Volume 4 • Issue 1 • 1000196J Food Process TechnolISSN:2157-7110 JFPT, an open access journal

Open AccessResearch Article

FoodProcessing & Technology

Salleh et al., J Food Process Technol 2013, 4:1http://dx.doi.org/10.4172/2157-7110.1000197

Keywords: Strobilanthes Crispus; Bioactive flavonoid; Supercritical carbon dioxide extraction; Response surface methodology; HPLC

IntroductionSupercritical fluid extraction with carbon dioxide (SC-CO2)

has gained much attention for fast and effective extraction of a wide variety of compounds owing to the fact that carbon dioxide is an environmentally benign solvent, works under mild extraction conditions thus avoiding degradation of biological products and its solvating power is easily manipulated by changes in pressure and temperature. SC-CO2 is a good solvent for the extraction of non-polar compounds such as hydrocarbons [1]. On the other hand, the most frequent limitation of carbon dioxide as an extraction solvent is that its polarity is often too low to obtain efficient extraction for polar is compounds, either because the lack of sufficient solubility or the extraction has poor ability to displace the analytes from matrix site [2].

In order to overcome the problem, a polar co-solvent is usually added to increase compounds solubility to SC-CO2 extraction. Among all the polar co-solvents including methanol, ethanol, acetonitrile, acetone, water, ethyl ether and dichloromethane, methanol is the most commonly used because it is an effective polar co-solvent and is up to 20% miscible with CO2. However, ethanol may be a better choice in SFE of nutraceuticals because of its lower toxicity [3,4].

The conventional techniques to obtain plant extracts, such as steam distillation and organic solvent extraction, usually require several hours or even days as such consumed a large volume of solvent for the extraction process. Apart from difficulties in the extraction step, the solute/solvent separation may result in degradation of the thermolabile components and traces of the solvent used may be present in the product, thus reducing the quality assessment of the extraction yield [5,6]. Flavonoids are polyphenolic compounds that are widely distributed in fruits, vegetables, teas and medicinal plants and most commonly known for their antioxidant activity. There are now growing interest in these compounds as they are reported to play a role in the control and prevention of cancer and tumerogenesis, possibly as a result of their antioxidant activity [7].

Strobilanthes crispus (Pecah Kaca) has been used traditionally as antidiabetic, diuretic, antilytic, and laxative. It also has been proven scientifically to possess high antioxidant activity, anti-AIDS, and anticancer properties due to its phytochemical constituent especially mineral contents, antioxidant vitamin as well as catechin [8,9]. It is commonly consumed in the form of herbal tea. Recent investigations on ethnopharmacological studies demonstrate that the S. crispus leaves extract was an effective antioxidant in which their ability responded as antihyperglycemic and antilipidemic agent. The extract has the effect on minimizing the glucose level in blood and also reduces the risk of blood vessels and heart muscle/ cardiovascular ailments [10]. Therefore, it is interesting to find an effective method to prepare bioactive flavonoid compounds from S. crispus.

In this study the main focus will be looking at three important independent variables that influence the SC-CO2 extraction, namely pressure, temperature and co-solvent (modifier). The effect on SC-CO2 extraction yield and identification of the extractable bioactive flavonoid compounds will be carried out by manipulating the three independent variables stated above. Therefore the objective of this work is to optimize the effect of pressure, temperature and co-solvent flow rate on the extraction yield and bioactive flavonoid compounds of S. crispus by SC-CO2 extraction.

*Corresponding author: Liza Md Salleh, Faculty of Chemical Engineering, University of Technology, Malaysia, 81310 Skudai, Johor, Malaysia, E-mail: syukriah_org@yahoo.com

Received October 10, 2012; Accepted November 28, 2012; Published December 07, 2012

Citation: Salleh LM, Rahman RA, Selamat J, Hamid A, Islam Sarker MZ (2013) Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface Methodology. J Food Process Technol 4: 197. doi:10.4172/2157-7110.1000197

Copyright: © 2013 Salleh LM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractSupercritical carbon dioxide (SC-CO2) extraction was used to obtain the bioactive flavonoid compounds of

