IT Analysis of Discrete and Combined Effect of Solvent, Extraction Time, and Extraction Temperature on Polyphenol Compounds Extraction from Roxburgh Fig (Ficus auriculata Lour.) Fruit Using Response Surface Methodology

Gayathri Jagadeesan, Kasipandi Muniyandi, M. Ashwini Lydia, Gayathri Nataraj, Suman Thamburaj, Saikumar Sathyanarayanan, and Parimelazhagan Thangaraj

5.1 INTRODUCTION

The advancement in the custom of using medicinal plant- based bioactive compounds influenced the international market for further exploration of important ethnopharma- cological plants (Mendomja-Filho 2006). Despite all the nutrition from different diets, plant-based products are rich in antioxidants and therapeutic potentials. Amidst countless bioactive compounds, the plant secondary metabolites are utilized in pharmacological research for drug development and also used as therapeutic agents against oxidative stress, metabolic disorder, and apoptosis (Chandran et al. 2015; George et al. 2016; Muniyandi et al. 2017; Sathyanarayanan et al. 2017; Suman et al. 2018; Manoharan et al. 2019). The phenolic compounds are the natural antioxidants with dietary elements, which play an outstanding role against oxidative stress by defending the endogenous reactive oxygen species (Sivaraj et al. 2018; Sreeja et al. 2018). Therefore, the screening and extraction of phytocompounds have become decisive in the development of nutraceuticals, dietary ingredients, and pharmaceutical components.

The family Moraceae, generally known as the fig family, and the fig fruits are being utilized for therapeutic effects against liver diseases, menstrual disorders, diabetes, and respiratory problems in the Indian traditional systems (Upadhyay et al. 2007; Arunachalam and Parimelazhagan 2013). F. auriculata, commonly distributed all over India, and the fruits are edible either raw or in the form of jam, roasted fruits, and juices also cooked as curries for the medication of diarrhea and dysentery. Ethnobotanically,

F. auriculata fruits were also used for wound healing, mumps, cholera, and jaundice (Gairola and Biswas 2008). The fruits were reported to contain sterols, terpenes, fla- vonoids, coumarins, laxative substances, sugars, and vitamins A and C with significant antioxidant, antihepatotoxic, antimicrobial, anticancer, antidiabetic, antipyretic, and anti-inflammatory activities (Gairola and Biswas 2008). The phenolic compounds from F. auriculata fruit have significant therapeutic properties; therefore, the voluminous extraction of phenolic compounds would be effective for the pharmaceutical industries for the advancement of drug and nutraceutical development.

The phytochemical extraction may be affected by many factors, such as solvent composition, extracting time, extraction temperature, and solvent to sample ratio (Jagadeesan et al. 2019), therefore optimization should be emphasized to improve the extraction process by considering all the factors. The response surface methodology technique certainly influenced the field of extraction of phytochemicals from medicinal plants (Cacace and Mazza 2003; Rajha et al. 2014; Jagadeesan et al. 2019). Response surface methodology has an extensive application in the design, development, and formulation of new products by optimizing. The technique improves the performance of the systems and increases the yield of the process without increasing the cost of the study (Bezerra et al. 2008). Hence, with all the above detail, this study was designed using response surface methodology to determine the discrete and combined effect of solvent, extraction temperature, and extraction time on the polyphenol extraction from F. auriculata fruits.

  • 5.2 MATERIALS AND METHODS
  • 5.2.1 Chemicals and Solvents

The analytical grade chemicals and reagents used in this study were purchased from HiMedia Laboratories (Mumbai, India); Sisco Research Laboratories (Mumbai, India); Sigma-Aldrich (St. Louis, Missouri, USA); and Merck (Bengaluru, India). The assays were carried out using water obtained from a Milli-Q water system (Merck Millipore, Billerica, MA, USA).

