# High-Performance Green Extraction of Polyphenolic Antioxidants from Salvia fruticosa Using Cyclodextrins: Optimization, Kinetics, and Composition

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## Abstract

**:**

_{CD}), pH, and liquid-to-solid ratio (R

_{L/S}), a Box–Behnken design was chosen, with three central points. Temperature effects on the extraction yield were also considered, by carrying out kinetics. The results showed that m-β-CD was the most effective extraction booster, providing total polyphenols yields that amounted to 98.39 mg gallic acid equivalents g

^{−1}dry mass. The kinetic assay demonstrated that extraction was highly effective at 80 °C, increasing significantly polyphenol yield, as well as the ferric-reducing power and antiradical activity of the extracts. It was also proven that extraction with m-β-CD was the least energy-demanding process. Liquid chromatography-tandem mass spectrometry examination revealed that m-β-CD might possess higher affinity for luteolin 7-O-glucuronide extraction, but β-CD for rosmarinic acid extraction.

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Chemicals

#### 2.2. Plant Material

#### 2.3. Batch Stirred-Tank Solid–Liquid Extraction

#### 2.4. Experimental Design

_{CD}), pH, and liquid-to-solid ratio (R

_{L/S}) on the performance of aqueous extraction of polyphenols from S. fruticosa. To accomplish this, a response surface methodology was employed, using the Box–Behnken experimental design including three central points, which enables determination of the first- and second-order coefficients of the mathematical model with high reliability [9]. Total polyphenol yield (Y

_{TP}) was the screening response and codification of the variables chosen (C

_{CD}, pH, R

_{L/S}) between −1 (lower limit) and 1 (upper limit), and was performed as follows:

_{i}is the dimensionless value of the independent variable i, and X

_{i}is its actual value. X

_{0}represents the actual value of variable i at the central point of the design, and ΔX

_{i}is the step change of X

_{i}, corresponding to a change by a unit of the dimensionless value (Table 1). The ranges used for the independent variables were chosen on the basis of critical evaluation of literature data. The significance of the model, each polynomial coefficient, and the model coefficient R

^{2}were acquired by performing analysis of variance (ANOVA). On the basis of this analysis, insignificant dependent terms (p > 0.05) were not included in the mathematical equations (models). The desirability function enabled the determination of the optimal extraction conditions for maximizing Y

_{TP}and visualization of the independent variable effect on Y

_{TP}was delivered as 3D response surface plots. Model validation was done by comparing predicted and experimental response values, after carrying out experiments under optimal extraction conditions.

#### 2.5. Determination of Total Polyphenols (TP)

_{2}CO

_{3}solution (20% w/v) was added and the samples were left to react for 60 min, in the dark. The absorbance at 740 nm was then obtained, using appropriate blank, and the concentration in total polyphenols (C

_{TP}, mg L

^{−1}) was calculated from a calibration curve, constructed with gallic acid as standard. Total polyphenol yield (Y

_{TP}) was estimated by the following equation and expressed as mg gallic acid equivalents (GAEs) g

^{−1}dry mass (dm):

#### 2.6. Antiradical Activity (A_{AR}) Measurement

_{AR}, a DPPH assay was used [10]. Samples were diluted 1:20 with methanol before each analysis. A volume of 0.025 mL of diluted sample was mixed with 0.975 mL of DPPH solution (100 μM in methanol) and the absorbance was immediately recorded at 515 nm (A

_{515(i)}). The mixture was allowed to react for 30 min and then recording of absorbance at 515 nm was repeated (A

_{515(f)}). A

_{AR}was calculated as follows:

_{DPPH}is the DPPH concentration (μM) and C

_{TP}is the and total polyphenol concentration (mg L

^{−1}) in the reaction mixture; A

_{515(f)}is the A

_{515}at t = 30 min and A

_{515(i)}the A

_{515}at t = 0; and Y

_{TP}is the total polyphenol yield (mg g

^{−1}) of the extract. A

_{AR}was expressed as μmol DPPH g

^{−1}dm.

#### 2.7. Ferric-Reducing Power (P_{R}) Determination

_{R}of the extracts was assayed as described previously [10]. A volume of 0.05 mL of extract, diluted 1:20 with methanol, was combined with 0.05 mL FeCl

_{3}(4 mM in 0.05 M HCl) and the mixture was incubated in a thermostated water bath, set at 37 °C, for 30 min. After incubation, 0.9 mL TPTZ solution (1 mM in 0.05 M HCl) was added, and after exactly 10 min, the absorbance at 620 nm was measured. P

_{R}was determined from an ascorbic acid calibration curve (50–300 μM) and given as μM ascorbic acid equivalents (AAEs) g

^{−1}dm.

