Optimization of the extraction process for flavonoids from basil ( Ocimum basilicum ) using response surface methodology

Basil (Ocimum basilicum), an edible and medicinal plant with high nutritional value and therapeutic efficacy, was used as a potential source of total flavonoids in this study. Heat reflux extractions were performed using aqueous ethanol. The optimized extraction conditions of total flavonoids from O. basilicum were determined by Box-Behnken design with response surface methodology. Response surface plots showed that the optional four independent variables significantly influenced the extraction yield of total flavonoids. The extraction parameters for the highest total flavonoids yield were optimized as: extraction temperature of 79.74 °C, ethanol concentration of 77.63%, ratio of liquid to material of 29.72:1 (mL/g), and extraction time of 2.06 h. The average yield of total flavonoids under above optimum parameters was 42.61 mg of rutin equivalents per g of extract dry matter, which was in good agreement with the predicted value of 40.23 mg/g. These optimized conditions could be useful for the extraction of flavonoids from O. basilicum.


INTRODUCTION
Flavonoids, a group of polyphenolic compounds and one of the most important classes of secondary metabolites, are widely distributed throughout the plant kingdom.In recent decades, flavonoids have attracted considerable interest due to their various biological and pharmaceutical activities including anticancer, antibacterial, antioxidant, neuroprotective, anti-diabetic, anti-inflammatory and anti-ulcer potential in medicine and health-care food [1][2][3][4] .Flavonoids have complicated structures with multiple chiral centers, making chemical synthesis both difficult and commercially unfeasible, the extraction and purification of bioactive compounds like flavonoids from natural resources are of vital importance in food and medicinal industry, especially the preparation of nutraceuticals, dietary supplements, functional food ingredients and additives to food, cosmetic and pharmaceutical products [5][6][7] .
Ocimum basilicum L. (Lamiaceae), commenly known as sweet basil is an annual herb, native to Asia, South America, Africa and the Mediterranean and widely cultivated in many regions 8 .O. basilicum is an important medicinal and culinary herb 9 .This plant is well known for its diverse pharmacological properties and has been widely used in traditional medicine for the treatment of various ailments such as diabetes, inflammation, cardiovascular diseases, headaches, cough, anxiety, convulsion, fevers, cold, digestive disorders and migraines, insect bites, menstrual cramps, sinusitis, and a variety of neurodegenerative disorders [10][11][12][13][14][15] .
Previous investigations indicated that enriched flavonoids extract of O. basilicum showed strong biological activities and may be responsible for the pharmaceutical and biological effects of the plant [16][17][18][19] .Therefore, it becomes important that how to economically extract the natural flavonoids in O. basilicum.
Box-Behnken design, a widely used form of response surface methodology (RSM), was first introduced by Box and Wilson 16 .It can explore the relationships between the response values and the independent parameters and optimize the process or products influenced by multiple variables.One of the most significant advantages of this method is its ability to take the interactions among different variables into accounts, which are ignored in the traditional one-variable at a time analyses [20][21][22] .
There is little exploitation on the extraction of total flavonoids from the whole aerial part of O. basilicum, though the total flavonoids content of O. basilicum have ever been studied.In this study, the total flavonoids content was considered as response value, and achieved by using RSM.Ethanol concentration, ratio of liquid to material, extraction temperature and extraction time were resigned as optimization parameters.Box-Behnken design was employed to optimize the process parameters of total flavonoids extraction from O. basilicum followed by canonical and ridge analyses.The present study aims to provide technical support for better extraction and utilization of flavonoids from O. basilicum.

Materials and equipments
The aerial part of O. basilicum was collected from Zoucheng County, Shandong Province, China, in October 2012 and authenticated by Dr. Dan Wang from the Institute of Chemical Industry of Forest Products, Chinese Academy of Forestry, China.A voucher specimen (CMSCE-121010) has been deposited at the herbarium of Tianjin Key Laboratory of Pulp and Paper, College of Materials Science and Chemical Engineering, Tianjin University of Science and Technology, China.Rutin was obtained from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).Aluminum nitrate, Ethanol, Sodium hydroxide, Sodium nitrite, other chemicals and solvents were of analytical grade and purchased from Tianjin Chemical Reagent Company (Tianjin, China).The total flavonoids analysis was measured by UV-2500 spectrophotometer (Shimadzu Corporation, Japan).For concentration of samples, the N-1100V-WD rotavapour (EYELA, Japan) was used.Samples were freeze dried with a LGJ-12 lyophilizer (Songyuan Huaxing Technology Development Co., Ltd, Beijing, China).

