extraction of allicin by ethanol

based onethanol/ammonium sulfate in laboratory and pilot scale Fenfang Lib, Qiao Lib, Shuanggen Wub, Zhijian Tana,⇑ a Institute of Bast Fiber Crops and Center of Southern Economic Crops,

Salting-out extraction of allicin from garlic (Allium sativum L.) based on

ethanol/ammonium sulfate in laboratory and pilot scale

Fenfang Lib, Qiao Lib, Shuanggen Wub, Zhijian Tana,⇑

a

Institute of Bast Fiber Crops and Center of Southern Economic Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China

b

College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China

a r t i c l e i n f o

Article history:

Received 13 June 2016

Received in revised form 24 August 2016

Accepted 24 August 2016

Available online 25 August 2016

Keywords:

Salting-out extraction

Allicin

Purification

Biological activities

a b s t r a c t

Salting-out extraction (SOE) based on lower molecular organic solvent and inorganic salt was considered

as a good substitute for conventional polymers aqueous two-phase extraction (ATPE) used for the extrac-tion of some bioactive compounds from natural plants resources In this study, the ethanol/ammonium sulfate was screened as the optimal SOE system for the extraction and preliminary purification of allicin from garlic Response surface methodology (RSM) was developed to optimize the major conditions The maximum extraction efficiency of 94.17% was obtained at the optimized conditions for routine use: 23% (w/w) ethanol concentration and 24% (w/w) salt concentration, 31 g/L loaded sample at 25°C with pH being not adjusted The extraction efficiency had no obvious decrease after amplification of the extrac-tion This ethanol/ammonium sulfate SOE is much simpler, cheaper, and effective, which has the poten-tiality of scale-up production for the extraction and purification of other compounds from plant resources

Ó 2016 Elsevier Ltd All rights reserved

1 Introduction

Garlic (Allium sativum L.) is a well-known edible and medicinal

plant since ancient China Allicin (diallylthiosulfinate) is an

organosulfur compound, and it is one major biological active

sub-stance in garlic (Tyagi, Pradhan, Srivastava, & Mehrotra, 2014)

Actually, allicin is converted from alliin after crushing of the garlic

clove under the action of alliinase (Amagase, Petesch, Matsuura,

studied for its antibacterial properties in the middle of 20th

cen-tury (Cavallito & Bailey, 1944), then its other pharmacological

actions of anti-oxidant, antifungal, antihypertensive,

anti-inflammatory, and inhibition of tumor were also found (

El-Kashef, El-Kenawi, Suddek, & Salem, 2015; Hirsch et al., 2000;

using water or ethanol aqueous solution (Arzanlou & Bohlooli,

2010; Bocchini, Andalo, Pozzi, Galletti, & Antonelli, 2001; Wang

Glatzel, & Martinez, 2012; Liang, Qiao, Bi, Zou, & Zheng, 2012;

for the extraction of allicin from garlic in laboratory, pilot or large

scale However, the solvent extraction can obtain the crude extract

and the samples need further purification; SFE requires sophisti-cated instrument and high cost

Aqueous two-phase extraction (ATPE) was first introduced by Albersson, and the most commonly used two aqueous two-phase systems (ATPSs) were PEG/salt and PEG/dextran (Albertsson,

1986) ATPS based on low molecular organic solvents (e.g metha-nol, ethametha-nol, acetone, and n-propanol) and inorganic salts had been developed in recent years, which can also be called salting-out extraction (SOE) system (Dong et al., 2016) Compared with poly-mer ATPS, SOE has the advantages of lower cost, lower viscosity, quicker phase separation time, relatively lower environmental tox-icity and easier to scale up (Amid, Shuhaimi, Sarker, & Manap, 2012; Fu, Yang, & Xiu, 2015; Liu, Zou, Gao, Gu, & Xiao, 2014; Ooi

been used to extract various bioactive compounds from different plants resources, such as anthocyanins from grape juice (Wu

2015), phenolic compounds from Ficus carica L (Feng et al.,

2015), rutin from acerola waste (Reis et al., 2014), lignans from Zanthoxylum armatum (Guo, Su, Huang, Wang, & Li, 2015), and polysaccharides from Semen Cassiae (Chen et al., 2016)

The objective of this study is to use SOE for the extraction and preliminary purification of allicin from garlic powder The allicin

is extracted into alcohol-rich phase, while partial impurities are extracted into the salt-rich phase The extraction conditions were

http://dx.doi.org/10.1016/j.foodchem.2016.08.092

0308-8146/Ó 2016 Elsevier Ltd All rights reserved.