#### 2.8. High-Performance Liquid Chromatography (HPLC)

^{−1}.

#### 2.9. Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS)

^{−1}. Eluents and elution program were as described above. Mass spectra were acquired in negative ionization mode, with the following settings: sheath gas pressure, 30 mTorr; capillary temperature, 300 °C; collision pressure at 1.5 mTorr; auxiliary gas pressure, 15 mTorr. Quantification was accomplished with external standard methodology, using a luteolin 7-O-glucoside (5–1500 μg L

^{−1}, R

^{2}= 0.9982) and a rosmarinic acid (50–3000 μg L

^{−1}, R

^{2}= 0.9985) calibration curve. The standards were dissolved in HPLC grade methanol and stored at −17 °C.

#### 2.10. Statistical Analysis

^{™}Pro 13. Linear and non-linear correlations, as well as curve fittings, were performed at least at a 95% significance level, with SigmaPlot

^{™}12.5.

## 3. Results and Discussion

#### 3.1. Optimisation of the Extraction Performance

_{CD}, R

_{L/S}, and pH, and to identify possible synergistic functions between them. Evaluation of the fitted model and the suitability of response surface were based on the ANOVA and lack-of-fit test (Table 2), by considering the closeness of the predicted and measured values (Table 3). The second-degree polynomial equations (mathematical models), considering only the significant terms, are presented in Table 4, along with the square correlations coefficients (R

^{2}) of the models, which are indicators of the total variability around the mean determined by the model. Because all total R

^{2}of the models were equal or higher than 0.97, and the p-value for lack of fit (assuming a confidence interval of 95%) was highly significant for all models, it can be argued that equations exhibited excellent fit to the experimental data. The contour plots constructed on the basis of the models, which are presented on a comparative arrangement in Figure 2, provide an at-a-glance image of how the experimental variables affected response (Y

_{TP}), but also illustrate the differences between the three CDs used.

_{CD}was found to exert a non-significant effect, suggesting that any shift in C

_{CD}within the range tested cannot impact Y

_{TP}. The same was observed for HP-β-CD, but for m-β-CD, this variable was highly significant (p = 0.0047). This outcome strongly indicated that the nature of the CD used may play a key role in extraction. On the other hand, no cross term between m-β-CD concentration and either R

_{L/S}or pH was significant, showing that combined effects did not occur. Contrary to those, for the extractions performed with any CD, both R

_{L/S}and pH were significant.

_{L/S}and pH were significant too, demonstrating that combinations of these two variables may have either a negative (HP-β-CD) or positive (m-β-CD) influence on the extraction yield.

_{TP}achieved with HP-β-CD and m-β-CD were identical and significantly higher than that obtained with β-CD (p < 0.05). This finding highlighted the prominent role of the nature of the CD used for the extraction. In support of this are pertinent results on the extraction of olive pomace polyphenols, where HP-β-CD exhibited superior extraction capacity compared with either m-β-CD or γ-CD [11]. Data on polyphenol extraction from pomegranate fruit were in the same line [12], stressing the superiority of HP-β-CD against β-CD as a polyphenol extraction booster. In opposition, anthocyanin extraction was more efficient with β-CD rather than HP-β-CD [13]. Such discrepancies might emerge from the different encapsulating capacity of the CDs used towards structurally unrelated polyphenolic constituents. Indeed, examinations with pure polyphenols (catechin) demonstrated a more efficient encapsulation with β-CD than HP-β-CD or m-β-CD [14]. Therefore, the higher-performance extraction of S. fruticosa polyphenols observed with m-β-CD might reflect the manifestation of such phenomena. Given that the modelling performed revealed significant effect of C

_{CD}only for m-β-CD, then it could be postulated that m-β-CD interacted more strongly with S. fruticosa polyphenols than β-CD or HP-β-CD within the C

_{CD}limits tested.

