OPTIMIZATION OF THE Ocimum basilicum L. EXTRACTION PROCESS REGARDING THE ANTIOXIDANT ACTIVITY
Senka S. Vidovića*, Zoran P. Zekovića,Žika D. Lepojevića, Marija M. Radojkovića, Stela D. Jokićb and Goran T. Anačkovc
a
University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
b
Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology, Franje Kuhača 20, 31000 Osijek, Croatia
cUniversity of Novi Sad, Faculty of Natural Sciences, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia
The levels of input variables (temperature and extraction solvent) that optimize a par-ticular response (total phenols content, total flavonoids content and antioxidant activity) of the Ocimum basilicum L. extraction process were determined by the response surface methodology (RSM). The influence of theextraction temperature on extraction process was investigated in the range from 33.8ºC to 76.2ºC, as well as of extraction solvent ethanol, in the range of concentrations from 21.7% to 78.3%. For the preparation of basil dry extract, characterized with minimal IC50 value, the calculated optimal values of temperature and ethanol concentration were: 75.33ºC and 73.66% (w/w).
KEY WORDS: basil, extraction, antioxidants, RSM.
INTRODUCTION
Sweet basil(Ocimum basilicum L.) is a widely used herb with many properties and applications. This herb is used not only for the cooking, but also in commercial frag-rances, flavourings and for increasing the shelf life of food products (1). It is distributed in different folk medicines for a treatment of insomnia, kidneys inflammation, cough, asthma, inflammation of urinary tract, etc. Basil extract has a sedative and anticonvulsant properties (2), as well as important antimicrobial and antifungal activity (3). Herbs are considered as sources of different antioxidant compounds. These compounds are of great importance in terms of their use for preventing oxidative stress that may cause several degenerative diseases. Many epidemiological studies, prove the existence of a link bet-ween diets rich in antioxidants and a reduced risk of diseases, particularly of cancer and cardiovascular diseases. Beside their importance due to their medicinal properties anti-oxidants are important as preservatives in the food industry.
The high antioxidant activity of basil and its extracts, and the majority of its medicinal properties, have been attributed primarily to rosmarinic acid (4), but some other like
feic acid derivates, such as cichoric acid (5, 6), are also found in substantial concentra-tions. Rosmarinic acid belongs to phenolic compounds which have been marked as main antioxidant agent.
In a past few years, much attention has been paid to the extraction of antioxidants and the creation of antioxidant supplements from different natural sources (herbs, fruits and vegetables). This attention increases since some of synthetic antioxidants used in the food and other industries have been reported to be toxic. Natural antioxidants could replace synthetic once, which is a tendency in the modern pharmaceutical and food industries. Extracts from natural plants, as carriers of antioxidant compounds and antioxidant acti-vity, could be obtained by different methods, by using different solvents and at different process conditions (temperature, time, pressure, etc.). Extraction with organic solvents offers good recovery of polyphenols, which is related to the preparation of extracts with high antioxidant activity. As solvent extraction can be performed by different solvents and at different process conditions, to obtain the extracts with appropriate properties, the analysis and selection of optimal process parameters is necessary. To find the levels of input variables that optimize a particular response (total phenols content, total flavonoids content, and antioxidant activity) response surface methodology (RSM), as one of the best optimization tools, can be applied. The objective of this study was to employ RSM to assess the effect of different combinations of temperature, in the range from 33.8ºC to 76.2ºC, and aqueous solution of ethanol as extraction solvent, in a range from 21.7% to 78.3%, on the total phenols content (TP), total flavonoids content (TF), and antioxidant activity of O. basilicum extracts.
EXPERIMENTAL
Plant material and sample preparation
Basil samples were collected in Bosnia and Herzegovina, Republika Srpska, near Banja Luka, in July 2009. The collected plant material has been naturally dried under sun and then stored in paper bags at room temperature. Voucher specimens (Ocimum basili-cum L. 1753 No 2-1792, Bosnia and Herzegovina, Banja Luka, Česma, ruderal habitats, 08.2008. det.: Goran Anačkov) were confirmed and deposited at the Herbarium of the Department of Biology and Ecology (BUNS Herbarium), Faculty of Natural Sciences, University of Novi Sad, Serbia (7). The material was ground in a blender just before the extraction. The particle size, 0.726±0.13 mm, was determined using sieve sets (Erweka, Germany). The ground plant material (10 g) was extracted by aqueous solution of ethanol (100 ml) of specific concentration at different extraction temperatures. The extraction process was carried outfor 90 minutes. After filtration, the solvent was evaporated and obtained dry extract was analyzed for the total phenols, total flavonoids and antioxidant activity.
Determination of total phenolics and total flavonoids
g of dry O. basilicum extract. The total flavonoids content (TF) was determined by alumi-nium chloride colorimetric assay (10) and it was expressed as mg of catehin equivalents (CE) per g of dry O. basilicum extract.