Strobilanthes crispus (Pecah Kaca). Process variables namely pressure, temperature and co-solvent flow rate were studied for optimization of the extraction yield and bioactive flavonoid compound of S. crispus by response surface methodology following a Box Behnken design of experiments. HPLC was performed to determine the major bioactive flavonoid compounds from S. crispus. The optimum value of yield extraction (6.55%) was obtained at pressure 200 bar, 50°C temperature and co-solvent flow rate of 5 g/min. Under the optimum conditions, eight flavonoid compounds were identified namely (+)-catechin, (-) epicatechin, rutin, myricetin, luteolin, apigenin, naringenin and kaempferol. The experimental extraction yield is in good agreement with the predicted one. The response surface methodology used in this study was able to predict the optimal extraction conditions for the extraction yield of bioactive flavonoid compounds from S.crispus leaves extract.

Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface MethodologyLiza Md Salleh1*, Russly Abdul Rahman2, Jinap Selamat2, Azizah Hamid2 and Md Zaidul Islam Sarker3

1Faculty of Chemical Engineering, University of Technology, Malaysia, 81310 Skudai, Johor, Malaysia2Faculty of Engineering, Universiti Putra Malaysia 43400 Serdang, Selangor, Malaysia3Kulliyah of Pharmacy, Universiti Islam Antarabangsa, Kuantan Campus, 25200 Kuantan, Malaysia

Citation: Salleh LM, Rahman RA, Selamat J, Hamid A, Islam Sarker MZ (2013) Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface Methodology. J Food Process Technol 4: 197. doi:10.4172/2157-7110.1000197

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Volume 4 • Issue 1 • 1000196J Food Process TechnolISSN:2157-7110 JFPT, an open access journal

Materials And MethodsMaterials and reagents

The leaves of S. crispus were harvested from the herbal garden of the Faculty of Medicine and Health Sciences, University Putra Malaysia. The clean dry leaves were grounds in a dry mill blender (MX-335, Panasonic, Malaysia) to produce powder. The dried leaves were stored in a dark place at room temperature for 20 days. Immediately prior to the extraction process, the dried leaves were grounded in a dry mill blender (MX-335, Panasonic, Malaysia) to form a powder in order to increase the surface area of sample. The particle size of the powder was determined through a sieve analysis (Endecotts Minor, USA) with an approximate size of 0.50 mm [11].

Commercial grade liquid carbon dioxide (purity 99.99%), supplied in cylinder with dip tube, was purchased from Malaysian Oxygen (MOX) Malaysia. Ethanol (EtOH, 99.5%, analytical grade) was obtained from Scharlau Chemical, European Union and methanol (MeOH, HPLC grade) was purchased from Fisher Scientific Chemical, USA. Tri-Flouro- Acetic acid (TFA ≥98%) was obtained from Sigma-Aldrich, Germany. The flavonoid standards including ( +)-catechin, ( -)-epicatechin, apigenin, rutin, luteolin, kaempferol, myricetin and naringenin were purchased from Sigma-Aldrich, Germany.

Supercritical Carbon Dioxide (SC-CO2) extraction

The SC-CO2 system used in this study is shown in figure 1. SC-CO2 extraction was performed using 500ml extractor (ABRP200, Pittsburg, PA, USA). The SC-CO2 extraction system and components were acquired from the Thar Designs, Inc. (Pittsburgh, PA), which includes the following: 500 ml extraction vessel, model P-50 high-pressure pump, automated back pressure regulator model BPR-A-200B, and PolyScience brand water bath with pump unit (model 9505). Circulated deionized water at 5°C was used for cooling different zones in the SC-CO2 extraction apparatus. The independent variables

were temperature (40, 50 and 60°C), pressure (100, 150 and 200 bar) and dynamic extraction time (40, 60 and 80 min).

Thirty gram of ground plant material was well mixed with 2.0 mm diameter glass beads were placed into the extractor vessel. The introduction of rigid material such as glass bead or sea sand, pack with ground sample was able to maintain proper flow rate of CO2 in the extraction vessel and at the same time maintain the desired permissibility of the particle during extraction process [12,13].