_{L/S}varied closely within 93–100 mL g

^{−1}(Table 5). This outcome showed that the influence exerted by R

_{L/S}on the extraction performance was not significantly affected by the structure of CD. The magnitude of R

_{L/S}is related with the concentration gradient between the liquid phase (extraction medium) and the surface of the solid particle, which is directly involved in mass transfer. If R

_{L/S}is below a certain limit, then the equilibria established may not favor fast diffusion of the solute during extraction, owing to non-negligible resistance to mass transfer [15]. Several examinations on polyphenol extraction from plant tissues using conventional organic solvents suggested R

_{L/S}optima between 81 [16] and 100 mL g

^{−1}[17,18,19]. Considering that the average R

_{L/S}value in this study was 96 mL g

^{−1}, it could be argued that an aqueous medium containing any of the CDs assayed would behave as a common solvent in this regard.

_{a}may lie well above 7 for several substituted phenolics [21], for some flavonols such as quercetin, which are frequently encountered in plant tissues, pK

_{a1}may vary within 5.06 to 7.36 [22]. In any case, at pH > 5, even limited dissociation of the most acidic phenolic hydroxyls could occur, provoking a significant increase in polyphenol polarity. This issue was also addressed in previous studies on polyphenol extraction with water/ethanol mixtures [23,24,25], where optimal extraction pH for total polyphenols was always <5. In these cases, increased extraction yield was ascribed to higher solubility of non-dissociated polyphenols in ethanol-containing solvents and increased polyphenol stability at acidic pH.

_{CD}concentration within the limits tested for β-CD and HP-β-CD were shown to exert non-significant impact on Y

_{TP}, but it was not clear whether the presence of any CD used could affect Y

_{TP}. To examine this, extractions were performed with each CD under optimised conditions, as well as with aqueous solutions under the same R

_{L/S}and pH, without the addition of CD (Table 6). In every case, it was demonstrated that addition of CDs provoked significantly higher Y

_{TP}, highlighting the importance of the CDs used as aqueous extraction boosters. The highest difference in Y

_{TP}was found for m-β-CD (22.06%), followed by HP-β-CD (19.32%) and β-CD (11.28%).

#### 3.2. Extraction Kinetics and Temperature Effects

_{TP(t)}and Y

_{TP(s)}represent the TP yield at any time t and at equilibrium (saturation), respectively. k is the second-order extraction rate constant. When t approaches 0, the initial extraction rate, h, given as Y

_{TP(t)}/t, is defined as follows:

_{TP(s)}and h, using SigmaPlot

^{™}12.5, can be seen. For all CDs, the three kinetic parameters exhibited an increase as a response to raising the temperature up to 80 °C.

_{TP(s)}was achieved with m-β-CD (98.39 mg GAE g

^{−1}dm) at 80 °C, and it was only 5.3% lower than that achieved with 60% methanol (Figure 5). Both β-CD and HP-β-CD were significantly less effective, giving Y

_{TP(s)}75.85 and 81.93 mg GAE g

^{−1}dm, respectively. To further assess the impact of temperature, samples obtained at the end of each treatment (180 min) were also assayed for antioxidant activity (Figure 6). In line with Y

_{TP(s)}, extracts obtained with m-β-CD exhibited the highest A

_{AR}(1112.51 μmol DPPH g

^{−1}dm), followed by HP-β-CD (836.17 μmol DPPH g

^{−1}dm) and β-CD (824.08 μmol DPPH g

^{−1}dm). The results for P

_{R}were in concurrence, giving corresponding values of 241.88, 210.72, and 185.74 μmol AAE g

^{−1}dm. This outcome suggested that, using m-β-CD, polyphenol-enriched extracts with improved antioxidant characteristics may be produced at 80 °C.

_{0}

_{0}to a pre-exponential factor. In Table 8, the parameters k

_{0}, a, and b, calculated by SigmaPlot

^{™}12.5, are given analytically. Extraction with m-β-CD displayed the lowest b value, which suggested that it was the least affected by temperature, as opposed to the extraction with HP-β-CD. This finding evidenced that m-β-CD provided the most effective and the least energy-demanding extraction of polyphenols. To ascertain this and obtain a tentative estimation of the barriers required for the extraction with each CD tested, the activation energy was determined as follows [28]:

_{ref}was chosen as the mean temperature of testing (60 °C) and T = 40 °C. k

_{ref}and k were the corresponding second-order extraction rate constants. E

_{a}is the activation energy (J mol

^{−1}) and R the universal gas constant (8.314 J K

^{−1}mol

^{−1}). E

_{a}thus estimated for the extraction with β-CD, HP-β-CD, and m-β-CD were 7.18, 9.50, and 5.64 kJ mol

^{−1}, respectively. This finding did confirm that the extraction with m-β-CD was the least energy-demanding process.