Preparation of O. basilicum extracts
Air-dried O. basilicum samples were ground in a Willey mill to obtain a fine powder (particle diameter: 0.2-0.5 mm).Then 5.0 g of the powder materials were extracted by aqueous ethanol solvent used for the heat reflux extraction at a designed temperature, extraction time, ratio of liquid to raw material and ethanol concentration.The extraction solution was separated from insoluble residue by centrifugation (12857×g for 3 min).The filtrate obtained after filtration of the extraction solution was collected and diluted to 100 mL for the determination of flavonoids contents.

Proximate composition
Chemical compositions of the samples, namely moisture, ash, crude protein, fat, total dietary fiber were determined according to the Association of Official Analytical Chemist methods 23 .The moisture content was determined by drying at 105 °C until constant weigh.The ash content was determined by the cineration of 2 g sample in a muffle furnace at 500 °C for 2 h.Soxhlet extraction technique using petroleum ether (40-50 °C) was used to evaluate the fat contents of the samples.Kjedahl method was used to determine the crude protein contents of the samples.The content of total dietary fiber (TDF) in O. basilicum was determined according to the AOAC enzymaticgravimetric method.The carbohydrate contents of the samples were estimated by difference (% carbohydrate = 100% -sum of percentage of moisture, ash, fat, total dietary fibercrude fibre and crude protein contents).All analyses were done in duplicate and averaged.

Determination of total flavonoids content (TFC)
Total flavonoids were analyzed using aluminium nitrate colorimetric method 24 .One-milliliter diluted sample solution was mixed with 0.3 mL 5% NaNO 2 solution.The mixture was allowed to stand at room temperature for 6 min with intermittent shaking.Then, 4 mL 4% NaOH solution was added into the mixture and the total volume was made up to 10 mL with distilled H 2 O.With sufficient mixing and 15 min standing, the absorbance was measured at 510 nm, and the solution was determined with a UV-2500 spectrophotometer, and the blank was the solution without the sample.The calibration curve (y = 12.9200x + 0.0328, where y was absorbance value of sample, and x was the sample concentration) ranged from10 to 50 µg/mL (R 2 = 0.9997).

Experimental design
In order to evaluate the extraction parameters and optimize the conditions of total flavonoids extraction, a Box and Behnken design was used in the present study.The effect of the independent variables including extraction temperature (°C), ethanol concentration (%), ratio of liquid to material (mL/g) and extraction time (h) were evaluated in the extraction process 25 .the four factors chosen for this work were designated as X 1 , X 2 , X 3 , and X 4 and divided into three levels: the high, intermediate and low, and the code value was +1, 0, and −1, respectively.The four variables were coded according to the following equation: (1) where X i was a coded variable value; x i was the actual variable value; x 0 was the actual independent variable value at the center point; and Δx was the variable step change.The total flavonoids content value in each trial was the average of triplicates.All the data were expressed as mean ± standard deviation (SD).The extraction conditions were optimized to obtain the maximum ratio of total flavonoids by Stat-Ease Design-Expert 8.0.5b.The behavior of the system was explained by the following quadratic equation: (2) where Y was the typical response function; β 0 was a constant; β i , β ii , and β ij were the linear, quadratic and interactive coefficients, respectively; X i was the coded independent variables level.The terms X i X j and X i 2 represented the interaction between different variables and quadratic terms, respectively

Chemical composition analysis of O. basilicum
Chemical composition of O. basilicum was demonstrated in Table 1.Results showed that total dietary fiber was the highest in O. basilicum, accounting for 43% of the dry basis (43 g/100 g dry weight).Previous studies have shown that increase in fibre consumption might have contributed to the reduction in the incidence of certain diseases such as diabetes, coronary heart disease, colon cancer and various digestive disorders 26 .Results of this work indicated that O. basilicum is suitable as a dietary fibre source for high-valued functional products.Crude lipid and protein were 1.85% and 1.61% of total amount, respectively.The moisture content in O. basilicum was 7.70% and the total ash content was high in O. basilicum (17.00%).The results indicated that O. basilicum could be a source of rich mineral elements bearing nutritional importance 27 .

The influence of extraction temperature on total flavonoids yield
Powdered samples of 5.00 g were used for heat reflux extraction with 100 mL 60% ethanol solution at 50-100°C for 1.0 h.Total extract yield could be determined according to the methods described above and the results were shown in Fig. 1a.It could be seen from Fig. 1a that the extraction yield started to increase with increasing temperature and reached a plateau at 80 °C followed by a slight drop with further increase of temperature.It can be concluded that a higher temperature might cause decomposition of flavonoids and increase the solubility of impurities 28 .In addition, the solvent (ethanol) was also lost at high temperature.Taking these factors into account, the center point of the optimal temperature was considered to be 80 °C for further RSM experiment.