⇑ Corresponding author.

E-mail address: tanzhijiantgzy2010@aliyun.com (Z Tan).

Contents lists available atScienceDirect Food Chemistry

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / f o o d c h e m

optimized at laboratory scale, SOE was scaled up to the pilot scale

under the optimized conditions The phase-forming components of

ethanol and ammonium sulfate were recycled and reused

2 Materials and methods

2.1 Materials and reagents

The garlic samples were originated in Shandong province and

bought from the Vanguard supermarket in Changsha City, Hunan

Province The allicin standard was purchased from National

Insti-tutes for Food and Drug Control (Beijing, China) with HPLC purity

larger than 98% HPLC grade acetonitrile was purchased from

TEDIA Company, Inc (Fairfield, OH, USA) The analytical reagents

of different organic solvents (ethanol, n-proanol, isopropanol,

ace-tone and acetonitrile) and salts (ammonium sulfate, sodium

dihy-drogen phosphate, sodium sulfate, and potassium phosphate) were

provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai,

China) 2,2-diphenyl-1-picpicrylhydrazyl (DPPH), ferrous sulfate,

salicylic acid hydrogen peroxide were purchased from Alladin

Reagent Co., Ltd (Shanghai, China) Escherichia Coli DH5awas

pro-vided by Tiangen Biotech Co., Ltd (Beijing, China) All other

reagents used in this study were analytical grade and no further

treatments were processed for them

2.2 Preparation of crude extract

The garlic was peeled, then smashed into powder In order to

make complete converting of alliin to allicin, a grinding time of

30 min was employed Then the garlic powder was extracted by

absolute ethanol with ultrasonic assisted extraction for 20 min in

an ultrasonic bath (model KQ-5200 DE, Kunshan Ultrasound Co

Ltd., Kunshan, China) The mixture was centrifugated and filtrated

to remove the insoluble substance The supernate was evaporated

to remove the ethanol, the crude extract of allicin was obtained

2.3 Phase diagram

The phase diagrams indicate the information of the

concentra-tion of each component is required to form the ATPS, which were

drawn by the similar turbidimetric titration reported in our

previ-ous work (Tan, Li, & Xu, 2013) Firstly, organic solvent of known

mass was added into a tube Then, a salt solution of known mass

fraction was added dropwise, and the solution in the tube was well

mixed The solution became turbid after adding of the salt solution

then separated into two phases The mass fraction of each added

component was calculated Lastly, a few drops of water was added

to make the mixture clear again The above procedures were

repeated to obtain sufficient data to construct the phase diagrams

2.4 Salting-out extraction

SOE system was formed by adding salt aqueous solution into a

tube containing a certain amount of organic solvents, then crude

allicin extract was added ATPS was used for the SOE of allicin after

complete stirring Allicin was extracted into the top phase (organic

solvent-rich phase), while some hydrophilic substances, such as

alliin, lysine, glutamic, etc., tended to partition in the bottom phase

(salt-rich phase) (Jiang, Lu, Tan, & Cui, 2014) Centrifugation

was performed to accelerate the phase-forming and achieve

complete extraction, then two clear phases formed and the volume

of each phase was noted down HPLC analysis was used for

the quantitative determination of the allicin concentration The

extraction efficiency (E/%) of allicin in top phase was calculated

by the Eq.(1)

E¼CtVt

where Ctwas the allicin concentration in top phase, and Vtwas the volume of top phase, Coand Vowere the concentration and volume, respectively of crude allicin extract added into the system

2.5 HPLC conditions The allicin samples were analyzed using a Dionex UltiMate

3000 HPLC system (Dionex, Sunnyvale, California, USA) coupled with a LPG-3400 pump, a VWD-3400 UV–vis detector, and a TCC-3000 column compartment A Promosil C18chromatographic column (250 4.6 mm i.d., 5lm particle size) was used to analyze the samples A PC coupled with a Chameleon software was used to collect and analyze the data The mobile phase was composed of acetonitrile, H2O, and acetic acid (75:25:0.6; v/v/v) The flow rate was 1.0 mL/min with isocratic elution and the effluent was moni-tored at the wavelength of 240 nm The oven temperature was set at 30°C All the samples of 20lL were injected into the HPLC for analysis The mobile phase and samples solution were filtered through microfiltration membrane (0.45lm) before analysis The standard curve for analysis of allicin is A = 292.67C + 0.34 (R2= 0.9995), where A is the peak area and C is the allicin concen-tration Standard solutions of allicin were diluted in the range from 0.024 to 0.114 mg/mL

