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

Abstract

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.

1. 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 lipid-lowering [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 such as common complications after coronary intervention included bleeding, pulmonary embolism, infection and so on. Besides, long term use of some medicines as nitroglycerin would also generate drug resistance and migraine pain [11]. Therefore, prevention CHD has become the trend of medical development.

The famous French paradox proved that red wine can prevent CHD [3]. Accumulated studies demonstrated that extracts from red wine possessed antioxidant activity, anti-coagulation and lipid-lowering efficacy, which corresponded to the pathogenesis of CHD. Traditional methods for isolating and evaluating components were most carried out on animal and cell models, which were time-consuming, labor-intensive, expensive and hardly to be used for the direct screening [12]. The developed methods for screening active compounds were extracted, the single component from sample and evaluated the biological activity by pharmacological means. Animal model was the most important screening manner in the part of bioactive components evaluation, which can clearly response to the efficacy of ingredients in the whole body. However, the method was high cost and low efficiency. Besides, compared with the animal model, cellular model was relatively cheap and convenient, but it had higher requirement on instrumentation [13]. Therefore, it was urgent to establish a fast, portable and accurate method to screen and identify the bioactive ingredients from red wine. Ultra high performance liquid chromatography (UHPLC) was used to separate the components of food with appropriate chromatographic conditions and the fraction collector was used to enrich compounds automatically in the fixed-time interval according to the retention time in the UHPLC chromatogram. Compared with HPLC, UHPLC exhibited higher separation and narrower chromatographic peak. At the same time, ACQUITY UPLC column (HSS T3, 1.8 μm, 2.1 mm × 100 mm, Waters, Ireland) could withstand 100% water mobile phase and the particle size was only 1.8 microns, which was adapted to separation of complex samples especially for the strong polar compounds from red wine. Indeed, some researches showed that ultra high performance liquid chromatography-fraction collector (UHPLC-FC) could quickly separate and enrich compounds. Moreover, ultra fast liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UFLC-Q-TOF/MS) was used to identify the components for obtaining structural information [12]. Accordingly, integrated the UHPLC-FC and UFLC-Q-TOF/MS could be used for quickly separating, enriching and identifying the potential active compounds from complex samples.

In this study, UHPLC-FC was used to separate, collect and enrich the fractions from red wine. Then antioxidant activity, inhibitory activity against thrombin and lipase activities of all fractions were performed for evaluating activities. Furthermore, UFLC-Q-TOF/MS was used to identify the components in fractions from red wine (Figure 1). As the results, 77 potential active components from red wine were screened and 12 ingredients out of them were selected for quantification based on integration of the multi-activity. Accordingly, the multi-activity integrated strategy was significant for the rapid and effective discovery of the potential bioactive components from red wine to prevent CHD, which provided reference for exploring the health care function of food.

2. Result and Discussion

2.1. Establishment of Multi-Activity Integrated Strategy

In the study, an ultra high performance liquid chromatography-photo-diode array-fraction 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 data of antioxidant activity (expressed in Trolox), thrombin inhibitory activity (expressed in inhibition) and lipase inhibitory activity (expressed in inhibition).

	Antioxidant Activity Inhibition (Trolox (%))	Thrombin Inhibitory Activity Inhibition (%)	Lipase Inhibitory Activity Inhibition (%)
F1	31.07	37.82	69.67
F2	no	44.42	87.30
F3	45.35	24.65	no
F4	55.64	26.68	no
F5	29.63	29.77	no
F6	42.56	26.61	no
F7	no	no	no
F8	no	no	no
F9	24.08	21.21	no
F10	23.10	no	no
F11	no	no	87.35
F12	no	no	no
F13	25.70	no	no
F14	no	22.44	no
F15	no	no	no
F16	36.79	no	100.20
F17	55.64	no	66.44
F18	29.63	no	no
F19	42.56	no	124.96
F20	no	no	70.68
F21	no	no	113.43
F22	19.34	19.22	136.53
F23	30.56	no	94.14
F24	23.59	no	103.15
F25	42.87	18.56	66.12
F26	54.32	35.06	97.89
F27	48.31	36.21	67.39
F28	43.88	48.47	68.79
F29	39.03	29.44	127.97
F30	40.83	18.07	90.74
F31	32.31	24.49	no
F32	30.82	no	no
F33	no	no	no
F34	no	no	no
F35	no	no	no
F36	30.36	35.12	no
F37	24.07	no	no
F38	no	no	no
F39	19.44	no	no
F40	19.48	no	no
F41	no	no	no
F42	22.28	no	71.92
F43	22.64	no	77.88
S44	25.36	no	84.26
F45	no	27.81	no
F46	25.08	no	no
F47	no	no	no
F48	no	no	79.38
F49	no	no	68.49
F50	no	no	no
F51	no	19.33	no
F52	no	no	no
F53	no	no	no
F54	27.09	no	no
F55	25.94	no	73.85
F56	25.27	27.66	72.88
F57	36.68	20.91	66.70
F58	no	20.65	76.42
F59	22.80	no	69.65
F60	23.21	no	67.84

