Bioresource Technology 101 (2010) 2537–2544

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Bioresource Technology

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Supercritical carbon dioxide extraction of seed oil from yellow horn (Xanthocerassorbifolia Bunge.) and its anti-oxidant activity

Su Zhang a,b,1, Yuan-Gang Zu a,b,1, Yu-Jie Fu a,b,*, Meng Luo a,b, Wei Liu a,b, Ji Li a,b, Thomas Efferth c

a Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR Chinab Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR Chinac Department of Pharmaceutical Biology, Institute of Pharmacy, University of Mainz, 55099 Mainz, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 June 2009Received in revised form 12 November 2009Accepted 20 November 2009Available online 22 December 2009

Keywords:Yellow hornSeed oilSupercritical carbon dioxide extractionAnti-oxidant activityResponse surface methodology

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.11.082

* Corresponding author. Address: Key LaboratorMinistry of Education, Northeast Forestry UniversitTel./fax: +86 451 82190535.

E-mail address: yujie_fu2002@yahoo.com (Y.-J. Fu1 These authors contributed equally to this work.

Supercritical fluid carbon dioxide (SF-CO2) extraction (SFE) of seed oil from yellow horn and its anti-oxi-dant activity were investigated. The effects of CO2 flow rate and particle size were firstly optimized, and acentral composite design (CCD) combined with response surface methodology was used to study theeffects of extraction pressure, temperature and time on the extraction yields. A maximal extraction yieldof 61.28% was achieved under optimal conditions of extraction pressure 30 MPa at 45.68 �C, 2.08 h andCO2 flow rate 12 kg/h with 0.5 mm particle size. By analyzing the chemical composition of the seed oil,we found that the content of unsaturated fatty acids was approximately 90%. Furthermore, the anti-oxi-dant activity of seed oil was assessed by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assayand a b-carotene bleaching test. Yellow horn seed oil possessed notable concentration-dependent anti-oxidant activity with IC50 values of 0.151 and 0.195 g/mL, respectively.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Yellow horn (Xanthoceras sorbifolia Bunge.), which belongs tothe Sapindaceae family, is a shrub with a life span of more than200 years (Wang and Wang, 2002). It is indigenous to China andextensively distributed from north latitude 32�300 to 45�, east lon-gitude 100–127�. Yellow horn is a native tree in north of China,which has very well developed adaptability (drought, cold and saltresistance) and fastness ability. Especially, this plant can live wellbelow �40 �C except saline-alkali land or waterlogged fields. Thestem and fruit of yellow horn are used in folk medicine for thetreatment of rheumatoid arthritis (Kuang et al., 2001), nocturnalenuresis (Gao et al., 2002), etc. Anti-HIV (Ma et al., 2002) andanti-dementia (Sun and Zhang, 2001) effects have also been re-corded for the fruit of this plant.

Yellow horn is an important kind of oil crop in China because ofits abundant oil (55–70%) in the seeds and seed oil’s richness (85–93%) in the unsaturated fatty acids. Moreover, yellow horn seed oilis also a rich source of saponins. Recently, attention has beenincreasingly attracted to yellow horn seed oil as an excellent healthcare product, because it is effective for the treatment of hyperlipa-

ll rights reserved.

y of Forest Plant Ecology,y, Harbin 150040, PR China.

).

emia, arteriosclerosis, coronary heart disease and it can enhancethe microcirculation (Cui et al., 1987; Zhu et al., 1989). In addition,the high amount of unsaturated fatty acids may possess potentiallynotable anti-oxidant activity, hence, developing the utilization ofseed oil of yellow horn is of great significance. In fact, an interest-ing approach to enhance the value of seed oils from herbs is theirusage as potential sources of natural anti-oxidants (Simonetti et al.,2002). Yellow horn seed oil possesses the pharmaceutical potentialas high-quality healthy edible oil and as a valuable natural anti-oxidant. Therefore, the objectives of the present study are to inves-tigate the seed oil extraction procedure from yellow horn and toevaluate its anti-oxidant activity.

Generally, the conventional industrial methods of producingyellow horn seed oil include expeller pressing and conventionalsolvent extraction (mainly hexane). The former process is ofhigh-quality, but, in most cases, the yield is lower and it canmake the active component thermal degradation. The latterachieves almost complete recovery of the oil, however, causessolvent contamination and unacceptable as it is quite harmfulto human health and the environment, which may restrict itsuse in food, cosmetic and pharmaceutical industries. Supercriticalfluid carbon dioxide (SF-CO2) extraction (SFE) has been an excel-lent alternative for seed oil extraction to replace conventionalindustrial methods (Gomes et al., 2007; Lu et al., 2007). SFE be-comes the focus of attention especially in food, pharmaceuticaland cosmetic industries due to its chemical and physical proper-ties: lower critical pressure (74 bar) and temperature (32 �C),

