Based on Multi-Activity Integrated Strategy to Screening, Characterization and Quantification of Bioactive Compounds from Red Wine

According to French Paradox, red wine was famous for the potential effects on coronary heart disease (CHD), but the specific compounds against CHD were unclear. Therefore, screening and characterization of bioactive compounds from red wine was extremely necessary. In this paper, the multi-activity integrated strategy was developed and validated to screen, identify and quantify active compounds from red wine by using ultra high performance liquid chromatography-fraction collector (UHPLC-FC), ultra fast liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UFLC-Q-TOF/MS) and bioactive analysis. UHPLC-FC was employed to separate and collect the components from red wine, which was further identified by UFLC-Q-TOF/MS to acquire their structural information. Furthermore, the active fractions were tested for antioxidant activity, inhibitory activity against thrombin and lipase activities in vitro by the activity screening kit. As the results, there were 37 fractions had antioxidant activity, 22 fractions had thrombin inhibitory activity and 28 fractions had lipase inhibitory activity. Finally, 77 active components from red wine were screened and 12 ingredients out of them were selected for quantification based on the integration of multi-activity. Collectively, the multi-activity integrated strategy was helpful for the rapid and effective discovery of bioactive components, which provided reference for exploring the health care function of food.


Introduction
As a nutritious drink recommended by World Health Organization (WHO), red wine was widely recognized for its role in health care. According to Ben Cao Gang Mu (Compendium of Materia Medica) record, the famous classic book of traditional Chinese medicine, red wine could treat cold coagulation and blood stasis. Modern pharmacological studies showed that red wine had the function of antioxidant, anticoagulant and lipidlowering [1,2], which can prevent cardiovascular and cerebrovascular diseases such as coronary heart disease (CHD) [3,4].
It was widely believed that the mechanisms of CHD were the result of the interaction of many complex factors, among them the "response to injury hypothesis" had been recognized by researchers [5,6], which stated that the initial damage was the arterial endothelium, further leading to endothelial dysfunction [7]. Indeed, the occurrence of CHD was caused by many factors such as oxidative stress response, hyperlipidemia and platelet aggregation [8]. There were three main methods to treat CHD including pharmacotherapy, interventional therapy and operation [9,10]. Although these methods hold the potential effects for CHD, these methods would also lead some side effects

Establishment of Multi-Activity Integrated Strategy
In the study, an ultra high performance liquid chromatography-photo-diode array-

Establishment of Multi-Activity Integrated Strategy
In the study, an ultra high performance liquid chromatography-photo-diode arrayfraction collector (UHPLC-PDA-FC) system was used to establish multi-activity integrated strategy for screening and evaluating the potentially bioactive components from red wine. The sample was divided into sixty fractions (60 s per fraction) by UHPLC-PDA-FC. Sixty fractions were used to screen the antioxidant activity, thrombin inhibitory activity and lipase inhibitory activity. The multi-activity integrated chromatogram consisted of chromatogram and multi-targets integrated column diagram of antioxidant activity, inhibitory activity of thrombin and lipase were established ( Figure 2) and the detail data was listed in Table 1. The multi-activity integrated strategy could discover bioactive components rapidly and efficiently, which could be used for the identification and screening the active compounds from nutraceutical. According to the activities results, the activities were higher at 20 to 30 min, indicating that these kinds of ingredients may be the main active compounds from red wine.

Establishment of Multi-Activity Integrated Strategy
In the study, an ultra high performance liquid chromatography-photo-diode arrayfraction collector (UHPLC-PDA-FC) system was used to establish multi-activity integrated strategy for screening and evaluating the potentially bioactive components from red wine. The sample was divided into sixty fractions (60 s per fraction) by UHPLC-PDA-FC. Sixty fractions were used to screen the antioxidant activity, thrombin inhibitory activity and lipase inhibitory activity. The multi-activity integrated chromatogram consisted of chromatogram and multi-targets integrated column diagram of antioxidant activity, inhibitory activity of thrombin and lipase were established ( Figure 2) and the detail data was listed in Table 1. The multi-activity integrated strategy could discover bioactive components rapidly and efficiently, which could be used for the identification and screening the active compounds from nutraceutical. According to the activities results, the activities were higher at 20 to 30 min, indicating that these kinds of ingredients may be the main active compounds from red wine. The multi-activity integrated chromatogram of antioxidant activity, inhibitory activity of thrombin and inhibitory activity of lipase. The chromatogram of red wine was shown in superstratum, substratum was a column diagram of each fraction for the three activities. Table 1. The data of antioxidant activity (expressed in Trolox), thrombin inhibitory activity (expressed in inhibition) and lipase inhibitory activity (expressed in inhibition).