DPPH assay
The free radical scavenging activity of O. basilicum extracts was determined as des-cribed by Espin(11). Briefly, dry O. basilicum extract was mixed with methanol (96%) and 90 M of 2,2-diphenyl-1-picryl-hydrazyl (DPPH) solutionto give different final con-centration (from 0.0025 mg/ml to 0.008 mg/ml) of extract. After 60 min at room tempe-rature, the absorbance was measured at 517 nm and expressed as radical scavenging ca-pacity. Radical scavenging capacity (%RSC) was calculated by the following equation:
blank sample
A A
RSC 100 100
% [1]
where: Asampleis the absorbance of the sample solution and Ablank is the absorbance of the blank control.
This activity was also expressed as the inhibition concentration at 50% (IC50), the concentration of test solution required to obtain 50% of radical scavenging capacity.
Experimental design
Response surface methodology (RSM) was employed to analyze the effects of two factors on three responses and to identify the combination that will optimize the extrac-tion process. The five-level design (Table 1) was used for fitting a second-order response surface, and it was rotatable design.
Table 1. Investigated levels and coded values for each of the independent variable
Independent variable
Coded levels
(-1.414) (-1) (0) (1) (+1.414)
Ethanol (X1, %) 21.7 30 50 70 78.3
Temperature (X2, ºC) 33.8 40 55 70 76.2
The experiments were carried out to study the effect of the solvent concentration (ethanol-water mixture) and extraction temperature on the extraction of antioxidant com-pounds and on the antioxidant activity of O. basilicum extracts. Three responses in the form of different components and antioxidant activity of the extracts were evaluated: total phenols content (TP), Y1, total flavonoids content (TF), Y2, and antioxidant activity (IC50),
temperature and X1 is the concentration of the ethanol solution. Y is the analyzed response (total phenols content, total flavonoids content and antioxidant activity).
b biXi biiXii bijXiXj
Y 2
0 [2]
RESULTS AND DISCUSSION
In this study, the RSM was used to examine the functional relationship between the investigated variables, solvent concentration and extraction temperature, and the outputs, or responses: content of antioxidant compounds, total phenols and flavonoides, and the extract antioxidant activity. The range of tested extraction temperature was between 33.8ºC and 76.2ºC. The ethanol-water solution was used in the range of concentration from 21.7% to 78.3% (Table 1). To find the interactions between these two parameters (temperature and ethanol concentration) on the targeted compounds a statistical analysis was applied. Experimental data were obtained according to the design of the response surface methodology (RSM) presented in Table 2.
Table 2. Total phenols content (TP), total flavonoids content (TF) and antioxidant activity (IC50) of extracts
Run number
Temperature (°C)
Ethanol (%)
TP (g GAE/100 g)
TF (g CE/100 g)
IC50 · 10-3
(mg/ml)
1 40 30 10.39 4.48 5.05
2 40 50 14.20 4.98 4.70
3 40 70 16.83 4.28 4.44
4 55 30 12.58 4.79 4.43
5 55 50 15.98 5.48 4.27
6 55 70 17.99 5.58 3.97
7 70 30 11.88 4.62 4.63
8 70 50 13.61 5.53 4.10
9 70 70 17.31 5.10 3.87
10 55 21,7 10.40 4.09 5.24
11 55 78.3 17.04 5.58 4.15
12 33.8 50 13.56 4.51 4.98 13 76.2 50 12.61 5.38 4.01
14 55 50 15.98 5.48 4.28
15 55 50 15.98 5.47 4.28
16 55 50 15.98 5.47 4.25
17 55 50 15.99 5.48 4.27
18 55 50 15.98 5.47 4.28
Statistical analysis was performed using STATISTICA 8.0, StatSoft (Europe) Gmbh, Hamburg, Germany. The estimated coefficients (bi) of the second-order response model, generated from the statistical analysis for all responses, are shown in Table 3. Measured fit of the model data (R2) for all responses were high. The R2 values for TP, TF and IC50 was 0.976, 0.932, and 0.964, respectively.
Table 3. Estimated coefficient (bi) of second-order polynomial models for the investigated responses
Coefficient TP (Y1)
p-value for Y1
TF (Y2)
p-value for Y2
IC50 (Y3)
p-value for Y3
b0
Linear b1
b2
Quadratic b3
b4
Interaction b5
R2
15.90145
0.00219 2.66758
-1.36558 -0.83433
-0.24990 0.970
0.000000
0.991513 0.000000
0.000097 0.003945
0.448965
5.50691
0.273790 0.31741
-0.33577 -0.39453
0.16925 0.932
0.000000
0.003905 0.001444
0.002441 0.000773
0.182691
0.004237
-0.000295 -0.000338
0.000125 0.000164
0.000037 0.964
0.000000
0.000014 0.000004
0.019102 0.004188
0.570562
According to the data shown in the Table 3 linear term of ethanol concentration (b2) show a positive effect on the investigated responses, e.g. TP (p<0.05). Both quadratic terms, of temperature (b3) and ethanol concentration (b4), affect negatively on the TP (p<0.05). The interaction between these two parameters has no significant influence on the obtained response. The effects of temperature and ethanol concentration on TP of O. basilicum can be described by equation (3),which can be used for calculation of optimal extraction parameters.