2538 S. Zhang et al. / Bioresource Technology 101 (2010) 2537–2544

relatively non-toxic, non-flammable, lower costs, and easy elimi-nation from the extracts. In the supercritical state, CO2 revealspolarity comparable to liquid pentane and it is best suited forlipophilic compounds (Eikani et al., 1999). Furthermore, the ex-tracted product is of good quality and sparsely needs particularrefining operations. Therefore, CO2 extracts are generally recog-nized as safe (GRAS) to be used in food production (Gerard andMay, 2002). The SF-CO2 technology has been applied in the oilextraction from a large number of materials in food and pharma-ceutical processing with a high potential for future applications(King, 2000; Reverchon and Marrone, 2001).

In the present study, SFE of seed oil from yellow horn was stud-ied. The effects of main operating parameters, namely extractionpressure, extraction temperature, extraction time, CO2 flow rateand particle size, on the extraction yields of yellow horn seed oilwere investigated. The chemical composition of the seed oil wasanalyzed by GC–MS. Furthermore, anti-oxidant activity of seedoil under optimized conditions was determined by means of a2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assayand a b-carotene bleaching test.

2. Methods

2.1. Materials

Seeds of Xanthoceras sorbifolia Bunge. (Yellow horn) were col-lected during autumn in 2008 from Inner Mongolia AutonomousRegion, China, and identified by Prof. Shao-Quan Nie, Key Labora-tory of Forest Plant Ecology, Ministry of Education, Northeast For-estry University, Harbin, China. The age of plant materials wasseven years old. Seeds with hull were dried at 60 �C for 24 h inan oven. The moisture content of the seeds was determined as6.68%, and then milled to the desired particle size. All solvents usedin the analysis were of analytical grade.

2.2. SF-CO2 extraction

The SF-CO2 extraction (SFE) of seed oil from yellow horn wasperformed on an HA121-50-01 SFE device (Hua’an SupercriticalFluid Extraction Corp., Nan-tong, China) by means of a previouslydescribed procedure (Wang et al., 2007). Carbon dioxide (purity99.99%) was purchased from Liming Gas Corp. (Harbin, China).The operating methodology was as follows: liquid CO2 suppliedfrom a gas cylinder was cooled by ethanol to �5 �C before beingpressurized to the desired pressure and passed into the device.The entire device was also pre-pressurized. For each experiment,approximately 300 g seeds of yellow horn with the chosen particlesize were loaded into a steel cylinder equipped with mesh filters(13 lm) on both ends to prevent the particles being flushed out.The loaded cylinder was then introduced into the extraction vessel,and CO2 was let in. During the extraction process, the extractionpressure, extraction temperature and CO2 flow rate were con-trolled by adjusting the regulating valves on the front panel. Whenthe scheduled time was achieved, the extraction vessel wasdepressurized and the oil was collected from the separation vessel.The oil obtained under the optimal conditions was used for the fol-lowing tests. The amount of extracted oil was determined gravi-metrically after collection, and then the extraction yield isexpressed as the percent ratio of the mass of extracted oil to themass of yellow horn seed loaded in the extraction vessel, asfollows:

Extraction yield of seed oil ð%Þ¼ ðmass of extracted oil=mass of dried materialÞ � 100

2.3. Soxhlet extraction

A conventional method of Soxhlet extraction (SE) was per-formed to compare the extraction performances with SFE. Yellowhorn seeds (50 g) were added into a Soxhlet extractor. Approxi-mately, 400 mL n-hexane was added into the flask, which was con-nected to the extractor and condenser. The solvent flow rate wasmanually adjusted to 6 min/cycle, and the extraction process wasterminated after 100 cycles. The extraction was employed at opti-mal conditions (90 �C for 10 h, 0.5 mm particle size). After extrac-tions, n-hexane was removed at 50 �C under reduced pressureusing a rotary evaporator. Subsequently, the flasks were dried at105 �C under nitrogen stream protection in a drying oven for 1 h.The oils obtained were weighed and the yields were calculated.

2.4. Experimental design and statistical analysis

A 23 factorial portion central composite design (CCD) combinedwith response surface methodology was used for optimizingextraction pressure (X1), temperature (X2) and time (X3). In theCCD test, 14 experiments and six replicates at the center were em-ployed to fit the full quadratic equation model. The general equa-tion is:

Y ¼ b0 þXk

j¼1

bjXj þXk

j¼1

bjjX2j þ

XX

i<j

bijXiXj ðk ¼ 3Þ

where Y is the predicted response, b0, bj, bjj and bij are the regressioncoefficients for intercept, linearity, square and interaction, respec-tively, while Xi and Xj are the independent coded variables. The vari-ables of each factor were transferred to a range between �1 and 1for the appraisals, while the dependent variable was the extractionyield of oil. Coded variables were used according to the equation:

X0i ¼Xi � X0i

DXiði ¼ 1;2;3Þ

where Xi‘ is the coded value of the independent variable. Xi was the

real value of the independent variable. X0i was the real value of theindependent variable at the center point. DXi was the step change ofthe real value of variable ‘i’. The actual and coded levels of theindependent variables used in the experimental design are shownin Table 1.

