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Optimisation of extraction conditions of phenolic compounds and antioxidant activity of Ruta chalepensis L. using response surface methodology

  • Yassine BenchikhEmail author
  • Amina Zaoui
  • Rihab Derbal
  • Mostapha Bachir bey
  • Hayette Louaileche
Original Paper
  • 45 Downloads

Abstract

In the present study, the extraction conditions of phenolic compounds from the Ruta chalepensis L. (a medicinal plant) and the antioxidant activity of its extract were determined. The response surface methodology (RSM) flowing Box–Behnken design was used to study and optimise the effect of three factors [ethanol concentration (40–80%), sample to solvent ratio (0.05/10–0.5/10 g/mL) and extraction time (30–90 min)] on the total phenolic content (TPC) and the antioxidant activity (AA). The optimal extraction conditions predicted by the models were 50.33%, 0.28 g/10 mL, and 59.86 min for the ethanol concentration, the sample to solvent ratio and the extraction time, respectively. These factors gave the optimal predicted values of 521.10 mg GAE/g DW and of 60.48 mg AAE/g DW for the total phenolic content and the antioxidant activity, respectively. Total flavonoids (TFC), flavonols (TFlC), and ortho-diphenols (ODC) contents were further determined by evaluating the extract obtained by the validated optimal conditions of phenolic compounds. The obtained contents were 129.72 ± 1.09 mg QE/g DW, 24.98 ± 0.38 mg RE/g DW and 45.93 ± 0.37 mg CAE/g DW for TFC, TFlC, and ODC, respectively. The data of this report revealed that the extract of Ruta chalepensis L. is an excellent source of natural antioxidants that can be used or incorporated, as natural additives, into different food products in order to develop novel functional food products.

Keywords

Antioxidant activity Box–Behnken design Optimisation Phenolic compounds Response surface methodology Ruta chalpensis L. 

Introduction

Ruta chalpensis L. is a medicinal plant that is known as Fijel in the Maghreb countries. It is a shrub cultivated and mostly spread in the Mediterranean basin as well as the Tropical Atlantic Forest [1, 2, 3]. This plant is traditionally used as a medicinal plant, and it has large therapeutic effects against fever, menstrual problems, rheumatism, convulsion, dropsy, neuralgia, nervous and mental disorders, bleeding etc. In addition, it has antioxidant, anthelmintic, anti-inflammatory, anti-microbial and anti-cancer proprieties [1, 2, 4, 5, 6]. For example, the citric extract of this medicinal plant leaves obtained by infusion is given to children in order to treat nervous disorders and convulsions [7, 8]. It seems that Ruta chalpensis contains bioactive molecules of interest, such as phenolic acids, flavonoids, alkaloids, etc., which are responsible for the therapeutic effects, previously cited, and food protection against oxidation and microbiological alterations [3, 4, 8]. Among these molecules, phenolic compounds content of this plant was reported to have an effective antioxidant activity [3, 5, 9]. These natural molecules can be, indeed, used as alternative to synthetic additives in food products, like producing new functional foods that would procure a good health. Among the methodologies used in order to optimise the extraction of these compounds, response surface methodology (RSM) was reported as interesting tool to determine exactly the influence of several factors on the phenolic extraction and to determine the optimal extraction conditions [10, 11, 12]. This methodology reduces measurements, improves the statistical interpretation, and indicates the quadratic and interaction effects between factors [13, 14]. Many factors such as solvent concentration, sample to solvent ratio, extraction time and temperature can affect the extraction of phenolic compounds from plants and fruits [11, 15, 16]. In the large literature, there is scarcity in the studies that deal with the extraction conditions of the phenolic compounds from the Ruta chalepensis L. using response surface methodology. Thus, in the present study, we have fixed as objectives the modelling and optimisation of the extraction process of phenolic compounds from Ruta chalpensis using response surface methodology to maximise first the studied responses [total phenolic content (TPC) and antioxidant activity (AA)], and second to valorise this medicinal and endemic plant of Algeria. By performing the extraction process using the optimal parameters, the total flavonoids, flavonols and ortho-diphenols contents were selected to determine their contents in the optimal phenolic extract.

