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Enzyme and Microbial Technology 48 (2011) 100–105

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

nzyme-assisted extraction of flavonoids from Ginkgo biloba leaves:mprovement effect of flavonol transglycosylation catalyzed by Penicilliumecumbens cellulase

huo Chena,1, Xin-Hui Xinga, Jian-Jun Huangb, Ming-Shu Xub,∗

Department of Chemical Engineering, Tsinghua University, Beijing 100084, ChinaMarine College, Shandong University at Weihai, Shandong, Weihai 264209, China

r t i c l e i n f o

rticle history:eceived 31 August 2010ccepted 28 September 2010

eywords:nzyme-assisted extractionenicillium decumbens cellulaseransglycosylation

a b s t r a c t

We report a novel enzyme-involved approach to improve the extraction of flavonoids from Ginkgo biloba,in which the enzyme is employed not only for cell wall degradation, but also for increasing the solubilityof target compounds in the ethanol–water extractant. Penicillium decumbens cellulase, a commercial cellwall-degrading enzyme with high transglycosylation activity, was found to offer far better performancein the extraction than Trichoderma reesei cellulase and Aspergillus niger pectinase under the presence ofmaltose as the glycosyl donor. TLC, HPLC and MS analysis indicated that P. decumbens cellulase couldtransglycosylate flavonol aglycones into more polar glucosides, the higher solubility of which led to

lavonoidinkgo bilobaolubility

improved extraction. The influence of glycosyl donor, pH, solvent and temperature on the enzymatictransglycosylation was investigated. For three predominant flavonoids in G. biloba, the transglycosyla-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 enzymaticbifunctionality described here, naming enzymatic modification of target compounds and facilitation of

vides

cell wall degradation, pro

. Introduction

Enzyme-assisted extraction of natural functional compoundsrom plants is widely investigated in recent years for its advantagesn easy operation, high efficiency, and environment friendship [1].

ost of the works in this field utilize cellulase, pectinase and �-lucosidase to hydrolyze and degrade plant cell wall constituentso improve the release of intracellular contents [2–4]. However,nother important factor in the extraction – the intrinsic prop-rty especially the solubility of the target compound – has seldomeen concerned according to our knowledge. Low solubility of tar-et compounds in the extractant leads to low extraction yield andequire large amount of solvents, which largely impedes the eco-

omic efficiency in industry. Therefore, here we propose a novelnzyme-assisted extraction approach, in which the extraction ismproved not only from enhanced cell wall degradation, but alsorom the increased solubility of target compounds in the extractant.

∗ Corresponding author. Tel.: +86 631 5688303; fax: +86 631 5688303.E-mail address: xms@sdu.edu.cn (M.-S. Xu).

1 Current address: Department of Chemistry and Biotechnology, School of Engi-eering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2010.09.017

a novel approach for the extraction of natural compounds from plants.© 2010 Elsevier Inc. All rights reserved.

The extraction of plant flavonoids, which generally have poor sol-ubility in mild solvents such as ethanol–water solution, is selectedas an example to validate our proposal.

Flavonoids are natural compounds showing high physiologicalactivities in therapies for inflammations, heart diseases and cancers[5,6]. There are three predominant flavonol aglycones, quercetin,kaempferol, and isorhamnetin (Fig. 1), in Ginkgo biloba, a Chinesemedicinal plant well known for its high content of flavonoids [7].Extract from G. biloba is among the most popular phytomedicinesand herbal dietary supplements [8], whose primary active compo-nents are flavonoids (24%) and small amount of terpene lactones(6%) [9].