During the entire experimental process, CO2 flow rate rangedfrom 2 to 20 kg/h. The extraction pressure varying from 15 to35 MPa and temperature varying from 25 to 65 �C were controlledby adjusting valves according to the meter on the front panel. Oncethe scheduled time was achieved, the extraction vessel wasdepressurized and the extracts were collected from the separationvessel. The accumulated product samples were collected and con-centrated to give the extracts mass, which was used for the follow-ing tests. All the experiments were repeated three times, and theextraction yields were given as average values.

The data collected from SFE tests were analyzed using a re-sponse surface analysis procedure (Design-Expert 7.1.3 Trial,State-Ease, Inc., Minneapolis MN, USA). Analysis of variance wasperformed for calculations and modeling of optimal conditionsfor SFE of yellow horn seed oil. Values of P < 0.01 were regardedas significant.

2.5. Preparation of fatty acid methyl esters (FAMEs)

Yellow horn seed oil (10 g) was added into a 100 mL three-neckreaction flask with magnetic stirrer and mixed with methanol at amethanol/oil molar ratio of 6:1. Then, the flask was placed in awater bath at 60 �C, and allowed to react for 40 min with KOH as

Table 1Results of the central composite design for the extraction of seed oil from yellow horn.

Runs Factors Extraction yieldof oil (%)

X1 (Pa, MPa) X2 (temp.b, �C) X3 (tc, h) Exp.d Pred.e

1 �1 (20) �1 (35) �1 (1.5) 40.55 40.722 �1 (20) �1 (35) 1 (2.5) 50.87 50.813 �1 (20) 1 (55) �1 (1.5) 49.35 49.644 �1 (20) 1 (55) 1 (2.5) 54.56 55.275 1 (30) �1 (35) �1 (1.5) 50.85 50.716 1 (30) �1 (35) 1 (2.5) 53.74 54.017 1 (30) 1 (55) �1 (1.5) 58.76 59.388 1 (30) 1 (55) 1 (2.5) 57.82 58.219 �1.682 (16.59) 0 (45) 0 (2) 48.37 47.98

10 1.682 (33.41) 0 (45) 0 (2) 59.25 58.8511 0 (25) �1.682 (28.18) 0 (2) 45.56 45.6912 0 (25) 1.682 (61.82) 0 (2) 50.66 56.7413 0 (25) 0 (45) �1.682 (1.16) 50.25 49.9714 0 (25) 0 (45) 1.682 (2.84) 57.98 57.4715 0 (25) 0 (45) 0 (2) 60.86 59.9916 0 (25) 0 (45) 0 (2) 59.95 59.9917 0 (25) 0 (45) 0 (2) 60.82 59.9918 0 (25) 0 (45) 0 (2) 59.98 59.9919 0 (25) 0 (45) 0 (2) 58.35 59.9920 0 (25) 0 (45) 0 (2) 59.83 59.99

a P is expressed as the extraction pressure (MPa).b Temp. is expressed as the extraction temperature (�C).c t is expressed as the extraction time (h).d Exp. is expressed as experimental value.e Pred. is expressed as predicted value.

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catalyst at a concentration of 1% wt. of oil. After reaction, the mix-ture was brought to room temperature and treated with chloro-form (2 mL) and 1 mL of water. The mixture was vigorouslyshaken. The organic phase was separated, 1.0 g Na2SO4 was added,and then analyzed by GC–MS.

2.6. Gas chromatography–mass spectrometry analysis

GC–MS analysis was performed using an Agilent HP6890N/5973 gas chromatography/mass spectrometer (Agilent, Santa Clara,CA, USA), equipped with an HP-5 silica capillary column(30 m � 0.32 mm i.d.; film thickness 0.2 lm). The column temper-ature was initially at 40 �C (held for 10 min) and then increased to200 �C at 15 �C/min, held for 2 min, and finally increased to 230 �Cat 10 �C/min. The mass spectrometer was operated in positive ionmode with ionization energy of 70 eV. Injector and detector tem-peratures were 280 and 290 �C, respectively, and the ion sourcetemperature was 230 �C. Helium was used as the carrier gas, andthe split ratio was 1:40. Mass units were monitored from m/z 35to 425. The components of oil were identified on the basis of com-parison of their retention indices and mass spectra with publishdata and computer matching with National Institute of Standardsand Technology (NIST, 3.0) libraries provided with computer con-trolling the GC–MS system (Sandra and Bicchi, 1987).