Liquid carbon dioxide and co-solvent (ethanol) were pumped into the extraction vessel after the desired temperature was achieved. The flow rate of CO2 and co-solvent were maintained at 10 and 1 g/min respectively. Static extraction was performed for 30 min after desired pressure and temperature were reached. The dynamic extraction was initialized by opening the exit valve for the SC-CO2 extraction system. The static interval allows the sample of S.crispus to soak in the CO2 and co-solvent in order to equilibrate the mixture at the desired pressure and temperature. During the dynamic interval, CO2carrying the crude extract flowed out of the extraction vessel unit and into a collection vessel, where the CO2 was vented to a fume hood.

The main objective of this study was to optimize the operating conditions which includes pressure, temperature and co-solvent flow rate for SC-CO2 extraction of S.crispus extraction yield using Response Surface Methodology (RSM). Total extraction time was set for 90 min, with an initial static extraction period of 30 min, followed by a dynamic extraction for 60 min. Supercritical extraction were performed at three difference level s of pressure (100, 150 and 200 bar), temperature (40, 50 and 60°C) and co-solvent flow rate (1, 3 and 5 g/min). The powdered plant material (30g) was mixed with 90 g glass beads (2.0 mm) and place into the extractor vessel. Ethanol was chosen as co-solvent. The extraction was performed under various experimental and level in accordance with the Box Behnken Design (BBD).

High Performance Liquid Chromatography (HPLC) analysis

The extracts obtained from selected supercritical extraction conditions were analyzed using a Hi g h Performance L i qu i d Chromatography (HPLC). The H P L C ana lys es we re performed with a Water 600 pump Controller, 9486 tunable absorbance UV detector and equipped with an Eclipes XDR- C18 reversed-phase column (25 cm× 4.6 mm × 5 μm, Supelco, USA). The compounds were eluted with a gradient elution of mobile phase, solvent A of consisted of deionize water adjusted to pH 2.5 with TFA (triflouroacetic acid) and solvent B consisted of 100% methanol (HPLC grade). The injection volume was 20 μl and each run was followed by an equilibration period of 15 min. The temperature, flow rate and wavelength were set to room, 1.0 ml/min and 280 nm respectively.

Experimental design

In this study, the Box Behnken Design (BBD) was employed for the experimental design chosen with a fraction factorial for three independent variables at three levels. This design was preferred because relatively few experimental combinations of the variables are adequate to estimate complex response function.

The various process parameters involved in the total extraction

Figure 1: Flow diagram of supercritical carbon dioxide (SC-CO2) extraction system.

Co-solventPump

Wet GasMeter

SampleCollection

ExtractionVessel

Cylinder

CO2

CO2

CO2

Pump

Release

Citation: Salleh LM, Rahman RA, Selamat J, Hamid A, Islam Sarker MZ (2013) Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface Methodology. J Food Process Technol 4: 197. doi:10.4172/2157-7110.1000197

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Volume 4 • Issue 1 • 1000196J Food Process TechnolISSN:2157-7110 JFPT, an open access journal

yield of S.crispus were pressure (X1), temperature (X2) and co-solvent flow rate (X3) and coded as -1 (low), 0 (central point or middle) and +1 (high) respectively. Table 1 shows the actual experimental parameters and the coded experimental BBD levels used.

RSM was applied to the experimental data using statistical software, Design -expert V6 (trial version). Statistical terms and their definitions used in the Design-expert software are w e l l d e f i n e d e l s e w h e r e . Linear a n d s e c o n d o r d e r p o l y n o m i a l s w e r e f i t t e d t o t h e experimental data to obtain the regression equations. The sequential F-test, lack-of-fit test and other adequacy measures were used in selecting the best model [14].

For each of the step done, the total extraction yield (%) of S.crispus was determined: m Y (%) X 1 00

m extractextract

feed

= (1)

Where Yextract is percentage of extraction yield, mextract is the crude extract mass (g) and mfeed is the feed mass (g).