. 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.

2.2. Identification of 94 Components from Red Wine

The components from red wine with antioxidant activity or inhibitory potential against thrombin and lipase activities were analyzed by UFLC-Q-TOF/MS to obtain structural information. The molecular compositions of ingredients were identified according to the literature and exact molecular weight. The peak number, retention time, MS/MS fragmentation ions information and identification of 94 components from red wine were listed in

Table 2. UFLC/Q-TOF-MS data and identification 94 compounds from red wine.

Peak No.	RT (min)	Ion	Found at Mass	MS/MS Fragmentation Ions	Formula	Identification	ppm
1	0.70	[M+H]+	116.0702	70.0647	C5H9NO2	D-proline	0.8
2	0.76	[M+H]+	175.1189	70.0652	C6H14N4O2	L-arginine	−0.3
3	0.79	[M−H]−	179.0562	99.0087, 71.0137, 59.0139	C6H12O6	D-glucose	0.3
4	0.88	[M-H]−	181.0720	83.0143, 71.0140, 59.0140	C6H14O6	D-mannitol	1.5
5	0.96	[M−H]−	191.0199	85.0296, 59.0134	C6H8O7	citric acid	1.0
6	0.99	[M−H]−	149.0091	105.0217, 87.0085, 72.9928	C4H6O6	tartaric acid	−0.7
7	1.04	[M+H]+	138.0548	120.0433, 92.0495, 78.0339	C7H7NO2	4-aminobenzoic acid	−1.5
8	1.12	[M−H]−	341.1088	161.0459, 71.0137, 59.0138	C12H22O11	trehalose	−0.3
9	1.15	[M−H]−	149.0455	75.0221, 59.0133	C5H10O5	D-ribose	−0.2
10	1.31	[M−H]−	133.0142	115.0018, 71.0136	C4H6O5	L-malic acid	−0.5
11	1.33	[M+H]+	118.0862	72.0809	C5H11NO2	L-valine	−0.4
12	1.48	[M−H]−	173.0454	137.0225, 93.0343	C7H10O5	shikimic acid	−1.0
13	1.71	[M+H]+	150.0586	74.0243, 61.0108	C5H11NO2S	L-methionine	1.8
14	2.03	[M+H]+	124.0393	80.0506, 55.9346	C6H5NO2	niacin	0.2
15	3.27	[M−H]−	117.0192	99.9254, 73.0291	C4H6O4	succinic acid	−1.2
16	3.51	[M+H]+	165.0547	119.0479, 95.0503, 77.0388, 59.9302	C9H8O3	trans-4-hydroxycinnamic acid	0.7
17	3.85	[M+H]+	137.0913	121.0616, 103.0616, 95.0561, 91.0556, 77.0391	C8H11NO	tyramine	−0.5
18	5.97	[M−H]−	169.0141	125.0244, 107.0156, 79.0186	C7H6O5	gallic acid	−1.0
19	8.77	[M+H]+	141.0182	123.0080, 95.0127, 67.0187, 55.9453	C6H4O4	coumalic acid	−0.1
20	11.49	[M−H]−	153.0558	123.0085, 108.0221, 95.0139, 78.9590	C8H10O3	hydroxytyrosol	0.2
21	11.57	[M−H]−	153.0194	109.0298, 91.0298, 65.0029	C7H6O4	protocatechuic acid	0.3
22	13.85	[M−H]−	305.0664	179.0360, 165.0194, 137.0243, 125.0246	C15H14O7	(+)-gallocatechin	−1.0
23	14.27	[M−H]−	179.0349	135.0560, 89.0394, 79.0550	C9H8O4	acetylsalicylic acid	−0.7
24	14.27	[M−H]−	179.0348	161.0263, 135.0454, 117.0351	C13H12O9	caftaric acid	−1.0
25	15.05	[M−H]−	153.0194	123.0061, 109.0298, 91.0298	C7H6O4	gentisic acid	0.4
26	15.21	[M−H]−	137.0255	108.0213	C7H6O3	protocatechualdehyde	−2.7
27	15.31	[M−H]−	181.0506	135.0453	C9H10O4	homovanillic acid	0.0
