# Optimization of Baicalin, Wogonoside, and Chlorogenic Acid Water Extraction Process from the Roots of Scutellariae Radix and Lonicerae japonicae Flos Using Response Surface Methodology (RSM)

^{1}

^{2}

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## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Ethical Statement

#### 2.2. Samples and Chemicals Reagents

#### 2.3. Preparation of Standard Solution of Baicalin, Wogonoside, and Chlorogenic Acid

#### 2.4. Sample Preparation and Extraction Protocol

#### 2.5. HPLC Quantification and Method Validation

#### 2.6. Single-Factor Test Design

_{1}represents the content of chlorogenic acid or baicalin or wogonoside in the sample solution determined by HPLC and calculated via standard curve, which is the number of milligrams of each component contained in the sample solution per milliliter, and C

_{2}represents the concentration of the sample solution as stated above (5 mg/mL), which is the number of milligrams of Scutellariae Radix and L. japonicae Flos in the sample solution per milliliter.

_{i}is the actual measurement value, Y

_{min}and Y

_{max}are the minimum and maximum of all values measured in the group. After calculating the d

_{i}value, the OD value is computed according to the following equation:

#### 2.7. Box–Behnken Design (BBD) for Optimization of Extraction Conditions

_{0}was a constant, β

_{i}(linear), β

_{ii}(quadratic) and β

_{ij}(interactive) were the coefficients; X

_{i}denotes independent variables. The terms X

_{i}X

_{j}and X

_{i}

^{2}represented the quadratic and interaction terms, respectively.

#### 2.8. Data Analysis

## 3. Results

#### 3.1. High Performance Liquid Chromatography (HPLC) and Validation of Extraction Process

#### 3.2. Standard Curve Determination and Range of Linearity

#### 3.3. Precision Experiments

#### 3.4. Stability, Reproducibility, and Recovery of Baicalin, Wogonoside, and Chlorogenic Acid

#### 3.5. Single-Factor Experiments

#### 3.6. Optimization of the Extraction Parameters by Box–Behnken Design and Response Surface Methodology

_{1}+ 0.25780X

_{2}− 0.16021X

_{3}+ 1.35500E − 004X

_{1}X

_{2}+ 0.011285X

_{1}X

_{3}+

0.011285X

_{1}X

_{3}− 1.29500E − 003X

_{2}X

_{3}− 3.65725E − 003X

_{1}

^{2}− 5.29400E − 003X

_{2}

^{2}− 0.17628X

_{3}

^{2}

_{1}, X

_{3}, X

_{1}X

_{3}, X

_{1}

^{2}, X

_{2}

^{2}, and X

_{3}

^{2}were significant model terms and there could be a chance of 0.01% that model ªF-valueº increased due to noise. Lack of fit F-value (5.104) was not statistically significant (p > 0.05). 7.47% chance of error was found in the lack of fit F-value due to noise signals. Therefore, the model was fit and appropriate and could be employed for experimental extraction.

_{1}) and extraction time (X

_{3}) on OD values at the liquid–solid ratio of 25:1 (mL/g). OD value first enhances and then decreases at different extraction temperature. The longer or shorter extraction time causes marked changes in OD value. The results showed that (Figure 4A) the impact of interaction was significant between extraction temperature and extraction time on OD value as the two-dimensional contour plot was oval in shape. Figure 4C,D displays the effect of extraction temperature (X

_{1}) and liquid–solid ratio (X

_{2}) at an extraction time of 3 h. The interaction effect of extraction temperature and liquid–solid ratio was significant on OD value ((Figure 4C, oval in shape). The interaction effect of extraction time (X

_{3}) and liquid–solid ratio (X

_{2}) on OD value shown in Figure 4E,F at an extraction temperature of 90 °C. It has been noted that OD value first enhances and then decreases at different liquid–solid ratio or extraction time. The interaction effect of extraction time and liquid–solid ratio on OD value was found non-significant (round in shape) as shown in Figure 4E. The interaction of extraction time and temperature is the most significant. While, the interaction of liquid–solid ratio and extraction temperature, and the interaction of liquid–solid ratio and extraction time is the least significant, and these results were consistent with the results of variance (Table 6.). The optimum values of extraction parameters such are extraction time, extraction temperature, and liquid–solid ratio were 2.42 h, 92.58 °C, and 25.23:1 (mL/g), respectively, and the predicted OD value calculated under the above condition was 0.741. Noticeably, the above conditions were modified for easiness to an extraction time of 2.4 h, extraction temperature of 93 °C, and liquid–solid ratio of 25:1 (mL/g). Based on modified conditions, the concentrations of baicalin, wogonoside, and chlorogenic acid were 0.078 mg/mL (extraction yield was 15.6 mg/g), 0.031 mg/mL (extraction yield was 6.2 mg/g), and 0.013 mg/mL (extraction yield was 2.6 mg/g), respectively, and the experimental OD value was 0.759 ± 0.09 (n = 3), higher than the value obtained from non-optimum conditions. Interestingly, it was found that the model was fit and reliable to forecast the overall indexes of extraction process from L. japonicae Flos and Scutellariae Radix due to only slight deviation (2.44%) from the true value (Table 7).

