
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00293-1
10.1016/j.ultsonch.2024.107045
107045
Separation Process
A novel ultrasound-assisted enzyme extraction method of total flavonoids from Viticis Fructus and processed Viticis Fructus: Comparison of in vitro antioxidant activity
Li Yuman a1
Zhang Qing b1
Fang Qi a1
Zhu Hui c
Zong Xuelin a
Gao Xun a
Shi Yun syun1@163.com
a⁎
Qin Kunming qinkm123@126.com
a⁎
a School of Pharmacy, Jiangsu Ocean University, Lianyungang 222005, China
b Lianyungang Chinese Medicine Hospital, Lianyungang 222004 , China
c Engineering Research Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, China
⁎ Corresponding authors. syun1@163.comqinkm123@126.com
1 Equal contributors.

24 8 2024
11 2024
24 8 2024
110 10704510 7 2024
15 8 2024
23 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Graphical abstract

In this study, it is the first that the Viticis Fructus (VF) was used as the raw material for extracting total flavonoids using the ultrasound-assisted enzyme extraction (UAE) method. Response surface methodology was employed to determine the optimal extraction parameters. The optimal conditions were as follows: 60 % ethanol solution as the extract solvent, material–liquid ratio of 1:25, pH value of 4, enzyme addition amount of 1.5 %, enzymatic hydrolysis time of 30 min, enzymatic hydrolysis temperature of 40 ℃, and ultrasonic time of 50 min. Comparing the total flavonoid yield of VF and processed VF (PVF) extracted using different methods, it was observed that UAE resulted in a higher total flavonoid yield compared to traditional ultrasound extraction and enzyme extraction. Additionally, the total flavonoid yield of PVF extracted by all three methods was generally higher than that of VF. The PVF solution extracted by UAE also demonstrated better in vitro antioxidant activity compared to VF. These results suggest that UAE is an effective method to enhance the activity of natural total flavonoids. The study of the physicochemical properties and in vitro antioxidant activity of VF and PVF showed that the total flavonoid yield and antioxidant activity significantly increased after VF stir-frying, indicating that their efficacy can also be enhanced.

Keywords

Viticis Fructus
Ultrasound-assisted enzyme extraction
Response surface methodology
Total flavonoids
Antioxidant activity
==== Body
pmc1 Introduction

Viticis Fructus (VF) is a traditional Chinese medicine (TCM), which can also be regarded as food as a dietary supplement. The VF is the dried ripe fruit of V. trifolia L. (VT) or Vitex trifolia L. var. simplicifolia Cham. (VTS) (Family Verbenaceae) [1]. In China, the main place of production of VTS is Jiangxi Province, while VT is primarily produced in Yunnan, Guangxi, and Hainan [2]. VF was first recorded in “The Divine Husbandman’s Classic of Materia Medica (Shen Nong Ben Cao Jing)”. Traditional processing methods for VF mainly involve stir-frying. The taxonomy of VF’s botanical origins has always been controversial. The 2020 edition of the Pharmacopoeia of the People’s Republic of China considers VTS a variety of VT. In addition, the VTS is also identified as V. rotundifolia L. f. (VR), a separate species recognized by its usually single leave [3]. The VF in this study are all dried ripe fruit from VR.

In the traditional Japanese medicine, particularly within the framework of Kampo formulations, Viticis Fructus (VF) is recognized for its therapeutic virtues, including analgesic, sedative, antitumor and anti-inflammatory properties, as documented in the “Japanese Standards for Non-Pharmacopeial Crude Drugs” (2012) [4]. These medicinal effects are largely attributed to VF’s rich composition of flavonoids, phenolic acids, and volatile compounds, with flavonoids playing a important role [5]. Contemporary pharmacological and clinical investigations have further elucidated VF’s broad spectrum of bioactivities, including analgesic, antipyretic, antioxidant, anti-inflammatory, antitumor, anti-aging, and hypotensive effects, primarily attributed to its flavonoid compounds [6], [7]. Recent researches demonstrate the enhanced efficacy of processed VF (PVF), which emphasize the significance of exploring flavonoids extraction and activity from both VF and PVF, the VF and PVF are shown in Fig. 1. Tradition extraction methods, methanol, ethanol and water extraction, are time-inefficient and offer low yields [8]. In contrast, ultrasound-assisted enzymolysis extraction (UAE) emerges as a potent technique to improve flavonoid yields, enhancing the mechanical and cavitation effects of ultrasound to disrupt biological structures like cell walls and membranes, promoting intracellular mass transfer. Enzymes further accelerate cell wall and cell membrane breakdown, facilitating flavonoid release and extraction [9], [10]. Nevertheless, UAE presents several limitations, including the high temperatures during enzymatic hydrolysis, substantial enzyme addition, and extended durations of enzymatic hydrolysis (the steps of UAE method). These factors may detrimentally affect the integrity of flavonoid structures or complicate their separation processes, consequently diminishing the overall yield. Therefore, optimizing UAE to achieve higher yields is essential.Fig. 1 Photographs of VF (A) and PVF (B) samples.

Response surface methodology (RSM) stands as a experimental strategy designed for the optimization and analytical discernment of processes, with the primary objective of determining the ideal set of parameters to culminate in the desired outcomes of a given process [11]. This methodology posits a hypothetical response surface to articulate the intricate interplay between various experimental variables and the resultant process response, encompassing aspects such as product quality, efficiency, and cost-effectiveness. The surface is represented through polynomial equations or alternative mathematical models, facilitating the prediction of response outcomes across diverse factorial permutations. Moreover, RSM enables a more efficient experimental regimen by utilizing a reduced number of trials to methodically evaluate the interrelations among different parameters [12]. In the context of establishing the optimal UAE conditions for VF and its processed variant PVF, the Box–Behnken design was employed as a strategic approach to refine the process. This included the meticulous adjustment of variables such as the amount of enzyme addition, enzymolysis temperature and duration, extraction solvent, and their interactive effects.