5.2.2 Effect of Ethanol Concentration,

Extracting Temperature, and Extraction Time on the Polyphenol Extraction

The main aim for the present investigation was to determine the discrete and combined effects of solvent, extraction temperature, and extracting time on the polyphenolic content extraction from F. auriculata fruits. The fruits were collected from Western Ghats, Coimbatore, Tamil Nadu, India, and identified by the Botanical Survey of India, Southern Regional Centre, Coimbatore, Tamil Nadu, India. Before shade drying, the sample was rinsed with distilled water, after shade drying, the fruits were finely grounded and used for extraction. The central composite design was used to determine the effects of three parameters, such as ethanol concentration, extraction temperature, and extracting time, and the design parameters were given in our previously published paper (Jagadeesan et al. 2019). The total phenolic content, total flavonoid content, and antioxidant activity were selected as responses. The central composite design for the experiment parameters was presented in Table 5.1. F. auriculata fruit extracts were prepared by adding the fruit powder with various combinations of parameters as tabulated in Table 5.1. The prepared extracts were centrifuged at 3000 x g for 10 minutes, and then the supernatant was collected and dried at room

TABLE 5.1

Summary of Response Surface Methodology Design for Optimizing the F. auriculata Fruit Polyphenol Extracting Conditions

Design Expert—Trial Version 11.0.3.0 Study: Response Surface Method (Central Composite Design)

Model: Quadratic Model Total Experiments: 16

Variables

Coded Variables Levels

Responses/Units

-1

1

X-Ethanol concentration (%)

30

50

R1-Total phenolic content (mg gallic acid equivalents/g fruit extract)

Y-Extraction temperature (°C)

30

55

R2-Total flavonoid content (mg rutin equivalents/g fruit extract)

Z-Extraction time (minutes)

40

120

R3-2,2-diphenyl-1-picryl hydrazyl radical scavenging activity (% inhibition)

temperature. After some time, the dried extract was collected and used for the proposed studies. The optimal conditions for the extraction of the fruit polyphenolic contents were determined by comparing the experimental and predicted values of three responses from the following regression equations.

where Y—predicted response, p0—central point response, pj, pu, and рц—linear, quadratic, and interaction coefficients, and Xj and Xj—variables levels.

5.2.3 Estimation of F. auriculata Fruit Extracts Total Phenoucs Contents

The extracted phenolics of F. auriculata fruit run order extracts were estimated by Thangaraj (2016). About 100 pL of the fruit’s extract was taken from the sample stock solution, and the total volume made up to 1 mL using distilled water with 1 N Folin-Ciocalteu phenol reagent of 0.5 mL added. After 5 minutes of dark incubation, 2.5 mL of 5% sodium carbonate was added and mixed well. After 40 minutes of dark incubation, the formed blue color was read at 725 nm against the reagent blank. The total phenolic content present in the fruit samples was quantified using a gallic acid calibration curve and expressed in milligram gallic acid equivalents per gram of fruit extract.

5.2.4 Estimation of F. auriculata Fruit Extracts Total Flavonoid Contents

The flavonoid contents of the F. auriculata fruit run order extracts were determined by using the method described in Thangaraj (2016). The fruit extracts (0.5 mL) were taken in triplicates and made up to an equal volume with distilled water and kept for 6 minutes of incubation after adding 0.15 mL of 5% sodium nitrite. After the incubation, 0.15 mL of 10% aluminum chloride was added including the reagent blank. Then 2 mL of 4% sodium hydroxide was added, and the final volume made up to 5 mL using distilled water and incubated for 15 minutes at room temperature. After the incubation, the developed pink color was read at 510 nm, and the results were expressed in milligrams of rutin equivalents in grams of fruit extract using a rutin calibration curve.

5.2.5 Analysis of 2, 2-Diphenyl-1-Picryl Hydrazyl Radical Scavenging Activity

The 2, 2-diphenyl-l—picryl hydrazyl radical scavenging ability of prepared F. auriculata fruit experimental extracts were analyzed by the method described by Thangaraj (2016). The desired series of concentration of the extract was taken from the prepared stock, and then the final tube volume was made to 100 pL by adding methanol. Then the 0.1 mM 2, 2-diphenyl-1—picry 1 hydrazyl methanol solution of 3 mL was mixed with the extracts, after the incubation period, the optical density was read at 517 nm against the reagent blank. The tube with 3 mL 2, 2-diphenyl-l-picryl hydrazyl metha- nolic solution alone served as the negative control. The radical inhibition was expressed in terms of inhibition percentage.