2.7. Determination of anti-oxidant activity

The seed oil obtained under optimal conditions was subjectedto analysis of its anti-oxidant activity using a 2,2-diphenyl-1-pic-rylhydrazyl (DPPH) radical-scavenging assay and a b-carotenebleaching test. All data were averages (± standard deviations) oftriplicate determinations of three independent tests.

2.7.1. DPPH radical-scavenging assayThe scavenging activity of seed oil towards DPPH radicals was

determined by the method of Amarowicz et al. (2004). An aliquotof seed oil (100 lL) was mixed with 1.4 mL of ethanol and then

added to 1 mL of 0.004% DPPH (Sigma–Aldrich) in ethanol. Themixture was shaken vigorously and immediately placed in anUNICO UV-2100 spectrophotometer (UNICO, Shanghai, China) tomonitor the decrease in absorbance at 517 nm. Monitoring wascontinued for 70 min until the reaction reached a plateau. Ascorbicacid (Sigma–Aldrich), a stable anti-oxidant, was used as a syntheticreference. The radical-scavenging activities of samples, expressedas percentage inhibition of DPPH, were calculated according tothe formula:

Inhibition percentage (Ip) = 100(AB � AA)/AB (Yen and Duh,1994)

where AB and AA are the absorbance values of the blank and of thetested samples, respectively, checked after 70 min.

2.7.2. b-Carotene bleaching testThe b-carotene bleaching test was conducted as described by

Taga et al. (1984) with minor modifications. Approximately10 mg of b-carotene (type I synthetic, Sigma–Aldrich) was dis-solved in 10 mL chloroform. The carotene–chloroform solution(0.2 mL) was pipetted into a boiling flask containing 20 mg linoleicacid (Sigma–Aldrich) and 200 mg Tween 40 (Sigma–Aldrich). Chlo-roform was removed under vacuum using a rotary evaporator at40 �C. Then, 50 mL distilled water were added to the residue undervigorous shaking to form an emulsion. The emulsion (5 mL) wasadded to a tube containing 0.2 mL of seed oil, and the absorbancewas immediately measured at 470 nm against a blank, consistingof an emulsion without b-carotene. The tubes were placed in awater bath at 50 �C and the oxidation of the emulsion was moni-tored spectrophotometrically by measuring the absorbance at470 nm over a 60 min period. Control samples contained 200 lLwater instead of seed oil. Butylated hydroxytoluene (BHT, Sigma–Aldrich), a stable anti-oxidant, was used as a reference. The anti-oxidant activity was expressed as inhibition percentage with refer-ence to the control after a 60 min incubation using the followingequation:

AA ¼ 100ðDRC � DRSÞ=DRC

(AA = anti-oxidant activity; DRC = degradation rate of the con-trol = [ln(a/b)/60]; DRS = degradation rate in the presence of thesample = [ln(a/b)/60], a = absorbance at zero time; b = absorbanceat 60 min).

3. Results and discussion

3.1. Optimization of SFE procedure

3.1.1. Effect of particle size and CO2 flow rateThe effect of particle size on the extraction yield was investi-

gated using mean particle sizes of 0.3, 0.5, 0.7, 1.0 or 2.0 mm at atemperature of 45 �C, a pressure of 25 MPa, and a CO2 flow rateof 12 kg/h. Fig. 1 shows the extraction time kinetics for each meanparticle size resulting in extraction yields of 62.05%, 57.98%,57.58%, 55.77% and 52.03%, respectively. This indicates that theparticle size had little influence on the global extraction yield. Dur-ing the initial extraction phase, a large slope was obtained for allfive extractions. At this stage only the operational conditions oftemperature and pressure were important, and the flow rate em-ployed was sufficiently low to promote solvent saturation. The glo-bal extraction yields showed that a reduction in particle size onlyslightly increased the extraction yields. Hence, a particle size of0.5 mm was selected for subsequent tests.

The effect of CO2 flow rate was studied at a temperature of45 �C, a pressure of 25 MPa, a particle size of 0.5 mm and an extrac-

Fig. 1. Effect of particle size on extraction yields versus different extraction time.Fig. 3. Effect of CO2 flow rate on the extraction yields versus the amount of CO2.