2.6 Antioxidant activity of allicin obtained by SOE The free-radical scavenging activity of allicin was assessed using DPPHradical according to the method with moderate mod-ification (Athukorala, Kim, & Jeon, 2006) Two milliliter allicin solu-tion was added into 2.0 mL DPPH ethanol solution (40lg/mL) Another two samples were prepared with 2.0 mL ethanol being added into 2.0 mL DPPH(as blank) and 2.0 mL allicin concentra-tion soluconcentra-tion being added into 2.0 mL ethanol (as control), respec-tively The mixture was shaken vigorously and left to stand for

40 min at ambient temperature in darkness Absorbance was determined at 517 nm by a UV–Vis spectrophotometer (UV-2100, Unico, USA), vitamin c (Vc) was used as a comparison to allicin The results were expressed as percentage inhibition of the DPPH radical on Eq.(2)

Scavenging capacityð%Þ ¼ ½1  ðAs AcÞ=Ao ð2Þ

where Ac, Asand Aorepresent the absorbance of control, sample and blank, respectively The antioxidant capacity of test compounds was expressed as IC50, the concentration necessary for 50% reduction of DPPH

The scavenging ability of hydroxyl radical (OH) was evaluated according to the modified Fenton method (Wang & Jiao, 2000)

To a colorimetric tube, 4.0 mmol/L FeSO4, 10.0 mmol/L H2O2, 5.0 mmol/L salicylic acid ethanol solution and a certain concentra-tion of allicin sample were added The proporconcentra-tion of each compo-nent was indicated as follow: Ac(1.0 mL FeSO4+ 0.5 mL salicylic acid + 7.5 mL H2O + 1.0 mL H2O2), As (1.0 mL FeSO4+ 0.5 mL sali-cylic acid + 7.5 mL allicin sample + 1.0 mL H2O2), and Ao (1.0 mL FeSO4+ 0.5 mL H2O + 7.5 mL allicin sample + 1.0 mL H2O2) Each mixture was vigorously shaken and left to stay for 30 min at ambi-ent temperature in darkness The absorbance was determined at

510 nm by a UV–Vis spectrophotometer (UV-2100, Unico, USA) and the scavenging radical activity was calculated according to the identical equation (5) used in DPPHdetermination The antiox-idant capacity of test compounds was expressed as IC50, the con-centration necessary for 50% reduction ofOH

