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J. of Supercritical Fluids 95 (2014) 318–324

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

j o ur na l ho me page: www.elsev ier .com/ locate /supf lu

henolic and flavonoid content and antioxidants capacity ofressurized liquid extraction and perculation method from roots ofcutellaria pinnatifida A. Hamilt. subsp alpina (Bornm) Rech. f.

. Golmakania, A. Mohammadib, T. Ahmadzadeh Sanic, H. Kamalib,∗

Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, IranResearch Center of Natural Products Health, North Khorasan University of Medical Sciences, Bojnurd, IranSchool of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

r t i c l e i n f o

rticle history:eceived 12 July 2014eceived in revised form5 September 2014ccepted 15 September 2014vailable online 28 September 2014

a b s t r a c t

The extract was separated from roots of Scutellaria pinnatifida using perculation and pressurized liquidextraction (PLE). A circumscribed central composite (CCC) was used to optimize the effective extractionvariables. For achieving maximum extraction yield via PLE the temperature, pressure, static time, dynamictime, and the solvent flow rate were adjusted 65.8 ◦C, 39.2 bar, 12.9 min, 18.9 min, and 0.76 mL/min,respectively. Ferric reducing antioxidant power (FRAP) (mmol/g) and 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical scavenging activity (mg/mL) were evaluated and the highest antioxidant activity wasobserved from the PLE extract. The total phenolic and flavonoid content was calculated and a good corre-

eywords:cutellaria pinnatifidaressurized liquid extractionhenoliclavonoiderric reducing antioxidant power

lation founded between phenolic content and antioxidant activity. The results indicated the root of thisplant is a potential source of natural antioxidants and flavonoids. The PLE method is quicker and it hasmore extraction yield than perculation.

© 2014 Elsevier B.V. All rights reserved.

PPH scavenging activity

. Introduction

Pressurized liquid extraction (PLE, Dionex trade name of accel-rated solvent extraction (ASE)) is an efficient, rapid, selective, andeliable extraction method [1]. PLE technology and its applicationsave been developed and used by various scientists [2–6]. More-ver, it has been applied for the quantitative extraction of differentamples of environmental organic compounds from soils and lipids,nd also for the analysis of food and biological samples [6]. Theossibility of changing several extraction variables such as temper-ture, pressure, and volume of solvent is a promising characteristicf available automated PLE instruments [7]. Using a modified super-ritical fluid extraction (SFE) apparatus, we have developed a PLEethod, mainly as an analytical tool, to determine oil content or

s a sample preparation method for the oil quality measurementsith full capability of automation when the workload is high [8].

pecial emphasis is placed on obtaining a rapid, selective, efficient,nd reliable extraction process.

∗ Corresponding author. Tel.: +98 584 2248004; fax: +98 584 2247202.E-mail addresses: h.kamali@nkums.ac.ir, h.kamali62@yahoo.com (H. Kamali).

ttp://dx.doi.org/10.1016/j.supflu.2014.09.020896-8446/© 2014 Elsevier B.V. All rights reserved.

The genus of Scutellaria belong to the Lamiaceae family containsmore than 300 species spread throughout the world [9,10] and has20 species and two hybrids in Iran [11] 10 species and two hybridsare endemic in this country [12]. One of the Iranian species ofScutellaria is Scutellaria pinnatifida that has two subspecies: Scutel-laria pinnatifida A. Hamilt. ssp. pinnatifida and Scutellaria pinnatifidaA. Hamilt. ssp. alpina [10,13] and the Persian name of this plant isBoshghabi [13]. This genus is well known for using in treatmentof hypertension, arteriosclerosis, inflammatory, hepatitis, allergy,cancer, anxiety and sleeplessness [13], this genus also is a seda-tive, tonic, and anti-spasmodic agent [14–16]. The dried roots ofthis genus administered in Chinese traditional herbal medicine [17]and they have antioxidant [18], anti-inflammatory [19], sedative[20], antiviral [21] and antithrombotic [22] activity. Flavonoids arethe main components of this genus [23]. The major bioactive com-ponents in roots of Scutellaria baicalensis were baicalein, baicalin,wogonin [24]. Baicalin has anti-inflammatory, anti-allergic, antiox-idant and hepatoprotective activities [25]. The flavonoids suchas baicalin, baicalein and wogonin extracted from the genus of