Y1=15.09145+0.00219X1+2.66758X2-1.36558X12-0.83433X22-0.2499X1X2 [3]
Fig
B cally total conce noids no in secon
wher temp T
Figu
gure 1. Influence o
Both linear terms ( significant influe flavonoids yield. entration, influenc s. The interaction nfluence on the ob nd -order equation
Y2=5.50691+0.2
e Y is the flavono erature.
he influence of th
ure 2. Influence of
of temperature an
(b1 and b2), of tem ence (p<0.05) and Also, both quadr ced significantly between these tw btained response. n:
27379X1+0.31741
oids content, X1 is
he parameters on T
f temperature and
nd ethanol concent
mperature and etha d influencee posit
atic terms (b3 and (p<0.05), but neg wo parameters, as
The response surf
1X2-0.33571X12-0.
the ethanol conce
TF is illustrated by
ethanol concentra
tration on total ph
anol concentration tively the investig d b4), of temperatu gatively the yield in the case of tota face was generate
.39453X22-0.1692
entration and X2 i
y chart presented i
ation on total flavo
enols content
n, have statisti-gated response-ure and ethanol
of total flavo-al phenols, had ed based on the
5X1X2 [4]
s the extraction
in Figure 2.
A in the The T Furth The o ration from U This set pa Fu have contr and b tion b T extra tratio (max culate mg/m and 7 mg/m
Figu
As can be seen from e temperature, bu TF yield increases her increase of eth optimal process v n of the O. basilic
Eq [4] were 63.07 Under these condit
calculation was ex arameters at 63.1º
urthermore, both a significant influ rast to the privious b4) exhibited statis between these two
Y3=0.00424-0.0
The influence of t cts is presented in on needed for the p ximal antioxidant
ed value of IC50,u ml. This calculatio 73.7%), and the o ml.
ure 3. Influence o
m Figure 2, the to ut after reaching a s with the increase hanol concentratio values of tempera
cum extract with 7ºC and 60.35%. tions, the calculat
xperimetally conf ºC and 60.3%) the linear terms (b1 a uence (p<0.05) on s cases, linear term stically significan o parameters had n
00029X1-0.00034X
the process param n Figure 3. The op preparation of an activity), calculat using obtained val on was experimen obtained O. basil
f temperature and
otal flavonoids con a certain point, aro e of ethanol conce on leads to a dec ture and ethanol a maximal conte
ted TF yield was firmed, i.e. under t e obtained TF yield and b2), of temper n the antioxidant a ms had a negative t positive effect o no influence on ob
X2+0.00012X12+0.
meters on the anti ptimal values for t
extract characteri ted from Eq (5), i lues for the invest ntally confirmed licum dry extract
d ethanol concentr
ntent increases wi ound 65ºC, it sho entration, from ab crease of the flavo
concentration nee nt of total flavon
5.66 g CE/100 g these conditions ( d was 5.66 g CE/1 rature and ethanol activity of O. basil e, while both quad on IC50 value. Aga btained response (
.00016X22-0.00004
oxidant activity o temperature and e ized with the mini s 75.33ºC and 73 tigated parameters
(at the condition had the IC50 valu
ation on the antiox
ith the increase ows a decrease. bout 20 to 60%. onoids content. eded for prepa-noids calculated
of dry extract. (experimentally 100 g.
l concentration, lium extract. In dratic terms (b3 ain, the interac-(Eq. [5]).
4X1X2 [5]
of O. basilicum ethanol concen-imal IC50 value
.66%. The cal-s, was 3.84·10-3 s set at 75.3ºC ue of 3.83·10-3
CONCLUSION
To find the levels of input variables, temperature and solvent concentration that opti-mize a particular response (content of total phenols and total flavonoids, as well as anti-oxidant activity of the extract), surface methodology has been used as an efficient tool. In the investigated range of temperatures (from 33.8ºC to 76.2ºC) and ethanol concentration (from 21.7% to 78.3%) for the preparation of an O. basilicum extract with highest content of total phenols the determined optimum values were 52.79ºC and 82.41%. Under these conditions the content of total phenols was calculated to be 18.07 g GAE/100 g of dry ex-tract. On the other hand, the optimal values corresponding to the highest content of total flavonoids are 63.07ºC and 60.35%. Under these conditions the content of total flavono-ids was 5.66 g CE/100 g of extract. Finally, the optimal values of the process parameters corresponding to the minimal IC50 value were 75.33ºC and 73.66%.
Acknowledgement
Financial support of this work by the Serbian Ministry of Education and Science, Project No. TR 31013 is gratefully acknowledged. We are grateful to Department of Bio-logy and EcoBio-logy, Faculty of Natural Sciences, University of Novi Sad, for support in term of confirmed and deposited investigated herb at the BUNS Herbarium.
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11.Espin J. C., C. Soler-Rivas and Wichers, H. J.: Characterization of total free radical scavenger capacity of vegetables oils and oils fractions using 2,2-diphenyl-1-pycril-hydrazil. J. Agric. Food Chem. 48, 3 (2000) 648-656.
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33,8ºC
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21,7% 78,3%.
IC50 : 75,33ºC 73,66%
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Ʉʂɭɱɧɟɪɟɱɢ:Ocimum basilicum, , , , RSM