This study compared the effects of three different extraction methods on the total flavonoid yield of VF and PVF: ultrasound extraction (UE), enzyme extraction (EE), and UAE. We selected the optimal extraction method to extract VF and PVF from 14 different regions and studied the total flavonoid components’ scavenging effect on free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, and hydroxyl radicals. This exploration of antioxidant activity in vitro aims to provide some reasonable reference for better development of VF and PVF.

2 Materials and methods

2.1 Pretreatment

Fourteen batches of VF samples (No. S1-S14; Table 1) were obtained from pharmaceutical companies in China. To gain PVF, VF was fried over a gentle heat until completely dry and cooled. A voucher specimen is deposited at the School of Pharmacy Jiangsu Ocean University, Lianyungang, China.Table 1 Details of the 14 batches of VF samples.

No.	Origin	Batch Number	
S1	Jiangxi, China	230,907	
S2	Shandong, China	230,419	
S3	Yunnan, China	230,625	
S4	Guangxi, China	230,717	
S5	Chongqing, China	230,823	
S6	Hunan, China	230,729	
S7	Guangdong, China	230,817	
S8	Guangdong, China	230,827	
S9	Shandong, China	230,731	
S10	Jiangsu, China	230,725	
S11	Anhui, China	230,615	
S12	Henan, China	230,819	
S13	Sichuan, China	230,728	
S14	Guangdong, China	230,621	

2.2 Single-factor experiment

VF powder (1 g) from batch S2 (a randomly selected batch of VF from the 14 areas) was weighed and conducted single factor experiments, testing the effects of solid–liquid ratio, ultrasonic time, solution pH, enzyme addition amount, enzymolysis temperature, enzymolysis time, and extract solvent. The effects of these factors on the total flavonoid yield were evaluated, and the optimal value of each factor was used as the fixed condition for subsequent single-factor experiments.

The single-factor experimental conditions were as follows: 1:5, 1:10, 1:15, 1:20, and 1:25 for solid–liquid ratio; 10 min, 20 min, 30 min, 40 min, and 50 min for ultrasonic time; 2, 3, 4, 5, and 6 for solution pH; 0.5 %, 1.0 %, 1.5 %, 2.0 %, and 2.5 % for cellulase addition amount; 30 ℃, 40 ℃, 50 ℃, 60 ℃, and 70 ℃ for enzyme digestion temperature; 0.5 h, 1.0 h, 1.5 h, 2.0 h and 2.5 h for enzymatic hydrolysis time; and extract solvents of 60 % ethanol, 70 % ethanol, 80 % methanol, 90 % methanol, and 100 % methanol. Each single-factor experiment was conducted at an ultrasound power of 150 W, as this power level is relatively stable and conducive to the extraction of flavonoids in VF and PVF.

2.3 Experimental design for optimal conditions

Based on the principles of the Box–Behnken design, this study selected four influential factors: the enzyme addition amount (A), the enzymatic hydrolysis temperature (B), the enzymatic hydrolysis time (C), and the extract solvent (D). To explore the interaction among these parameters, a 4-factor, 3-level experimental design using the Box-Behnken central combination was implemented. The specifics of this design are systematically shown in Table 2.Table 2 Box–Behnken test factors and levels.

	Factor	
	A	B	C	D	
	Enzyme addition amount(%)	Enzymatic hydrolysis temperature(℃)	Enzymatic hydrolysis time(h)	Extract solvent(%)	
Level	0.5	40	0.5	60 % ethanol	
1.0	50	1.0	70 % ethanol	
1.5	60	1.5	80 % methanol	

2.4 Different extraction methods of 14 batches of VF and PVF

2.4.1 UE method for 14 batches of VF and PVF

VF and PVF powder (1 g) from each of the 14 batches were weighed and placed in conical flasks. Subsequently, 25 mL of 60 % ethanol was added to each flask, and the resultant mixtures were then exposed to an ultrasound extraction process, adhering to predefined temperature and duration parameters. After ultrasonication, the extracts were allowed to cool before being decanted into 25 mL volumetric flasks, which were subsequently filled to the calibration mark with the identical ethanol solution. This procedure culminated in the preparation of the UE solution devoid of enzymes for the 14 batches of VF and PVF.

2.4.2 EE method for 14 batches of VF and PVF

VF and PVF powder (1 g) from each of the 14 batches were weighed and placed in conical flasks. Subsequently, 25 mL of 60 % ethanol (pH=4) was added along with a certain ratio of cellulase. Enzymatic digestion was conducted under specified temperature and time conditions. After digestion, the enzyme was inactivated in a high-temperature water bath for 5 min. Once cooled, transfered the extracts to 25 mL volumetric flasks and filled to the calibration mark with the same solvent to obtain an ultrasound free EE solution from 14 batches of VF and PVF.

2.4.3 UAE method for 14 batches of VF and PVF

VF and PVF powder (1 g) from each of the 14 batches were weighed and placed in conical flasks. Subsequently, 25 mL of 60 % ethanol solution, adjusted to a pH of 4, along with a predetermined proportion of cellulase, was added to each flask. The process proceeded with enzymatic hydrolysis at controlled temperatures, which was then succeeded by an UAE phase, conducted under defined temperature and duration parameters. After ultrasonication, to stop the enzyme activity, the mixture was subjected to a high-temperature water bath for a duration of 5 min. When it cooled, the extract solution were decanted into 25 mL volumetric flasks and filled to the calibration mark with the initial solvent, culminating in the preparation of the UAE solutions derived from the 14 batches of VF and PVF. The reason for the choice of specific process parameters for different extraction methods, such as 60 % ethanol and pH 4 is that such parameters derive from optimal conditions in single-factor experiment.