Inhibition percentage =

5.2.6 Statistical Analyses

The in vitro studies calculations were carried out using Microsoft Excel 2007 (Microsoft Corporation, Washington, DC, USA). The Response Surface Methodology (RSM) experiments were designed by Design-Expert, Trial Version 11.0.3.0 (Stat-Ease Inc., Minneapolis, MN, USA). One-way Analysis of Variance (ANOVA) was used to produce lack of fit, coefficient of determination (R2), and F-test, and it was used to evaluate the adequacy of the model and response surface plots with multiple linear regressions with three responses to fit the model.

  • 5.3 RESULTS AND DISCUSSION
  • 5.3.1 Validation of the Model

For the optimization of phenolic compound extraction from plants, the second-order polynomial model is often used with the variables such as extraction time, extraction temperature, and solvent composition, and so on (Cacace and Mazza 2003; Rajha et al. 2014; Jagadeesan et al. 2019). And the proposed model is significantly fitted for the phenolic compounds extraction from F. auriculata fruit, and the ANOVA results were presented in Table 5.2. The model was validated by insignificant lack of fit (P > 0.05), which indicated the precision of the proposed model and the accurate prediction of the variables, which effects the polyphenol extraction with the experimental data. Also, the coefficient of determination (R2) was ranging from 0.8 to 0.9 for the studied responses (Table 5.2), indicating the adequacy of this model. The Folin-Ciocalteu reagent was used to quantify phenolics using a gallic acid calibration graph (y = 0.0276x - 0.011; R2 = 0.9913). The total phenolic content of F. auriculata showed significant P (0.0010) and F (10.20) values and the lack of fit was found to be an insignificant P (0.4916) value, which proved that the model was accurate (Table 5.2). The P and F values of the parameters and the linearity between the predicted and actual values showed that the model has a significant effect on the phenolic extraction with the slight variations in the graph (Figure 5.1a). The flavonoid content in F. auriculata fruit was determined by the rutin calibration curve, and the results were expressed in milligram

TABLE 5.2

Analysis of Variance for Response Surface Quadratic Model for the Extraction of Phenolic Compounds from

F. auriculata

R2 of Total Phenolic Content = 0.9107

R2 ofTotal Flavonoid Content = 0.9073

R2 of 2, 2-Diphenyl-1-Picryl Hydrazyl Radical Scavenging Activity = 0.8449

Sum of Square

df

Mean Square

F Value

P Value

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

Model

859.85

9223.88

482.46

9

95.54

1024.88

53.61

10.20

9.78

5.45

0.0010***

0.0011***

0.0094***

X

142.65

0.6033

13.00

1

142.65

0.6033

13.00

15.23

0.006

1.32

0.0036***

0.9412

0.2800

Y

4.45

1330.44

104.68

1

4.45

1330.44

104.68

0.474

12.70

10.64

0.5082

0.0061***

0.0098***

Z

56.59

259.54

2.83

1

56.59

259.54

2.83

6.04

2.48

0.287

0.0363*

0.1499

0.6046

XY

130.98

853.81

30.55

1

130.98

853.81

4.40

13.99

8.15

3.10

0.0046***

0.0189

0.1119

XZ

72.37

1019.79

24.33

1

72.37

1019.79

7.89

7.73

9.74

2.47

0.0214*

0.0123*

0.1503

YZ

328.73

2366.58

115.91

1

328.73

2366.58

39.70

35.11

22.59

11.78

0.0002***

0.0010***

0.0075***

Residual

84.28

942.69

88.58

9

9.36

104.74

9.84

Lack of Fit

48.01

448.26

25.32

5

9.60

89.65

5.06

1.06

0.725

0.320

0.4916*

0.6396»

0.8783»

Pure Error

36.27

494.43

63.25

4

9.07

123.61

15.81

Cor Total

944.12

10166.57

571.04

18

Notes: R2—coefficient of determination; X, Y, Z—selected variables for the present study; X—ethanol concentration; Y—extraction temperature; Z—extraction time; X. Y, Z—the linear effects of selected variables on polyphenol extraction; XY, XZ, YZ—combined effects of selected variables on polyphenol extraction; R1, R2, R3—selected responses for the present study; R1 —total phenolic content; R2—total flavonoid content; R3—2,2-diphenyl-1 -picryl hydrazyl radical scavenging activity.