28	15.43	[M−H]−	183.0299	124.0234, 95.0136	C8H8O5	methyl gallate	0.0
29	15.90	[M−H]−	137.0244	93.0340	C7H6O3	4-hydroxybenzoic acid	0.4
30	16.32	[M−H]−	353.0877	191.0616, 179.0341, 135.0439	C16H18O9	chlorogenic acid	−0.2
31	17.36	[M−H]−	295.0458	163.0400, 119.0503, 87.0094	C13H12O8	p-coutaric acid	0.1
32	18.08	[M−H]−	163.0401	119.0498, 93.0347	C9H8O3	m-coumaric acid	0.2
33	18.08	[M−H]−	295.0458	163.0400, 119.0498	C13H12O8	coutaric acid	−0.4
34	18.15	[M−H]−	145.0507	73.0273, 55.0191	C6H10O4	dimethyl succinate	0.6
35	19.24	[M−H]−	121.0296	108.0212, 92.0265	C7H6O2	benzoic acid	0.8
36	19.66	[M−H]−	577.1346	425.0864, 407.0775, 289.0714, 161.0238, 125.0243	C30H26O12	procyanidin B1	−0.9
37	19.89	[M−H]−	305.0664	245.0462, 219.0652, 139.0401, 125.0244	C15H14O7	(−)-epigallocatechin	0.2
38	19.98	[M−H]−	577.1346	407.0768, 289.0709	C30H26O12	procyanidin B3	0.0
39	20.01	[M−H]−	167.0350	108.0212	C8H8O4	vanillic acid	0.2
40	20.21	[M−H]−	163.0400	119.0502, 93.0343	C9H8O3	2-Hydroxycinnamic acid	−0.1
41	20.47	[M−H]−	289.0713	245.0843, 203.0713, 151.0401, 109.0294	C15H14O6	catechin	−1.5
42	20.58	[M−H]−	325.0563	193.0490, 117.0341	C14H14O9	fertaric acid	−0.5
43	20.98	[M−H]−	179.0349	135.0449, 89.0393	C9H8O4	caffeic acid	−1.0
44	21.90	[M−H]−	441.0186	289.0735, 217.0167, 191.0337, 151.0386	C22H18O10	epicatechin gallate	2.2
45	22.06	[M−H]−	197.0454	182.0256, 167.0014, 138.0317, 123.0090, 95.0142	C9H10O5	syringic acid	−0.3
46	23.01	[M−H]−	577.1346	425.0893, 407.0782, 289.0721	C30H26O12	procyanidin B2	−0.7
47	23.11	[M−H]−	325.0926	163.0394, 145.0294, 117.0343	C15H18O8	p-coumaric acid glucoside	−0.5
48	23.37	[M−H]−	189.0769	71.0503	C8H14O5	diethyl malate	0.3
49	23.69	[M−H]−	165.0556	119.0527, 103.0565	C9H10O3	desaminotyrosine	−0.5
50	24.37	[M−H]−	137.0244	93.0470, 65.0473	C7H6O3	2-hydroxybenzoic acid	2.2
51	24.38	[M−H]−	289.0713	203.0710, 187.0396, 151.0401, 123.0449, 109.0290	C15H14O6	epicatechin	−0.7
52	24.68	[M−H]−	167.0350	123.0436, 108.0212	C8H8O4	3-hydroxy-4-methoxybenzoic acid	2.5
53	25.16	[M−H]−	197.0454	169.0142, 125.0239, 79.0184	C9H10O5	ethyl gallate	−0.9
54	25.32	[M]+	493.1341	331.0822, 316.0553	C23H25O12	malvidin-3-O-glucoside	0.1
55	25.74	[M−H]−	163.0401	119.0503, 93.0347	C9H8O3	p-coumaric acid	−0.6
56	30.75	[M−H]−	167.0350	123.0450, 65.0024	C8H8O4	homogentisic acid	0.8
57	30.80	[M−H]−	577.1346	425.0886, 407.0789, 289.0734	C30H26O12	procyanidin B7	0.6
58	34.06	[M−H]−	479.0826	316.0225, 271.0249, 151.0034	C21H20O13	myricetin-3-O-galactoside	−1.0
59	35.93	[M−H]−	389.1242	227.0716, 185.0606, 143.0505	C20H22O8	trans-piceid	−0.2
60	38.70	[M−H]−	300.9987	257.011, 229.0126, 145.0295	C14H6O8	ellagic acid	−0.9
61	38.84	[M−H]−	181.0506	109.0286, 65.0032	C9H10O4	dihydrocaffeic acid	0.5