Materials and methods

Standards and reagents

Folin–Ciocalteu was from VWR Prolabo (Fantenay-sous-Bois, France); gallic acid, sodium carbonate, sodium acetate, sodium hydroxide and methanol were from Biochem, Chemopharma (Cosne-sur-loire, France); ethanol was from Biochem, Chemopharma (Quebec, Canada); aluminium chloride and sodium molybdate were from VWR Prolabo (Leuven, Belgium); 1,1-diphenyl-2-picrylhydrazyl (DPPH), quercetin, rutin and caffeic acid were from Sigma Chemical (Sigma–Aldrich GmbH, Germany).

Plant material

The medicinal plant of Ruta chalepensis L. was collected from the mountain of Milia (Jijel, Algeria) on February 2018. The aerial parts of this plant (secondary stems, leaves and flowers) were air-dried at room temperature in darkness. After 5 days of drying, the parts of the plant were ground by using a grander (SilverCrest, Canada) and the powder was passed through sieve, by using a sieve shaker (Retsch, AS 200, Germany), under 250 µm of porosity.

Selection of the appropriate extraction conditions

According to the results of a preliminary study using one-factor/one-method in order to evaluate the effect of each factor independently of the others, on the extraction of the phenolic compounds, the solvent concentration of 40–80%, the sample to solvent ratio of 0.05/10–0.5/10 g/mL and the extraction time of 30–90 min were selected to optimise antioxidants extraction using response surface methodology.

Extraction procedure

An aliquot of sample was placed in a flask of 50 mL with 10 mL of ethanol. The extraction was carried out under magnetic stirring at 500 rpm by using the magnetic stirrer (AGIMATIC-S, P-SELECTA, Spain), at different times and solvent concentration. The temperature of extraction was fixed at 25 °C. The extract was centrifuged at 1700×g (Sigma 2–16 P centrifuge, Germany) for 20 min and the supernatant was passed through a filter paper (F1001, Chem, Chmlab Group, Barcelona, Spain). The filtrate was then used as the extract to determine the studied responses.

Determination of phenolic contents and antioxidant activity

Determination of total phenolic content

The total phenolic content (TPC) was determined according to the protocol of Singleton and Rossi [17] as described by Ouerghemmi et al. [9]. Briefly, an aliquot of 0.13 mL of Ruta chalepensis L. extract was mixed with 0.5 mL of distilled water and 0.13 mL of Folin–Ciocalteu reagent. After 6 min of incubation at room temperature, 1.25 mL of sodium carbonate (7%) was added. The mixture was let 30 min in darkness at room temperature, and then the absorbance was measured at 760 nm with spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). The gallic acid was used as standard and the TPC was expressed as milligrams gallic acid equivalents per one gram of Ruta chalepensis L. dry weight (mg GAE/g DW). The mathematical formula by which the TPC was calculated is as follow:
$${\text{TPC=}}\frac{(Y{\text{*10*}}DF/m)}{4.173}$$

where, TPC is the total phenolic content (mg GAE/g); Y is the absorbance of the reaction mixture containing phenolic compounds; 10 is the volume of the solvent; DF is the dilution factor; m is the mass of the sample; 4.173 is the value of the linear regression line.

Determination of total flavonoid content

The total flavonoid content (TFC) was measured according to the method described by Dewanto et al. [18]. An aliquot of 0.3 mL of the extract was mixed with 0.3 mL of sodium nitrite (5%). After 6 min, 0.3 mL of aluminium chloride (10%) was added and after five minutes, 2 mL of sodium hydroxide (1 M) was added to the mixture. The mixture was adjusted to 10 mL with distilled water. The absorbance of the mixture reaction was measured immediately at 510 nm. The quercetin was used as standard and the TFC was expressed as milligrams quercetin equivalents per one gram of Ruta chalepensis L. dry weight (mg QE/g DW).