2.5 Total flavonoids determination of 14 batches of VF and PVF

The quantification of the total flavonoid content was performed utilizing a derivative of the methodology proposed by Saeed et al. [13]. Briefly, a precise mass of 4.0 mg of rutin was dissolved in 5 mL of methanol (100 % v/v), establishing a rutin standard stock solution with a concentration of 0.8 mg/mL. Subsequently, aliquots of the rutin standard stock solution, 0.125, 0.15, 0.5, 0.6, 0.7, and 0.8 mL were transferred into seven separate test tubes respectively. To each of these test tubes, 0.3 mL of a 5 % NaNO2 solution was introduced and thoroughly mixed. Waitting an interval of 6 min, an addition of 0.3 mL of a 10 % Al(NO3)3 solution was made to each mixture and again ensured thorough mixing. After a subsequent period of 6 min, 4.0 mL of a 4 % NaOH solution was added, and the volume was completed to the mark with methanol (100 % v/v), well-mixed, and the solution was allowed to stand for 15 min.

The absorbance of each solution was meticulously measured at a wavelength of 506 nm. A standard curve of rutin was constructed, the concentration of rutin against the measured absorbance (Fig. 2). Using this calibration curve to calculate the total flavonoid content, expressed in milligrams of rutin equivalent per gram of dried portion. The formula employed for the determination of the total flavonoids yield is as follows:Yields(mg/g)=weightoftotalflavonoidsweightofVForPVFpowder

Fig. 2 Rutin standard curve.

2.6 HPLC analysis of UE, EE, and UAE extracts from VF and PVF

The HPLC content determination was performed using a method modified by Wu et al. [14]. HPLC separation was conducted on a Shimadzu system (Shimadzu, Kyoto, Japan), equipped with a photodiode array (PDA) detector. The chromatographic column used was a YMC-Pack ODS-A C18-001 column (250 mm × 4.6 mm, 5.0 μm), operated at 30 ℃. The mobile phase consisted of a gradient elution of solvent A (0.1 % Formic acid) and solvent B (Acetonitrile). The gradient program was: 0–15 min from 10 % to 15 % of B, 15–20 min at 15–18 % of B, 20–45 min at 18–35 % of B, 45–55 min at 35–40 % of B, and 55–65 min at 40–62 % of B, at a flow rate of 1 mL/min. The injection volume was 10 μL, and the detection wavelength was 258 nm. Quantify using standard curves and calculate the final concentration in μg/g. The analyte linear equations for VF are shown in Table 3.Table 3 Linear equations for six flavonoid components of VF.

Number	Analyte	Calibration Curve Equation	R2	Limit of detection (μg/mL)	Limit of quantification (μg/mL)	Linear Range (μg/mL)	
1	Isoorientin	y = 19590x-4115.9	0.9997	0.02	0.06	2.40 ∼ 76.92	
2	Orientin	y = 17673x + 21670	0.9994	0.02	0.06	2.40 ∼ 192.30	
3	Vitexin	y = 10981x-2899.8	0.9999	0.03	0.12	0.60 ∼ 76.92	
4	Taxifolin	y = 4160x-795.82	0.9998	0.12	0.40	0.60 ∼ 76.92	
5	Luteolin	y = 27021x-9752.1	0.9997	0.02	0.06	0.60 ∼ 76.92	
6	Casticin	y = 33582x-40599	0.9996	0.02	0.03	2.40 ∼ 153.84	

2.7 Determination of radical scavenging ability of 14 batches of VF and PVF

2.7.1 Determination of DPPH radical scavenging ability

The assay to quantify the DPPH radical scavenging activity utilized a protocol that was adapted from the approach described by Baliyan et al. [15]. Solutions of VF and PVF were prepared at varying concentrations (0.078125 mg/mL, 0.15625 mg/mL, 0.3125 mg/mL, 0.625 mg/mL, 1.25 mg/mL, and 2.5 mg/mL), with 0.10 mL of each solution subsequently mixed with an identical volume of a 0.2 mmol/L DPPH solution in ethanol. The mixtures were then incubated for 40 min shielded from light, ensuring reaction completion. Absorbance readings were taken at 517 nm to gauge the scavenging activity. Each concentration was tested in triplicate to ensure reliability and accuracy of the results.

2.7.2 Determination of ABTS radical scavenging ability

The capacity to scavenge ABTS radicals was evaluated using an adapted method from the study by Rumpf et al. [16]. Solutions of VF and PVF at the concentrations (0.078125 mg/mL, 0.15625 mg/mL, 0.3125 mg/mL, 0.625 mg/mL, 1.25 mg/mL, and 2.5 mg/mL) were each combined with 0.10 mL of the ABTS working solution. After a 10 min incubation period under conditions shielded from light, the absorbance was measured at 734 nm to assess the scavenging activity. This procedure was meticulously repeated in triplicate for each concentration to ensure the reliability of the results. The efficacy of VF and PVF from the 14 batches in scavenging DPPH and ABTS radicals was then quantified based on these absorbance measurements.DPPHandABTSradicalscavengingactivity%=1-A1-A2A0×100

(A1) represents the absorbance observed for the sample under investigation, (A2) denotes the absorbance recorded for the VF or PVF solution when combined with anhydrous ethanol, and (A0) corresponds to the absorbance of the control, which consists of deionized water mixed with DPPH or ABTS.