Scutellaria [23] have been shown to possess antilipoperoxidantand antiinflammatory activities [26,27] and demonstrated cyto-toxic to various human tumor cell lines in vitro and in vivo [28].In last years, flavonoids in Scutellaria have been widely studied for

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heir chemical properties and biological activities [29]. The hydro-istilled essential oil from dried aerial parts of S. pinnatifida A.amilt. ssp. alpina grown in Khorassan province was analyzed and

he major components of the oil were germacrene-D and beta-aryophyllene [30]. Free-radical-scavenging activity, antibacterialctivity and brine shrimp toxicity of different extracts of aerialarts of S. pinnatifida were assessed and the dichloromethane andethanol extracts exhibited free-radical-scavenging activity [31].Utilization of effective antioxidants with natural origin is

esired and some chemical compounds are safe antioxidants. Therere no reports about phytochemical and biological studies carriedut on roots of S. pinnatifida A. Hamilt. ssp. alpina native fromran and the aim of the present study focuses on determinationotal phenolic and flavonoids from roots of S. pinnatifida A. Hamilt.sp. alpina and investigation of their antioxidant activities via PLEnd perculation method. The antioxidant properties were investi-ated using two tests: ferric reducing antioxidant power (FRAP)nd 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging. Aircumscribed central composite method (CCC) was used to guidexperimentation of extraction and a mathematical model was con-tructed to investigate the relationship between the extractionield, total phenolic, total flavonoid, and antioxidants activity andhe variables influencing extraction such as temperature, pressure,tatic time, dynamic time, and flow rate. The validity of the modelas evaluated through the analysis of variance (ANOVA) technique

y using Minitab software [32,33].

. Materials and methods

.1. Chemicals and reagents

Methanol (CHROMASOLV, ≥99.9%, Sigma–Aldrich), Folin-iocalteau reagent (F9252, Sigma–Aldrich), Na2CO3 (451614,nhydrous powder, 99.999%, Sigma–Aldrich), Gallic acid91215, Fluka), Aluminum Chloride (563919, anhydrous powder,9.999%, Sigma–Aldrich), 2,2-diphenyl-1-picrylhydrazyl (257621,igma–Aldrich), Quercetin (Q4951, ≥95%, Sigma–Aldrich),scorbic acid (A1300000, European Pharmacopoeia (EP) Ref-rence Standard, Fluka), Butylated Hydroxy Toluene (BHT)W218405 ≤ 99, Sigma–Aldrich), 2,4,6-tripyridyl-s-triazine (TPTZ)T1253 for spectrophotometric, ≥98%, Sigma), FeSO4.7H2O (21542,99.0%, Sigma–Aldrich)

.2. Preparation of Scutellaria pinnatifida

The roots of S. pinnatifida A. Hamilt. ssp. alpina were collectedrom Tighbal mountain (2700 m) in Darkesh valley in Bojnurd,orth Khorasan province of Iran in Jun 2013. The plant was identi-ed from the Ferdowsi University of Mashhad Herbarium. Voucherpecimen (No. 11868) was deposited in the Herbarium of Schoolf Pharmacy, Mashhad University of Medical Sciences. The rootsere dried at 40 ◦C for a period of 3 h prior to extraction. In the

nd of the normal drying process of roots, the water residue wasround 10.2%. Following the extraction procedures flowers werenely grinded using laboratory equipments [33]. Since extractioninetics in this study was controlled by the kernel particle size,n important sieving step was carried out to achieve reproduciblextraction yield in which the samples were passed through a sieveith mesh sizes between 20 and 30 (particle diameters ranging

ver 0.60–0.85 mm). The dried samples were kept within sealedag in the cold and dry place until they were used.

.3. Perculation extraction

The dried roots (100 g) were perculated with 1.0 L of methanolMeOH) at room temperature. The whole extract was filtered and

al Fluids 95 (2014) 318–324 319

solvent was evaporated under vacuum at 40 ◦C to afford 10 g extract(yield 10%) [34].