The response function used was a quadratic polynomial equation as given below: Yield = A0 + A1X1 + A2X2 + A3X3 + A4X1X2 + A5X2X3 + A6 X1X3 + A7X1

2+ A8X22 + A9X3

2 (2)

Where:

A0 = constant

A1, A2 and A3 = linear coefficients

A4, A5 and A6 = cross product coefficients

A7, A8 and A9 = quadratic coefficients

Results and DiscussionModel fitting

In order to develop response surface equation, the final mathematical equation in terms of actual factors as determined by Design-Expert software is given below:

Y = 5.23 + 1.10X1 – 0.19X2 + 0.52 X3 + 0.008 X1 X2 + 0.17 X1 X3+ 0.007 X2 X3+0.051X1

2 – 0.40 X22 – 0.38X3

2 (3)

Where Y is the flavonoid extraction yield of S. crispus, and X1, X2 and X3 are the coded variables for pressure, temperature and co-solvent flow rate, respectively.

The design of the experiments in the uncoded (actual) and coded levels of variables for SC-CO2 extraction together with the experimental data for response are presented in table 2. The highest value of yield extraction (6.55%) was given at pressure of 200 bar, temperature at 50 °C and co-solvent flow rate of 5 g/min. On the other hand, the lowest value of yield percentage (3.57%) was observed at 100 bar 50 °C and 3g/min. In genera l , exploration and optimization of a fitted response surface may produce poor or misleading results, unless the model exhibits a good fit, which makes checking the model adequacy essential [15].

Therefore the model adequacy was checked by an F-test and the determination of R2. By Analysis of Variance (ANOVA) as shown in table 3, the R2, correlation coefficient value of this model was determined to be 0.9713, which implied that the sample variation of more than 95% was attributed to the variables and only less than 5% of the total variance could not be explained by the model. The R2value

Figure 2: Response surface plots showing of pressure and temperature on the extraction yield and their interaction. The co-solvent flow rate was constant at 3 g/min.

6.40

5.70

5.00

4.30

3.60

60.00

55.00

50.00

45.00

40.00 100.00

125.00150.00

175.00200.0

yiel

d(%

)

temperature(oC)pressure (bar)

Independent variable Coded Factor LevelLow (-1) Center (0) High (1)

Pressure (bar) X1 100 150 200Temperature (°C) X2 40 50 60Co-solvent flow rate (g/min) X3 1 3 5

Table 1: Independent variables and their coded levels chosen for Box Behnken design.

Run P code T (ºC) code T(min) code Yield (mg/g)(Bar) Observed Predicted

1 100.00 -1 50.00 0 3.00 0 3.73 3.972 200.00 +1 60.00 +1 3.00 0 6.02 6.173 100.00 -1 50.00 0 3.00 0 3.57 3.604 200.00 +1 50.00 0 3.00 0 6.01 5.785 100.00 -1 50.00 0 1.00 -1 3.59 3.456 200.00 +1 40.00 -1 1.00 -1 5.17 5.077 100.00 -1 60.00 +1 5.00 +1 4.29 4.158 200.00 +1 50.00 0 5.00 +1 6.55 6.689 150.00 0 40.00 -1 1.00 -1 4.23 4.13

10 150.00 0 60.00 +1 1.00 -1 3.64 3.7311 150.00 0 50.00 0 5.00 +1 3.73 5.1612 150.00 0 50.00 0 5.00 +1 4.68 4.79

13 (C) 150.00 0 50.00 0 3.00 0 5.15 5.2314 (C) 150.00 0 50.00 0 3.00 0 5.10 5.2315 (C) 150.00 0 50.00 0 3.00 0 5.16 5.2316 (C) 150.00 0 50.00 0 3.00 0 5.13 5.2317 (C) 150.00 0 50.00 0 3.00 0 5.59 5.23

Table 2: Box-Behnken design with experimental and predicted values of extraction yield of s.crispus by SC-CO2.

Source Sum ofSquares

Degree of freedom

Mean Square

F Value P Value

Model 13.49 9 1.50 26.34 0.0001esidual 0.40 7 0.057Lack of Fit 0.23 3 0.076 1.77 0.2924Pure Error 0.17 4 0.0Total 14.29 23

Table 3: Analysis of variance (ANOVA) for the response surface quadratic model f or the extraction yield of Strobilanthes crispus.