“ Not significant. Statistically significant at *P < 0.05; * * P < 0.01; and ***P < 0.001.

rutin equivalents per gram of extract (y = 0.0022x - 0.0089; R" = 0.9926). The results of the quadratic polynomial model (Table 5.2) suggested that this model has high response for the flavonoid extraction from fig fruits. The ANOVA of the model showed F and P values of 9.78 and 0.0011, respectively (Table 5.2), which explained the significance of the model with the R2 value of 0.9073 in total flavonoid content extraction. The 2, 2-diphenyl-l-picryl hydrazyl radical scavenging activity of the F. auriculata fruit extract experimental values were given in Table 5.3, and the insignificant lack of fit P value 0.8783 showed that the model was fit. So, these antioxidant phenolic and flavonoid compounds could be obtained by the enhanced solvent composition with temperatures under various extracting times (Cacace and Mazza 2003; Rajha et al. 2014; Jagadeesan et al. 2019). The relationship between the significant independent variables and responses was described by equations 5.3 through 5.5.

2, 2-diphenyl-l-picryl hydrazyl radical

From equations 5.3 through 5.5, X—discrete effect of ethanol concentration; Y—discrete effect of extraction temperature; Z—discrete effect of extraction time; XY—combined effect of ethanol concentration and extraction temperature; XZ—combined effect of ethanol concentration and extraction time; YZ—combined effect of extraction temperature and

TABLE 5.3

Central Composite Design Data and Triplicate Determination Results of Total Phenolic Content, Total Flavonoid Content, and 2, 2-Diphenyl-l-Picryl Hydrazyl Radical Scavenging Activity from the Obtained Experimental Data

Experiment

X

Y

Z

R1

R2

R3

1

50

30

120

31.20 ± 2.18

46.30 ± 3.97

26.39 ±4.91

2

40

42.5

147

41.83 ±2.38

64.73 ± 2.64

28.06 ± 3.00

3

40

42.5

13

29.47 ± 0.84

51.78 ±5.50

28.57 ± 0.83

4

23

42.5

80

40.70 ± 10.60

71.55 ±0.70

39.64 ±2.71

5

40

63.5

80

42.75 ± 3.82

123.49 ±24.17

43.91 ±3.38

6

57

42.5

80

29.83 ±2.41

72.38 ± 4.90

36.03 ± 1.76

7

50

30

40

47.83 ± 6.77

90.97 ± 2.64

34.86 ± 0.63

8

30

55

40

38.16 ±9.57

58.64 ± 1.75

32.88 ± 1.04

9

40

21

80

44.57 ± 2.54

99.45 ± 3.69

38.81 ±0.21

10

40

42.5

80

39.67 ± 2.54

82.68 ± 1.85

35.51 ±1.17

II

30

55

120

59.21 ±5.44

127.94 ±7.35

46.61 ±0.38

12

50

55

120

38.18 ±4.01

122.08 ±4.38

44.36 ± 0.28

13

30

30

40

40.64 ± 14.19

92.99 ±6.13

37.96 ± 1.53

14

30

30

120

35.13 ±3.70

87.74 ± 11.00

34.74 ± 1.42

15

50

55

40

30.08 ± 9.89

103.70 ± 10.65

39.32 ± 3.59

16

40

42.5

80

36.23 ± 2.38

78.64 ± 4.86

36.37 ± 1.67

Notes: X. Y, Z—selected variables for the present study; X—ethanol concentration (%); Y—extraction temperature (°C); Z-extraction time (minutes); Rl, R2. R3—selected responses for the present study; Rl—total phenolic content (mg gallic acid equivalents/g fruit extract); R2—total flavonoid content (mg rutin equivalents/g fruit extract); R3—2, 2-diphenyl-l-picryl hydrazyl radical scavenging activity (% inhibition); values are mean of triplicate determination (n = 3) ± standard deviation.

extraction time; X2—integrative effect of ethanol concentration; Y2—integrative effect of extraction temperature; and Z2—integrative effect of extraction time.