2540 S. Zhang et al. / Bioresource Technology 101 (2010) 2537–2544

tion time of 2 h. The CO2 flow rates were 2, 4, 6, 8, 10, 12, 14, 16, 18and 20 kg/h, respectively. It is apparent that the increase of the CO2

flow rate increased the amount of oil extracted (Fig. 2) within therange of 2–14 kg/h. After a CO2 flow rate of 12 kg/h was achieved,the extraction yields did not increase much further with increasingof CO2 flow rates. The mass transfer parameter increased withsupercritical fluid flow rates, until it reached a constant value. Sal-gin (2007) reported comparable results for SFE of jojoba seed oil.On the other hand, the amount of extracted oil per kg CO2 washigher at lower flow rates (Fig. 3) due to intra-particle diffusionresistance. This may explain the smaller slope of the extractioncurve at higher flow rates (Fig. 3).

Fig. 3 shows that the extraction kinetics was characterized bythree phases. The first phase (constant extraction rate) was charac-terized by extraction of oil more accessible to solvent at the super-ficial layers of the seed particles. The intermediate phase, theamount of solute available on the surface decreased, and the soluteinside the particle started diffusing to the surface. Finally, in thethird phase, exhaustion of the solute on the surface limited extrac-tion. Due to diffusion of solute from inside the particles, the extrac-tion rates decreased. Almost 70% of the total oil content wasrecovered during the constant extraction rate phase (linear),employing approximately 40% of the CO2. Subsequently, the

Fig. 2. Effect of CO2 flow rate on the extraction yields of seed oil from yellow horn.

remaining 30% oil content was recovered using 60% of the totalCO2 consumption. In the first phase, the surface of the materialwas completely covered by oil. Therefore, there was no resistanceto mass transfer, and the solubility of the oil was the only limitingfactor. In the second and third phases, intra-particle diffusion con-trolled oil transfer from the interior to the surface of the particles,the extraction is mass transfer controlled and the extraction rate islow. Under certain pressure, the slower the fluid rate, the deeper itpenetrates the matrix, and extraction process would take longertime. With the higher CO2 flow rate, the extraction time is shorter,but the consumption of CO2 would be increased, which is depictedin Fig. 3. Therefore, suitable CO2 flow rate is necessary while con-sidering the efficiency and cost of extraction. From the above re-sults, considering the oil yield, production cycle, andmanufacture cost, extraction at a CO2 flow rate of 12 kg/h was cho-sen as optimal condition in this study.

3.1.2. Optimization of SFE operating parametersThe objective of the present study was to optimizing the oper-

ating conditions to achieve an efficient extraction of seed oil fromyellow horn. Because various parameters potentially influence theSFE process, the optimization of the operating conditions plays acritical role in the development of a SFE method. Central compositedesign (CCD) combined with response surface methodology wasused for optimizing. According to the preliminary tests, the particlesize of material was sieved to 0.5 mm and a CO2 flow rate of 12 kg/h was employed. Three parameters including extraction pressure,temperature and time were optimized by a 23 factorial portioncentral composite design. All results obtained from 20 experimen-tal runs and the predicted data from the model based on the exper-imental data were summarized in Table 1. Three-dimensionalprofiles of multiple non-linear regression models were used to de-pict the interactive effects of operational parameters for the seedoil (Fig. 4A–C).

The effects of pressure and temperature are shown in Fig. 4A.Four parameters are critical to understand the solute behavior insupercritical media and, thus, to successfully perform supercriticalfluid extractions: (1) the miscibility or threshold pressure (Gid-dings et al., 1968, 1969), which corresponds to the pressure atwhich the solute partitions into the supercritical fluid, (2) the pres-sure at which the solute reaches its maximum solubility, (3) thefractionation pressure range, which is the pressure region betweenthe miscibility and solubility maximum pressures (in this intervalit is possible to selectivity extract one solute by choosing the cor-

Fig. 4. Response surfaces representations for oil (A–C) from yellow horn seed. (A)Varying extraction temperature and pressure; (B) varying extraction pressure andtime; (C) varying extraction temperature and time.

S. Zhang et al. / Bioresource Technology 101 (2010) 2537–2544 2541

rect pressure) and (4) a knowledge of the physical properties of thesolute, particularly its melting point (in fact most solutes dissolvebetter when they are in their liquid state, i.e. above their meltingpoint).

The extraction pressure is the main parameter that influencesthe extraction efficiency. It was observed that the yield of oil signif-icantly increased with increasing pressure at a given temperature,especially at low pressure and temperature (Fig. 4A). If the giventemperature was higher than a certain value (about 45 �C), whilepressure was rising, the oil yield increased at low-pressure levels.Once the pressure reached high levels, the oil yield slightly de-creased. This can be explained by an increase in supercritical CO2

density resulting from elevation of this pressure, which means anenhanced solubility of solutes. However, high pressure is not al-

ways recommended due to increased repulsing solute–solventinteractions resulting from highly compressed CO2 at high-pres-sure levels, which potentially induce complex extraction and diffi-cult analysis (Clifford, 1999; Pourmortazavi and Hajimirsadeghi,2007). On the other hand, it has to be taken into consideration thatthe presence of co-extracted solutes can dramatically change thesolubility level of the solute of interest.