62	39.28	[M]+	465.1036	303.051, 229.0488	C21H21O12	delphinidin-3-O-glucoside	1.9
63	39.31	[M−H]−	463.0884	301.0379, 271.0245, 243.0291, 151.0031	C21H20O12	hyperoside	0.4
64	39.71	[M−H]−	449.1090	285.0413, 151.0041	C21H22O11	quercitrin	0.2
65	39.83	[M]+	627.1462	303.0351	C27H31O17	delphinidin-3,5-O-diglucoside	1.1
66	40.08	[M−H]−	477.0670	301.0352, 255.0296, 151.0034	C21H18O13	quercetin-3-O-glucuronide	−0.9
67	41.28	[M−H]−	493.0985	330.0381, 315.0155, 271.0249, 151.0034	C22H22O13	laricitrin-3-O-glucoside	−0.6
68	42.86	[M−H]−	447.0936	301.0372, 271.0256, 255.0295, 151.0020	C21H20O11	luteolin -3’-glucoside	1.2
69	42.98	[M−H]−	449.1090	285.0413, 151.0041	C21H22O11	astilbin	0.9
70	43.45	[M−H]−	463.0884	301.0379, 255.0305, 151.0039	C21H20O12	isoquercitrin	0.8
71	44.78	[M−H]−	447.0936	301.0372, 255.0293, 151.0016	C21H20O11	astragalin	0.8
72	45.19	[M−H]−	389.1242	227.0994, 185.0606	C20H22O8	cis-piceid	0.0
73	45.72	[M−H]−	317.0300	245.0504, 137.0241, 109.0292, 151.0035	C15H10O8	myricetin	−0.9
74	45.77	[M]+	479.1193	317.0648, 302.0429, 285.0362, 153.0184	C22H23O12	petunidin-3-O-glucoside	1.9
75	45.80	[M−H]−	477.1041	315.0530, 300.0263, 271.0255, 243.0306, 151.0036	C22H22O12	isorhamnetin-3-O-glucoside	0.6
76	47.17	[M−H]−	227.0715	185.0587, 143.0501, 115.0556	C14H12O3	trans-resveratrol	0.6
77	47.42	[M−H]−	435.1300	273.0769, 179.0349, 167.0352	C21H24O10	phloridzin	0.7
78	49.16	[M]+	507.1141	303.0767, 187.0626, 113.0234	C23H23O13	delphindin-3-O-(6-O-acetylglucoside)	1.6
79	51.13	[M−H]−	207.0662	179.0433, 135.0446	C11H12O4	ethyl caffeate	−0.6
80	51.94	[M]+	639.1719	477.1176, 419.0853, 331.0874	C32H31O14	malvidin-3-O-(6-O-coumaroylglucoside)	1.7
81	52.60	[M+H]+	303.0497	285.0398, 257.0456, 229.0492, 153.0188	C15H10O7	morin	−0.8
82	52.60	[M]+	303.0497	229.0492, 153.0188	C15H11O7	delphinidin	−0.8
83	52.63	[M−H]−	301.0351	245.0447, 229.0513, 151.0034, 121.0292	C15H10O7	quercetin	−1.0
84	52.67	[M−H]−	227.0715	143.0504	C14H12O3	resveratrol	−0.9
85	53.23	[M−H]−	331.0459	316.0231, 271.0251, 178.9991, 151.0039	C16H12O8	laricitrin	0.0
86	55.82	[M−H]−	271.0613	229.0500, 177.0190, 151.0048, 119.0618	C15H12O5	naringenin	0.4
87	57.10	[M]+	287.0549	213.0559, 153.0180, 137.0233	C15H11O6	cyanidin	−0.3
88	57.12	[M−H]−	285.0403	239.0356, 187.0403, 151.004	C15H10O6	kamepferol	−0.6
89	57.32	[M−H]−	345.0618	330.0385, 315.0153, 259.0252, 187.0394	C17H14O8	syringetin	0.6
90	57.39	[M]+	317.0656	229.0498, 153.0188	C16H13O7	petunidin	0.0
91	57.41	[M−H]−	315.0508	300.0270, 271.0241, 255.0299, 227.0241, 151.0036	C16H12O7	isorhamnetin	−0.6
92	58.32	[M+H]+	193.1588	56.9419	C13H20O	ionone	0.6
93	60.04	[M+H]+	173.1536	76.0221	C10H20O2	ethyl caprylate	0.2
94	60.96	[M+H]+	195.1748	177.1634, 107.0855, 77.0387	C13H22O	theaspirane	2.1