The influence of ethanol concentration on total flavonoids yield
Five grams powdered O. basilicum samples were used for heat reflux extraction with 100 mL ethanol solution of different concentrations ranging from 0% (water, without ethanol) to 100% (ethanol, without water) at 80 °C for 1 h.As displayed in Fig. 1b, the extraction yield began to increase with the raise of ethanol concentration and reached the maximum value at the ethanol concentration of 80%.After that, the extraction yield declined with further increase in ethanol concentration.Besides, the amount of fat-soluble substance increased with increasing ethanol concentration, which would bring hurdles for the further purification 21 .Considering the extraction yield and subsequent purification cost, the center point of concentration of ethanol should be controlled at 80%.

The influence of the ratio of liquid to material on total flavonoids yield
In this work, effects of different ratio of liquid to material (5:1, 10:1, 15:1, 20:1, 25:1 and 30:1) on the extraction yield were investigated under following conditions: extraction temperature at 80°C, ethanol concentration at 80% and extraction time of 1 h.Total extract yield of flavonoids affected by ratio of liquid to material was demonstrated in Fig. 1c.There was a smooth rise in the extraction yield of flavonoids as ratios of liquid-material increased from 5:1 to 30:1, as presented in Fig. 1c.Taking the solution cost and further concentration of extracts into consideration, the center point of the ratio of liquid to material of 20:1 was adopted in this work.

The influence of extraction time on total flavonoids yield
Extraction time is another factor that will influence the extraction efficiency and selectivity of solution.The different extraction time were carried out at 0.5, 1.0, 1.5, 2.0, 2.5 and 3 h with other reaction conditions fixed as follows: extraction temperature at 80°C, ethanol concentration at 80%, and the ratio of liquid to material of 20:1.The total extract yields were shown in Fig. 1d, which indicated that the extract yields increased with the increase of extraction time from 0.5 to 2.5 h and reached a peak at 2.5 h.After 2.5 h, it significantly dropped.An adequate extraction time would increase the dissolve of target components; while more prolonged treatment time might cause loss of activities 20 .Thus, for the single factor experiment of extraction duration, 2.0 h was chosen as the center point of extraction time.

Multiple regression analysis of extraction parameters on TFC
In this investigation, RSM was used to optimize the extraction of total flavonoids from O. basilicum.All of the 29 designed experiments were carried out for optimizing four individual parameters in this Box-Behnken design.Considering that extraction number was also an important factor for extraction of active components from plant and taking into account of extraction efficiency 29 , the extraction number 2 was employed for the 29 designed trials in this work.According to the factorial design, the experimental conditions and the results of total flavonoids yield were shown in Table 2. Multiple regression analysis is applied for the analysis of the experimental data by the response variable and the test variables were related by the following second-order polynomial equation: where X 1 , X 2 , X 3 and X 4 represent the coded values of extraction temperature, ethanol concentration, the ratio of liquid to material and extraction time, respectively.
ANOVA was used to evaluate the model for significance and suitability, and a statistical summary was shown in Table 3.The model F-value of 11.41 with a low probability P-value indicates the high significance of the model which was suitable in this experiment 28 .The coefficient of determination (R 2 = 0.9194) indicated the satisfactory correlation between the actual values and the predicted ones and supported above conclusion.The Adj. R 2 values was 0.8388, which meant most variation (>83.88%) of the total flavonoid content could be predicted by the models, while only 17% variation could not be explained by the model.From the P-values of each model term, the linear variables X 2 , X 3 were statistically significant at P < 0.0001; the linear variable X 1 and two-variable interaction X 4 2 had significant influences (P < 0.05) on the yield of flavonoids, whereas the linear variables X 4 and the two-variable interaction X 1 X 2 , X 1 X 3 , X 1 X 4, and X 3 X 4 had no significant influence (P > 0.1) on flavonoids extraction.By comparing linear and quadratic coefficients, it can be concluded that the ethanol concentration and the ratio of liquid to material were the most significant parameters influencing the total flavonoids yield followed by extraction temperature and extraction time.) extraction number 2 was adopted in this work.