2.4. PLE procedure

The equipment used for PLE is very similar to that used in super-critical fluid extraction. Since CO2 must be liquefied by using coolercirculator device prior to its pumping. It would be easier to operatewith methanol than CO2. Most published studies described a similararrangement for the PLE equipment [32,33]. For this research, theapparatus was modified using a switching valve after the pumps toenable pumping of the liquid solvent and CO2, alternatively into theextraction vessel, and the back pressure regulator [33]. The appara-tus used for PLE is shown in Fig. 1. The prepared S. pinnatifida sample(∼30 g) was loaded into the 100 mL cylindrical stainless steel cell.Commonly used PLE and SFE methods Cotton wool was packed atthe exit end of the cell to prevent transfer of solid samples to thetubing and clogging of the system [32]. The PLE method was per-formed dynamically by passing methanol at different flow rates,temperatures, pressures, and times through the extraction cell. Themethanol residue in the cell, tubing, and back pressure regulatorwas removed with purging the PLE system with CO2 at the end ofeach extraction. To reach the mentioned goal, firstly the CO2 shouldbe converted into the supercritical state and after that, it will dis-solve any contamination remaining in PLE system. Methanol waspumped into the system to wash the tubing, every time the systemtubing was clogged [33].

2.5. DPPH radical scavenging assay

The antioxidant activity of the extracts was described by 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging capacityof the extracts [35]. DPPH radical scavenging ability of the extractswas evaluated by the method of Singh et al. [36]. DPPH wasfreshly prepared in methanol to 0.1 mM. Extracts were dissolvedin methanol to several concentrations (8, 4, 2, 1, 0.5, 0.25 mg/mL)and 0.1 mL of the solution and 3.9 mL of DPPH solution weremixed and were kept in darkness for 30 min and the absorbancewas read at 517 nm. The experiment was carried out in tripli-cate. The percentage of radical scavenging activity was calculatedfrom this equation: % DPPH radical scavenging = [(Absorbance ofcontrol − Absorbance of Sample)/(Absorbance of control)] × 100.Methanol was used as blank, ascorbic acid and butylated hydroxytoluene (BHT) and Quercetin used as positive controls. AbsorbanceInhibitor (AI) was calculated as IC50 values were calculated usingGraphPad Prism version 5.01 software [37].

2.6. Ferric reducing antioxidant power (FRAP) assay

The ferric reducing antioxidant power (FRAP) reagent con-tained 10 mM TPTZ solution in 40 mM HCl, 20 mM FeCl3·6H2O and0.3 M acetate buffer with pH 3.6 [38]. Three mL freshly preparedFRAP reagent mixed with 100 �L of each sample was incubated at37 ◦C for 10 min in a water bath. After incubation, the absorbancewas measured at 593 nm. Aqueous solutions of known Fe(II) con-centration, in the range of 0–1 mM (FeSO4·7H2O), were used forcalibration. One milliliter of several concentrations of FeSO4 plus1 mL of 10 mM TPTZ and 10 mL of 300 mM acetate buffer (pH 3.6)were used for a calibration curve. FRAP values were expressed asmean ± standard error (SE) mmol Fe(II) per gram.

2.7. Determination of total phenolic content

The total phenolic content in the extract from roots of S. pinnat-ifida was determined using Folin-Ciocalteu phenol reagent method[39]. Briefly, 100 �L extract (1000 mg/L) was mixed with diluted

320 E. Golmakani et al. / J. of Supercritical Fluids 95 (2014) 318–324

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ig. 1. Schematic diagram of PLE system: (1) CO2 tank; (2) molecular sieve filter; (alves; (6) water vessel; (7) high-pressure piston pump; (9) three ways valve; (10) p15) sample collection vessel.

olin-Ciocalteau reagent (1/10, 500 �L). After 1 min of reaction,odium carbonate (Na2CO3) (20%, 1.5 mL) was mixed to each tube,hen tubes were vortexed and incubated for 120 min at room tem-erature. The absorbance was read at 760 nm using Ultra Violetisible (UV–vis) spectrophotometer. The analyses were performed

n triplicates. The standard curve was prepared using 50 to 500 mg/Lolutions of gallic acid in methanol. Total phenol values werexpressed as gallic acid equivalents (mg gallic acid: (GA) per dryeight of extract).