2.7.3 Determination of hydroxyl radical scavenging ability

The evaluation of hydroxyl radical scavenging capacity was conducted following a modified version of the protocol described by Lin et al. [17]. Solutions of VF and PVF were prepared at various concentrations, including 0.078125 mg/mL, 0.15625 mg/mL, 0.3125 mg/mL, 0.625 mg/mL, 1.25 mg/mL, and 2.5 mg/mL. To each of these solutions, 0.05 mL of FeSO4 (6 mmol/L), 0.05 mL of a salicylic acid solution in ethanol (6 mmol/L), and 0.05 mL of H2O2 (6 mmol/L) were sequentially added. The resultant mixtures were incubated at 37℃ for 30 min, after which the absorbance (AW) was recorded at 510 nm. The background absorbance (AY) was determined using deionized water in place of H2O2, whereas the reference absorbance (A0) was obtained by substituting deionized water for the VF or PVF solution. Each absorbance measurement was carried out in triplicate. The formula for calculating the hydroxyl radical scavenging rate was applied as follows:Hydroxylradicalscavengingrate%=1-AW-AYA0×100

3 Results and discussion

3.1 Influences of different factors on UAE

As shown in Fig. 3, the highest yield was achieved using 60 % ethanol as the solvent. In extractions conducted with ethanol concentrations below 60 %, there is observed to be inefficient powder filtration, leading to a reduced extract volume and a total flavonoid yield inferior to that achieved with ethanol extractions at concentrations of 60 % or higher. This underperformance justifies the selection of 60 % to 80 % ethanol as the solvent concentration range for response surface methodology experiments. This decision is further supported by the outcomes of single-factor experiments, wherein extractions utilizing 90 % methanol and 100 % methanol resulted in significantly lower total flavonoid yields compared to those obtained through ethanol extraction, thereby excluding the higher methanol concentrations from consideration in the response surface methodology experiments. Under ultrasonic conditions, the yield increased gradually from 10 min to 20 min, plateaued between 20 min and 40 min, and then increased again from 40 min to 50 min, reaching the highest yield at 50 min. Extending the ultrasonic conditions within appropriate limits can enhance flavonoid yield by allowing better penetration of the extraction material. Ultrasound effectively disrupts the structural organization of VF and PVF, promoting the dissolution of flavonoid macromolecules. However, it has been documented that the primary objective of ultrasonic extraction is to minimize the duration and associated costs of experimentation, while endeavoring to maximize the extraction of active compounds within a reduced timeframe. Therefore, after comparing the five levels set for ultrasound time as a factor, there was no further extension of ultrasound time for research. Further research will be conducted with the requirements of production. Excessive ultrasound power can lead to the breakdown of flavonoid glycosidic bonds and reducing yield [18], [19]. However, there are many studies using hydrodynamic cavitation applied to the extraction of natural products, which the reason is that it can increase the yield of effective ingredients in natural products [20], [21].Fig. 3 Effects of extract solvent (A), material–liquid ratio (B), pH value (C), enzyme addition (D), enzymatic digestion time (E), enzymatic digestion temperature (F), and ultrasonic time (G) on total flavonoid yield.

The results indicate that the enzyme dosage is increased from 0.5 % to 1.5 % and the enzymatic hydrolysis temperature is increased from 40 ℃ to 60 ℃, the total flavonoid yield shows an upward trend. However, when the enzyme dosage is higher than 1.5 %, the total flavonoid yield significantly decreases. Considering the economic cost of the experiment, the enzyme dosage of 0.5 % to 1.5 % was selected for response surface experiments. Due to the significant decrease in total flavonoid yield when the enzymatic hydrolysis temperature is higher than 60 ℃, considering that too low an enzymatic hydrolysis temperature cannot effectively activate enzyme activity, and too high an enzymatic hydrolysis temperature can cause the enzyme to lose activity, a temperature range of 40 ℃ to 60 ℃ was chosen for response surface experiments. When the enzyme amount was 0.50 % to 1.50 %, flavonoid yield gradually increased due to increased enzyme-substrate contact, which damaged the cell wall and increased cell membrane permeability. This reduction in mass transfer resistance enhanced the diffusion coefficient of flavonoids, thus increasing yield. However, at 2.0 % cellulase addition, the yield decreased, probably because of excessive enzyme-hydrolyzed flavonoid glycosidic bond. Additionally, when enzyme molecules saturated the reaction system, some enzymes remained unbound to the substrate [22].

The yield of flavonoids increased from 30 ℃ to 60 ℃ but decreased at 70 ℃ due to reduced cellulase activity at higher temperatures, preventing effective enzymatic digestion of VF powder [23]. The maximum yield was reached after 2.5 h of enzymatic digestion. Beyond this time, the yield did not increase, probably because the enzyme had already fully reacted with the available substrate in the early stages [24]. In the single factor experiment, the three factors of enzymatic hydrolysis time of 0.5 h, 1.0 h, and 1.5 h were more significant. At the same time, due to considerations of experimental time cost and subsequent production time cost, the total flavonoid yield at enzymatic hydrolysis of 2.0 h and 2.5 h was not higher than that at enzymatic hydrolysis of 0.5 h in the single factor experiment. Therefore, the longer enzymatic hydrolysis time of 2.0 h and 2.5 h was not selected in the response surface experiment.

3.1.1 Statistical analysis and model fitting

Table 2 shows that the statistical regression analysis of the total flavonoid yield, and conducting by Design Expert 13 software for both model fitting and statistical evaluation. The correlation between the response values and the influencing factors was modeled in the following manner:Yield%=0.792+0.0058∗A-0.0067∗B+0.0058∗C-0.075∗D-0.005∗AC-0.0225∗AD+0.01∗BC-0.0075∗CD-0.0272∗A2-0.006∗B2+0.0052∗C2-0.006∗D2

The results of the statistical analysis for the model are presented in Table 4.Table 4 Box–Behnken response surface design and corresponding response values.