2.7 Antibacterial property of allicin obtained by SOE

An agar-well diffusion method was used to assess the

antibac-terial ability of allicin obtained by SOE (Shan, Cai, Brooks, &

coli was selected as a strain and subcultured, and allicin samples

of different concentration ranges were prepared Secondly,

inocu-lated suspension was spread onto the surface of every plate

equally Thirdly, three wells with diameter of 6 mm were cut from

the agar using a hole puncher Fourthly, 50lL allicin solution was

pipetted into each well Ethanol and deionized water acted as the

negative controls Lastly, the inoculated plates were left

undis-turbed and incubated at 35°C for 24 h Following these processes,

antibacterial activity was estimated by measuring the diameter of

growth inhibition zone using a Vernier caliper Each sample was

tested in triplicate and all the equipments and tested reagents used

in this experiment were sterilized

2.8 Statistical analysis

Design Expert 7.0 (DE, Stat-Ease, Inc., Minneapolis, USA) was

used to analyze the experimental data and obtain the response

models The lack of fit and coefficient of determination (R2) were

used to judge the suitability of model The analysis of variance

(ANOVA) was carried out with the comparison of the actual and

predicted values, and the optimized conditions for three major

variables were acquired by statistical analysis The statistical

anal-ysis for screening the SOE system and single factor experiments

3 Results and discussion

3.1 Selection of the optimum SOE system

To screen the optimal SOE system for the extraction and

isola-tion of allicin, various organic solvents (ethanol, n-propanol,

iso-propanol, acetone and acetonitrile) and salts (ammonium sulfate,

sodium dihydrogen phosphate, sodium sulfate, and potassium

phosphate) were considered as the phase-forming components

The binodal curves were shown inFig 1, which imply that the

clo-ser the binodal curves to the coordinates, the less salt (organic

sol-vent) required for forming two phases under the same organic

solvent (salt) concentration

It can be seen inFig 1(a) that the phase-forming ability of all

the organic solvents in the organic solvents/ammonium sulfate

n-propanol > ison-propanol > ethanol > acetone The reasons for

influ-encing the phase-forming ability of organic solvents are

solubility, boiling-point, and kosmotropicity of salts of organic

phase-forming ability of the four types of salt in the ethanol/salt

ATPSs followed the order: potassium phosphate > sodium

sul-fate > ammonium sulsul-fate > sodium dihydrogen phosphate The

phase-forming ability (salting-out ability) of salt influencing

differ-ent ATPSs is mainly related to the ions’ Gibbs free energy of

2012) The phase diagrams can be used for selecting the organic

solvent and salt concentrations in the RSM experiments

The extraction efficiencies using different SOE systems were

shown inFig 2 As it can be seen that two ATPSs formed by

ammo-nium sulfate and sodium sulfate had relatively higher extraction

efficiency than other two ATPSs formed by sodium dihydrogen

phosphate and potassium phosphate It can also be seen that the alkaline system (ethanol/potassium phosphate) was not suitable for the allicin extraction Considering to the reasons that ammo-nium sulfate can lead to acidic condition and has better solubility than sodium sulfate, ammonium sulfate was chosen as the phase-forming salt As to the systems formed by different organic sol-vents and ammonium sulfate, the SOE system based on ethanol had the highest extraction efficiency Therefore, ethanol/ammo-nium sulfate was chosen as the optimum SOE system

3.2 Single factor experiments There are many factors, such as ethanol and ammonium sulfate concentration, loaded sample, temperature, pH etc., can influence the SOE Therefore, it is necessary to optimize these conditions to obtain the maximum extraction efficiency In the single factors experiments, when one factor was investigated, the other factors were set at fixed values

3.2.1 Effect of ethanol and ammonium sulfate concentration The ethanol and ammonium sulfate concentration can be con-sidered as the most influencing factors in majority SOE systems

In order to optimize the ethanol concentration, the concentration ranges of 18%–26% (w/w) were studied The results in Fig 3(a) showed that the maximum extraction efficiency was obtained at the ethanol concentration of 24% (w/w)

The change of salt concentration was behaved as the change of salting-out effect To study the ammonium sulfate concentration, the salt concentration in the range of 17%–25% (w/w) was chosen The results in Fig 3(b) showed that the extraction efficiency increased with increasing of salt concentration, then decreased with further addition of salt, and the maximum extraction

0 1 2 3 4 5 6 7 8 9 10 11 10

20 30 40 50 60 70 80 90

(a)

Mass fraction of ammonium sulfate (%)

iso-propanol acetone n-propanol acetonitrile ethanol

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 20

30 40 50 60 70 80 90

Mass fraction of salt (%)

Potassium phosphate Sodium sulfate Sodium dihydrogen phosphate Ammonium sulfate

(b)

Fig 1 Binodal curves for the different organic solvent and salt ATPS at 298.15 K.

efficiency was obtained at the ammonium sulfate concentration of

23% (w/w) In fact, the increasing of salt concentration can increase

the salting-out ability (Liu, Mu, Sun, Zhang, & Chen, 2013), which is

beneficial for the extraction, however the salt concentration will

reach saturation and more water will be pulled into salt-rich phase

phase become not good solvent for dissolving allicin

3.2.2 Effect of loaded sample

After the extraction experiments being designed, as much

alli-cin as possible will be added into the system in order to achieve

biggest economic benefit As it was reported that the crude load

can alter the partition behavior of target molecules (Amid et al.,

2012) The crude extract was dissolved into the ethanol to prepare

concentration ranges of 20–60 g/L in this study, and then salt was

added to form ATPS for SOE of allicin The results in Fig 3(c)

showed that when 40 g/L allicin sample was prepared, the maxi-mum extraction efficiency was obtained, and then decreased with increasing of loaded allicin That can be interpreted the excessive addition of allicin will make the saturation of extraction

3.2.3 Effect of temperature and pH The extraction temperature and system pH are also key factors affecting the extraction to some extent The effect of temperature was studied in the range of 25–50°C The results in Fig 3(d) showed that the extraction efficiencies decreased with increasing

of temperature In our previous studies, it was also found that the SOE was better to be operated around the room temperature because this procedure was spontaneous and exothermic (Tan

done at room temperature without heating

Fig 2 SOE of allicin using different ATPSs formed by 25% (w/w) organic solvents and 12% (w/w) salts at room temperature, the loaded sample was 40 g/L with system pH being not adjusted Different letters in the same series indicate significant difference at P < 0.05 level.