.8. Determination of total flavonoid content

Flavonoid quantification was done using aluminum chloride col-rimetric method [40]. Plant extracts (0.5 mL) were mixed with.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 Motassium acetate and 2.8 mL of distilled water and were incubatedt room temperature for 30 min and then the absorbance of theeaction mixture was measured at 415 nm. All experiments wereepeated three times and values were expressed in mean standardeviation in terms of flavonoid content (Quercetin equivalent: QEer dry weight of extract). The calibration curve was prepared using2.5–100 �g/mL of Quercetin in methanol.

.9. Experimental design

A statistical experimental design based on “circumscribed cen-ral composite” was planned [32] and the extraction yield were

easured for different variables such as temperature, pressure,tatic time, dynamic extraction time, and flow rate of methanoloded as x1, x2, x3, x4, and x5, respectively. These variables werenvestigated at five levels (−2, −1, 0, 1, 2) and the dependent vari-bles were Y. We used the Minitab software package to designnd evaluate these five independent variables at five levels on theesponses according to the Eq. (1). The experimental extractionield, total phenolic, total flavonoid, antioxidants capacity (DPPHC50 and FRAP value) for different selected levels of variables ishown in Table 1 for 32 runs.

= ˇ0 + ˙ˇj · Xi + ˙ˇjj · X2j + ˙ˇjk · Xj · Xk (1)

here Y = response, ˇ0 = intercept, ˇj = linear coefficients,jj = squared coefficients, ˇjk = interaction coefficients, Xi, X2

j,

j, Xk = level of independent variables.

2 �m pore size filter; (4) carbon dioxide transfer pump; (5, 8, 13) two-way needleing coil; (11) extraction cell; (12) thermostated oven; (14) back-pressure regulator;

3. Results and discussion

3.1. PLE optimum conditions

For maximum PLE yield (12.21%), maximum total phenolic(396.94 mg/g), maximum total flavonoid (127.78 mg/g), minimumDPPH IC50 (0.86 mg/mL), and maximum FRAP value (34.9 mmol/g)the temperature, pressure, static time, dynamic time, andmethanol flow rate were 65.8 ◦C, 39.2 bar, 12.9 min, 18.9 min, and0.76 mL/min, respectively.

A second-order polynomial equation is proposed for the predic-tion of PLE yield, total phenolic, total flavonoid, minimum DPPHIC50, FRAP value as a function of different variables as follows:

Yy = −40.9363 − 30.2900 T + 0.6129 P + 1.3106 ts + 1.9705 td

+ 2.4576 f − 4.4391 T2 − 0.9240 P2 − 0.6252 t2s

− 1.0381 t2d − 1.1375 f 2 (2)

Ytp = −1330.80 − 984.70 T + 19.93 P + 42.61 ts + 64.06 td

+ 79.89 f − 144.31 T2 − 30.03 P2 − 20.32 t2s

− 33.75 t2d − 36.98 f 2 (3)

Ytf = −430.590 − 318.605 T + 6.477 P + 13.785 ts + 20.727 td

r + 25.850 f − 46.693 T2 − 9.719 P2 − 6.576 t2s

− 10.919 t2d − 11.964 f 2 (4)

Yic = 8.09191 + 4.12121 T − 0.08393 P − 0.17888 ts + 0.26871 td

− 0.33463 f + 0.60402 T2 − 0.12593 P2 − 0.08507 t2s

+ 0.14132 t2d + 0.15482 f 2 (5)

Yfr = −117.553 − 86.980 T + 1.760 P + 3.763 ts + 5.658 td

+ 7.057 f − 12.747 T2 − 2.653 P2 − 1.795 t2s

− 2.981 t2d − 3.266 f 2 (6)

where y is extraction yield, tp is total phenolic, tf is total flavonoids,ic is DPPH IC50, fr is FRAP value, T is extraction temperature, P is

extraction pressure, ts is static time, td is dynamic time, and f ismethanol flow rate.

