefore used for the enzyme-assisted extraction.
The extraction yield turned to be 28.3 mg/g of dw, 31% higher than that under the pre-optimized con-
ditions, and 102% higher than that under the conditions without enzymes. The utilization of enzymatic
bifunctionality described here, naming enzymatic modification of target compounds and facilitation of
cell wall degradation, provides a novel approach for the extraction of natural compounds from plants.
© 2010 Elsevier Inc. All rights reserved.
1. Introduction The extraction of plant flavonoids, which generally have poor sol-
ubility in mild solvents such as ethanol–water solution, is selected
Enzyme-assisted extraction of natural functional compounds as an example to validate our proposal.
from plants is widely investigated in recent years for its advantages Flavonoids are natural compounds showing high physiological
in easy operation, high efficiency, and environment friendship [1]. activities in therapies for inflammations, heart diseases and cancers
Most of the works in this field utilize cellulase, pectinase and - [5,6]. There are three predominant flavonol aglycones, quercetin,
glucosidase to hydrolyze and degrade plant cell wall constituents kaempferol, and isorhamnetin (Fig. 1), in Ginkgo biloba, a Chinese
to improve the release of intracellular contents [2–4]. However, medicinal plant well known for its high content of flavonoids [7].
another important factor in the extraction – the intrinsic prop- Extract from G. biloba is among the most popular phytomedicines
erty especially the solubility of the target compound – has seldom and herbal dietary supplements [8], whose primary active compo-
been concerned according to our knowledge. Low solubility of tar- nents are flavonoids (24%) and small amount of terpene lactones
get compounds in the extractant leads to low extraction yield and (6%) [9].
require large amount of solvents, which largely impedes the eco- Various methods have been used to assist the extraction of
nomic efficiency in industry. Therefore, here we propose a novel flavonoids from plants, such as ultrasonication [10], supercriti-
enzyme-assisted extraction approach, in which the extraction is cal fluids [11], microwave [12], membrane adsorption [13] and
improved not only from enhanced cell wall degradation, but also molecular imprinting [14]. There are also several reports on the
from the increased solubility of target compounds in the extractant. enzyme-assisted extraction of plant favonoids. Pectinase and pro-
tease were employed for the extraction of anthocyanins from black
currant juice press residues [15]. Luteolin and apigenin were enzy-
matically extracted from pigeonpea leaves with pectinase, cellulase
∗ Corresponding author. Tel.: +86 631 5688303; fax: +86 631 5688303.
and -glucosidase [16]. In another study, extraction of antimicro-
E-mail address: [email protected] (M.-S. Xu).
1
Current address: Department of Chemistry and Biotechnology, School of Engi- bial and antioxidant phenolics, mainly anthocyanins, from berries
neering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. was studied, and the enzyme was found to effectively hydrolyze
0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2010.09.017
S. Chen et al. / Enzyme and Microbial Technology 48 (2011) 100–105 101
Table 1
TLC analysis for enzymatic catalysis and extraction of flavonol aglycones.
3.3.2. pH
Effects of pH in the range of 2–9 on the transglycosylation was
Fig. 3. Chromatograms of flavonoid samples before and after treatment with P.
examined by preparing the reaction solutions with different initial decumbens cellulase: (A) quercetin; (B) kaempferol; (C) isorhamnetin.
pHs. As shown in Fig. 4B, P. decumbens cellulase showed transg-
lycosylation activities within a wide pH range. Around pH 6 the
catalytic rate was the highest for all three substrates.
the substrates. When ethanol concentration was higher than 60%,
3.3.3. Solvent ethanol–water ratio the transglycosylation rate decreased rapidly with the increase of
Substrate solubility generally has large effects on the catalytic ethanol, which was presumably due to the enzyme inactivation
reaction. Flavonol aglycones have very low solubility in water, and caused by excess ethanol in the solution.
the solubility rises as the ethanol concentration increases in water
solution. So the effects of solvent ethanol concentrations from 0 to
90% (v/v) on the enzymatic transglycosylation were examined. As 3.3.4. Temperature
shown in Fig. 4C, the transglycosylation rate reached the highest As shown in Fig. 4D, the transglycosylation rate reached the peak
at the ethanol concentration of 50% for kaempferol and isorham- at 60 ◦ C for all of quercetin, kaempferol and isorhamnetin. When
netin, and 60% for quercetin. When ethanol concentration was the temperature was higher than 60 ◦ C, the catalysis was notably
between 20 and 50%, the catalysis was facilitated with the increase inhibited, probably due to the inactivation of P. decumbens cellulase
of ethanol, probably resulted from the increase of the solubility of at a high temperature.
104 S. Chen et al. / Enzyme and Microbial Technology 48 (2011) 100–105
Fig. 4. Effect of the glycosyl donor (A), pH (B), ethanol concentration (C), and temperature (D) on the enzymatic transglycosylation of quercetin, kaempferol, and isorhamnetin.
3.3.5. Transglycosylation kinetics by P. decumbens cellulase thermore, the kinetics of the enzymatic transglycosylation was
According to the above results, quercetin, kaempferol and studied. The initial rate of transglycosylation showed a good rela-
isorhamnetin have very similar optimal conditions for their tionship with the initial substrate concentration, which fits to the
transglycosylation by P. decumbens cellulase. Time dependence Michaelis–Menten equation. Table 2 gives the kinetic parameters
experiment showed that the transglycosylation reached the for the three flavonoid substrates under the optimal catalytic con-
plateau after 30 h of enzymatic treatment under the optimal con- ditions.
ditions of pH 6, 50% ethanol, 60 ◦ C and 0.5 g/L maltose (Fig. 5).
The transglycosylation rates of quercetin, kaempferol and isorham- 3.4. Extraction under optimal transglycosylation conditions
netin reached 45%, 50% and 41%, respectively (Table 2), compared
with 25%, 27% and 27% under the pre-optimized conditions. Fur- We further examined whether the extraction of flavonoids is
improved when the transglycosylation reaches its highest under
the optimal catalytic conditions of pH 6, 50% ethanol and 60 ◦ C.
As shown in Table 3, total flavonoid content in the extracts
was improved under the optimal transglycosylation conditions
Table 2
Transglycosylation rates and kinetic parameters of flavonol aglycones catalyzed by
P. cellulase under optimal conditions (pH 6, 50% ethanol, 0.5 g/L maltose, 60 ◦ C, 30 h).
Table 3
Extraction yield of total flavonoids from G. biloba.
as expected. The extraction yield turned to be 28.3 mg/g of dw, [8] Tang J, Sun J, Zhang Y, Li L, Cui F, He Z. Herb–drug interactions: effect of Ginkgo
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