#### 3.3. Polyphenolic Profile

## 4. Conclusions

## Author Contributions

## Funding

## Conflicts of Interest

## Nomenclature

A_{AR} | antiradical activity (μmol DPPH g^{−1}) |

C_{CD} | cyclodextrin (β-CD, HP-β-CD, m-β-CD) concentration (%, w/v) |

dm | dry mass (g) |

E_{a} | activation energy (kJ mol^{−1}) |

k_{0} | pre-exponential factor (g mg^{−1} min^{−1}) |

k | second-order extraction rate constant (g mg^{−1} min^{−1}) |

k_{ref} | second-order extraction rate constant at reference T (g mg^{−1} min^{−1}) |

P_{R} | reducing power (μmol AAE g^{−1}) |

R | universal gas constant (8.314 K^{−1} mol^{−1}) |

R_{L/S} | liquid-to-solid ratio (mL g^{−1}) |

t | time (min) |

T | temperature (°C) |

T_{ref} | reference T (°C) |

Y_{TP} | yield in total polyphenols (mg GAE g^{−1}) |

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**Figure 1.**Cyclodextrin structures tested in this study. Assignments: m-β-CD, methyl β-cyclodextrin; HP-β-CD, hydroxypropyl β-cyclodextrin; β-CD, β-cyclodextrin.

**Figure 2.**Contour graphs presenting the effect of simultaneous variation of independent variables on the response. Assignments: β-CD, β-cyclodextrin; HP-β-CD, hydroxypropyl β-cyclodextrin; m-β-CD, methyl β-cyclodextrin.

**Figure 3.**Desirability function for each of the CDs tested, displaying optimal conditions and maximum predicted response values. Assignments: m-β-CD, methyl β-cyclodextrin; HP-β-CD, hydroxypropyl β-cyclodextrin; β-CD, β-cyclodextrin.

**Figure 4.**Kinetics of polyphenol extraction from S. fruticosa, with each of the CDs tested, within a range from 40 to 80 °C. Extractions were performed under optimized conditions. Assignments: m-β-CD, methyl β-cyclodextrin; HP-β-CD, hydroxypropyl β-cyclodextrin; β-CD, β-cyclodextrin.

**Figure 5.**Plot showing Y

_{TP}achieved using each of the CDs tested, under optimized conditions, at 80 °C, after 180 min.

**Figure 6.**Diagram illustrating the A

_{AR}and P

_{R}of the extracts produced using each of the CDs tested, under optimized conditions, after 180 min, at 80 °C. Assignments: m-β-CD, methyl β-cyclodextrin; HP-β-CD, hydroxypropyl β-cyclodextrin; β-CD, β-cyclodextrin.

**Figure 8.**HPLC trace recorded at 330 nm, of a S. fruticosa extract, obtained with m-β-CD under optimized conditions, at 80 °C, after 180 min.

Independent Variables | Code Units | Coded Variable Level | ||
---|---|---|---|---|

−1 | 0 | 1 | ||

C_{CD} (%, w/v) | X_{1} | 0.60 | 1.00 | 1.40 |

R_{L/S} (mL g^{−1}) | X_{2} | 20 | 60 | 100 |

pH | X_{3} | 3 | 5 | 7 |

**Table 2.**Statistical data associated with the mathematical models, built using response surface methodology. m-β-CD, methyl β-cyclodextrin; HP-β-CD, hydroxypropyl β-cyclodextrin.