Response surface analysis of extraction parameters on TFC
Two-dimensional contour plots and three-dimensional response surfaces were schematic representations of the regression function.The independent variables were obtained by keeping two of the variables constant.As presented in Figs. 2 and 3, it was evident that there were interaction influences between factors on the responses.As could be seen from Figs. 2b and 3b, there was an obvious rise in the flavonoids yield with the ratio of liquid to material increasing from 15:1 to 20:1 at the extraction temperatures below 80 °C.After that, the influence of temperature on the flavonoids extraction ratio was not significant.The ratio curve reached a plateau at the ratio of liquid to material of 20:1, which manifested that a ratio of liquid to material of 20:1 was required to achieve maximum extraction amount.
The influences of extraction temperature interaction with extraction time on the flavonoids yield were displayed in Figs.2c and 3c.According to the data, the effect of extraction temperature was more significant than that of extraction time on response value.The extraction yield of flavonoids increased rapidly when the extraction temperature increased from 70 °C to 80 °C.However, decreased yield was observed after 80 °C.
In Figs.2d and 3d, it was clear that the influence of the liquid-solid ratio on the extraction rate of flavonoids was not obvious at the given ethanol concentration range.When the solid-liquid ratio was prepared at a fixed value of 20:1, the flavonoids yield increased with the increasing of extraction concentration and then decreased after the concentration was reaching around 85 °C.
The effects of ethanol concentration and extraction time on the yield of flavonoids were shown in Figs.2e  and 3e.The yield was mainly depended upon ethanol concentration rather than extraction time, which resulted in a curvilinear increase until the ethanol concentration of 80% and then slightly decreased.The increase of extraction time resulted in a substantial increase of extraction yield and the peak at extraction time of 2.06 h, indicating that 2.06 h was required to achieve the maximum yield.
The influence of the ratio of liquid to material and extraction time on the flavonoids extraction was displayed in Figs.2f and 3f.Experimental data indicated that the effect of extraction time was less important than the ratio of liquid to material.It was noticed that the yield of flavonoids increased with the increasing of the ratio of liquid to material from 15:1 to 20:1, while extraction time of more than 2.06 h appeared to be adverse on the extract, which suggested that a suitable extraction time was important to the yield of flavonoids.
Based on the above results and analysis, it was concluded that the sequence of each factor for improving the response value of flavonoids extraction yield was as follows: ethanol concentration > liquid-solid ratio > temperature > extracting time, which was in good agreement with our findings in ANOVA.In this work, the optimal conditions to reach the maximum yield of flavonoids were investigated using Design Expert 8.0 software, and the optimum condition was obtained and recommended as a practical optimum: temperature of 79.74 °C, ethanol concentration of 77.63%, liquid-solid ratio of 29.72:1 (mL/g), and extraction time of 2.06 h.The estimated value for Y was optimized to 40.23 mg of rutin equivalents per g of extract dry matter under this condition.

Optimization of extracting parameters and validation of the model b a
To further test the validation of the optimal analytical model, the extraction experiment was carried out with the extraction parameters designed in the model.The optimal conditions was modified as follows: extraction temperature of 80 °C, ethanol concentration of 78%, the ratio of liquid to material of 30:1 and extraction time of 2 h, respectively.Under the condition, the experimental yield of flavonoids was 42.61 mg of rutin equivalents per g of dry matter, which was close to the predicted value and better than the results of single-factor analyses.Consequently, this indicated that the model was successful and adequate to the prediction of the extraction yield.

Fig. 1 .
Fig. 1.Effect of different extraction parameters (extraction temperature, °C; ethanol concentration, %; the ratio of liquid to material, mL/g and extraction time, h) on yield of total flavonoids.

Figs. 2a and
3a showed the influence of extraction temperature and ethanol concentration on the flavonoids extraction yield.Results demonstrated that the extraction yield was minimal at low and high levels of extraction temperature and ethanol concentration.The extraction yield increased greatly with the increase of the extraction temperature when the ethanol concentration ranged from 60% to 80%.However, when the extraction temperature increased up to around 80 °C, ethanol concentration played an important role for improving flavonoids yield.

Fig. 2 .
Fig. 2. Three-dimensional response surface exhibiting the effect of different extraction parameters (X 1 : extraction temperature; X 2 : ethanol concentration; X 3 : the ratio of liquid to material and X 4 : extraction time) added on the response Y (extraction yield of flavonoids).

Fig. 3 .
Fig. 3. Contour plots displaying the influence of different extraction parameters (X 1 : extraction temperature; X 2 : ethanol concentration; X 3 : the ratio of liquid to material and X 4 : extraction time) added on the response Y (extraction yield of flavonoids).
Results of this work indicated that O. basilicum is suitable as a dietary fibre source for high-valued functional products.This work clearly shows that the extraction of flavonoids from O. basilicum was improved significantly by optimizing several key extraction parameters.The extraction conditions obtained by RSM were accurate and reliable.This research opens a new route in extracting and utilizing flavonoids from O. basilicum as an inexpensive source of health-promoting additives in food and medicinal industry.

Table 2 .
The Box-Behnken experimental design with four independent variables.

Table 3 .
ANOVA for response surface quadratic model analysis of variance table.