. The extracted ion chromatogram of positive and negative ions was illustrated in Figure 3. Peak 1 gave the mass spectrum ion at 116.0702 (C₅H₉NO₂) with MS/MS fragment of 70.0647 (C₄H₇N) [M+H-HCOOH]⁺, which was detected to be D-proline [14,15,16]. Peaks 11 (C₄H₉N) [M+H-HCOOH]⁺ and 14 (C₅H₃N) [M+H-HCOOH]⁺ were based on the same regular. Therefore, peaks 11 and 14 were identified as L-valine (C₅H₁₁NO₂) [14] and niacin (C₆H₅NO₂) [17]. Peak 2 had the mass spectrum ion at 175.1189 (C₆H₁₄N₄O₂) with MS/MS fragment of 70.0652 [M+H-HCOOH-2NH₂-CN]⁺, which was detected to be L-arginine [14]. Peak 3 had a molecular weight of 179.0562 (C₆H₁₂O₆) with fragments of 99.0087 (C₄H₄O₃) [M-H-3H₂O-C₂HO]⁻, 71.0137 (C₃H₄O₂) [M-H-3H₂O-C₂HO-CO]⁻ and 59.0139 (C₂H₄O₂) [M-H-3H₂O-C₂HO-CO-CH₂]⁻. Therefore, it was identified as D-glucose. Peak 4 had mass spectrum ion at 181.072 (C₆H₁₄O₆) with MS/MS experiment gave fragmentation information for 83.0143 [M-H-C₃H₂O₃]⁻ and 71.0140 [M-H-C₃H₆O₃-H₂O]⁻, which could be identified as D-mannitol [18]. Peak 5 had major ions at m/z 191.0199 (C₆H₈O₇) and the MS/MS fragments were 85.0296 (C₄H₆O₂) [M-H-2CO₂-H₂O]⁻ and 59.0134 (C₃H₈O) [M-H-3CO₂]⁻. Therefore, it was identified as citric acid [19]. Peak 6 was identified as tartaric acid by its characteristic fragmentation pattern, which MS/MS spectra ions m/z 105.0217 (C₃H₆O₄) [M-H-CO₂]⁻ and 87.0085 (C₃H₄O₃) [M-H-CO₂-H₂O]⁻ were observed [20]. By the similar regular pattern, peak 40 (C₉H₈O₃) had the MS/MS fragment of m/z 119.0502 (C₈H₈O) [M-H-CO₂]⁻. Therefore, Peak 40 was identified as 2-hydroxycinnamic acid [21]. Peak 7, the molecular weight was 138.0548 (C₇H₇NO₂) and the fragment ions observed included m/z 120.0433 (C₆H₇N) [M+H-H₂O]⁺, 78.0339 (C₆H₆) [M+H-HCOOH-NH₂]⁺, which was identified to be 4-aminobenzoic acid [22]. Peak 8 gave a mass spectrum ion at 341.1088 (C₁₂H₂₂O₁₁) with MS/MS fragments of 161.0459 (C₆H₉O₅) [M-H-C₆H₁₁O₅-H₂O]⁻ and 71.0137 (C₃H₄O₂) [M-H-C₉H₁₄O₈-H₂O]⁻, which was detected to be trehalose [23]. The molecular weight of peak 9 was 149.0455 (C₅H₁₀O₅). In the MS/MS experiment, the predominant ions appeared at 75.0221 (C₃H₇O₂) [M-H-C₂H₃O₃]⁻ and 59.0133 (C₂H₂O₂) [M-H-C₃H₈O₂]⁻. By comparing the characteristic fragmentation pattern with the reported in references, it can be identified as D-ribose. Peaks 10 (C₄H₆O₅) was identified as L-malic