Citation: Salleh LM, Rahman RA, Selamat J, Hamid A, Islam Sarker MZ (2013) Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface Methodology. J Food Process Technol 4: 197. doi:10.4172/2157-7110.1000197

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Volume 4 • Issue 1 • 1000196J Food Process TechnolISSN:2157-7110 JFPT, an open access journal

of more than 0.75 is statistically considered accurate for developing a model or equation [16]. From this R2 value it shows clearly that the regression model defined well the true behavior of the system. The adjusted determination coefficient (Adj. R2) value was 0.9344 which was also satisfactory to confirm the significance of the model.

Analysis of response surface

The best way to visualize the effect of the independent variables on the dependent ones is to draw surface response plots of the model, which were done by varying two variables within the experimental range and holding the other one constant at the central point. Figures 2-4 are the response surface plots and the regression coefficients of response function with statistical analysis are given in table 4.

Figure 2 is a response surface plot showing the effect of pressure and temperature on the extraction yield at fixed co-solvent flow rate of 3 g/min. Pressure had a positive linear effect on extraction yield at low-pressure levels. An increase of pressure can result in an increase of fluid

density, which alters solute solubility. This is most likely due to the improvement of oil solubility resulted from the increased CO2 density with the rise of pressure [16]. At high- pressure levels, however, the negative quadratic effect of pressure on the oil yield also became important. This is probably a reflection of the increased repulsive solute –solvent interactions resulted from the highly compressed CO2 at high-pressure levels [17]. Based on this observation, the volatility and polarity of extracted analytes might be responsible for the result.

From table 4, according to this test the temperature at 95% confidence is not considered to be statistically insignificant. Temperature showed a negative linear effect t, however, the negative quadratic effect of temperature on extraction yield is significant. This is because to temperature has dual effects on extraction. Increasing the temperature from 40 to 50°C drastically accelerates the mass transfer in the supercritical fluid, with a consequent increase in the solubility of analytes and improves the extraction yield. However, increasing the temperature from 50 to 60°C decreased the efficiency of the extraction yield due to the decrease of SC-CO2 density which can be seen clearly from Figure 2 and 3. It is important to note that, the solubility of analytes in a solid matrix in supercritical fluid extraction is a major factor determining the extraction efficiency. The solubility is generally controlled by the volatility of substance, which is a function of temperature and solvation effect of the supercritical fluid, which is a function of fluid density [18,19]. Therefore, it is difficult to predict the effect of temperature.

Figure 3 and 4 are the response surface and contour plots that showed showing the effect of co-solvent flow rate on extraction yield at fixed pressure of 150 bar and temperature of 50°C. Similar to the effect of pressure, co-solvent has a positive linear effect on the extraction yield. The co-solvent improves SC-CO2 extractability by increasing the polarity of solute in the sample. It has been observed that the addition of methanol [19] and ethanol can drastically increase the extraction of compound from plant matrices. In this study similar effect was observed in the effect of co-solvent (ethanol) flow rate. Co- solvent exerts it effect mainly in two basic ways namely by interacting with analyte complex to promote rapid desorption into the supercritical fluid and enhancing the solubility properties of solute in SC-CO2 extraction. Various co-solvent flow rates used exhibited different effects in changing the fluid polarity and thus had diverse effects on solubility enhancement of extraction yield.

The application of co-solvent increased the solubility of analytes compound in SC-CO2. The enhancement of yields of extracts, due to the effect of co-solvent, may be the result of physical interaction of solutes with the mixed solvent, possibly owing to an increase in the

Figure 3: Response surface plots showing of pressure and co-solvent flow rate on the extraction yield and their interaction. The temperature was constant at 50°C.

6.69

5.88

5.07

4.26

3.45

5.00

4.00

3.00

2.00

1.00 100.00125.00

150.00

175.00200.00

pressure (bar)Co-solvent flow rate

(g/min)

yiel

d(%

)

Figure 4: Response surface plots showing of co-solvent flow rate and temperature on the extraction yield and their interaction. The pressure was constant at 150 bar.