Fig 3 Single factor experiments for studying the effect of (a) ethanol concentration (the ammonium sulfate concentration was 25% and the loaded sample was 40 g/L at room temperature with pH being not adjusted), (b) ammonium sulfate concentration (the ethanol concentration was 20% and the loaded sample was 40 g/L at room temperature with pH being not adjusted), (c) loaded sample (the ethanol concentration was 20% and the ammonium sulfate concentration was 25% at room temperature with pH being not adjusted), (d) temperature (the ethanol concentration was 20%, the ammonium sulfate concentration was 25%, and the loaded sample was 40 g/L with pH being not adjusted), and (e) pH (the ethanol concentration was 20%, the ammonium sulfate concentration was 25%, and the loaded sample was 40 g/L at room temperature) on the SOE of allicin Different letters in the same series indicate significant difference at P < 0.05 level.

The change of pH can influence the electrical charge of target

compound, thus to influence its hydrophobicity/hydrophilicity

and furtherly influence its partition in SOE (Wang et al., 2010)

The system pH was adjusted by hydrochloric acid and sodium

hydroxide aqueous solution The effect of pH was investigated at

pH 4.0–8.0 It can be seen fromFig 3(e) that the pH 6.0 was most

suitable for the extraction The aqueous solutions of allicin lead to

a pH of approximately 6.5 (Cavallito & Bailey, 1944; Freeman &

favored This optimal ethanol/ammonium sulfate ATPS can

gener-ate an acidic system with a pH of approximgener-ately 6.5, and the

alka-line condition was not suitable for SOE, thus it is unnecessary to

adjust the system pH at the extraction procedure

3.3 Optimization of the SOE conditions by RSM

The SOE procedure is very complex, the interactions between

influencing factors should also be considered Therefore, it’s better

to study the interaction between the three independent process

variables (ethanol concentration, ammonium sulfate

concentra-tion, and loaded sample), RSM based on Box–Behnken design

(BBD) was developed to optimize the extraction conditions.Table 1

showed the obtained results by the RSM models

indicates regression model was significant The Model F-value of

16.74 implies that the model is significant There is a 0.06%

proba-bility that a ‘‘Model F-Value” occurs due to noise The model terms

can be considered to be significant if the P-values are less than

0.05 According to this principle, the model terms of X1, X2, X2X3,

X1, X2can be considered as significant terms

The ‘‘Lack of Fit F-value” of 5.23 implies the ‘‘Lack of Fit” is not significant relative to the pure error and that there is a 7.2% chance that a ‘‘Lack of Fit F-value” this large could occur due to noise The regression model was generated by the software, which was pre-sent in Eq.(3):

Y¼ 2:24 þ 0:57X1 0:14X2 0:060X3þ 0:091X1X2

þ 0:26X1X3 0:081X2X3 0:59X2 0:10X2 0:23X2ðR2

where Y is the extraction efficiency (%), X1is the ethanol concentra-tion (%, w/w), X2is the ammonium sulfate concentration (%, w/w), and X3is the loaded sample (g/L) The coefficient of determination (R2) is 0.9556, implying that more than 95.56% of the variations in the process efficiency could be explained by the model

The response surfaces for the effects of the independent vari-ables on the average extraction efficiency of allicin were shown

conditions for the extraction of allicin are an ethanol concentration

of 22.57% (w/w), salt concentration of 24.11% (w/w), and the loaded sample of 30.96 g/L The yield, averaged over triplicate runs, was 94.17% under the optimized conditions [23% (w/w) ethanol concentration, 24% (w/w) salt concentration, and 31 g/L loaded sample], which was very close to the predicted value of 94.68% This demonstrates that the model is adequate for predicting the expected optimization The purity of the obtained allicin under the optimized conditions was determined by quantitative analyses using HPLC The result indicated that the purity of allicin reached

to 68.4%, having noteworthy increase compared with the crude extract (purity of 31.8%)

3.4 Antioxidant activity and antibacterial property The DPPH and OH assays presented different antioxidant capacity It can be seen fromFig 5(a) that the radical scavenging capacity of allicin for the DPPHassay was poorer than that of Vc, the IC50 values for allicin and Vc were approximate 5.0lg/mL and 15lg/mL, respectively.Fig 5(b) appeared the radical scaveng-ing ability for the Fenton reaction assay, it can be seen that the rad-ical scavenging ability ofOH increased with increasing of allicin concentration The IC50 for allicin was approximate 10lg/mL, while that for Vcwas approximate 80lg/mL, indicating that the allicin had very strong scavenging ability ofOH