Active Ingredients of Red Wine
The analytical data of active fractions with antioxidant activity, inhibitory activity of thrombin or lipase and 94 compounds of mass spectrometry were correlated according to retention times and peaks order. In all, 77 compounds were screened for the prevention and treatment of CHD. The corresponding relationship between fraction number and peak number of the potential active components was shown in Table 3. These active components were concentrated in phenolic acids and flavonoids. Phenolic acids were retained during 20-30 min, which had higher antioxidant and inhibitory activity against thrombin and lipase. Flavonoids were retained during 42-60 min, which had stronger antioxidant and inhibitory activity against lipase. Besides, five ensemble approaches were considered to screen the quantitative composition. The ingredients had multi-activity at the same time; the compounds had a strong single activity; the reference substance was easy to obtain; the activity of this ingredient has been reported many times in literature; the compound was abundant, respectively. The five ensemble approaches were applicated for selecting active ingredients to the quality evaluation of red wine. 63 compounds had multiple activities; 13 compounds had a strong single activity, which were protocatechuic acid, (+)-catechin gallate, (+)-epigallocatechin, acetylsalicylic acid, caftaric acid, chlorogenic acid, benzoic acid, procyanidin B7, trans-piceid, astragalin, cis-piceid, ethyl caffeate and malvidin-3-O-(6-O-coumaroylglucoside). According to the reference substance was easy to obtain and the activity of this ingredient has been reported many times in literature, a total of 19 compounds were screened, which were succinic acid, gallic acid, coumalic acid, procyanidin B1, (-)-epigallocatechin, vanillic acid, catechin, caffeic acid, syringic acid, epicatechin, p-coumaric acid, quercetin-3-O-glucuronide, isoquercitrin, isorhamnetin-3-Oglucoside, trans-resveratrol, quercetin, protocatechuic acid, (+)-catechin gallate and (+)epigallocatechin. Finally, based on the content of the compound from red wine, 12

Active Ingredients of Red Wine
The analytical data of active fractions with antioxidant activity, inhibitory activity of thrombin or lipase and 94 compounds of mass spectrometry were correlated according to retention times and peaks order. In all, 77 compounds were screened for the prevention and treatment of CHD. The corresponding relationship between fraction number and peak number of the potential active components was shown in Table 3. These active components were concentrated in phenolic acids and flavonoids. Phenolic acids were retained during 20-30 min, which had higher antioxidant and inhibitory activity against thrombin and lipase. Flavonoids were retained during 42-60 min, which had stronger antioxidant and inhibitory activity against lipase. Besides, five ensemble approaches were considered to screen the quantitative composition. The ingredients had multi-activity at the same time; the compounds had a strong single activity; the reference substance was easy to obtain; the activity of this ingredient has been reported many times in literature; the compound was abundant, respectively. The five ensemble approaches were applicated for selecting active ingredients to the quality evaluation of red wine. 63 compounds had multiple activities; 13 compounds had a strong single activity, which were protocatechuic acid, (+)-catechin gallate, (+)-epigallocatechin, acetylsalicylic acid, caftaric acid, chlorogenic acid, benzoic acid, procyanidin B7, trans-piceid, astragalin, cis-piceid, ethyl caffeate and malvidin-3-O-(6-O-coumaroylglucoside). According to the reference substance was easy to obtain and the activity of this ingredient has been reported many times in literature, a total of 19 compounds were screened, which were succinic acid, gallic acid, coumalic acid, procyanidin B1, (−)-epigallocatechin, vanillic acid, catechin, caffeic acid, syringic acid, epicatechin, p-coumaric acid, quercetin-3-O-glucuronide, isoquercitrin, isorhamnetin-3-O-glucoside, trans-resveratrol, quercetin, protocatechuic acid, (+)-catechin gallate and (+)-epigallocatechin. Finally, based on the content of the compound from red wine, 12 compounds were screened for further determination, which were gallic acid, coumalic acid, proanthocyanidin B1, catechin, caffeic acid, isoquercitrin, protocatechin, syringic acid, epicatechin, p-coumaric acid, isorhamnetin-3-O-glucoside and quercetin.