Various methods have been used to assist the extraction offlavonoids from plants, such as ultrasonication [10], supercriti-cal fluids [11], microwave [12], membrane adsorption [13] andmolecular imprinting [14]. There are also several reports on theenzyme-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, cellulaseand �-glucosidase [16]. In another study, extraction of antimicro-bial and antioxidant phenolics, mainly anthocyanins, from berrieswas studied, and the enzyme was found to effectively hydrolyze

S. Chen et al. / Enzyme and Microbial

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Fig. 1. Chemical structures of three predominant flavonol aglycones in G. biloba.

lycosides to their corresponding aglycones [17]. Although thenzyme-assisted approach has largely improved the extractionate, due to the intrinsic low polarity of flavonol aglycones, thextraction after enzymatic treatment still has to be performed witholvents like hexane, acetone and butanone in order to achieveigh productivity. Nevertheless, regarding toxicological limita-ions, there is a clear trend in the industry to substitute theserganic solvents for alternative nontoxic solvents, particularly inroducts for human consumption, with the first option being waterr alcohols [18]. Therefore, large amount of high concentrationthanol–water solution is often used. However, this unfavorablyeads to a high production cost, making it desirable to increasehe solubility of flavonoids without affecting their physiologicalctivities.

We come up with an idea to utilize the transglycosylationctivity of glycosidases to introduce hydrophilic groups, e.g. gly-osides, into flavonoids to improve their polarity in the extractionrocess, while utilizing the enzyme’s activity in cell wall hydrol-sis at the same time. Although commercial cellulases are oftenelected and modified to minimize their transglycosylation activ-ty for more complete degradation of cellulose chains in biomassransformation [19], many cellulase systems in microorganismshow both hydrolysis and the reverse transglycosylation activ-ties [20]. Enzymatic transglycosylation by various glycosidases,ncluding glucosidases [20–22], galactosidases [20], xylosidases23], rhamnosidases [24], etc., has already gained importance toe an efficient and low cost way to synthesize oligosaccharides,lkyl glucosidases, and glycoconjugates. In a study by Gao et al.25], catechin, a flavonoid, was successfully glycosylated into itslycoside form by several glycosidases. In our case, the trans-lycosylated product of ginkgo flavonoids, naming the flavonollycosides, present physiological activities on almost the same levelith flavonol aglycones, and can be much more readily absorbed by

he human body [6] directly in the small intestine or after hydrol-sis by bacterial enzymes in the intestine [5]. A commercial cellall-degrading enzyme, cellulase from Penicillium decumbens, wassed in our research for flavonol glycosylation. It is composedf endoglucanase, cellobiohydrolase and �-glucosidase [26,27].esides high activity for cellulose degradation, P. decumbens cel-

ulase shows high transglycosylation activity probably originatingrom its �-glucosidase activity as indicated in our previous research28].

In this work, we aim to make it clear whether the enzymaticransglycosylation of flavonoids could improve their extractionrom ginkgo leaves. For this purpose, the effect of P. decumbensellulase on the extraction yield was compared with several otherell wall degrading enzymes with little transglycosylation activi-ies under the addition of glycosyl donors. After validated for itsmproving effect on the extraction, flavonol transglycosylation by. decumbens cellulase was confirmed by analyzing the catalyticroducts of the three predominant flavonoids in G. biloba. Thene optimized the enzymatic conditions, studied the transglyco-

ylation kinetics, and examined whether the extraction could bemproved under the optimal transglycosylation conditions. To theest of our knowledge, this is the first report to utilize enzymaticifunctionality – for cell degradation and target product transfor-ation – in the extraction of natural compounds from plants. This

Technology 48 (2011) 100–105 101

is also the first report on enzyme-assisted extraction of flavonoidsfrom ginkgo leaves.

2. Materials and methods

2.1. Plant material

Dried leaves of G. biloba were bought from Henan Medical Lim-ited (Zhengzhou, China) and ground into powder. The particle sizewas controlled within 280 and 600 �m using sifters.

2.2. Chemicals and enzymes

Quercetin, kaempferol and isorhamnetin were purchased fromSigma–Aldrich (Steinheim, Germany). Cellulase from Trichodermareesei ATCC 26921 (Celluclast 1.5 L, ≥700 U/g) was bought fromNovozymes A/S (Bagsvaerd, Denmark). Pectinase from Aspergillusniger (>1 U/mg) was bought from Sigma–Aldrich (Steinheim, Ger-many). P. decumbens cellulase was provided by Ningxia CellulasePreparation Plant, China. All other reagents were of analytical gradeand commercial available.