Determination of total flavonol content

The total flavonol content (TFlC) of plant extract was determined according to the method used by Yermakov et al. [19] as described by Kacem et al. [3]. One milliliter of extract was added to the mixture of 1 mL of aluminium chloride (2%) and 3 mL of sodium acetate (5%). After 2 h of incubation, the absorbance was measured at 440 nm. The rutin was used as standard, and the TFlC was expressed as milligrams rutin equivalents per one gram of Ruta chalepensis L. dry weight (mg RE/g DW).

Determination of total ortho-diphenol content

The total ortho-diphenol content (ODC) of Ruta chalepensis L. was measured by using the method of Mateos et al. [20] as described by Kacem et al. [3]. Thus, one milliliter of the sodium molybdate (5%) was added to 4 mL of the extract. After 15 min of the incubation, the absorbance was measured at 370 nm. The caffeic acid was used as standard and the ODC was expressed as milligrams caffeic acid equivalents per one gram of Ruta chalepensis L. dry weight (mg CAE/g DW).

Evaluation of antioxidant activity

The free radical scavenging activity or antioxidant activity was evaluated according to the method by Brand-Williams et al. [21]. A hundred microliters of the extract were added to 1 mL of DPPH solution (60 mM). After 30 min of incubation in the darkness, the decrease in absorbance was determined at 517 nm. Ascorbic acid was used as a standard, and the antioxidant activity was expressed as milligrams ascorbic acid equivalents per one gram Ruta chalepensis L. dry weight (mg AAE/g DW).

Statistical analyses

Experimental design

The experimental approach used in the present study was the Box–Behnken design that includes three variables and three factorial levels. As stated above, the independent variables used were solvent concentration (x1, ethanol/water, %), the sample to solvent ratio (x2, g/10 mL), and the extraction time (x3, min). The coded and real independent variables and the experimental design were mentioned in the Table 1. The lowest, the central and the highest levels of the variables were coded as − 1, 0, + 1, respectively.

Table 1

Matrix of Box–Behnken design mentioning the factors and the coded and uncoded levels of three variables, experimental (observed) and predicted data of total phenolic content and antioxidant activity

Run

Variable levelsa

TPCb

Antioxidant activityb

x 1

x 2

x 3

Observed

Predicted

Observed

Predicted

1

40 (− 1)

0.05 (− 1)

60 (0)

365.73

337.03

36.99

35.94

2

40 (− 1)

0.5 (+ 1)

60 (0)

452.78

457.82

37.39

39.97

3

80 (+ 1)

0.05 (− 1)

60 (0)

349.68

344.64

35.64

33.06

4

80 (+ 1)

0.5 (+ 1)

60 (0)

154.36

183.06

16.37

17.42

5

60 (0)

0.05 (− 1)

30 (− 1)

408.26

399.95

45.94

44.42

6

60 (0)

0.05 (− 1)

90 (+ 1)

214.99

257.03

19.85

25.01

7

60 (0)

0.5 (+ 1)

30 (− 1)

322.63

280.59

25.63

20.47

8

60 (0)

0.5 (+ 1)

90 (+ 1)

327.29

335.60

35.82

37.35

9

40 (− 1)

0.275 (0)

30 (− 1)

432.26

469.27

43.23

45.81

10

80 (+ 1)

0.275 (0)

30 (− 1)

299.48

312.82

36.62

40.72

11

40 (-1)

0.275 (0)

90 (+ 1)

415.78

402.44

56.27

52.17

12

80 (+ 1)

0.275 (0)

90 (+ 1)

328.74

291.74

34.41

31.83

13

60 (0)

0.275 (0)

60 (0)

488.98

502.08

56.02

59.45

14

60 (0)

0.275 (0)

60 (0)

502.44

502.08

58.97

59.45

15

60 (0)

0.275 (0)

60 (0)