The influence of temperature on extraction was more difficult topredict than that of pressure, because of its two counter effects onthe yield of oil. First, the temperature elevation decreased the den-sity of CO2, leading to a reduction in the solvent power to dissolvethe solute. Second, the temperature rise increased the vapor pres-sure of the solutes, bringing about the elevation in the solubility ofoils in SF-CO2. Consequently, the solubility of the solute was likelyto decrease, kept constant, or increased with rising temperatures atconstant pressure (Thana et al., 2008), which depended on whetherthe solvent density or the solute vapor pressure was the predomi-nant one. As presented in Fig. 4A, an increase in oil yield was ob-served by increasing the temperature in an early stage ofextraction, because the change of solvent density was less effectivethan that of solute vapor pressure. The trend was, however, re-versed, when the temperature reached a certain value under therange of pressure in this work. Similar phenomena were also re-ported for the extraction of other lipids by SF-CO2 (Wang et al.,2008). Since the solubility of lipids depended largely on the bal-ance between fluid density and solute vapor pressure, both con-trolled by fluid pressure and temperature, an extractiontemperature and pressure in the range from 42 to 50 �C, 25 to35 MPa, respectively, was practical during SFE of yellow horn oil.

Fig. 4B shows the three-dimensional plot of the response sur-face for the oil yield as related to extraction pressure and time.Fig. 4C describes the interactive effect of extraction temperatureand time. Extraction time exhibited an important effect on theoil yield, but the influence of this parameter was not as significantas that of extraction pressure and temperature. It did not continueto significantly increase until the extraction time was over120 min. Although prolonging the contact time led to a slight in-crease in the oil yield, further increasing the extraction times upto 150 min resulted in little change in the yield of oil.

For increased pressure from 20 to 30 MPa together with an in-crease of extraction time from 1.5 to 2.5 h, the oil extraction yieldwas enhanced in different slopes. As shown in Fig. 4B, it was ob-served that the yield of oil significantly increased with increasingtime at a lower pressure, but slightly enhanced at higher pressure.This can be explained by an increase in supercritical CO2 densityresulting from an elevation of this pressure, which means an en-hanced solubility of solutes. If this occurred at the initial phase,the reaction time was reduced.

Fig. 4C shows that with increasing temperatures from 35 to55 �C along with an increase in extraction time from 1.5 to 2.5 h,the oil extraction yields were not further enhanced. When the tem-peratures were accelerated, the amount of oil extracted first in-creased, but began to decrease when the temperature surpassed50 �C. This indicates that an increase in temperature acceleratedthe mass transfer ratio from plant material to solvent until a cer-tain value, thus increasing the extraction yield. However, theextraction yields of seed oil decreased slightly after 2 h extractionwith a temperature of 55 �C. The oil yield can be adjusted bychanging the temperature because of its effect both on decreasingthe CO2 density and on increasing the vapor pressure of the solutes,both of which are detrimental and advantageous consequences,respectively.

As shown in Table 1, the extraction yields of oil were similar tothose expected, and the results demonstrated the accuracy of thepredictive model. According to the analysis of variance (ANOVA),correlation coefficients (R2) of 0.953, were obtained for oil with

2542 S. Zhang et al. / Bioresource Technology 101 (2010) 2537–2544

the calculated model. In addition, highly significant levels(P < 0.001) were obtained by statistical analysis. Eq. (1) showsthe relationship between extraction yield and extraction pressure,temperature and time:

Y ¼ �196:9812þ 6:1878X1 þ 4:3913X2 þ 62:7883X3

� 0:0013X1X2 � 0:6790X1X3 � 0:2235X2X3 � 0:0825X21

� 0:0408X22 � 7:8230X2

3 ð1Þ

where Y is the extraction yield (% dry weight of seed), X1 is theextraction pressure (MPa), X2 is the extraction temperature (�C)and X3 is the extraction time (h).

The optimum levels of the tested parameters were obtained bysolving Eq. (1). The predicted values were a pressure of 30 MPa, atemperature of 45.68 �C and a time of 2.08 h. The maximum pre-dicted extraction yield was 61.28%. Verification experiments werecarried out for five times under these optimal conditions and a CO2

flow rate of 12 kg/h and a particle size of 0.5 mm. The resultingmean extraction yield was 61.16% with a relative standard devia-tion (RSD) of 1.06%.

3.2. Comparison of SFE with SE

When the optimization tests were completed, the SFE wasshown to be an efficient method with high extraction yield(61.2%) at low pressure (30 MPa), short reaction time (2.08 h)and low temperature (45.68 �C) without any toxic solvent.Although SFE has received considerable attention for the extractionof seed oils, SFE of seed oil from yellow horn has not been reportedyet. Hence, a comparison of SFE and SE regarding their perfor-mances of seed oil extraction from yellow horn is indispensable.