Term | Standard Error | t Ratio | Probability > t | Sum of Squares | F Ratio |
---|---|---|---|---|---|

β-CD | |||||

Intercept | 0.846624 | 51.50 | <0.0001 * | 6.31901 | - |

C_{CD} | 0.518449 | 1.71 | 0.1471 | 239.91451 | 2.9386 |

R_{L/S} | 0.518449 | 10.56 | 0.0001 * | 252.90005 | 111.5718 |

pH | 0.518449 | −10.84 | 0.0001 * | 12.14523 | 117.6107 |

C_{CD} R_{L/S} | 0.733198 | −2.38 | 0.0634 | 0.11560 | 5.6481 |

C_{CD} pH | 0.733198 | −0.23 | 0.8258 | 0.65610 | 0.0538 |

R_{L/S} pH | 0.733198 | −0.55 | 0.6045 | 1.93408 | 0.3051 |

C_{CD}C_{CD} | 0.763136 | −0.95 | 0.3865 | 53.14168 | 0.8994 |

R_{L/S} R_{L/S} | 0.763136 | −4.97 | 0.0042 * | 82.82608 | 24.7134 |

pH pH | 0.763136 | −6.21 | 0.0016 * | 6.31901 | 38.5181 |

Lack-of-Fit | 0.1067 | 9.972175 | 8.5298 | ||

HP-β-CD | |||||

Intercept | 1.05319 | 44.02 | <0.0001 * | 1.88180 | - |

C_{CD} | 0.644945 | 0.75 | 0.4859 | 298.77901 | 0.5655 |

R_{L/S} | 0.644945 | 9.48 | 0.0002 * | 130.81531 | 89.7874 |

pH | 0.644945 | −6.27 | 0.0015 * | 3.18623 | 39.3119 |

C_{CD} R_{L/S} | 0.912089 | 0.98 | 0.3728 | 6.94323 | 0.9575 |

C_{CD} pH | 0.912089 | 1.44 | 0.2082 | 22.94410 | 2.0865 |

R_{L/S} pH | 0.912089 | −2.63 | 0.0468 * | 1.93186 | 6.8950 |

C_{CD}C_{CD} | 0.949333 | 0.76 | 0.4805 | 9.20776 | 0.5806 |

R_{L/S} R_{L/S} | 0.949333 | −1.66 | 0.1571 | 80.32413 | 2.7671 |

pH pH | 0.949333 | −4.91 | 0.0044 * | 1.88180 | 24.1386 |

Lack-of-Fit | 0.1153 | 15.332875 | 7.8313 | ||

m-β-CD | |||||

Intercept | 0.547155 | 87.27 | <0.0001 * | 34.56961 | |

C_{CD} | 0.273577 | 7.60 | 0.0047 * | 195.89960 | 57.7357 |

R_{L/S} | 0.335062 | 18.09 | 0.0004 * | 193.40255 | 327.1775 |

pH | 0.335062 | −17.97 | 0.0004 * | 0.18923 | 323.0072 |

C_{CD} R_{L/S} | 0.386897 | −0.56 | 0.6133 | 3.27610 | 0.3160 |

C_{CD} pH | 0.386897 | 2.34 | 0.1013 | 17.09663 | 5.4715 |

R_{L/S} pH | 0.547155 | 5.34 | 0.0128 * | 6.91763 | 28.5536 |

C_{CD}C_{CD} | 0.47385 | 3.40 | 0.0425 * | 20.80413 | 11.5533 |

R_{L/S} R_{L/S} | 0.47385 | −5.89 | 0.0097 * | 79.95325 | 34.7456 |

pH pH | 0.47385 | −11.56 | 0.0014 * | 34.56961 | 133.5322 |

Lack-of-Fit | 0.8547 | 0.4840687 | 0.1844 |

**Table 3.**Experimental design points and the corresponding predicted and measured Y

_{TP}values for the extractions carried out with each of the CDs used. GAE, gallic acid equivalent.

Design Point | Independent Variables | Response (Y_{TP}, mg GAE g^{−1} dw) | |||||||
---|---|---|---|---|---|---|---|---|---|