514.83

502.08

63.35

59.45

ax1, Solvent concentration (%); x2, Sample to solid ratio (g/10 mL); x3, Extraction time (min)

bTotal phenolic content (TPC) and antioxidant activity were expressed as mg GAE/g DW and mg AAE/g DW, respectively

Data analysis

The JMP 14.0.1 software (SAS Institute, Inc., Cary, NC, USA) was used to analyse the experimental data. Experimental data were fitted to a second order polynomial model and the regression coefficients were obtained. The generalised second-order polynomial model used in the response surface analysis was as given in the following equation (Eq. 1):
$$Y={a_0}+\mathop \sum \limits_{{i=1}}^{3} {a_i}{x_i}+\mathop \sum \limits_{{i=1}}^{3} {a_{ii}}x_{i}^{2}+\mathop \sum \limits_{{i=1}}^{3} \mathop \sum \limits_{{j=i+1}}^{3} {a_{ij}}{x_i}{x_j} \ldots$$
(1)

where a0, ai, aii, and aij are the regression coefficients of intercept, linear, quadratic and interaction terms, respectively, and xi and xj are the independent variables.

Fisher’s test was used to determine the type of the model equation and Student’s t test was performed to determine the significance of regression coefficients.

Validation of the model

The optimal conditions for the extraction of phenolic compounds of Ruta chalepensis L. were obtained by using the predictive validated equation of RSM. The experimental and predicted responses were then compared in order to determine the accuracy of the model.

Results and discussions

Analysis of the models

The levels of three studied factors, the total phenolic contents, and the antioxidant activities results (experimental and predicted values) were showed in Table 1. The results indicated that the experimental responses were close to the predicted results by the models. This was well confirmed by the significant correlation between the experimental and predicted results (p < 0.05). This is also supported by the values of root mean square error (RMSE) which was weak, with values of 41.95 for the total phenolic content and of 5.43 for the antioxidant activity.

The coefficient of determination (R2) was calculated by using the data of variance analysis. This coefficient gives information about the predictive power of the model; if its value is close to 1 and the lack of fit is not significant, then the model can be validated. In the present study, the coefficients of determination for total phenolic content and antioxidant activity were 0.94 and 0.95, respectively. This explains well the significance of the two models, so the models can be validated (Table 2).

Table 2

Analysis of variance of the models and the lack of fit for the total phenolic content (TPC) and antioxidant activity (AA)

Source

DF

Sum of squares

Mean squares

F Ratio

TPC

 Model

9

139811.06

15534.6

8.8265

 Error

5

8799.97

1760.0

Prob. > F

 Total adjusted

14

148611.03

 

0.0137*

 Lack of fit

3

8465.67

2821.89

16.8823

 Pur error

2

334.30

167.15

Prob. > F

 Total error

5

8799.97

 

0.0564

R2 = 0.94

AA

 Model

9

2590.10

287.79

9.7517

 Error

5

147.56

29.51

Prob. > F

 Total adjusted

14

2737.66

 

0.0110*

 Lack of fit

3

120.35

40.12

2.9493

 Pur error

2

27.21

13.60

Prob. > F

 Total error

5

147.56

 

0.2634

R2 = 0.95

DF degrees of freedom

*P < 0.05

The regression analysis of the two models (total phenolic content and antioxidant activity) indicated that their mean squares were higher than the mean squares of residues (Table 2). This indicated that the variance of the two models was higher than the variance of residues. Furthermore, the comparison of these two variances, according to the Fisher ratio, showed the highest values, which were 8.826 for the phenolic compounds and 9.751 for the antioxidant activity, corresponding to the probabilities of 0.0137 and 0.0110, respectively.

On the other hand, the ratio of the mean squares of lack of fits and those of pure errors (experimental error) were respectively 2821.89 and 167.15 for the phenolic compounds and 40.12 and 13.60 for the antioxidant activity, corresponding to the probabilities of 0.056 and 0.263, respectively. These results clearly indicated the non significant of the lack of fit for the two models (Table 2).

According to the previous results, the two models have the highest significance; thereby they were validated to explain the experimental results and used to predict the responses.