#### 3.7. Effect of Extraction Cycle on Baicalin, Wogonoside, and Chlorogenic Acid Content

## 4. Discussion

## Author Contributions

## Funding

## Conflicts of Interest

## References

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**Figure 1.**High performance liquid chromatography (HPLC) chromatograms of (

**A**) mixture sample of S. baicalensis Georgi and L. japonica Thunb, and (

**B**–

**D**) standard solution of baicalin, wogonoside, and chlorogenic acid.

**Figure 2.**Standard curve of baicalin (

**A**), wogonoside (

**B**), and chlorogenic acid (

**C**). Horizontal axis represents concentration of baicalin, wogonoside, and chlorogenic acid (mg/mL) and vertical axis represents peak area ((μAU (absorption unit) * S (time in sec)).

**Figure 3.**Effect of extraction temperature (

**A**), extraction time (

**C**), and liquid–solid ratio (

**E**) on the extraction yield of baicalin, wogonoside, and chlorogenic acid and OD values (

**B**,

**D**,

**F**).

**Figure 4.**A-F showing response surface plots three-dimensional (3D) and contour plots two-dimensional (2D) showing the effects of different extraction parameters (X

_{1}: Extraction temperature, °C; X

_{2}: Liquid–solid ratio, mL/g; X

_{3}: Extraction time, h) added on the response Y.

**Figure 5.**Effect of extraction number on the yield of baicalin, wogonoside, and chlorogenic acid. (

**A**) shows the effect of extraction cycle on extraction yield (mg/g) of the three compounds, whereas (

**B**) shows the obtained OD values at different extraction cycles.

Independent Variables | Symbols | Coded Levels | ||
---|---|---|---|---|

−1 | 0 | 1 | ||

Extraction temperature (°C) | X_{1} | 80 | 90 | 100 |

Liquid–solid ratio (mL/g) | X_{2} | 20:1 | 25:1 | 30:1 |

Extraction time (h) | X_{3} | 2 | 3 | 4 |

Concentrations (mg/mL) | $\mathbf{Peak}\text{}\mathbf{Areas}\text{}(\overline{\mathit{x}}\text{}\pm \text{}\mathbf{SD})$ | RSD% | |
---|---|---|---|

0.04 | 1,400,829.8 ± 11,486.7 | 0.82 | |

Baicalin | 0.12 | 4,176,222.6 ± 11,373.6 | 0.27 |

0.20 | 6,904,145.2 ± 17,260.3 | 0.25 | |

0.02 | 399,412.4 ± 3708.5 | 0.93 | |

Wogonoside | 0.06 | 1,196,842.2 ± 8566.3 | 0.72 |

0.10 | 2,065,237 ± 11,709.9 | 0.57 | |

0.01 | 56,175.3 ± 876.3 | 1.56 | |

Chlorogenic acid | 0.03 | 165,526.9 ± 2284.9 | 1.38 |

0.05 | 478,578.2 ± 4508 | 0.94 |

**Table 3.**Stability and reproducibility of baicalin, wogonoside, and chlorogenic acid determined by HPLC.

Stability | Reproducibility | |||
---|---|---|---|---|

$\mathbf{Peak}\text{}\mathbf{Areas}\text{}(\overline{\mathit{x}}\text{}\pm \text{}\mathbf{SD})$ | RSD | $\mathbf{Peak}\text{}\mathbf{Areas}\text{}(\overline{\mathit{x}}\text{}\pm \text{}\mathbf{SD})$ | RSD | |

Baicalin | 3,922,371 ± 44,077.5 | 1.12% | 3,969,422 ± 83,402.7 | 2.10% |

Wogonoside | 1,582,977 ± 25,327.6 | 1.60% | 1,552,831 ± 34,628.1 | 2.23% |

Chlorogenic acid | 235,760 ± 2678.3 | 1.14% | 236,604 ± 6881.4 | 2.91% |

Weight of Compounds in the Extract Samples (mg) | Weight of Standard Substances Added (mg) | Measured Quantity (mg) | $\mathbf{Recovery}\text{}\mathbf{Rate}\text{}(\overline{\mathit{x}}\text{}\pm \text{}\mathbf{SD})\%$ | RSD% | |
---|---|---|---|---|---|

Baicalin | 1.093 ± 0.0262 | 1 | 1.1937 ± 0.019 | 100.69 ± 1.23 | 1.22 |

Wogonoside | 0.448 ± 0.0103 | 0.45 | 0.4938 ± 0.003 | 101.68 ± 2.05 | 2.02 |

Chlorogenic acid | 0.153 ± 0.0032 | 0.15 | 0.3024 ± 0.006 | 99.6 ± 1.56 | 1.57 |

No. | X_{1} (°C) | X_{2} (ml/g) | X_{3} (h) | Extraction Ratio (mg/g) | OD | ||
---|---|---|---|---|---|---|---|