5.43

5.00

4.58

4.16

3.73

5.00

4.00

3.00

2.00

1.00 40.00

45.00

50.00

55.00

60.00

temperature (oC)

yiel

d(%

)

co-solvent flow rate

(g/min)

Model term Coefficient estimation Standard error F-value Prob>FIntercept 5.230 0.120 - -Pressure (X1) 1.100 0.084 168.510 <0.0001Temperature (X2) -0.190 0.084 5.130 0.0580Co-solvent flow rate (X3) X1:X2X1:X3X2: X3X1

2

X22

X32

0.5200.0070.1700.0060.051-0.400-0.380

0.0840.1200.1200.1200.1200.1200.120

37.9300.0042.0400.0030.19011.63010.570

0.00050.95000.19660.95650.67700.01130.0140

R2=0.9713; Adj R2 = 0.9344, SD=0.24; CV=4.88%X1, X2 and X3 are the main effects; X1X2 , X1X3 and X2 X3 are the interaction effects Table 4: Regression coefficient analysis of response function to predict extraction yield of S.crispus.

Citation: Salleh LM, Rahman RA, Selamat J, Hamid A, Islam Sarker MZ (2013) Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface Methodology. J Food Process Technol 4: 197. doi:10.4172/2157-7110.1000197

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Volume 4 • Issue 1 • 1000196J Food Process TechnolISSN:2157-7110 JFPT, an open access journal

0.800

0.600

0.400

0.200

0.000

0.00 10.00 20.00 30.00 40.00Minutes

AU

Cat

echi

nE

plca

tech

in

Rut

in

Myr

icet

in

Lute

olin

Api

geni

n

Nar

inge

nin

Kam

pter

ol

Figure 8: HPLC Chromatogram SC-CO2 extraction at pressure: 200 bar, temperature: 50˚C and co-solvent flow rate: 3 g/min.

solvent density [20]. This result was in agreement with the previous studies that modifier increased the yield of ginseng oil and the effect was dose-dependent [21].

From table 4, regression coefficient analysis of response concluded that the principal effects of pressure and co-solvent flow rate were statistically significant (P<0.05) for the extraction yield response while temperature has less significant effect than the two others. Result also showed that the interaction between the independent variables had insignificant influence on the extraction yield.

Optimization

The SC-CO2 extraction condition of S.crispus would be considered optimum if the extraction yield reached maximum values. Desirability function approach was used to achieve this goal. Therefore, the criteria applied were maximum pressure, temperature in the range and maximum value of co-solvent flow rate. The criteria outlined above produced an optimum region in combination contour plot as shown in figure 5. The model predicted pressure, temperature and co-solvent flow rate as 200 bar, 50°C and 5 g/min respectively where these points were located within the experimental ranges, implying that the analytical techniques could be used to identify the optimal conditions.

Identification and quantification of the extracted compounds

The extraction at optimum conditions was analyzed by HPLC in order to determine the contents of main bioactive flavonoid

compounds. The standard mixtures of eight flavonoid compounds are prepared in order as a comparison to extracted bioactive compounds in this work. All flavonoid compounds from the extraction yield were identified by matching the retention time and their spectral characteristics against those of standards curve. The summary of calibration curve (correlation curves). Figure 6 illustrates the HPLC chromatogram of a standard mixture of for eight flavonoid standards including (+)-catechin, (−)-epicatechin, rutin, myricetin, luteolin, apigenin, naringenin and kaempferol. Figure 7-9 represent three chromatographs showing the flavonoid compounds in the yield when extracted with SC-CO2 extraction at (200 bar, 50°C, 1 g/min), at (200 bar, 50°C, 3 g/min) and at maximum condition (200 bar, 50°C, 5 g/min) were also analyzed by HPLC for comparison Detail profile of under optimum condition, kaempferol (21.54 mg/g) obtained at highest concentration compare with other flavonoids compound and even upon analysis by HPLC for all other SC-CO2 extraction

Figure 5: Contour plot of optimum condition of co-sovent flowrate, temperature and pressure.

60.00

55.00

50.00

45.00

40.00

100.00 125.00 150.00 175.00 200.00

Pressure (bar) ; x;,

Combine

Co-solventflow rate = 5.00g/min

Tem

pe ra

ture

(o q

; x;

4.00

4.494.98

5.475.96

Figure 6: HPLC chromatogram of mix standard flavonoids compound.