5.3.2 Discrete Effect of Solvent, Extraction Time and Extraction Temperature on Polyphenol Extraction

The preparation of extracts is the important process for separating phytochemicals, and there are various factors which affect the extraction conditions, such as the sample nature, solvent, extraction temperature, and extraction time (Bezerra et al. 2008). In the present work, F. auriculata fruit extracts were obtained by using different concentrations of aqueous ethanol ranging from 30% to 50% for effective extraction (Jagadeesan et al. 2019). The obtained results indicated that the increasing ethanol concentration increases the extraction of phenolic and flavonoid content. The total phenolic content of the run order extracts was calculated using the Folin-Ciocalteu method and the values range from 29.47 ± 0.84 to 59.21 ± 5.44 mg gallic acid equivalents/g sample (Table 5.3). In the case of flavonoid, the estimated amount ranged from 51.78 ± 5.50 to 127.94 ± 7.35 mg rutin equivalents/g extract, whereas in the case of free radical scavenging activity, the scavenging percentage varies from 26.39% ±4.91% to 46.61% ± 0.38% (Table 5.3). From the ANOVA results, the variable ethanol concentration contributed significantly to the better extraction of the phenolic content from fruit samples with the P value of 0.0036 and F value of 15.23 (Table 5.2). But in the case of flavonoid, the ethanol concentration revealed moderate significant values (F = 0.006; P = 0.9412), and the antioxidant results supported that the solvent effect was significant with higher radical inhibition (F = 1.32; P = 0.2800) (Table 5.2). The extractions of phenolic and flavonoid compounds from a fruit are highly associated with the concentration of the solvent concentration (Zhang et al. 2007; Do et al. 2014; Vajic et al. 2015), this conclusion is supported by the obtained results of this study that were the ethanolic concentrations greatly influenced the phenolic and flavonoid content extraction. These results were highly correlated with our earlier result (Jagadeesan et al. 2019), where the phenolic and flavonoid contents were higher at moderate ethanol concentration. The statistical analysis showed that the extracting temperatures highly influenced the flavonoids extraction (P = 0.0061; F = 12.70), as well as the antioxidant activity (P = 0.0098; F = 10.64) (Table 5.2). The significant extraction of the flavonoids could be responsible for the higher antioxidant activity of the fruit extract. The higher temperature increases the extracting ability of the solvent by enhancing polyphenol diffusion and the solubility (Al-Farsi and

Lee 2008). The previous reports (Wang et al. 2007; Zhang et al. 2007; Al-Farsi and Lee 2008; Do et al. 2014; Vajic et al. 2015; Jagadeesan et al. 2019) also supported the present results that the extraction temperature could favor the breakdown of bound polyphenol in the sample. The extraction time is also important in polyphenol extraction (Wang et al. 2007; Zhang et al. 2007; Al-Farsi and Lee 2008; Do et al. 2014; Vajic et al. 2015; Jagadeesan et al. 2019), in the ANOVA analysis, the extraction time showed significant values for phenolic (P = 0.0363; F = 6.04) and flavonoid (P = 0.1499; F = 2.48) content (Table 5.2).

5.3.3 Combined Effect of Solvent, Extraction Time, and Extraction Temperature on Polyphenol Extraction

The extraction combined effect of solvent, extraction time, and extraction temperature on the phenolic and flavonoid content was given in Table 5.2 and Figures 5.1 through 5.3. All of the selected variables significantly contributed toward the extraction of polyphenol from F. auriculata fruits. Based on the ANOVA results, solvent and temperature (XY) were combined and influenced the extracting ability (P = 0.0046; F = 13.99) (Table 5.2). Also, the interactive effect (YZ) of extraction temperature and extraction time highly established the phenolic extraction condition with the P value of 0.0002 and F value of 35.11. Whereas, the flavonoid (P = 0.0010; F = 22.59) and antioxidant activity (P = 0.0075; F = 11.78) were significantly influenced by the combined effect of extraction temperature and extraction time (Table 5.2). Moreover, the ethanol concentration and extraction time also slightly influenced the phenolic and flavonoid content extraction with the significant value of P < 0.05 (Table 5.2). Therefore, the respective and combined parameters of the ethanol concentration, extraction temperature, and extraction time were highly supported for

Response surface plots for the combined effect of variables on total phenolic content of F

FIGURE 5.1 Response surface plots for the combined effect of variables on total phenolic content of F. auriculata. (a) Correlation between experimental and predicted value, (b) effect of ethanol concentration and extraction temperature, (c) effect of ethanol concentration and incubation time, and (d) effect of extraction temperature and incubation time.

