315 OPTIMIZATION OF THE Ocimum basilicum L ... - doiSerbia

APTEFF, 43, 1-342 (2012) DOI: 10.2298/APT1243315V

UDC: 66.011:[66.061:635.71 BIBLID: 1450-7188 (2012) 43, 315-323 Original scientific paper

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 Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology, Franje Kuhača 20, 31000 Osijek, Croatia c University of Novi Sad, Faculty of Natural Sciences, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia

b

The levels of input variables (temperature and extraction solvent) that optimize a particular 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 fragrances, 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 between diets rich in antioxidants and a reduced risk of diseases, particularly of cancer and cardiovascular diseases. Beside their importance due to their medicinal properties antioxidants 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 caf* Corresponding author: Senka S. Vidović, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, e-mail: [email protected]

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APTEFF, 43, 1-342 (2012) DOI: 10.2298/APT1243315V


feic acid derivates, such as cichoric acid (5, 6), are also found in substantial concentrations. 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 activity, 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 basilicum 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 out for 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 The content of total phenolics (TP) in the O. basilicum extract was determined by the Folin-Ciocalteu procedure (8, 9) and expressed as mg of gallic acid equivalent (GAE) per 316

APTEFF, 43, 1-342 (2012) DOI: 10.2298/APT1243315V


g of dry O. basilicum extract. The total flavonoids content (TF) was determined by aluminium 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 described by Espin (11). Briefly, dry O. basilicum extract was mixed with methanol (96%) and 90 M of 2,2-diphenyl-1-picryl-hydrazyl (DPPH) solution to give different final concentration (from 0.0025 mg/ml to 0.008 mg/ml) of extract. After 60 min at room temperature, the absorbance was measured at 517 nm and expressed as radical scavenging capacity. Radical scavenging capacity (%RSC) was calculated by the following equation:

% RSC  100 

Asample  100

[1]

Ablank

where: Asample is 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 extraction 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 Ethanol (X1, %) Temperature (X2, ºC)

(-1.414) 21.7 33.8

(-1) 30 40

Coded levels (0) 50 55

(1) 70 70

(+1.414) 78.3 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 compounds 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), Y3. Nineteen runs, with six replications at the central point, were performed to cover as more as possible combinations of the factor levels. The coded and uncoded independent variables used in the RSM design and obtained responses are shown in Table 2. Experimental data were analyzed by RSM to fit the second order polynomial model (Eq. [2]), where b0, bi, bii and bij are the regression coefficients; Xi are the coded variables; X2 is the 317

APTEFF, 43, 1-342 (2012) DOI: 10.2298/APT1243315V


temperature and X1 is the concentration of the ethanol solution. Y is the analyzed response (total phenols content, total flavonoids content and antioxidant activity).

Y  b0   bi X i   bii X ii2   bij X i X j

[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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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Temperature (°C) 40 40 40 55 55 55 70 70 70 55 55 33.8 76.2 55 55 55 55 55 55

Ethanol (%) 30 50 70 30 50 70 30 50 70 21,7 78.3 50 50 50 50 50 50 50 50

TP (g GAE/100 g) 10.39 14.20 16.83 12.58 15.98 17.99 11.88 13.61 17.31 10.40 17.04 13.56 12.61 15.98 15.98 15.98 15.99 15.98 15.98

TF (g CE/100 g) 4.48 4.98 4.28 4.79 5.48 5.58 4.62 5.53 5.10 4.09 5.58 4.51 5.38 5.48 5.47 5.47 5.48 5.47 5.48

IC50 · 10-3 (mg/ml) 5.05 4.70 4.44 4.43 4.27 3.97 4.63 4.10 3.87 5.24 4.15 4.98 4.01 4.28 4.28 4.25 4.27 4.28 4.28

APTEFF, 43, 1-342 (2012) DOI: 10.2298/APT1243315V


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 b0 Linear b1 b2 Quadratic b3 b4 Interaction b5 R2

15.90145

p-value for Y1 0.000000

5.50691


0.00219 2.66758

0.991513 0.000000

0.273790 0.31741

-1.36558 -0.83433

0.000097 0.003945

-0.24990 0.970

0.448965

TP (Y1)

0.004237


0.003905 0.001444

-0.000295 -0.000338

0.000014 0.000004

-0.33577 -0.39453

0.002441 0.000773

0.000125 0.000164

0.019102 0.004188

0.16925 0.932

0.182691

0.000037 0.964

0.570562

TF (Y2)

IC50 (Y3)

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