C_{CD} (X_{1}) | R_{L/S} (X_{2}) | pH (X_{3}) | β-CD | HP-β-CD | m-β-CD | ||||

Measured | Predicted | Measured | Predicted | Measured | Predicted | ||||

1 | −1 | −1 | 0 | 30.74 | 30.97 | 39.03 | 39.80 | 38.16 | 38.21 |

2 | −1 | 1 | 0 | 46.97 | 45.41 | 49.94 | 50.24 | 51.03 | 50.77 |

3 | 1 | −1 | 0 | 34.68 | 36.24 | 39.29 | 38.99 | 42.54 | 42.80 |

4 | 1 | 1 | 0 | 43.94 | 43.70 | 53.77 | 53.00 | 54.54 | 54.49 |

5 | 0 | −1 | −1 | 35.63 | 34.81 | 34.64 | 35.66 | 42.68 | 42.37 |

6 | 0 | −1 | 1 | 25.35 | 24.38 | 33.85 | 32.36 | 26.50 | 24.47 |

7 | 0 | 1 | −1 | 45.60 | 46.57 | 51.18 | 52.67 | 48.64 | 48.64 |

8 | 0 | 1 | 1 | 33.70 | 34.52 | 40.81 | 39.79 | 42.13 | 42.44 |

9 | −1 | 0 | −1 | 42.12 | 42.70 | 49.09 | 47.30 | 48.47 | 48.73 |

10 | 1 | 0 | −1 | 45.56 | 44.82 | 46.35 | 45.63 | 51.03 | 51.08 |

11 | −1 | 0 | 1 | 31.06 | 31.80 | 35.86 | 36.58 | 34.93 | 34.88 |

12 | 1 | 0 | 1 | 33.82 | 33.24 | 38.39 | 40.18 | 41.11 | 40.85 |

13 | 0 | 0 | 0 | 43.93 | 43.60 | 45.72 | 46.36 | 48.56 | 47.75 |

14 | 0 | 0 | 0 | 43.99 | 43.60 | 46.10 | 46.36 | 48.50 | 47.75 |

15 | 0 | 0 | 0 | 42.88 | 43.60 | 47.27 | 46.36 | 46.94 | 47.75 |

Cyclodextrin | 2nd Order Polynomial Equations | R^{2} | p |
---|---|---|---|

β-CD | 43.60 + 5.48X_{2} − 5.63X_{3} − 3.79${\mathrm{X}}_{2}^{2}$ − 4.74${\mathrm{X}}_{3}^{2}$ | 0.98 | 0.0006 |

HP-β-CD | 46.36 + 6.11X_{2} − 4.04X_{3}− 2.39X_{2}X_{3} − 4.66${\mathrm{X}}_{3}^{2}$ | 0.97 | 0.0025 |

m-β-CD | 47.75 + 2.08X_{1} + 6.06X_{2} − 6.02X_{3} + 2.92X_{2}X_{3} − 2.79${\mathrm{X}}_{2}^{2}$ − 5.48${\mathrm{X}}_{3}^{2}$ | 1.00 | 0.0024 |

**Table 5.**Values of the optimal predicted conditions and maximum predicted Y

_{TP}(± sd) for S. fruticosa polyphenol extraction by the CDs tested.

Cyclodextrin | Maximum Predicted Response (Y_{TP}, mg GAE g^{−1} dw) | Optimal Conditions | ||
---|---|---|---|---|

C_{CD} (w/v, %) | R_{L/S} (mL g^{−1}) | pH | ||

β-CD | 47.48 ± 2.29 | 0.88 | 93 | 3.75 |

HP-β-CD | 54.40 ± 4.42 | 1.40 | 100 | 3.90 |

m-β-CD | 54.72 ± 2.11 | 1.40 | 94 | 4.54 |

**Table 6.**The effect of each of the CDs tested on the extractability of polyphenols from S. fruticosa, compared with equally buffered deionized water, under optimal R

_{L/S}.

Extraction Medium | Y_{TP} (mg GAE g^{−1} dw) | Extraction Conditions | ||
---|---|---|---|---|

C_{CD} (w/v, %) | R_{L/S} (mL g^{−1}) | pH | ||

β-CD | 45.75 ± 1.11 | 0.88 | 93 | 3.75 |

Buffered dH_{2}O | 40.59 ± 1.01 | - | 93 | 3.75 |

HP-β-CD | 50.52 ± 1.19 | 1.40 | 100 | 3.90 |

Buffered dH_{2}O | 40.76 ± 1.02 | - | 100 | 3.90 |

m-β-CD | 54.25 ± 1.35 | 1.40 | 94 | 4.54 |

Buffered dH_{2}O | 42.28 ± 1.05 | - | 94 | 4.54 |

**Table 7.**Kinetic parameters determined for the extraction of S. fruticosa polyphenols with the CDs tested. Extractions were accomplished under optimal C

_{CD}, R

_{L/S}, and pH.