The results of the statistical analyses including the estimatedregression coefficients, and p-values of the PLE yield, total phenolic,

E. Golmakani et al. / J. of Supercritical Fluids 95 (2014) 318–324 321

Table 1Yield, total phenolic, total flavonoid, FRAP, and DPPH IC50 radical scavenging activity for the different selected levels of variables.

Run T (◦C) P (bar) ts (min) td (min) f (mL/min) Yield (w/w %) Total phenolic(mg/g)

Total flavonoid(mg/g)

FRAP (mmol/g) DPPH IC50

(mg/mL)

1 40 20 5 10 0.8 1.839 59.784 19.343 5.280 2.2732 70 20 5 10 0.4 3.546 115.276 37.298 10.182 2.0413 40 40 5 10 0.4 0.443 14.401 4.659 1.271 2.4634 70 40 5 10 0.8 7.099 230.781 74.670 20.384 1.5575 40 20 15 10 0.4 0.352 11.443 3.702 1.010 2.4756 70 20 15 10 0.8 6.095 198.142 64.109 17.501 1.6947 40 40 15 10 0.8 3.550 115.406 37.340 10.193 2.0408 70 40 15 10 0.4 4.907 159.521 51.613 14.090 1.8559 40 20 5 20 0.4 2.480 80.622 26.085 7.121 2.186

10 70 20 5 20 0.8 8.209 266.866 86.345 23.572 1.40611 40 40 5 20 0.8 5.770 187.576 60.691 16.568 1.73812 70 40 5 20 0.4 6.655 216.347 70.000 19.110 1.61813 40 20 15 20 0.8 4.907 159.521 51.613 14.090 1.85514 70 20 15 20 0.4 6.735 218.948 70.841 19.339 1.60615 40 40 15 20 0.4 3.734 121.388 39.275 10.722 2.01516 70 50 15 20 0.8 11.665 379.217 122.697 33.496 0.93517 25 30 10 15 0.6 0.272 8.842 2.860 0.780 2.48618 85 30 10 15 0.6 7.802 253.635 82.064 22.403 1.46119 55 10 10 15 0.6 8.026 260.917 84.420 23.046 1.43120 55 50 10 15 0.6 9.696 315.207 101.986 27.842 1.20421 55 30 0 15 0.6 7.008 227.823 73.713 20.123 1.56922 55 30 20 15 0.6 8.849 287.672 93.077 25.410 1.31923 55 30 10 5 0.6 2.988 97.136 31.428 8.579 2.11724 55 30 10 25 0.6 9.566 310.981 100.619 27.468 1.22125 55 30 10 15 0.2 3.301 107.312 34.721 9.478 2.07426 55 30 10 15 1.0 8.458 274.961 88.965 24.287 1.37227 55 30 10 15 0.6 9.372 304.999 98.683 26.940 1.24628 55 30 10 15 0.6 10.389 337.736 109.276 29.832 1.10929 55 30 10 15 0.6 10.115 328.828 106.393 29.045 1.146

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30 55 30 10 15 0.6 9.5331 55 30 10 15 0.6 9.8132 55 30 10 15 0.6 9.25

otal flavonoid, minimum DPPH IC50, FRAP value were tabulated inable 2. The R2 adjusted of the yield, total phenolic, total flavonoid,PPH IC50, FRAP value were 0.9838. This means that the devel-ped models have been able to fully predict the extraction yield.he linear regression coefficients, R2 for the yield, total phenolic,

otal flavonoid, DPPH IC50, FRAP value were 0.9544 which showood performance of the model based on the observed and pre-icted extraction yield, total phenolic, total flavonoid, DPPH IC50,RAP value. The smaller the p-value, the more significant is the

able 2egression coefficients, significant p-values for the model estimated using Minitab softw

PLE yield Total phenolic Total fla

RC PV RC PV RC

*C −40.9363 0.000 −1330.80 0.000 −430.59*T −30.2900 0.000 −984.70 0.000 −318.60*P 0.6129 0.707 19.93 0.707 6.44*ts 1.3106 0.273 42.61 0.273 13.78*td 1.9705 0.111 64.06 0.111 20.72*f 2.4576 0.053 79.89 0.053 25.85*T2 −4.4391 0.000 −144.31 0.000 −46.69*P2 −0.9240 0.007 −30.04 0.007 −9.71*ts