Baicalin | Wogonoside | Chlorogenic Acid | |||||

1 | 80 | 20 | 3 | 8.46 | 4.58 | 2.54 | 0 |

2 | 100 | 20 | 3 | 17.02 | 5.24 | 1.90 | 0.39 |

3 | 80 | 30 | 3 | 8.46 | 7.58 | 2.52 | 0.00 |

4 | 100 | 30 | 3 | 17.30 | 5.34 | 1.92 | 0.41 |

5 | 80 | 25 | 2 | 8.52 | 4.00 | 2.36 | 0.06 |

6 | 100 | 25 | 2 | 18.58 | 3.78 | 1.74 | 0 |

7 | 80 | 25 | 4 | 8.54 | 4.10 | 2.62 | 0.09 |

8 | 100 | 25 | 4 | 17.10 | 5.58 | 1.98 | 0.48 |

9 | 90 | 20 | 2 | 12.88 | 4.72 | 1.88 | 0.25 |

10 | 90 | 30 | 2 | 14.18 | 4.94 | 1.90 | 0.31 |

11 | 90 | 20 | 4 | 13.38 | 5.62 | 2.18 | 0.48 |

12 | 90 | 30 | 4 | 13.44 | 5.72 | 2.22 | 0.51 |

13 | 90 | 25 | 3 | 14.64 | 6.30 | 2.38 | 0.66 |

14 | 90 | 25 | 3 | 15.04 | 7.04 | 2.42 | 0.75 |

15 | 90 | 25 | 3 | 14.92 | 6.38 | 2.40 | 0.69 |

16 | 90 | 25 | 3 | 15.44 | 6.56 | 2.44 | 0.73 |

17 | 90 | 25 | 3 | 14.38 | 6.26 | 2.40 | 0.65 |

Source | Sum of Squares | Degree of Freedom | Mean Square | F-Value | p-Value |
---|---|---|---|---|---|

Model | 1.160 | 9 | 0.129 | 25.800 | 0.0001 * |

X_{1} | 0.160 | 1 | 0.160 | 32.051 | 0.0008 * |

X_{2} | 0.001 | 1 | 0.001 | 0.292 | 0.6059 |

X_{3} | 0.111 | 1 | 0.111 | 22.271 | 0.0022 * |

X_{1} X_{2} | 0.000 | 1 | 0.000 | 0.0367 | 0.8535 |

X_{1} X_{3} | 0.051 | 1 | 0.051 | 10.191 | 0.0152 * |

X_{2} X_{3} | 0.000 | 1 | 0.000 | 0.034 | 0.8599 |

X_{1}^{2} | 0.563 | 1 | 0.563 | 112.67 | <0.0001 * |

X_{2}^{2} | 0.074 | 1 | 0.074 | 14.755 | 0.0064 * |

X_{3}^{2} | 0.131 | 1 | 0.131 | 26.175 | 0.0014 * |

Residual | 0.035 | 7 | 0.005 | ||

Lack of fit | 0.028 | 3 | 0.009 | 5.104 | 0.0747 |

Pure error | 0.007 | 4 | 0.002 | ||

Cor total | 1.196 | 16 |

Optimum Condition | Extraction Ratio (mg/g) | OD | |||||
---|---|---|---|---|---|---|---|

Extraction Temperature (°C) | Liquid–Solid Ratio (mL/g) | Extraction Time (h) | Baicalin | Wogonoside | Chlorogenic Acid | Experimental | Predicted |

93 (92.58) | 25 (25.23) | 2.4 (2.42) | 15.6 | 6.2 | 2.6 | 0.759 ± 0.09 | 0.741 |

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**MDPI and ACS Style**

Li, J.; Wang, R.; Sheng, Z.; Wu, Z.; Chen, C.; Ishfaq, M.
Optimization of Baicalin, Wogonoside, and Chlorogenic Acid Water Extraction Process from the Roots of *Scutellariae Radix* and *Lonicerae japonicae Flos* Using Response Surface Methodology (RSM). *Processes* **2019**, *7*, 854.
https://doi.org/10.3390/pr7110854

**AMA Style**

Li J, Wang R, Sheng Z, Wu Z, Chen C, Ishfaq M.
Optimization of Baicalin, Wogonoside, and Chlorogenic Acid Water Extraction Process from the Roots of *Scutellariae Radix* and *Lonicerae japonicae Flos* Using Response Surface Methodology (RSM). *Processes*. 2019; 7(11):854.
https://doi.org/10.3390/pr7110854

**Chicago/Turabian Style**

Li, Jichang, Rui Wang, Zunlai Sheng, Zhiyong Wu, Chunli Chen, and Muhammad Ishfaq.
2019. "Optimization of Baicalin, Wogonoside, and Chlorogenic Acid Water Extraction Process from the Roots of *Scutellariae Radix* and *Lonicerae japonicae Flos* Using Response Surface Methodology (RSM)" *Processes* 7, no. 11: 854.
https://doi.org/10.3390/pr7110854