The effects of the three studied factors on the extraction of phenolic compounds and antioxidant activity are shown in Table 3. When the value of the probability of the factor is less than 0.05, it indicates that this factor has a great impact. In addition, when the difference between the value of the estimation coefficient and the standard error of a factor is large, more the factor is judged influent. It is also noted that the more the t ratio (value of estimation coefficient divided by the value of standard error) is important, the more the response is influenced by the factor.

Table 3

Estimation coefficient, standard error and Student’s t test values of response surface for TPC and antioxidant activity

Source

Estimation coefficient

Standard error

t Ratio

Prob. > |t|

TPC

 Intercept

502.08

24.22

20.73

< 0.0001*

 Solvent concentration

− 66.79

14.83

− 4.50

0.0064*

 Sample to solid ratio

− 10.20

14.83

− 0.69

0.5222

 Extraction time

− 21.98

14.83

− 1.48

0.1985

 Solvent concentration*Sample to solid ratio

− 70.59

20.98

− 3.37

0.02*

 Solvent concentration*Extraction time

11.44

20.98

0.55

0.6091

 Sample to solid ratio*Extraction time

49.48

20.98

2.36

0.0648

 Solvent concentration*Solvent concentration

− 60.34

21.83

− 2.76

0.0397*

 Sample to solid ratio*Sample to solid ratio

− 111.11

21.83

− 5.09

0.0038*

 Extraction time*Extraction time

− 72.68

21.83

− 3.33

0.0208*

Antioxidant activity

 Intercept

59.45

3.14

18.95

< 0.0001*

 Solvent concentration

− 6.36

1.92

− 3.31

0.0213*

 Sample to solid ratio

− 2.90

1.92

− 1.51

0.1913

 Extraction time

− 0.63

1.92

− 0.33

0.7548

 Solvent concentration*Sample to solid ratio

− 4.92

2.72

− 1.81

0.1300

 Solvent concentration*Extraction time

− 3.81

2.72

− 1.40

0.2194

 Sample to solid ratio*Extraction time

9.07

2.72

3.34

0.0206*

 Solvent concentration*Solvent concentration

− 8.51

2.83

− 3.01

0.0297*

 Sample to solid ratio*Sample to solid ratio

− 19.34

2.83

− 6.84

0.0010*

 Extraction time*Extraction time

− 8.30

2.83

− 2.94

0.0324*

x1: Solvent concentration (%), x2: Sample to solid ratio (g/10 mL), x3: Extraction time (min)

*P < 0.05

According to the results of Table 3, the solvent concentration exerted significant effects on the extraction of phenolic compounds of Ruta chalepensis L. These effects were linear, quadratic, and interactive with sample to solid ratio. The latest have also a quadratic effect on the extraction of phenolic compounds. A quadratic effect have also exerted by the extraction time. For the antioxidant activity, the linear and quadratic effects of solvent concentration were exerted. The sample to solid ratio and the extraction time have exerted quadratic and interactive effects together. However, the sample to solid ratio and the extraction time have not exerted a linear effect on both total phenolic content and antioxidant activity.

The obtained effects on the extraction of the phenolic compounds and the antioxidant activity were classified in the following decreasing order:

Sample to solvent ratio*Sample to solvent ratio > Solvent concentration > Solvent concentration*Sample to solvent ratio > Sample to solvent ratio*Extraction time > Extraction time*Extraction time > Solvent concentration*Solvent concentration.

According to the results obtained previously, the fitted models by considering significant terms can be expressed as in the following equations Eq. 2 (total phenolic content) Eq. 3 (antioxidant activity):
$${\text{TPC}}={502.08}-{66.79}{x_1}-{70.59}{x_1}{x_2}-{60.34}{x_1}{x_1}-{111.11}{x_2}{x_2}-{72.68}{x_3}{x_3} \ldots$$
(2)
$${\text{AA}}={59.45}-{6.36}{x_1}+{9.07}{x_2}{x_3}-{8.52}{x_1}{x_1}-{19.34}{x_2}{x_2}-{8.30}{x_3}{x_3} \ldots$$
(3)

TPC: total phenolic content, AA: antioxidant activity, x1: solvent concentration, x2: sample to solvent ratio; x3: extraction time.