T (°C) | Kinetic Parameters | ||
---|---|---|---|

k (×10^{−3})(g mg ^{−1} min^{−1}) | h (mg g^{−1} min^{−1}) | Y_{TP(s)} (mg GAE g^{−1}) | |

β-CD | |||

40 | 4.95 | 11.76 | 48.74 |

50 | 5.59 | 14.45 | 50.86 |

60 | 5.83 | 21.51 | 60.75 |

70 | 7.53 | 33.70 | 66.89 |

80 | 8.46 | 48.68 | 75.85 |

HP-β-CD | |||

40 | 2.43 | 8.91 | 60.53 |

50 | 2.71 | 11.09 | 63.92 |

60 | 3.02 | 16.30 | 73.41 |

70 | 4.40 | 28.84 | 80.92 |

80 | 6.92 | 46.47 | 81.93 |

m-β-CD | |||

40 | 2.55 | 14.66 | 68.99 |

50 | 2.64 | 17.99 | 82.56 |

60 | 2.90 | 21.79 | 86.71 |

70 | 2.95 | 25.97 | 93.89 |

80 | 3.28 | 31.76 | 98.39 |

**Table 8.**Fitting parameter values determined by correlating second-order extraction rates (k) with T.

CD | Parameter Estimates | ||||
---|---|---|---|---|---|

k_{0} (×10^{−6}) | a (×10^{−5}) | b | R^{2} | p | |

β-CD | 3.409 | 0.4500 | 0.0305 | 0.97 | 0.0320 |

HP-β-CD | 2.242 | 0.0068 | 0.0816 | 1.00 | 0.0022 |

m-β-CD | 2.103 | 0.1727 | 0.0238 | 0.96 | 0.0351 |

**Table 9.**Spectral information pertaining to polyphenols detected in S. fruticosa extracts, obtained with either CD tested.

No | Rt (min) | UV/Vis (λ_{max}) | [M − H]^{−} (m/z) | Other Ions (m/z) | Tentative Identity |
---|---|---|---|---|---|

1 | 19.58 | 270, 340 | 593 | - | Unknown |

2 | 22.28 | 280, 344 | 477 | 301 | 6-Hydroxy luteolin 7-O-glucoside |

3 | 24.38 | 256, 352 | 461 | 285 | Luteolin 7-O-glucuronide |

4 | 25.25 | 258, 348 | 593 | 285 | Luteolin 7-O-rutinoside |

5 | 25.82 | 270, 352 | 491 | 299 | 6-Methoxyluteolin 7-O-glucoside (nepitrin) |

6 | 26.62 | 246, 316 | 359 | 161 | Rosmarinic acid |

7 | 27.50 | 264, 346 | 445 | 269 | Apigenin 7-O-glucuronide |

8 | 29.55 | 270, 352 | 475 | 299 | 6-Methoxyluteolin derivative |

9 | 30.12 | 274, 332 | 461 | 299, 283 | 6-Methoxyluteolin derivative |

**Table 10.**Quantitative data on the recovery of major S. fruticosa polyphenols with the CDs tested, under optimal conditions.

Extract | Yield (mg g^{−1} dm) | |
---|---|---|

Luteolin 7-O-glucuronide | Rosmarinic Acid | |

β-CD | 3.35 ± 0.02 | 7.12 ± 0.00 |

HP-β-CD | 2.38 ± 0.02 | 6.17 ± 0.10 |

m-β-CD | 3.63 ± 0.03 | 6.40 ± 0.20 |

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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**MDPI and ACS Style**

Grigorakis, S.; Benchennouf, A.; Halahlah, A.; Makris, D.P.
High-Performance Green Extraction of Polyphenolic Antioxidants from *Salvia fruticosa* Using Cyclodextrins: Optimization, Kinetics, and Composition. *Appl. Sci.* **2020**, *10*, 3447.
https://doi.org/10.3390/app10103447

**AMA Style**

Grigorakis S, Benchennouf A, Halahlah A, Makris DP.
High-Performance Green Extraction of Polyphenolic Antioxidants from *Salvia fruticosa* Using Cyclodextrins: Optimization, Kinetics, and Composition. *Applied Sciences*. 2020; 10(10):3447.
https://doi.org/10.3390/app10103447

**Chicago/Turabian Style**

Grigorakis, Spyros, Amina Benchennouf, Abedalghani Halahlah, and Dimitris P. Makris.
2020. "High-Performance Green Extraction of Polyphenolic Antioxidants from *Salvia fruticosa* Using Cyclodextrins: Optimization, Kinetics, and Composition" *Applied Sciences* 10, no. 10: 3447.
https://doi.org/10.3390/app10103447