As can be seen in Table 2, the extraction yield of SE reached atleast 62.3% under the optimized conditions. Meanwhile, we founda similar extraction yield by SFE for 2 h at 45 �C compared with

Table 2Main extraction conditions of yellow horn seed oil by different methods.

Method Solvent Particle size (mm) Pressure (MPa)

SFE CO2 0.5 30SE n-Hexane 0.5 –a

a SE was carried out under the atmospheric pressure.

Table 3Fatty acid profiles and relative contents of seed oil from yellow horn.

No. Component Relative content (%) Standard deviatio

1 Hexadecanoic acid 6.12 0.032 9,12-Octadecadienoic acid 38.62 0.113 9-Octadecenoic acid 25.34 0.124 Octadecanoic acid 3.25 0.055 11-Eicosenoic acid 10.61 0.136 Eicosanoic acid 0.42 0.017 13-Docosenoic acid 10.17 0.278 Docosanoic acid 0.72 0.029 15-Tetracosenoic acid 3.23 0.0710 Tetracosanoic acid 0.41 0.01

Table 4Comparison of main fatty acid contents of yellow horn oil by different methods.

Method C16:0 C18:0 C18:1 C18:2

SFEa 6.12 3.25 25.34 38.62SEa 6.87 4.13 23.66 37.23

a It was obtained under optimized conditions.

that of SE for 10 h at 90 �C, and there is no toxic solvent consumedin SFE. Although the cost of SFE would be a little bit higher thanthat of SE because it requires a mass of CO2 and the relatively highpressure. SFE, as a green extraction method, has attracted muchattention for its advantages including being non-explosive, non-toxic, and available in high purity with non-solvent residues. Inaddition, the CO2 is easily removed from the extract when pressureand temperature are reduced below its critical condition. Fromthese results, it was concluded that application of SFE offers a fast,environmentally friendly and easy route of oil production. Theadvantages of SFE were conductance in shorter time, relativelylow temperatures as well as no requirement for toxic solvents.Consequently, supercritical fluid carbon dioxide extraction repre-sents a promising alternative for the extraction of oils from naturalproducts.

3.3. Chemical composition of yellow horn seed oil

The fatty acid profiles of seed oil from yellow horn were ana-lyzed by GC–MS. The major fatty acid composition of yellow hornoil prepared by SFE is shown in Table 3, and the comparison ofmain fatty acid contents of yellow horn oil by different methodsis shown in Table 4. The results indicate that the yellow horn oilis rich in unsaturated fatty acids (nearly 90% of the total fattyacids), and the polyunsaturated fatty acids (PUFA) constitute52.46%. The yellow horn oil contains much higher proportion of11-eicosenoic acid (10.61%) than the normal edible oils such as oilsof soybeans and safflower (Han et al., 2009). The dietary fats abun-dant in PUFA can prevent disorders such as atherosclerosis coro-nary heart disease and high blood pressure. 9-octadecenoic acid(oleic acid), belonging to monounsaturated fatty acids (MUFA), isthe second most abundant in yellow horn oil (approximately25%). The main saturated acids in the yellow horn oil are hexadeca-noic acid (palmitic acid) and octadecanoic acid (stearic acid). It can

Temperature (�C) Reaction time (h) Conversion yield (%)

45.68 2.08 61.16 ± 0.6590 10 62.31 ± 0.59

n Molecular formula Molecular weight Degree of similarity (%)

C16H32O2 256 95C18H32O2 280 99C18H34O2 282 98C18H36O2 284 99C20H38O2 310 96C20H40O2 312 98C22H42O2 338 99C22H44O2 340 97C24H46O2 366 99C24H48O2 368 95

C20:1 SFA MUFA PUFA Others

10.61 10.92 35.51 52.46 1.1110.12 13.15 32.83 50.05 3.97

S. Zhang et al. / Bioresource Technology 101 (2010) 2537–2544 2543

be found that the fatty acids contents of yellow horn oil obtainedfrom SFE are similar to those from SE, but both contents of MUFAand PUFA in oil obtained by SFE are higher than those in oil ob-tained by SE. Meanwhile, the contents of saturated acids and otherimpurities in oil obtained by SE are higher than those in oil ob-tained by SFE. The results indicated that oil obtained by SFE is ofbetter quality than that obtained by SE.

Fig. 6. Anti-oxidant activity of seed oil from yellow horn assessed by a b-carotenebleaching test. BHT at a concentration of 4 mg/mL was regarded as control test.