UHPLC Analysis
The UHPLC conditions were considered to obtain better chromatographic separation. The different concentrations of acid (0.03%, 0.05% and 0.1% trifluoroacetic acid, 0.05% formic acid, 0.05% acetic acid and pure water), column temperatures (25, 30 and 35 • C) and detection wavelength (210, 220, 254 and 280 nm) were optimized. By comparing resolutions and the peak shapes, the better separation was achieved when 0.1% trifluoroacetic acid was selected as mobile phase, the column temperature and flow rate were optimal at 30 • C and 0.3 mL min −1 , respectively. The UHPLC chromatogram was shown in Figure 4.

Method Validation
The precision, linearity, stability, accuracy, LODs and LOQs were validated. The RSD values of intra-day and inter-day precision were all less than 2.2% and 4.8%, respectively. Overall, 12 components were stable within 24 h at room temperature, and RSDs were less than 2.9%. The calibration curves of the 12 compounds were of high correlation coefficient (r > 0.999) under the concentration ranges. The LODs were ranged from 0.04 to 0.75 µg/mL and LOQs were ranged from 0.12 to 2.0 µg/mL for the 12 compounds, respectively. The results were shown in (Table 4). As can be seen from Table 5, the recoveries of the 12 compounds were in the range of 83.4-106% and RSDs values were not more than 4.3%. All these values were found in an acceptable range, indicating that the method was accurate, reproducible and reliable for quantification of bioactive compounds from red wine. 0.05% formic acid, 0.05% acetic acid and pure water), column temperatures (25, 30 and 35 °C) and detection wavelength (210, 220, 254 and 280 nm) were optimized. By comparing resolutions and the peak shapes, the better separation was achieved when 0.1% trifluoroacetic acid was selected as mobile phase, the column temperature and flow rate were optimal at 30 °C and 0.3 mL min −1 , respectively. The UHPLC chromatogram was shown in Figure 4.  (6), syringic acid (7), epicatechin (8), p-coumaric acid (9), isoquercitrin (10), isorhamnetin 3-O-glucoside (11) and quercetin (12).

Method Validation
The precision, linearity, stability, accuracy, LODs and LOQs were validated. The RSD values of intra-day and inter-day precision were all less than 2.2% and 4.8%, respectively. Overall, 12 components were stable within 24 h at room temperature, and RSDs were less than 2.9%. The calibration curves of the 12 compounds were of high correlation coefficient (r > 0.999) under the concentration ranges. The LODs were ranged from 0.04 to 0.75 μg/mL and LOQs were ranged from 0.12 to 2.0 μg/mL for the 12 compounds, respectively. The results were shown in (Table 4). As can be seen from Table 5, the recoveries of the 12 compounds were in the range of 83.4-106% and RSDs values were not more than 4.3%. All these values were found in an acceptable range, indicating that the method was accurate, reproducible and reliable for quantification of bioactive compounds from red wine.  (6), syringic acid (7), epicatechin (8), p-coumaric acid (9), isoquercitrin (10), isorhamnetin 3-O-glucoside (11) and quercetin (12). Table 4. Precision, stability, linearity, LODs and LOQs of 12 compounds from red wine.

Confirmation the Activity of the 12 Compounds
In order to verify that the compounds in the obtained fractions, 12 active compounds mixture were contrasted for red wine in terms of antioxidant activity, thrombin inhibitory activity and lipase inhibitory activity. The results indicated that inhibition of selected 12 compounds had reached almost 90% of red wine. The inhibition ratios of 12 compounds mixture and red wine were displayed in Figure 6. Moreover, the difference between inhibition of red wine and 12 compounds mixture were also shown in Figure 6. Comparing the inhibition ratio of red wine and 12 compounds which concentrations were the same as red wine, it was shown that the inhibition ratios of 12 compounds were similar to red wine, which was verified that the screening 12 compounds could reflect the total activity of red wine in terms of antioxidant activity, thrombin inhibitory activity and lipase inhibitory activity. Accordingly, the blindness of selecting quantitative component was avoided, which could further provide reference for screening and quantification the active ingredients from nutraceutical and traditional Chinese medicine.