Analysis of response surfaces

To better highlight these two validated models, the variations of the total phenolic content and the antioxidant activity of Ruta chalpensis L. according to the two factors that varied into the experimental field, the optimal graphics were generated (Figs. 1, 2, 3).

Fig. 1

Surface response plots showing the effects of the solvent concentration and the sample to solid ratio on the total phenolic content (TPC, a) and the antioxidant activity (AA, b)

Fig. 2

Surface response plots showing the effects of the solvent concentration and the extraction time on the total phenolic content (TPC, a) and the antioxidant activity (AA, b)

Fig. 3

Surface response plots showing the effects the sample to solid ratio and the extraction time on total phenolic contents (TPC, a) and antioxidant activity (AA, b)

The Fig. 1 represented the effects of solvent concentration and sample to solvent ratio on total phenolic contents (TPC, Fig. 1a) and antioxidant activity (AA, Fig. 1b). According to these graphs, it seems that these two factors have the quadratic effects on the extraction of the phenolic compounds and the antioxidant activity of Ruta chalepensis L. This showed that the extraction of phenolic compounds was simultaneously influenced by the solvent concentration and the sample to solid ratio, and that the polarity of the solvent and the amount of the sample added in this solvent exerted the important effects on the extraction of the phenolic compounds. Beyond the optimal values (solvent concentration of 50.33% and sample to solvent ratio of 0.28 g/10 mL), the total phenolic content and the antioxidant activity decreased progressively. This can be explained by the fact that beyond a sample to solid ratio, the saturation of the solvent and the decreasing of the solubility of the phenolic compounds can be occurred and the phenolic compounds extraction would decrease; in this case, the solvent favoured the extraction of less polar phenolic compounds [22, 23]. This was also argued by the decrease in the antioxidant activity of Ruta chalepensis L. beyond these optimal values. However, it is worthwhile to note that the sample to solvent ratio has not exerted a linear effect on the extraction of phenolic compounds from Ruta chalepensis L.

Figure 2a represented the total phenolic contents values (TPC) according to the solvent concentration and the extraction time and Fig. 2b represented antioxidant activity values (AA) according to the same factors mentioned above. The data showed that these two factors have the quadratic effects on the extraction of the phenolic compounds and the antioxidant activity of Ruta chalepensis L., and they have not exerted an interaction effect. Thus, the extraction of phenolic compounds was independently influenced by the solvent concentration and the extraction time. Beyond the extraction time value of 59.86 min, the total phenolic compounds were started progressively to decrease because the phenolic compounds, previously extracted, with the risk to be altered during the prolonged time of the extraction may be due to the oxidation to quinines [24]. This phenomenon can be explained by the decrease in the polarity of the phenolic compounds at low solvent concentration which explains that they were not highly extracted [25]. This is also explained by the decrease of antioxidant activity of the extraction just after 59.86 min of extraction.

Figure 3 represents the sample to solid ratio and the extraction time according to the total phenolic contents (TPC, Fig. 3a) and antioxidant activity values (AA, Fig. 3b). These two factors have quadratic effects, but without any linear and interactive effects on the extraction of the phenolic compounds and antioxidant activity. Beyond the optimal value of the sample to solvent ratio of 0.28 g/10 mL, the phenolic compounds decreased progressively when the extraction time was prolonged more than 59.86 min. This can be explained by the saturation of the extraction solvent, the degradation of the phenolic compounds, which were extracted before, and the prolonged extraction time with its effect on the nature of these compounds [25]. This was clearly showed by the evolution of the antioxidant activity values, which decreased after the optimal values of the sample to solvent ratio and the extraction time.