2 −0.6252 0.001 −20.32 0.001 −6.57*td

2 −1.0381 0.000 −33.75 0.000 −10.91*f2 −1.1375 0.000 −36.98 0.000 −11.96T × P 0.2729 0.529 8.87 0.529 2.87T × ts 0.2088 0.492 6.79 0.492 2.19T × td 0.1076 0.721 3.50 0.721 1.13T × f 0.2386 0.434 7.76 0.434 2.51P × ts 0.1686 0.517 5.48 0.517 1.77P × td 0.1172 0.651 3.81 0.651 1.23P × f 0.4062 0.135 13.20 0.135 4.27td × ts 0.1296 0.478 4.21 0.478 1.36ts × f 0.0512 0.777 1.66 0.777 0.53f × td 0.1084 0.551 3.52 0.551 1.14

* Significant factor.

310.070 100.324 27.388 1.225319.010 103.217 28.178 1.188300.838 97.337 26.573 1.264

corresponding coefficient. Based on the statistical results (ANOVA)with confidence level of 95%, the effect of each term in the mod-els could be significant provided that its p-value be smaller than0.05 (p-value < 0.05). The results of the yield, total phenolic, totalflavonoid, DPPH IC50, FRAP value in Table 2 indicate that the terms

in linear forms and in quadratic forms have strong influence onthe extraction yield and the interaction terms have no effect onthe extraction yield. It is imperative to realize that even thoughp-value > 0.05 (Table 2) for the linear terms of P and ts but due

are.

vonoid DPPH FRAP value

PV RC PV RC PV

0 0.000 8.09191 0.000 −117.553 0.0005 0.000 4.12121 0.000 −86.980 0.0007 0.707 −0.08393 0.705 1.760 0.7075 0.273 −0.17888 0.272 3.763 0.2737 0.111 −0.26871 0.110 5.658 0.1110 0.053 −0.33463 0.053 7.057 0.0533 0.000 0.60402 0.000 −12.747 0.0009 0.007 0.12593 0.007 −2.653 0.0076 0.001 0.08507 0.001 −1.795 0.0019 0.000 0.14132 0.000 −2.981 0.0004 0.000 0.15482 0.000 −3.266 0.0001 0.529 −0.03734 0.527 0.784 0.5297 0.492 −0.02853 0.491 0.600 0.4922 0.721 −0.01478 0.719 0.309 0.7210 0.434 −0.03249 0.434 0.685 0.4344 0.517 −0.02297 0.517 0.484 0.5173 0.651 −0.01584 0.653 0.337 0.6513 0.135 −0.05540 0.135 1.166 0.1353 0.478 −0.01774 0.475 0.372 0.4788 0.777 −0.00687 0.780 0.147 0.7770 0.551 −0.01487 0.548 0.311 0.551

3 rcritical Fluids 95 (2014) 318–324

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Fig. 2. Response surface of PLE yield as a function of temperature and pressure atts = 12.9 min, td = 18.9 min, and f = 0.76 mL/min.

and matrix and the solvent strength is increased up to pressure of39.2 bar where the extraction yield was reduced [49]. The decreasein yield, total phenolic, total flavonoid, and FRAP value with

22 E. Golmakani et al. / J. of Supe

o hierarchy rule in which the p-value < 0.05 for the higher orderquadratic) of this variable, therefore, the effect of linear terms muste considered in the model [33].

.2. Total phenolic and flavonoid content

Antioxidant properties of plant extracts are associated withhe phenolic compounds to donate hydrogen to the radical41]. Standard curve for determination of total phenolic contentsas drawn and the regression equation was: y = 0.003x + 0.068

R2 = 0.98). The total phenolic content of the PLE (396.94 mg/g) anderculation (196.66 mg/g) extracts are given in Table 1. Total phe-olic content in the extracts was in the range of 8.842–396.94 mgallic acid equivalent (GAE)/g of extract. The total phenols of opti-um PLE extract was more than perculation extract as shown in

able 3. The regression equation for total flavonoid determinationas: y = 0.004612x + 0.006423 (R2 = 0.99), the amount of flavonoid

aried widely in extracts and ranged from 2.860 to 127.78 mg/g ofxtract are given in Table 1. The total flavonoid of optimum PLExtract was more than perculation extract as shown in Table 3.