Confirmation the Activity of the 12 Compounds
In order to verify that the compounds in the obtained fractions, 12 active compounds mixture were contrasted for red wine in terms of antioxidant activity, thrombin inhibitory activity and lipase inhibitory activity. The results indicated that inhibition of selected 12 compounds had reached almost 90% of red wine. The inhibition ratios of 12 compounds mixture and red wine were displayed in Figure 6. Moreover, the difference between inhibition of red wine and 12 compounds mixture were also shown in Figure 6. Comparing the inhibition ratio of red wine and 12 compounds which concentrations were the same as red wine, it was shown that the inhibition ratios of 12 compounds were similar to red wine, which was verified that the screening 12 compounds could reflect the total activity of red wine in terms of antioxidant activity, thrombin inhibitory activity and lipase inhibitory activity. Accordingly, the blindness of selecting quantitative component was avoided, which could further provide reference for screening and quantification the active ingredients from nutraceutical and traditional Chinese medicine.

The Application of Multi-Activity Integrated Strategy
Red wine contained many ingredients, which had different activities such as antioxidant, anticoagulant and lipid-lowering. CHD was the results of the interaction of many complex factors. Hence, the multi-activity of red wine was corresponding to the multiple pathogenesis of CHD. Indeed, the multi-activity integrated strategy of red wine was successfully established to quality evaluation of red wine based on the screened 12 bioactive compounds to prevent CHD. Additionally, the multi-activity integrated strategy may help to discover bioactive components rapidly and efficiently, which provided reference for exploring active ingredients in food.

Reagents and Chemical
HPLC-grade Acetonitrile (ACN) and methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Trifluoroacetic acid (TFA) was obtained from Guangzhou Chem- Figure 6. The inhibition ratios of red wine and 12 active compounds mixture with different dilution times and the difference between inhibition of red wine and 12 compounds mixture.

The Application of Multi-Activity Integrated Strategy
Red wine contained many ingredients, which had different activities such as antioxidant, anticoagulant and lipid-lowering. CHD was the results of the interaction of many complex factors. Hence, the multi-activity of red wine was corresponding to the multiple pathogenesis of CHD. Indeed, the multi-activity integrated strategy of red wine was successfully established to quality evaluation of red wine based on the screened 12 bioactive compounds to prevent CHD. Additionally, the multi-activity integrated strategy may help to discover bioactive components rapidly and efficiently, which provided reference for exploring active ingredients in food.

Sample and Standard Solution Preparation
The red wine was analyzed immediately after the bottle opening. The sample (2 mL) was filtrated through a 0.22 µm polyvinylidene fluoride membrane. Appropriate amounts of gallic acid, catechin, caffeic acid, epicatechin, isoquercitrin, coumalic acid, proanthocyanidin B1, syringic acid, p-coumaric acid and isorhamnetin-3-O-glucoside were precisely weighed and dissolved with methanol to prepare standards solutions of 1 mg/mL. All the samples and standard solutions were stored at 4 • C in the dark for analysis.

Preparation of the Red Wine Fractions
Fraction collector (BSZ-100, Shanghai Jiapeng Technology Instrument, Shanghai, China) was used to prepare fractions. The red wine sample was injected into the UH-PLC system for separation. Then fractions were collected every 60 s by setting the fraction collector and evaporated to dryness by nitrogen gas. The residues were dissolved by methyl alcohol:water (1:1).

UFLC/Q-TOF-MS Analysis
The identification was performed on a hybrid quadrupole time-of-flight tandem mass spectrometry (Exion-LC TM /X500R QTOF, AB SCIEX, Foster City, CA) equipped with an electrospray ionization (ESI) interface. The mass spectrometer was operated both in positive and negative ion mode. The parameters were set as following: the ion spray voltage of 5500/−4500 V; turbo spray temperature (TEM) of 550 • C; declustering potential (DP) of ±70 V; collision energy (CE) of ±40 V; nebulizer gas (gas 1) of 50 psi; heater gas (gas 2) of 50 psi and curtain gas of 30 psi [20,43]. Nitrogen was kept as the nebulizer and auxiliary gas. TOF/MS and TOF/MS-MS were scanned with the mass range of m/z 100-1200 and 50-1200, respectively. The experiments were run with 200 ms accumulation time for TOF/MS and 80 ms accumulation time for TOF/MS-MS. Continuous recalibration was carrying out at each 3 h. In addition, dynamic background subtraction (DBS) trigger information-dependent acquisition (IDA) was used to trigger acquisition of MS/MS of low-level constituents. The accurate mass and composition for the precursor ions and fragment ions were analyzed using the SCIEX OS software integrated with the instrument.