0 .3000

0 .2500

0 . 2000

0 .1500

0 .1000

0 .0500

0 .0000

0 .00 10 . 00 20 .00 30 .00

Catechin

Epicatechin

Rutin

Myricetin

Apigenin

Naringenin

kampferol

Minutes

AU

0.1400

0.1200

0.1000

0.0800

0.0600

0.0400

0.0200

0.0000

0 .00 10 . 00 20. 00 30 .00Minutes

Cat

echi

n

Epi

cate

chin

Rut

in

Myr

icet

in

Lute

olin A

pige

nin

Nar

inge

nin

Kam

pfer

ol

AU

Figure 7: HPLC chromatogram of SC-CO2 extraction at pressure: 200 bar, temperature: 50˚C and co-solvent flow rate: 1 g/min.

0.1500

0.1000

0.0500

0.0000

0.00 10.00 20.00 30.00Minutes

Cat

echi

n

Epi

cate

chin

Rut

in

Myr

icet

in

Lute

olin

Api

geni

n

Nar

inge

nin

Kam

pfer

ol

AU

Figure 9: HPLC Chromatogram of SC-CO2 extraction at pressure 200 bar, temperature 50˚C and co-solvent flow rate: 5g/min.

Citation: Salleh LM, Rahman RA, Selamat J, Hamid A, Islam Sarker MZ (2013) Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface Methodology. J Food Process Technol 4: 197. doi:10.4172/2157-7110.1000197

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Volume 4 • Issue 1 • 1000196J Food Process TechnolISSN:2157-7110 JFPT, an open access journal

condition. Table 5 clearly revealed that percent of flavonoid content has linear relationship with extraction yield, where the solubility of bioactive flavonoid compound are also increasing once the optimum condition were achieved. Moreover concentration of other flavonoid compounds increased at this optimum condition where 13.85 mg/g ((+) - catechin), 10.82 mg/g (apigenin) and 10.70 mg/g (naringenin). However under optimum condition level, the separation of other residue also exists, which may be due to the higher concentration of co-solvent and the diffusion ability of solute to be extracted. Therefore other compounds were also extracted under this extraction condition.

ConclusionThe current study showed that the high correlation of the

mathematical model indicated that a quadratic polynomial model was sufficient to describe and predicted the response of yield extraction of flavonoid bioactive compounds from S. crispus by SC-CO2 extraction. Pressure and co-solvent flow rate have a stronger effect on the yield extracted, compared with temperature. The optimum conditions for bioactive flavonoid compounds extraction using SC-CO2 were found at pressure, temperature and co-solvent flow rate at 200 bar, 50°C and 5 g/min. Kampferol (21.54 mg/g), was found as the highest flavanoid compound found under optimum condition.

Acknowledgments

The authors gratefully acknowledge the Malaysian government and Universiti Putra Malaysia for awarding the research grant for this project.

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PecahKaca

ExtractionYield (%)

FlavonoidContent d Contents of flavonoid (mg/g)

(S.crispus)extraction mode (%) Catechin

(mg/g)Epicatechin

(mg/g)Rutin(mg/g)

Myricetin(mg/g)

Luteolin(mg/g)

Apigenin(mg/g)

Naringenin(mg/g)

Kampferol(mg/g)

Condition a 2.37 4.95 4.83 4.55 8.47 4.13 19.44 3.75 4.06 12.54Condition b 5.34 8.66 11.90 12.64 18.62 8.72 7.77 9.96 9.37 11.19Condition c 6.55 10.04 13.85 11.50 17.27 2.19 12.49 10.82 10.70 21.54

Table 5: Identification and quantification of the flavonoid compounds by SC-CO2 extraction under different conditions.

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Salleh LM, Rahman RA, Selamat J, Hamid A, Islam Sarker MZ (2013) Optimization of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobhilantes crispus (Pecah Kaca) Leaves by Response Surface Methodology. J Food Process Technol 4: 197. doi:10.4172/2157-7110.1000197