.3. Antioxidant activity of extracts

The ability of antioxidants to scavenge DPPH is attributed toheir hydrogen donating activity [42] and this method often usedo evaluate the antioxidant activity of natural compounds [43]. Theadical scavenging effects of the tested extracts were concentra-ion dependent and all samples with the highest concentration ofxtracts had strong scavenging effect. The PLE optimum extractxhibited considerably highest DPPH free radical scavenging activ-ty with lowest IC50 and the value of the AI than perculation method.

In the FRAP assay, the antioxidant activities were expressed ashe concentrations of antioxidant having a ferric reducing abilityquivalent to that of 1 mM of FeSO4 and expressed as mmol fer-ous(II) iron equivalent per gram sample. The equation of FRAP fortandard solution was: y = 0.435x + 0.075 (R2 = 0.988). The FRAP ofLE optimum extract was more than perculation method. Moreoverhe PLE optimum and perculation methods showed higher thanower than BHT but had lower power than Ascorbic acid are given

n Table 3.We therefore tested a supposition that there is a correlation

etween the antioxidant effects and total phenolic content. Scatterlot analysis of antioxidant activities and total phenolic contentere performed and there was a significant positive correlation

etween the IC50 and FRAP value and the total phenolic contentR2 = 0.976, R2 = 0.994). Many researchers reported positive corre-ation between total phenolic content and antioxidant activity butn some studies were no correlation [44]. Thus these results con-rmed previous studies that showed the phenolic compounds playn important role in providing antioxidant activity in medicinalerbs [45].

.4. Variables affecting PLE

.4.1. The effect of extraction temperatureIncreasing the temperature of the extraction increased the %

xtraction yield (Fig. 2), total phenolic, total flavonoid, and FRAPalue and decreased DPPH IC50 (Fig. 3). (Total phenolic, totalavonoid, and FRAP value figures were similar to yield changes;hus there is no need to bring these figures.) An extraction temper-ture of 65.8 ◦C was sufficient to give maximum % extraction yield,otal phenolic, total flavonoid, and FRAP value and minimum DPPH

C50. The increase in yield, total phenolic, total flavonoid, and FRAPalue and decrease in DPPH IC50 with temperature is due to increasen methanol solvating power at higher temperatures [46,47]. Tem-erature is the main parameter influencing the physicochemical

Fig. 3. Response surface of DPPH EC50 as a function of temperature and pressure atts = 12.9 min, td = 18.9 min, and f = 0.76 mL/min.

properties of methanol and the compounds to be extracted, and ithas a great influence on the extraction rate, efficiency, and selectiv-ity in PLE. Enhancement of the extraction efficiency may be relatedto the increased vapor pressures and accelerated thermal desorp-tion of the compounds from the sample matrix [48].

3.4.2. The effect of the extraction pressureWhen increasing the extraction pressure partially up to 39.2 bar,

the % extraction yield, total phenolic, total flavonoid, and FRAPvalue increased significantly but later it started slightly to decreasetill the pressure reached the 50 bar as shown in Fig. 4, but theDPPH IC50 changes is different from the rest of the parameters isshown in Fig. 5. By increasing pressure, interaction between solvent

Fig. 4. Response surface of PLE yield as a function of static and dynamic extractiontime at T = 65.8 ◦C, P = 39.2 bar, and f = 0.76 mL/min.

E. Golmakani et al. / J. of Supercritical Fluids 95 (2014) 318–324 323

Table 3Extraction yield, total phenolic, total flavonoid content and antioxidant properties of PLE optimum and perculation extracts from roots of Scutellaria pinnatifida.

Extracts Extraction yield (%) Total phenolic (gallic acid equivalentsmg/g of dry extract)

Total flavonoid (quercetin equivalentsmg/g of dry extract)

IC50 (mg/mL) FRAP value (mmolFe2+/g dry extract)

Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

PLE optimum 12.21% ± 2.1 396.94 ± 2.3 127.78 ± 1.8 0.86 ± 3.2 34.9 ± 2.9Perculation 10% ± 1.4 196.66 ± 1.3 91.3 ± 1.4 3.853 ± 3.3 19.8 ± 3.1BHT 0.518 ± 2.2 14.3 ± 1.9Ascorbic acid 0.168 ± 2.3 81.6 ± 2.3

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Fig. 6. Response surface of PLE yield as a function of dynamic extraction time andflow rate at P = 39.2 bar, ts = 12.9 min, and td = 18.9 min.