Validation of the optimal conditions

The optimal conditions of the phenolic compounds extraction of Ruta chalepensis L. were determined by the prediction profiler approach. The optimal levels of the three factors (solvent concentration, sample to solvent ratio, and extraction time), which optimise the two studied responses (total phenolic content and antioxidant activity) were 50.33% ethanol, 0.28 g/10 mL, and 59.86 min, respectively. These factors gave the optimal predictive values of 521.10 mg GAE/g DW and of 60.48 mg AAE/g DW for the total phenolic content and the antioxidant activity, respectively.

According to the large literature, the obtained results were higher than those obtained by Kacem et al. [3], Fakhfakh et al. [5], and Ouerghemmi et al. [9] which have determined the contents of TPC and antioxidant activities of Ruta chalepensis L. from Tunisia. These differences can be due to the use of conventional methods for extraction without any modelling compared to our study.

Total flovonoids, flavonols and orto-diphenols contents

Under optimal conditions for the extraction of phenolic compounds, the total flavonoid content was 129.72 ± 1.09 mg QE/g DW, the total flavonol content was 24.98 ± 0.38 mg RE/g DW, and the total ortho-diphenol content was 45.93 ± 0.37 mg CAE/g. These results were lower than those reported by Kacem et al. [3] and Ouerghemmi et al. [9]. However, the total flovonoid content was higher than that reported by Fakhfakh et al. [5]. These differences can be due to the changing climate, the state of the soil, biotic and abiotic stress, etc.

Conclusion

The extraction conditions of phenolic compounds from Ruta chalepensis L. were optimised using response surface methodology. The high correlation of the model indicated that the second-order polynomial model may be successfully used for the optimisation of extraction conditions. The solvent concentration has a significant linear effect on the extraction of phenolic compounds, as well as the sample to solvent ratio and the extraction time. In addition, the present study demonstrated that the three studied factors have exerted quadratic effects on the TPC and antioxidant activity of Ruta chalepensis L. Thus, significant interactive effects between the solvent concentration, the sample to solvent ratio and the extraction time have been elucidated by the model developed in this study.

The optimal extraction conditions predicted by the models were 50.33%, 0.28 g/10 mL, and 59.86 min for the ethanol concentration, the sample to solvent ratio, and the extraction time, respectively. These factors gave the optimal predictive values of 521.10 mg GAE/g DW and of 60.48 mg AAE/g DW for the total phenolic content and the antioxidant activity, respectively. The total flavonoid (TFC), the total flavonol (TFlC) and the total ortho-diphenol contents (ODC) were determined by evaluating the extract obtained by the validated optimal conditions of phenolic compounds. The obtained contents were 129.72 ± 1.09 mg QE/g DW, 24.98 ± 0.38 mg RE/g DW, and the 45.93 ± 0.37 mg CAE/g CAE/g DW for the TFC, the TFlC, and the ODC, respectively.

Our data revealed that the extract of Ruta chalepensis L. is an excellent source of natural antioxidants that can be used as natural additives to food products or consumed as extract to treat fever, menstrual problems, rheumatism, convulsion, dropsy, neuralgia, nervous and mental disorders, bleeding etc. It seems also interesting to purify the extract of Ruta chalpensis in order to separate the functional constituents whether these active compounds do not exert a synergic effect.

Notes

Acknowledgements

The authors are grateful to the Algerian Ministry of Higher Education and Scientific Research for the financial support. They would also convey special thanks to Dr. Mohamed El-Hadef El-Okki for his valuable help concerning the response surface methodology modelling and to Dr. Mohammed Gagaoua for his support in the scientific opinion and English editing of the manuscript as well as Mrs. Lamia Elmechta for her grammar checking.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflict of interest.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Département de Biotechnologie, Institut de la Nutrition, de l’Alimentation et des Technologies Agro-Alimentaires (I.N.A.T.A.A.)Université Frères Mentouri -Constantine 1ConstantineAlgeria
  2. 2.Laboratoire de Biochimie Appliquée, Faculté des Sciences de la Nature et de la VieUniversité de BejaiaBejaiaAlgeria

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