2.3. Enzyme-assisted extraction of total flavonoids

Distilled water was added to enzymes to obtain stock solutionsat the concentration of 2 mg/mL. For each batch of enzyme-assistedextraction, 50 mL enzyme stock solution and certain amount ofglycosyl donor were diluted with ethanol–water and adjustedwith acetate buffer to obtain 500 mL extractant with the desiredpH, ethanol–water ratio and glycosyl donor concentration. 30 gginkgo leaf powder was added to the extractant in an Erlenmeyerflask covered with aluminum foil. The mixture was incubatedunder 200 rpm stirring for 30 h on a multichannel magnetic stirrerwith temperature controller (Guohua Electronics Co., Changzhou,China). After incubation, the mixture was filtered and the fil-trate was forwarded to analysis. For the comparison of extractionsassisted by different enzymes, incubation was done at 40 ◦C in theextractant with an ethanol–water ratio of 3:7 (v/v) and at pH 6.0.

2.4. Determination of total flavonoids

The aluminumchloride colorimetric method described by Changet al. [29] was used to determine the total content of flavonoids.0.5 mL extract or standard solution was mixed with 1.5 mLmethanol, 0.1 mL of 10% aluminum chloride (substituted with dis-tilled water in blank probe), 0.1 mL of 1 M potassium acetate, and2.8 mL of distilled water. After 30 min incubation, absorbance at415 nm was determined against a distilled water blank on a UV-1206 spectrophotometer (Shimadzu, Kyoto, Japan). All sampleswere made in triplicate, and mean values of total flavonoid con-tent are expressed as milligrams of quercetin equivalents per gramof dry weight (dw) calculated according to the standard calibrationcurve.

2.5. TLC analysis

Extract from ginkgo leaves or enzymatic reaction mixture wasloaded to a silica gel plate (10 cm × 20 cm, GF254, Qingdao HaiyangChemical Co. Ltd., China) and developed with the mobile phase ofbenzene–ethyl acetate–acetone–acetic acid (10:8:2:2, v/v/v/v).

2.6. Enzymatic transglycosylation reaction

The reaction system for the enzymatic transglycosylation consi-sted of 2 mL stock solution of P. decumbens cellulase, 25 mg flav-onoid substrate and 25 mg glycosyl donor in 48 mL ethanol–water

1 obial Technology 48 (2011) 100–105

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olvent. Prior to the addition of the enzyme, pH of the solution wasdjusted by acetate buffer. The enzymatic catalysis was carried outor 30 h under 200 rpm stirring.

To determine the transglycosylation rate, 50 �L enzymatic reac-ion mixture was loaded on a silica gel plate and developed withhe same mobile phase as described above. Under the exposure inhe ultraviolet light from a portable UV lamp, flavonoids and theeaction products at the corresponding Rf positions were scrapednd extracted in 10 mL ethanol through 0.5 h ultrasonication. Thextract was filtrated and the absorbance of the filtrate at 370 nmas measured on the UV-1206 spectrophotometer (Shimadzu,yoto, Japan). According to the standard curve prepared by theame method, the residual flavonoid concentration (C) after trans-lycosylation was obtained. Transglycosylation rate was calculatedy the following equation:

ransglycosylation rate (%) = C0 − C

C0× 100,

here C0 and C refer to the initial and residual flavonoid concen-ration (mg/mL), respectively.

To study the kinetics of the transglycosylation, a series of exper-ments is carried out with varying substrate concentrations underhe conditions of pH 6, 50% ethanol, 60 ◦C and 0.5 g/L maltose addi-ion. The substrate concentrations at 0 h and 0.5 h in the reactionystem were determined from TLC in the same way as describedbove. The reaction rate calculated from the concentration changef the substrate in the first 0.5 h was used as the initial velocity.he measurements were then plotted in a Lineweaver–Burk plot,n which the inverse of substrate concentration was plotted againsthe inverse of the initial velocity. The values of the desired constantsm and Vmax were read directly off the plot.