Fig. 7. Response surface of DPPH EC50 as a function of dynamic extraction time and

ig. 5. Response surface of DPPH EC50 as a function of static and dynamic extractionime at T = 65.8 ◦C, P = 39.2 bar, and f = 0.76 mL/min.

ncreasing pressure after a certain pressure might be due to thenteraction between the other parameters. In practice, the pres-ure is kept high enough to maintain the methanol in liquid formt all extraction temperatures.

.4.3. The effect of static and dynamic extraction timeIn static extraction mode, extraction efficiencies strongly

epend on the partition-equilibrium constant and the solubility ofompounds. This may cause problems, especially with highly con-entrated samples and/or low-solubility analytes. Using dynamicxtraction, the equilibrium is displaced to completeness as fresholvent is continuously pumped through the sample. However, thestablishment of a static extraction step before dynamic extrac-ion shortened the time required for complete extraction. It waseported that a 10–20 min static contact time prior to dynamicperation improved the pressurized fluid extraction recovery [50].hus in this study, samples were held in the static extractionode in the range of 0–20 min, followed by a dynamic extrac-

ion in the range of 5–25 min. The results of this study indicatedhat static extraction longer than 12.9 min did not increase extrac-ion yield (Fig. 4), total phenolic, total flavonoid, FRAP value andid not decreased DPPH IC50 (Fig. 5) due to the disappearancef mass transfer driving force. Increasing the dynamic extractionime, increased the extraction yield, total phenolic, total flavonoid,RAP value, and decreased the DPPH IC50 up to 18.9 min. A dynamicxtraction time of 18.9 min was sufficient to give maximum extrac-ion yield, total phenolic, total flavonoid, FRAP value and minimumPPH IC50.

.4.4. The effect of the flow rateIncreasing the flow rate increased the extraction yield (Fig. 6),

otal phenolic, total flavonoid, and FRAP value and decreased thePPH IC50 (Fig. 7). Methanol might be saturated at lower flow ratesnd the extraction yield, total phenolic, total flavonoid, and FRAPalue reduces. An extraction flow rate of 0.76 mL/min was suffi-

ient to achieve maximum extraction yield, total phenolic, totalavonoid, and FRAP value and minimum DPPH IC50, while further

ncreases up to 1.0 mL/min resulted in little change on dependentariables, thus the best result was obtained in 0.76 mL/min [51].

flow rate at P = 39.2 bar, ts = 12.9 min, and td = 18.9 min.

4. Conclusion

In the present study, the PLE operating conditions were opti-mized to achieve maximum extraction yield, total phenolic, totalflavonoid from Brachyscome multifida, and their antioxidant activ-ity. The high correlation of the statistical model indicated that aquadratic polynomial model could be employed to optimize theextraction conditions to obtain maximum yield, total phenolic,and total flavonoid, FRAP value, and DPPH IC50 by pressurizedliquid extraction. Phenolic compounds could be a main agent ofantioxidant potentials of plants and could be a natural source ofantioxidants. The compounds such as flavonoids, which containhydroxyls, are responsible for the radical scavenging effect in theplants. The result of the present study showed that the extractwhich contain highest amount of flavonoid and phenolic com-pounds (PLE optimum extract), exhibited the greatest antioxidantactivity from roots of S. pinnatifida A. Hamilt. ssp. alpina. The highscavenging of PLE extract may be due to hydroxyl groups existing inthe flavonoids. In conclusion, the results of the present study sug-gest that tested plant have moderate to potent antioxidant activity

and we could know what components in the PLE extract showedthese activities.

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24 E. Golmakani et al. / J. of Supe

onflict of interest

The authors have declared no conflict of interest.

cknowledgements

The financial support by research center of natural productsealth in North Khorasan University of Medical Sciences is grate-

ully acknowledged.

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