Run	A	B	C	D	Yield (%)	
Enzyme addition amount (%)	Enzymatic hydrolysis temperature (℃)	Enzymatic hydrolysis time (h)	Extract solvent (%)	
1	1	50	1	70	0.79	
2	0.5	50	1.5	70	0.78	
3	0.5	40	1	70	0.72	
4	0.5	60	1	70	0.71	
5	1.5	40	1	70	0.80	
6	1	60	1.5	70	0.79	
7	0.5	50	0.5	70	0.76	
8	1	40	1.5	70	0.82	
9	1	50	1	70	0.77	
10	1	50	1.5	60	0.89	
11	1.5	50	1.5	70	0.76	
12	1.5	50	1	80	0.68	
13	1	60	1	60	0.86	
14	1	50	0.5	60	0.89	
15	1	40	0.5	70	0.83	
16	1.5	50	0.5	70	0.76	
17	0.5	50	1	60	0.81	
18	1	50	1	70	0.78	
19	1	50	1	70	0.81	
20	1.5	60	1	70	0.79	
21	1	40	1	80	0.69	
22	0.5	50	1	80	0.76	
23	1.5	50	1	60	0.82	
24	1	60	1	80	0.71	
25	1	60	0.5	70	0.76	
26	1	50	1.5	80	0.70	
27	1	50	1	70	0.81	
28	1	50	0.5	80	0.67	
29	1	40	1	60	0.84	

In the regression analysis, the P-value associated with the model is less than 0.005, demonstrating a statistically significant impact on the results, as shown in Table 5, Table 6. The misfit term is not significant, with a P-value of 0.0622, suggests that the model provides an adequate fit to the experimental data, exhibiting low error and negligible effects from extraneous variables. Within this study, an adjusted R-squared (Adj R2) value of 0.6210 and a correlation coefficient (R2) of 0.8105 were observed, indicating a good agreement between the experimental results and the model’s predictions. In addition, a signal-to-noise ratio exceeding 4 indicates that the model has good reproducibility.Table 5 Regression model analysis of variance.

Source	Sum of Squares	df	Mean Square	F-value	P-value		
Model	0.0772	14	0.0055	4.28	0.0047	significant	
A-Enzyme addition amount	0.0004	1	0.0004	0.3166	0.5825		
B-Enzymatic hydrolysis temperature	0.0005	1	0.0005	0.4136	0.5306		
C-Enzymatic hydrolysis time	0.0004	1	0.0004	0.3166	0.5825		
D-Extract solvent	0.0675	1	0.0675	52.34	<0.0001		
AB	0.0000	1	0.0000	0.0000	1.0000		
AC	0.0001	1	0.0001	0.0775	0.7847		
AD	0.0020	1	0.0020	1.57	0.2307		
BC	0.0004	1	0.0004	0.3102	0.5864		
BD	0.0000	1	0.0000	0.0000	1.0000		
CD	0.0002	1	0.0002	0.1745	0.6825		
A2	0.0048	1	0.0048	3.73	0.0738		
B2	0.0002	1	0.0002	0.1811	0.6769		
C2	0.0002	1	0.0002	0.1386	0.7152		
D2	0.0002	1	0.0002	0.1811	0.6769		
Residual	0.0181	14	0.0013				
Lack of Fit	0.0168	10	0.0017	5.24	0.0622	not significant	
Pure Error	0.0013	4	0.0003				
Cor Total	0.0953	28					

Table 6 Reliability analysis of the regression model.

Source		Source		
Std. Dev.	0.0359	R2	0.8105	
Mean	0.7779	Adjusted R2	0.6210	
C.V. %	4.62	Predicted R2	−0.0351	
		Adeq Precision	7.7760	

The significance levels of each factor revealed that the order of influence on the total flavonoid yield was D>B>C>A. The factors A, B, C, and D, interaction terms (AC, AD, BC, CD), and quadratic terms (A2, B2, C2, D2) all reached good levels.

3.1.2 Interaction analysis of every factor in the model

The three-dimensional (3D) response surface methodology facilitates the prediction of the interactions between factor interactions and their impact on the extraction rate. The nature of these interactions is visually represented by contour lines, where an oval shape signifies a substantial interaction, and a circular shape denotes a negligible interaction. And the gradient of the response surface elucidates the degree of influence each factor exerts on the total flavonoids extraction rate, with a more pronounced slope indicating a heightened influence [25], [26].

The response surface analysis revealed pronounced slopes for both enzyme addition amount and extraction solvent concentration (Fig. 4), which indicating its good significance. Meanwhile, according to the response surface data results, the enzymatic hydrolysis temperature and enzymatic hydrolysis time also showed good significance. With minimal amounts of enzyme added, an increase in extraction solvent concentration led to a diminished yield of total flavonoids. Conversely, at higher enzyme concentrations, the flavonoid yield initially rose before declining as the reaction time extended. The marked steepness of the slopes and the elliptical nature of the contour lines for the enzyme addition amount and extraction solvent concentration underscore a significant interactive effect on the flavonoid yield.Fig. 4 Response surface and contour diagrams illustrating the interaction of various experimental factors.

In contrast, the interaction contours between extract solvent concentration and enzymatic hydrolysis temperature, as well as between extract solvent concentration and enzymatic hydrolysis duration, exhibited a smaller slope, suggesting these interactions exert a lesser impact on the total flavonoid yield compared to the interaction between enzyme addition amount and extract solvent concentration. Additionally, the response surfaces for the combinations of enzymatic hydrolysis temperature and enzyme addition amount, enzymatic hydrolysis duration and enzyme addition amount, and enzymatic hydrolysis duration and enzymatic hydrolysis temperature demonstrated flatter slopes with circular contours, nevertheless, these interactions still have an impact on the total flavonoid production.

3.1.3 Response surface result verification

Table 7 presents both the optimal total flavonoid extraction scheme as forecasted by the software and the corresponding results. Analysis of these data demonstrated a mere 0.035 % difference between the predicted values and the actual results, which proves that the model’s precision and dependability in predicting the yield of total flavonoids. In order to accommodate the practical considerations associated with the subsequent experimental and production processes, the precise quantification and measurement of an enzyme addition amount of 1.259 % is deemed impractical. Consequently, an enzyme dosage of 1.5 %—which was identified as yielding the highest total flavonoid output during the single-factor analysis—was selected as a substitute for 1.259 %. Results from the validation experiments exhibited negligible discrepancies in comparison to the outcomes determined by the response surface methodology experiments. This indicates the viability of employing a practical extraction process for the extraction of VF and PVF.Table 7 Verification of response surface results.