3.4. Anti-oxidant activity of seed oil

Anti-oxidant activities of yellow horn seed oil prepared by SFEwere tested by two complementary test systems, namely 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay and b-carotene bleaching test with the references of anti-oxidants ascor-bic acid and BHT, respectively. The concentration of sample pro-ducing a 50% reduction of the radical absorbance (IC50) was usedas an index to compare the anti-oxidant activity.

The samples were assayed over a range of dilutions and the re-sults of the DPPH radical-scavenging assay were presented inFig. 5. Seed oil from yellow horn exhibited notable DPPH radical-scavenging activity with an IC50 value of 0.151 g/mL, and the bestefficacy was slightly lower than that of the reference ascorbic acid(95.32 ± 1.03%, 4 mg/mL). The values for seed oil from yellow hornranged from 14.04 ± 1.07% to 89.04 ± 1.42% when the concentra-tions varied from 0.029 to 0.920 g/mL, accordingly. Moreover,decreasing the concentration of seed oil resulted in a reductionof its anti-oxidant ability. The DPPH radical-scavenging activityof seed oil from yellow horn obtained in the present study washigher than some reported oil (Miraliakbari and Shahidi, 2008).

We also assessed the lipid peroxidation inhibitory activity ofthe yellow horn seed oil by a b-carotene bleaching test (Fig. 6).The results were consistent with the data obtained from the DPPHtest. Compared with BHT (94.68 ± 2.12%, 4 mg/mL), the seed oilfrom yellow horn revealed a good activity (83.03 ± 2.56%) withthe IC50 value of 0.195 g/mL. The inhibition ratios of seed oil rangedfrom 11.13 ± 0.76% to 83.03 ± 2.56% when the concentrations var-ied from 0.029 to 0.920 g/mL, accordingly. It seemed the anti-oxi-dant activities of all the tested samples were mostly related to theirconcentrations. The lipid peroxidation inhibitory activity of seedoil was weaker than the synthetic anti-oxidant BHT. However,the inhibition ratio of seed oil from yellow horn obtained in thepresent study was higher than some reported oil which had been

Fig. 5. Anti-oxidant activity of seed oil from yellow horn assessed by a DPPHradical-scavenging assay. Ascorbic acid at a concentration of 4 mg/mL was regardedas control test.

studied with the b-carotene bleaching test (Miraliakbari and Shah-idi, 2008).

In both test systems, we can find the oil exhibited remarkableanti-oxidant activity. In general, the fatty acid composition of theoil makes it special, because it contains a large amount of unsatu-rated fatty acids, nearly 90% of the total oil. It can be found approx-imately 35.51% of MUFA and 52.46% of PUFA. PUFA is known asessential fatty acids from the diet for human health because it can-not be produced in the human body. Alpha-linolenic and linoleicacids are the typical PUFA obtained from vegetable oils. These fattyacids can be elongated and desaturated into their longer-chain der-ivates, arachidonic and eicosapentaenoic acids (EPA) and docosa-hexaenoic acid (DHA). Several studies have strongly suggestedthat these fatty acids and some trace components are importantin relation to the anti-oxidant activity of the oil (Lorgeril and Salen,2004; Simopoulos, 2002; Ramaprasad et al., 2006). However, it isvery difficult to attribute the anti-oxidant effect of the total oil toone or a few active constituents, because a kind of oil always con-tains a mixture of different chemical constituents. In addition tothe major constituents, also minor constituents may make a signif-icant contribution to the oil’s activity. From the above results, wecould infer that the seed oil from yellow horn extracted by SFE pos-sesses better anti-oxidant activity, which is attributed to the coop-erating results of their compositions.

4. Conclusions

In the present study, the green SFE process was optimized toachieve an efficient extraction of seed oil from yellow horn. Theanti-oxidant activities of the resulting extracts have been evalu-ated by a DPPH radical-scavenging assay and a b-carotene bleach-ing test. Based on the results, we conclude that SFE represents avaluable alternative to the traditional SE for the efficient extractionof oil from yellow horn. Meanwhile, the present investigation indi-cates the seed oil from yellow horn extracted by SFE may play a po-tential role as health-promoting anti-oxidant agent in human dietswith economical potential for the pharmaceutical industry.

Acknowledgements

The authors gratefully acknowledge the financial supports byNational Natural Science Foundation of China (30770231), Hei-longjiang Province Science Foundation for Excellent Youths

2544 S. Zhang et al. / Bioresource Technology 101 (2010) 2537–2544

(JC200704), Agricultural Science and Technology AchievementsTransformation Fund Program (2009GB23600514), Key Project ofChinese Ministry of Education (108049), Innovative Program forImportation of International Advanced Agricultural Science andTechnology, National Forestry Bureau (2006-4-75), and Key Pro-gram for Science and Technology Development of Harbin(2009AA3BS083).

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