Antioxidant Activity Assay
After collection fractions, the antioxidant activity was tested for the 60 fractions by Total Antioxidant Capacity Assay Kit (ABTS). ABTS was oxidized to ABTS cation radical (ABTS•+) with appropriate oxidizing agent. The red wine has inhibited the production of ABTS•+ that can be detected by the absorbance at 734 nm. Initially, 0.4 mL ABTS stock solution was mixed with 0.4 mL of the oxidizing agent solution for the preparation of the radical. This solution was maintained at room temperature for 12-16 h in an amber bottle. Subsequently, the mixture was diluted in PBS to obtain an absorbance of approximately 0.7 ± 0.05. Finally, 200 µM ABTS was mixed with different fraction. The absorbance change at 734 nm was measured after 5 min. The reaction solution with PBS instead of test sample was used as a control test. The total antioxidant activity was expressed as Trolox-equivalent Antioxidant Capacity (TEAC) equivalent [51,52].

Thrombin Inhibitory Activity Assay
The inhibitory activity against thrombin was assessed by Thrombin Inhibitor Screening Kit (Fluorometric). Thrombin inhibitor screening principle was utilized the ability of thrombin to cleave a synthetic AMC-based peptide substrate to release AMC, which can be detected by measuring fluorescence intensity at Ex/Em = 350/450 nm. In the presence of thrombin inhibitors, the extent of cleavage reaction was reduced or completely abolished. The loss in fluorescence intensity can be correlated to the amount of inhibitor present in the assay solution. At first, the thrombin enzyme solution was prepared as 48 µL thrombin assay buffer and 2 µL thrombin enzyme stock solution in each well. Then, fractions were dissolved by methyl alcohol:water (1:1) and diluted to 10 times with thrombin assay buffer. The 10 µL diluted fractions or thrombin inhibitor control were added into the thrombin enzyme containing wells. The thrombin inhibitor control was consisted of 1 µL thrombin inhibitor and 9 µL thrombin assay buffer to thrombin enzyme well. The sample was incubated at room temperature for 15 min. Finally, the thrombin substrate solution was added into each well, which was consisted of 35 µL thrombin assay buffer and 5 µL thrombin substrate. Fluorescence was measured in a kinetic mode for 30-60 min at 37 • C (Ex/Em = 350/450 nm). The time points (T 1 = 5 min, T 2 = 10 min) were chosen in the linear range of the plot and obtain the corresponding values for the fluorescence (RFU1, RFU2). Irreversible inhibitors that inhibit the thrombin activity completely at the tested concentration will have ∆RFU = 0 and will show 100% relative inhibition. The inhibition (%) was calculated by (slope of enzyme control − slope of fraction)/slope of enzyme control × 100% [53].

Lipase Inhibitory Activity Assay
In this study, the inhibition of each fraction was assayed by measuring the fluorescence intensity of 4-methylumbelliferyl oleate (4-MU) (the hydrolytic product of 4-MUO). The assay was performed according to the method described with slight modifications. Initially, the concentrations of trypsin (0.83-30,000 U/mL) and time were investigated to establish linear range with the concentration of 4-MUO at 0.1 mM. Then, 25 µL fractions and 25 µL lipase solutions were mixed together and 50 µL 4-MUO was added to the mixture. Finally, the amount of 4-MU was measured by microplate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm after incubating at 25 • C for 30 min. The inhibition of pancreatic lipase activity was calculated as follows: inhibition (%) = [(A control − A sample )/A control − (A control − A control sample )/A control ] × 100%, where A sample was the fluorescence of the reactions with added fraction, A control was the control, and A control sample was the fluorescence of the solvent of the fraction [12,54].

Statistical Analyses
The results of precision and recovery have been measured in triplicates and the stability have been measured in sextuplicate, which were expressed as mean and RSD. The content of 12 compounds have been measured in triplicates and expressed as mean.

Conclusions
In this study, the multi-activity integrated strategy was proposed to screen, identify and quantify components from red wine for prevention of CHD via UHPLC-FC and UFLC-Q-TOF/MS. The multi-activity integrated strategy of red wine was established and verified the availability of screening the preventing CHD compounds from the complex sample. Red wine exhibited potential antioxidant activity and inhibitory activity against thrombin and lipase, which could be used to prevent CHD. In addition, the system had advantages over traditional methods in screening potential active compounds. The multi-activity integrated strategy may help to discover bioactive components rapidly and efficiently, which provided reference for exploring the health care function in food.