	Enzyme addition amount (%)	Enzymatic hydrolysis temperature (℃)	Enzymatic hydrolysis time (h)	Extract solvent (%)	Yield (%)	
Predicted value	1.259	40.000	0.500	60.000	1.098	
Actual value	1.500	40.000	0.500	60.000	1.063	

3.2 Influence of different extraction methods on total flavonoid yield in 14 batches of VF and PVF

Flavonoids, crucial secondary metabolites found in a variety of food and medicinal plants such as fruits, vegetables, and tea, are encompassed into subclasses including flavonols, flavanones, isoflavones, and proanthocyanidins, etc. [27]. Many flavonoids including casticin, orientin, isoorientin, luteolin, and rutin, have been identified and quantified in VF and PVF, constituting the primary bioactive compounds. Consequently, the total flavonoid yield serves as a crucial metric for assessing the efficacy of various extraction techniques. This research explores the impact of three distinct extraction methods—UE, EE, and UAE—on the total flavonoid yield of 14 batches of VF and PVF.

Table 8 and Fig. 5 illustrate the variance in total flavonoid yield among the three extraction methods across fourteen batches of VF and PVF, under identical conditions of extract solvent, solid-to-liquid ratio, and ultrasonication duration. Table 9 shows the results of paired t-tests for each group, and the differences between the groups are statistically significant. The UE method yielded the lowest flavonoid content. In certain VF samples, the yield from UAE surpassed that obtained through EE. However, for all PVF samples, the total flavonoid content extracted by the UAE method was the highest compared to the other two methods.Table 8 Effects of three different extraction methods on the total flavonoid yield of 14 batches of VF and PVF.

	Ultrasound extraction yield (%)	Enzyme extraction yield (%)	Ultrasound-assisted enzymatic extraction yield (%)	
Origin	VF	PVF	VF	PVF	VF	PVF	
S1	0.67 ± 0.01	1.46 ± 0.02	0.97 ± 0.00	1.43 ± 0.00	0.94 ± 0.02	1.72 ± 0.02	
S2	0.63 ± 0.02	1.30 ± 0.06	0.98 ± 0.04	1.42 ± 0.01	0.95 ± 0.01	1.67 ± 0.02	
S3	0.88 ± 0.02	1.43 ± 0.03	1.07 ± 0.01	1.51 ± 0.04	1.06 ± 0.01	1.61 ± 0.04	
S4	0.86 ± 0.01	0.96 ± 0.02	1.08 ± 0.02	1.15 ± 0.01	1.08 ± 0.03	1.17 ± 0.01	
S5	0.95 ± 0.04	1.26 ± 0.00	1.16 ± 0.00	1.57 ± 0.05	1.13 ± 0.02	1.61 ± 0.08	
S6	0.85 ± 0.04	1.13 ± 0.01	1.17 ± 0.03	1.38 ± 0.03	1.18 ± 0.01	1.40 ± 0.02	
S7	0.87 ± 0.00	1.13 ± 0.01	1.12 ± 0.02	1.19 ± 0.01	1.24 ± 0.04	1.22 ± 0.01	
S8	0.84 ± 0.00	1.50 ± 0.02	1.05 ± 0.04	1.54 ± 0.05	1.26 ± 0.06	1.63 ± 0.04	
S9	1.19 ± 0.02	1.87 ± 0.01	1.51 ± 0.03	2.45 ± 0.01	1.56 ± 0.01	2.49 ± 0.06	
S10	1.28 ± 0.04	1.55 ± 0.06	1.47 ± 0.07	1.75 ± 0.01	1.59 ± 0.04	1.97 ± 0.01	
S11	1.34 ± 0.00	2.15 ± 0.05	1.65 ± 0.06	2.22 ± 0.06	1.60 ± 0.03	2.32 ± 0.03	
S12	1.29 ± 0.03	1.58 ± 0.02	1.50 ± 0.04	1.70 ± 0.04	1.60 ± 0.01	1.74 ± 0.07	
S13	1.22 ± 0.05	2.19 ± 0.05	1.52 ± 0.02	2.28 ± 0.01	1.62 ± 0.08	2.45 ± 0.01	
S14	1.64 ± 0.01	1.66 ± 0.04	1.94 ± 0.06	1.78 ± 0.05	1.93 ± 0.06	1.86 ± 0.01	

Fig. 5 Total flavonoid yield of 14 batches of VF (A) and PVF (B) extracted through UE, EE, and UAE methods.

Table 9 The results of paired sample t-test.

		Pairing difference	t	df	Significance	
	Mean	Standard Deviation	Standard Error Mean	95% CI	
Lower	Upper	
1	UE yield (%) VF – UE yield (%) PVF	-0.470	0.2924	0.0782	-0.6446	-0.3069	-6.087	13	0.000	
2	EE yield (%)VF – EE yield (%)PVF	-0.370	0.2875	0.0768	-0.5360	-0.2040	-4.816	13	0.000	
3	UAE yield (%) VF – UAE yield (%) PVF	-0.437	0.3304	0.0883	-0.6279	-0.2464	-4.950	13	0.000	
4	UE yield (%) VF – EE yield (%) VF	-0.263	0.0565	0.0151	-0.2955	-0.2302	-17.410	13	0.000	
5	UE yield (%) VF – UAE yield (%) VF	-0.302	0.0752	0.0201	-0.3455	-0.2588	-15.042	13	0.000	
6	EE yield (%) VF – UAE yield (%) VF	-0.039	0.0781	0.0209	-0.0844	0.0058	-1.882	13	0.082	
7	UE yield (%) PVF – EE yield (%) PVF	-0.157	0.1504	0.0402	-0.2440	-0.0703	-3.909	13	0.002	
8	UE yield (%) PVF – UAE yield (%) PVF	-0.264	0.1392	0.0372	-0.3429	-0.1832	-7.087	13	0.000	
9	EE yield (%) PVF – UAE yield (%) PVF	-0.106	0.0904	0.0242	-0.1586	-0.0542	-4.403	13	0.001	

The UAE method leverages ultrasound cavitation effects to enhance the diffusion of flavonoids out of plant cells. This method employs cellulase to break down cellulose and related compounds in the plant cell walls and membranes, thus expediting cell wall disruption and promoting flavonoid release. Consequently, the UAE method performs better than the traditional EE and UE methods in terms of flavonoid yield enhancement. As VF is mostly used in PVF, the UAE method markedly elevates the total flavonoid yield, the key bioactive components in PVF. Therefore, the UAE technique is deemed highly effective for extracting total flavonoids from VF and PVF.