3.5 Antibacterial property Allicin obtained by SOE was used to test the antibacterial abil-ity The result inFig 5(c) showed that allicin exhibited significantly antibacterial ability based on the measurement of the diameter of inhibition zone Allicin obtained by SOE had similar antibacterial ability compared with the standard allicin at the same concentra-tion (seeing plates A and C) The data showed that diameter of inhi-bition zone increased significantly from 1.80 to 2.72 cm with increasing of allicin concentration from 0.185 to 0.923 mg/mL The controls of ethanol solution and pure water indicated that they had no antibacterial ability

3.6 Amplification of SOE and recycling of the phase-forming components

SOE was scaled-up from 25 g to 5 kg, 10 kg, and 20 kg of the total mass under the optimized conditions for routine application (23% (w/w) ethanol concentration, 24% (w/w) salt concentration, and 31 g/L loaded sample) using a continuous stirred tank reactor (Model GR-20, Zhengzhou Greatwall Scientific Industrial and Trade

Co, Ltd, Zhengzhou, China) Salt was directly added into the crude

Table 1

Experimental results for the three-factor/three-level BBD and analysis of variance

(ANOVA) for the quadratic response surface model.

Run Factor X 1 : ethanol

concentration

(%, w/w)

Factor X 2 : salt concentration (%, w/w)

Factor X 3 : loaded sample (g/L)

Extraction efficiency (%)

squares

Degree of freedom

Mean square

prob > F

extract after removing the sediments using a centrifuge (Model

TL-1000, Jiangsu Taizhou Taida Mechanical Industrial Co, Ltd,

Changzhou, China) in the pilot scale The experimental results

indicated that the extraction efficiencies had no obvious

reduction from laboratory scale to pilot scale, the extraction

efficiency was 91.5% at 20 kg scale, showing minor difference

with 94.17% at 25 g scale

The study of recycling of ethanol and ammonium sulfate was

done using the reported method (Li, Teng, & Xiu, 2010; Tan et al.,

2013) in the laboratory scale The two phases were separated by

a separating funnel, the ethanol in top phase can be recycled by

evaporation, and the concentration of recycled ethanol was 84.6% Ammonium sulfate in salt-rich phase can be recycled by dilution crystallization, the recycling efficiency can reach to 91.64% using methanol at a volume ratio of 2:1 to salt aqueous solution

4 Conclusions

In this work, ethanol/ammonium sulfate ATPS was successfully applied for the SOE of allicin from garlic in laboratory and pilot scale The factors influencing the SOE were investigated in detail The obtained optimum results were as follows: 24% (w/w) ethanol concentration, 23% (w/w) ammonium sulfate concentration, 40 g/L loaded sample at room temperature with pH being not adjusted The major three influencing factors were further optimized by RSM The optimized conditions were 22.57% (w/w) ethanol con-centration, 24.11% (w/w) ammonium sulfate concon-centration, and 30.96 g/L loaded sample, 94.17% of allicin can be obtained in alcohol-rich phase under this condition The purity of allicin

Fig 4 Response surface curves for the extraction of allicin from garlic showing

interaction between (a) ethanol concentration and salt concentration, (b) ethanol

concentration and loaded sample, and (c) salt concentration and loaded sample.

0 10 20 30 40 50 60 70 80 10

20 30 40 50 60 70 80 90 100

Concentration (±J/mL)

allicin

Vc (a)

0 10 20 30 40 50 60 70 80 90 100 10

20 30 40 50 60 70 80 90 100

Concentration (±g/mL)

allicin

Vc (b)

Fig 5 (a) Test the antioxidant capacity of allicin obtained by SOE using DPPH; (b) Test the antioxidant capacity of allicin obtained by SOE usingOH; (c) Test the antibacterial ability of allicin (A—allicin standard; B—ethanol solution; C—allicin obtained by SOE; D—pure water).

obtained by SOE was 68.4%, which was much higher than that of

crude extract (31.8% purity) The bioactive tests indicated allicin

obtained by SOE had good antioxidant activity and antibacterial

property Finally, this SOE was scaled up in pilot scale under the

optimized conditions, no obvious decrease of extraction

efficien-cies was observed The scale-up and recycling experiments

demon-strated this ethanol/ammonium sulfate SOE had the potentiality

for use in industrial production of allicin

Acknowledgement

This work was financially supported by the National Natural

Science Foundation of China (No 21406262)

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