.7. HPLC analysis

HPLC analysis was carried out using a Symmetry C18 column5 �m, 250 mm × 4.6 mm, Waters Corp., Milford, USA) attached toShimadzu HPLC LC-10A system (Shimadzu, Kyoto, Japan) with ahotodiode array detector monitored at 370 nm. The mobile phaseas methanol–water (1:1, v/v), the flow rate was set at 0.5 mL/min

nd the column temperature was maintained at 40 ◦C.

.8. MS analysis

The enzymatic reaction mixtures were fractionated using aemi-preparative scale HPLC system consisting of the same type ofolumn (5 �m, 250 mm × 4.6 mm, Waters Corp., Milford, USA) at aow rate of 5.0 mL/min. The fractionated reaction product was iso-

ated and further analyzed by mass spectrometry. The mass spectralnalyses were performed on a PE Sciex API 3000 mass spectrome-er with ESI ion source (Applied Biosystems, USA) (m/z 100–1000;.3 amu; dwell: 1.0 ms; pause: 2.0 ms).

. Results and discussion

.1. Effect of enzymes with different transglycosylation activitiesn extraction yield

Extraction of total flavonoids from ginkgo leaves was conductedith three enzymes, Trichoderma reesei cellulase, A. niger pectinase

nd P. decumbens cellulase. As shown in Fig. 2, the extraction yieldsf total flavonoids increased with treatment by the three enzyme

reparations. The amounts of flavonoids extracted with A. nigerectinase were slightly higher than those with T. reesei cellulasend P. decumbens cellulase. Fu et al. also reported that pectinase hadigher improving effect on the extraction of luteolin and apigenin

rom pigeonpea leaves than cellulase or �-glucosidase, probably

Fig. 2. Effect of different enzyme and maltose additions on the extraction yields oftotal flavonoids.

due to its better performance in cell wall degradation with its com-prised activities from pectinesterase, polygalacturonase and pectinlyase [16].

In order to study the effect of transglycosylation on the extrac-tion, maltose was added as a glycosyl donor to each of the extractionsystems containing different enzymes. Adding glycosyl donors isgenerally considered to be an efficient way to enhance transglyco-sylation while inhibiting the reverse hydrolysis. It is interesting tofind that the yield of the extraction assisted by P. decumbens cellu-lase, in contrast to the other two enzymes, was largely increasedwith the addition of maltose. When there was 2 g/L maltose in theextractant, the extraction yield with treatment by P. decumbens cel-lulase achieved 21.6 mg/g of dw, much higher than those by theother two enzymes. This indicated that, glycosyl donor addition hasnotable improving effect on the extraction involving P. decumbenscellulase but almost no effect on that involving A. niger pectinase orT. reesei cellulase. It has been reported that P. decumbens cellulasehas high transglycosylation activity [28], while A. niger pectinasehas no transglycosylation activity, and T. reesei cellulase has littletransglycosylation activity due to loss of its �-glucosidase con-stituent in the production process [19]. Therefore, it is reasonableto consider that, it is the maltose-induced transglycosylation thatimproves the extraction assisted by P. decumbens cellulase.

3.2. Confirmation of transglycosylation by P. decumbens cellulase

In order to validate the transglycosylation of flavonoids inthe extraction process with P. decumbens cellulase, we analyzedthe profile of the extraction products. TLC analysis of the totalflavonoid extracts with P. decumbens cellulase treatment showedsize decrease of the spots at high Rf and size increase of the spotsat low Rf compared with that without enzyme treatment, revealingthe generation of compounds with higher polarity in the extractionprocess. In order to identify the generated products, three pre-dominant flavonoids in ginkgo leaves, quercetin, kaempferol andisorhamnetin, were used as model substrates to study the catalysisby P. decumbens cellulase.

TLC results for the three substrates and their catalytic prod-ucts were shown in Table 1. Generation of compounds with higherpolarity was confirmed after enzyme treatment. It is important tonote that, the spots for quercetin, kaempferol and isorhamnetin at

Rf 0.68, 0.87 and 0.70, together with the spots for their catalyticproducts at Rf 0.10, 0.10 and 0.14 after enzymatic reaction, couldall be clearly identified on the TLC for the extracts after enzyme-assisted extraction.