3.3 Effects of UE, EE, and UAE on different flavonoids of VF and PVF

Three representative batches (S2, S9, and S11) were selected for HPLC content determination. Fig. 6 shows the HPLC chromatograms of standards and UAE extract (S9, PVF), which is highly representative. Six flavonoids—isoorientin, orientin, vitexin, taxifolin, luteolin, and casticin—were identified by comparing relative retention times and fingerprints with those of reference standards. The chemical structures of these six flavonoids are presented in Fig. 7.Fig. 6 Chromatograms of standard sample (A) and UAE (S9, PVF) sample (B): (1) isoorientin; (2) orientin; (3) vitexin; (4) taxifolin; (5) luteolin, and (6) casticin.

Fig. 7 Chemical structures of phenolic compounds. (A) isoorientin; (B) orientin; (C) vitexin; (D) taxifolin; (E) luteolin, and (F) casticin.

Table 10 and Fig. 8 illustrate the effect of the three extraction methods on the content of different flavonoids in VF and PVF. Overall, the UAE method increased the content of individual flavonoid components in VF and PVF. However, it is worth noting that the content of the six flavonoid components was not always highest under the UAE method compared to the other two methods. This variability may be related to the quality of VF from different batches and the structure of different compounds.Table 10 Effects of three extraction methods on the content of different flavonoids of VF and PVF (μg/g).

			Isoorientin	Orientin	Vitexin	Taxifolin	Luteolin	Casticin	
S2	Ultrasound extraction	VF	25.00 ± 0.06	129.94 ± 0.72	3.93 ± 0.01	2.48 ± 0.04	1.25 ± 0.01	17.35 ± 0.06	
PVF	35.41 ± 1.00	113.59 ± 2.24	4.23 ± 0.30	3.68 ± 0.08	4.21 ± 0.11	29.90 ± 0.59	
Enzyme extraction	VF	26.42 ± 0.11	124.34 ± 0.71	3.97 ± 0.08	1.91 ± 0.01	1.28 ± 0.01	16.14 ± 0.07	
PVF	36.58 ± 0.40	102.71 ± 0.85	4.09 ± 0.12	3.31 ± 0.16	3.85 ± 0.03	26.29 ± 0.22	
Ultrasound-assisted enzymatic extraction	VF	27.72 ± 0.22	131.46 ± 1.50	4.27 ± 0.05	2.52 ± 0.08	1.27 ± 0.01	16.77 ± 0.24	
PVF	38.71 ± 0.19	108.86 ± 0.02	4.59 ± 0.02	3.47 ± 0.18	3.46 ± 0.06	28.36 ± 0.39	
S9	Ultrasound extraction	VF	58.46 ± 0.21	118.51 ± 0.39	6.01 ± 0.06	15.02 ± 0.11	2.72 ± 0.06	26.91 ± 0.06	
PVF	77.00 ± 1.32	117.96 ± 1.94	7.44 ± 0.12	16.18 ± 0.51	5.63 ± 0.06	49.01 ± 1.09	
Enzyme extraction	VF	65.70 ± 2.79	123.41 ± 5.32	6.87 ± 0.27	16.92 ± 0.75	3.81 ± 0.14	27.15 ± 1.07	
PVF	84.35 ± 0.13	109.19 ± 0.33	7.15 ± 0.18	24.28 ± 1.19	6.51 ± 0.17	45.41 ± 0.06	
Ultrasound-assisted enzymatic extraction	VF	68.15 ± 1.86	126.64 ± 3.27	7.44 ± 0.19	17.18 ± 0.34	3.30 ± 0.07	27.76 ± 0.53	
PVF	93.79 ± 0.54	122.70 ± 2.35	8.54 ± 0.51	23.75 ± 1.42	6.44 ± 0.16	50.03 ± 0.81	
S11	Ultrasound extraction	VF	66.40 ± 0.31	129.33 ± 0.54	7.07 ± 0.05	17.54 ± 0.08	3.54 ± 0.08	28.33 ± 0.15	
PVF	86.54 ± 1.07	118.45 ± 2.17	7.52 ± 0.12	23.79 ± 1.21	7.10 ± 0.14	55.15 ± 0.78	
Enzyme extraction	VF	71.02 ± 0.12	126.46 ± 0.27	7.45 ± 0.14	18.00 ± 0.17	4.00 ± 0.02	27.50 ± 0.01	
PVF	85.25 ± 1.76	107.10 ± 2.59	8.19 ± 0.02	21.82 ± 0.32	6.52 ± 0.09	48.90 ± 0.90	
Ultrasound-assisted enzymatic extraction	VF	76.63 ± 1.71	134.83 ± 3.31	8.13 ± 0.02	18.06 ± 0.68	4.24 ± 0.01	28.85 ± 0.17	
PVF	85.02 ± 1.53	104.30 ± 0.66	7.72 ± 0.22	22.99 ± 1.00	6.84 ± 0.25	48.90 ± 0.76	

Fig. 8 Content of different flavonoids in three representative batches of VF (A–F) and PVF (a–f).

The results showed that the content of five components, except orientin, increased significantly during processing. This increase may be due to the cleavage of glycosidic bonds in the orientin glycoside during the heating process, which converts into other flavonoids and phenolic acid components, such as luteolin and chlorogenic acid. Overall, the chemical properties of flavonoids vary significantly due to differences in hydroxyl groups and glycosides in these compounds.