Response surface plots for the combined effect of variables on total flavonoid content of F

FIGURE 5.2 Response surface plots for the combined effect of variables on total flavonoid content of F. auriculata. (a) Correlation between experimental and predicted value, (b) effect of ethanol concentration and extraction temperature, (c) effect of ethanol concentration and incubation time, and (d) effect of extraction temperature and incubation time.

the extraction of phenolic content (Figure 5.1). From this developed quadratic model, the higher flavonoid content was achieved at the temperature of 45°C-50°C at the extracting time of 80 minutes with the ethanol concentration of 45%-50% (Figure 5.2) and the antioxidant activity was increased with the ethanol concentration of 30%-40%, with the temperature of 55°C-60°C, and at the extraction time of 120 minutes (Figure 5.3). The obtained results were highly correlated with the previous reports on the phenolic extraction through optimized conditions (Wang et al. 2007; Zhang et al. 2007; Al-Farsi and Lee 2008; Hossain et al. 2012; Gomes et al. 2013; Do et al. 2014; Vajic et al. 2015; Jagadeesan et al. 2019). The obtained results indicated that the moderate ethanol concentration and extraction temperature with a higher extraction time were highly favored for the extraction of phenolic and fla- vonoids compounds. Therefore, the phenolic and flavonoid content was independently affected by ethanol concentration, extracting temperature, and extraction time. Finally, the results concluded that 40% of ethanol concentration with 80 minutes of extraction time at 42.50°C could be optimum for the F. auriculata fruit phenolic compounds extraction.

5.3.4 Confirmation of the Proposed Model

In conclusion, the 40% ethanol, 80 minutes, and 42.50°C was suggested for the efficient extraction of phenolic compounds from F. auriculata fruits. The prepared extracts from these optimized conditions resulted in 59.21 ± 5.44 mg gallic acid equivaients/g sample of phenolic content, 127.94 ± 7.35 mg rutin equivalents/g sample of flavonoid, and 46.61% ± 0.38% of DPPH radical scavenging activity (Table 5.4).

Response surface plots for the combined effect of variables on 2, 2-diphenyl-l-picryl hydrazyl radical scavenging activity of F

FIGURE 5.3 Response surface plots for the combined effect of variables on 2, 2-diphenyl-l-picryl hydrazyl radical scavenging activity of F. auriculata. (a) Correlation between experimental and predicted antioxidant activity, (b) effect of ethanol concentration and extraction temperature, (c) effect of ethanol concentration and incubation time, and (d) effect of extraction temperature and incubation time.

TABLE 5.4

Analysis of Total Phenolic Content, Total Flavonoid Content, and 2, 2-diphenyl-1-picryl Hydrazyl Radical Scavenging Activity of F. auriculata Fruit Extract Prepared from Optimized Conditions

Total Phenolic Content (mg Gallic Acid Equivalents/g Fruit Extract)

Total Flavonoid Content (mg Rutin Equivalents/g Fruit Extract)

2, 2-diphenyl-1-picryl Hydrazyl Radical Scavenging Activity (% Inhibition)

59.21 ±5.44

127.94 ±7.35

46.61 ±0.38

Notes: Values are mean of triplicate determination (n = 3) ± standard deviation.

5.4 CONCLUSION

Therefore, this study resolutely showed the optimizing condition for better extraction of phenolic compounds from F. auriculata fruit using response surface methodology. From the results, it was concluded that three variables significantly affected the extraction of polyphenols from F. auriculata fruit. Particularly, the ethanol concentration and extraction temperature highly influenced the fig polyphenols content extraction. The proposed model was found to be highly significant for better extraction, and the studied values were fitted well. Hence, this method would be better for the extraction of natural phenolics from F. auriculata fruits with optimized conditions.

CONFLICT OF INTEREST

The authors declare no conflict of Interest.

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