S. Chen et al. / Enzyme and Microbial Technology 48 (2011) 100–105 103

Table 1TLC analysis for enzymatic catalysis and extraction of flavonol aglycones.

TLC sample Rf value

Quercetin standard 0.68Quercetin after enzymatic catalysis 0.68, 0.10Kaempferol standard 0.87Kaempferol after enzymatic catalysis 0.87, 0.10Isorhamnetin standard 0.70Isorhamnetin after enzymatic catalysis 0.70, 0.14Total flavonoid extract without

enzyme treatment0.68, 0.87, 0.70

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0.68, 0.87, 0.70, 0.10, 0.10, 0.14

In order to identify the polar products generated in thenzymatic catalysis, HPLC and MS analyses were performed. Chro-atograms for the catalytic products of quercetin, kaempferol and

sorhamnetin all clearly indicated the generation of a new com-ound with a shorter retention time after catalysis (Fig. 3). The m/zatios of the new compounds were determined to be 464 [M+H]+

calcd for C21H20O12, 464), 448 [M+H]+ (calcd for C21H20O11, 448)nd 478 [M+H]+ (calcd for C22H22O12, 478) on MS, correspond-ng to the glucosides of quercetin, kaempferol and isorhamnetin,espectively. The results proved the transglycosylation of flavonolglycones into flavonol glucosides by P. decumbens cellulase dur-ng the extraction process. Flavonol glucosides, which have higherolarity, are much more soluble in the ethanol–water extractanthan flavonol aglycones. This could explain why after P. decum-ens cellulase treatment, the extraction efficiency was remarkablymproved.

.3. Optimal conditions for enzymatic transglycosylation

As suggested by the maltose addition study, enhanced flavonoidxtraction could be achieved through facilitating the transgly-osylation by P. decumbens cellulase. So the optimization of P.ecumbens cellulase catalyzed transglycosylation was studied.uercetin, kaempferol and isorhamnetin were used as the model

ubstrates.

.3.1. Glycosyl donorSoluble starch, maltose and dextrin were selected as candidate

lycosyl donors. The concentration of all the glycosyl donors wasontrolled at 0.5 g/L. As shown in Fig. 4A, for all three substrates,ighest transglycosylation rate was obtained when maltose wassed as the glycosyl donor.

.3.2. pHEffects of pH in the range of 2–9 on the transglycosylation was

xamined by preparing the reaction solutions with different initialHs. As shown in Fig. 4B, P. decumbens cellulase showed transg-

ycosylation activities within a wide pH range. Around pH 6 theatalytic rate was the highest for all three substrates.

.3.3. Solvent ethanol–water ratioSubstrate solubility generally has large effects on the catalytic

eaction. Flavonol aglycones have very low solubility in water, andhe solubility rises as the ethanol concentration increases in waterolution. So the effects of solvent ethanol concentrations from 0 to0% (v/v) on the enzymatic transglycosylation were examined. As

hown in Fig. 4C, the transglycosylation rate reached the highestt the ethanol concentration of 50% for kaempferol and isorham-etin, and 60% for quercetin. When ethanol concentration wasetween 20 and 50%, the catalysis was facilitated with the increasef ethanol, probably resulted from the increase of the solubility of

Fig. 3. Chromatograms of flavonoid samples before and after treatment with P.decumbens cellulase: (A) quercetin; (B) kaempferol; (C) isorhamnetin.

the substrates. When ethanol concentration was higher than 60%,the transglycosylation rate decreased rapidly with the increase ofethanol, which was presumably due to the enzyme inactivationcaused by excess ethanol in the solution.

3.3.4. TemperatureAs shown in Fig. 4D, the transglycosylation rate reached the peak

at 60 ◦C for all of quercetin, kaempferol and isorhamnetin. When

the temperature was higher than 60 ◦C, the catalysis was notablyinhibited, probably due to the inactivation of P. decumbens cellulaseat a high temperature.