3.4 Antioxidant activity of 14 batches of VF and PVF in vitro

3.4.1 DPPH radical scavenging ability

DPPH, characterized by its stable radical nature with unpaired electrons (N·), undergoes a transformation into a stable entity (N–H) upon interaction with electrons and radicals. Appearing as purple in an aqueous medium, DPPH undergoes a color change to a lighter hue within an ethanol solution upon reaction with antioxidant entities, leading to the formation of stable diamagnetic molecules. This decolorization process is directly proportional to the quantity of unpaired electrons neutralized [28], [15].

The extraction of 14 batches of VF and PVF by the UAE method demonstrated a quantifiable correlation with the DPPH radical scavenging activity (Fig. 9). Specifically, a positive correlation was observed between the concentration of the extraction solutions from these batches and their DPPH radical scavenging effects, within a concentration range of 0.078125 to 2.5 mg/mL. At the maximum tested concentration of 2.50 mg/mL, the antioxidant capacities of VF and PVF extracts were assessed against the stable DPPH radical. VF showed low antioxidant activity. After stir-frying, the DPPH radical-scavenging activity of PVF was significantly improved.Fig. 9 DPPH radical scavenging ability of 14 batches of VF and PVF.

3.4.2 ABTS radical scavenging ability

Similar to the DPPH radical, the ABTS radical cation test quantifies the antioxidative potential through the neutralization of the stable radical cation ABTS•+, resulting in the formation of a blue-green chromophore exhibiting peak absorption at 734 nm. The presence of antioxidants leads to a reduction in the absorption intensity of this chromophore [29], [30].

The UAE method applied to 14 batches of VF and PVF demonstrated a quantitative association with the ABTS radical scavenging capacity (Fig. 10). Specifically, there was a positive correlation between the concentration of the extraction solutions from these batches and their ability to scavenge ABTS radicals, within a concentration range of 0.078125 to 2.5 mg/mL. At the upper limit of this range, 2.50 mg/mL, the ABTS radical scavenging performance of the extracts, like their DPPH radical scavenging efficacy, was significantly enhanced following stir-frying.Fig. 10 ABTS radical scavenging ability of 14 batches of VF and PVF.

3.4.3 Hydroxyl radical scavenging ability

Hydroxyl radicals, recognized as the most detrimental and toxic free radicals generated in metabolic processes [31], were produced by the Fenton reaction in this investigation. The capability of 14 batches of VF and PVF samples to scavenge these radicals was assessed by observing the colorimetric changes occurring throughout the reaction [32].

The ability to scavenge hydroxyl radicals was positively correlated with the concentration of extraction solutions from 14 batches of VF and PVF within a range of 0.078125 ∼ 2.5 mg/mL. At the concentration of 2.5 mg/mL, the hydroxyl radical scavenging capacity of PVF, extracted utilizing the UAE method, exceeded that of VF, as depicted in Fig. 11. However, this enhancement in scavenging capacity was found to be statistically insignificant.Fig. 11 Hydroxyl radical scavenging capacity of 14 batches of VF and PVF. NOTE: VF: Viticis Fructus; PVF: Processed Viticis Fructus.

4 Conclusions

In this study, a Box–Behnken response surface methodology was employed to optimize the UAE of flavonoids from 14 batches of VF and PVF, building on preliminary single-factor experiments. The optimized UAE extraction procedure was determined as follows: 60 % ethanol solution as the solvent, material–liquid ratio of 1:25, pH value of 4, enzyme dosage of 1.5 %, enzymatic hydrolysis duration of 30 min, hydrolysis temperature of 40 ℃, and ultrasonic time of 50 min. The experimental results closely matched the theoretical yield of total flavonoids, indicating the model’s reliability and precision. UV spectrophotometry analysis compared the effects of UE, EE, and UAE methods on the total flavonoid yield. The UAE method provided the highest yield, followed by the EE method, with UE yielding the lowest. HPLC analysis confirmed that the UAE method is the most efficient for extracting flavonoids from VF and PVF. Comparing the antioxidant capacity of the samples, PVF consistently showed higher antioxidant activity than VF across all tested free radical scavenging assays (DPPH, ABTS, and hydroxyl radicals). This enhanced activity surpasses the mere improvement in flavonoid extraction yield. The underlying reason for this enhancement may be attributed to the process of stir-frying VF to obtain PVF, which not only augments the flavonoid content but also significantly increases the levels of phenolic acids. Phenolic acids have been documented as one of the main active ingredients to antioxidant activity. Consequently, the antioxidant efficacy of PVF is markedly higher than that of VF. Future research will profound the dynamic variations in phenolic acid content resultant from the stir-frying of VF, to further explain this phenomenon. However, it is also can suggest that frying VF enhances both the yield of effective substances and its antioxidant activity and other pharmacological effects. The limitation of the study is that the significant issue associated with flavonoids of VF is their limited stability and solubility, factors that are influenced by their structural characteristics, including subclass, molecular weight, glycosylation patterns, and esterification levels, ultimately leading to reduced bioavailability. Its in vitro, or ex/in-vivo analyses will be conducted for further research.

CRediT authorship contribution statement

Yuman Li: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Qing Zhang: Validation, Supervision. Qi Fang: Validation, Investigation. Hui Zhu: Validation, Supervision. Xuelin Zong: Validation, Investigation. Xun Gao: Validation, Supervision. Yun Shi: Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. Kunming Qin: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Data 1

Acknowledgements

The authors sincerely acknowledge the financial support of this study by Science and Technology Development Plan of Lianyungang Chinese Medicine Association (ZD202202, ZD202204), the National Nature Science Foundation of China (82374038, 82204621) and Major Project of Natural Science Foundation of Jiangsu Higher Education Institutions (21KJA360005).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2024.107045.
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