104 S. Chen et al. / Enzyme and Microbial Technology 48 (2011) 100–105

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ig. 4. Effect of the glycosyl donor (A), pH (B), ethanol concentration (C), and tempera

.3.5. Transglycosylation kinetics by P. decumbens cellulaseAccording to the above results, quercetin, kaempferol and

sorhamnetin have very similar optimal conditions for theirransglycosylation by P. decumbens cellulase. Time dependencexperiment showed that the transglycosylation reached the

lateau after 30 h of enzymatic treatment under the optimal con-itions of pH 6, 50% ethanol, 60 ◦C and 0.5 g/L maltose (Fig. 5).he transglycosylation rates of quercetin, kaempferol and isorham-etin reached 45%, 50% and 41%, respectively (Table 2), comparedith 25%, 27% and 27% under the pre-optimized conditions. Fur-

ig. 5. Time dependence of the transglycosylation rate of flavonol aglycones (25 mgn 50 mL solvent) under optimal enzymatic conditions (pH 6, 50% ethanol, 60 ◦C and.5 g/L maltose).

D) on the enzymatic transglycosylation of quercetin, kaempferol, and isorhamnetin.

thermore, the kinetics of the enzymatic transglycosylation wasstudied. The initial rate of transglycosylation showed a good rela-tionship with the initial substrate concentration, which fits to theMichaelis–Menten equation. Table 2 gives the kinetic parametersfor the three flavonoid substrates under the optimal catalytic con-ditions.

3.4. Extraction under optimal transglycosylation conditions

We further examined whether the extraction of flavonoids isimproved when the transglycosylation reaches its highest underthe optimal catalytic conditions of pH 6, 50% ethanol and 60 ◦C.As shown in Table 3, total flavonoid content in the extractswas improved under the optimal transglycosylation conditions

Table 2Transglycosylation rates and kinetic parameters of flavonol aglycones catalyzed byP. cellulase under optimal conditions (pH 6, 50% ethanol, 0.5 g/L maltose, 60 ◦C, 30 h).

Flavonol aglycone Transglycosylationrate (%)

Km (mM) Vmax (�M/h)

Quercetin 45 2.40 3.57Kaempferol 50 3.70 20.0Isorhamnetin 41 2.43 0.55

Table 3Extraction yield of total flavonoids from G. biloba.

Extraction condition Yield (mg/g dw)

Without enzymes 14.0 ± 0.3Under pre-optimized enzymatic conditions 21.6 ± 0.3Under optimized enzymatic conditions 28.3 ± 0.5

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s expected. The extraction yield turned to be 28.3 mg/g of dw,1% higher than that under the pre-optimized conditions, and02% higher than (or 2 folds of) that under the conditions withoutnzyme involvement.

As shown in Fig. 2, the extraction yield was increased to 1.3 foldsy treatment with pectinase resulting from its facilitating effect onell wall degradation. In the case of P. decumbens cellulase, evenhe cell wall degradation activity could be seen at the same levels pectinase, there was another 0.7 fold increase in the extractionield, which should be accounted for flavonol transglycosylation.s mentioned above, the flavonol transglycosylation rate was about0% under the optimal conditions, revealing that when half of theavonol aglycones were enzymatically glycosylated into more sol-ble glycosides, the extraction yield was increased by at least 70%.

The present study demonstrates P. decumbens cellulase to behigh efficient enzyme to assist the extraction of flavonoid com-ounds in mild solvents like ethanol–water. The high performancef P. decumbens cellulase originates from its high activity not onlyn facilitating cell wall degradation, but also in transglycosylat-ng flavonol aglycones into more polar glycosides which haveigher solubility in the extractant. In general, this study providesnovel view for enzyme-assisted extraction of plant secondaryetabolites that, besides focusing on enzyme-facilitated cell wall

egradation, targeting on enzymatic modification of the proper-ies of target compounds could be an alternative approach. In thispproach, the key point is to find an enzyme which could modifyhe properties, such as solubility, of target compounds for morefficient extraction without damaging their bioactivity. Moreover,y using bifunctional enzymes or mixed enzyme preparations, thewo approaches might be combined for even higher extraction per-ormance.

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