acid [24] which the H₂O was removed at first, and then the CO₂ was removed. MS/MS fragment of L-malic acid was 115.0018 (C₄H₄O₄) [M-H-H₂O]⁻ and 71.0137 (C₃H₄O₂) [M-H-H₂O-CO₂]⁻. Peaks 12 (C₇H₁₀O₅) was identified as shikimic acid. MS/MS fragment was 137.0225 (C₇H₆O₃) [M-H-2H₂O]⁻. The molecular weight of peak 13 was 150.0586 (C₅H₁₁NO₂S) and ion m/z 74.0243 (C₃H₈O₂) [M+H-NH₃-C₂S]⁺ was observed in MS/MS spectra, which could be identified as L-methionine [16]. Peak 15 had a molecular weight of 117.0192 (C₄H₆O₄), which produced the MS/MS ions as 99.9254 (C₃H₆O₂) [M-H-CO₂]⁻ and 73.0291 (C₃H₄O) [M-H-CO₂-H₂O]⁻. Hence, it can be identified as succinic acid [25]. Peak 33 had the same fragment (73.0273 [M-H-C₃H₅O₂]⁻) with peak 15. The other fragment was 55.0191 [M-H-C₃H₅O₂-H₂O]⁻, which can be identified as dimethyl succinate [25]. Peaks 18, 21, 23, 24, 25, 29, 31, 32, 33, 39, 42, 43, 49, 50, 52, 55, 56, 60 and 61 were in the similar regular pattern with peak 15, the parent ion was removed one or more CO₂ and H₂O. Peaks 18 (125.0244 [M-H-CO₂]⁻, 107.0156 [M-H-CO₂-H₂O]⁻ and 79.0189 [M-H-CO₂-H₂O-CO]⁻), 21 (109.0298 [M-H-CO₂]⁻ and 91.0298 [M-H-CO₂-H₂O]⁻), 23 (135.0560 [M-H-CO₂]⁻ and 89.0194 [M-H-CO₂-CH₃CO]⁻), 24 (135.0454 [M-H-CO₂]⁻ and 117.0351 [M-H-CO₂-H₂O]⁻), 25 (109.0298 [M-H-CO₂]⁻ and 91.0298 [M-H-CO₂-H₂O]⁻), 29 (93.0340 [M-H-CO₂]⁻), 31 (163.0400 [M-H-2CO₂]⁻ and 119.0503 [M-H-3CO₂]⁻), 32 (119.0498 [M-H-CO₂]⁻ and 93.0347 [M-H-CO₂-H₂O]⁻), 33 (163.0400 [M-H-2CO₂]⁻ and 119.0498 [M-H-3CO₂]⁻), 39 (108.0212 [M-H-CO₂-CH₃]⁻), 42 (193.0490 [M-H-2CO₂-C₂H₅O]⁻ and 117.0341 [M-H-3CO₂-C₂H₅O-CH₃-H₂O]⁻), 43 (135.0449 [M-H-CO₂]⁻), 49 (119.0527 [M-H-CO₂]⁻ and 103.0565 [M-H-CO₂-H₂O]⁻), 50 (93.0470 [M-H-CO₂]⁻ and 65.0473 [M-H-CO₂-CO]⁻), 52 (123.0436 [M-H-CO₂]⁻ and 108.0212 [M-H-CO₂-H₂O]⁻), 55 (119.0503 [M-H-CO₂]⁻ and 93.0347 [M-H-CO₂-H₂O]⁻), 56 (123.0450 [M-H-CO₂]⁻), 60 (257.0110 [M-H-CO₂]⁻ and 229.0126 [M-H-CO₂-CO]⁻) and 61 (109.0286 [M-H-CO₂-C₂H₂]⁻) were the same regular, which were identified as gallic acid [20], protocatechuic acid [23], acetylsalicylic acid [26], caftaric acid [20], gentisic acid [23], 4-hydroxybenzoic acid [27], p-coutaric acid [28], m-coumaric acid [28], coutaric acid [28], vanillic acid [27], fertaric acid [28], caffeic acid [28], desaminotyrosine [29], 2-hydroxybenzoic acid [28], 3-hydroxy-4-methoxybenzoic acid [30,31], p-coumaric acid [28], homogentisic acid [23], ellagic acid [32] and dihydrocaffeic acid [33]. Peak 79 had the major first-order mass spectrum at m/z 270.0662 (C₁₁H₁₂O₄). The MS/MS fragments were the same as peak 43 of 179.0433 [M-H-CH₃]⁻ and 135.0446 [M-H-CH₃-CO₂]⁻. Therefore, it was identified as ethyl caffeate [28]. Peak 16 showed the mass spectrum ion at m/z 165.0547 (C₉H₈O₃) and the product ion was 119.0479 [M+H-HCOOH]⁺, which could be identified as trans-4-hydroxycinnamic acid [20]. Peak 17 gave a mass spectrum ion at 138.0548 (C₈H₁₁NO) with the MS/MS fragments at 121.0616 [M+H-NH₃]⁺ and 95.0561 [M+H-NH₃-C₂H₄]⁺, which was detected to be tyramine [34,35]. Peak 19 showed the mass spectrum ion at 141.0182 with the MS/MS fragments at 95.0127 [M+H-HCOOH]⁺, which could be identified as coumalic acid. Peak 20 gave [M−H]⁻ ion at m/z 153.0558. In MS/MS mode, the produced ions were m/z 123.0085 [C₈H₁₀O₃-CH₂O]⁻ and 108.0221 [C₈H₁₀O₃-CH₂OH-CH₃]⁻, which can be identified as hydroxytyrosol [36]. Peaks 22 and 37 were a type of isomer which yielded the same ion at 305.0246 [M−H]⁻ in the first-order mass spectrum. They had the same prominent ion at 125.0246 [C₁₅H₁₄O₇-C₉H₈O₄]⁻. They were speculated to be (−)-epigallocatechin [25] and (+)-gallocatechin [25] based on their retention times. Peak 26 had the [M−H]⁻ ion at m/z 137.0255. The product ion was 108.0213 [M-H-CHO]⁻. Therefore, peak 26 was identified as protocatechualdehyde. Peak 27 had the mass spectrum ion at 181.0506 (C₉H₁₀O₄) with MS/MS fragment of 135.0453 [M-H-CH₃-H₂O]⁻, which was identified as homovanillic acid [34,37]. Peak 28 showed mass spectrum ion at 183.0299 (C₈H₈O₅). The product fragment ions were 124.0234 [M-H-CO₂-CH₃]⁻ and 95.0136 [M-H-CO₂-CH₃-CHO]⁻. Hence, it was identified as methyl gallate [38,39]. Peak 30 gave [M−H]⁻ ion at m/z 353.0877 and MS/MS produced ions were at m/z 191.0616 [M-H-C₆H₅O₅]⁻ and 135.0439 [M-H-C₆H₅O₅-CO]⁻, it can be identified as chlorogenic acid [27,40]. Peak 35 was identified as benzoic acid by the MS spectra ion 121.0296 and MS/MS spectra ions m/z 92.0265 [M-H-CHO]⁻ and 108.0212 [M-H-CH]⁻. Peaks 36, 38, 46 and 57 had same molecular weight of C₃₀H₂₆O₁₂, which illustrated that they were isomers. Base on the MS/MS spectra, peak 36 (407.0775 [M-H-C₈H₈O₃-H₂O]⁻, 289.0714 [M-H-C₁₅H₁₃O₆]⁻), 38 (407.0768 [M-H-C₈H₈O₃-H₂O]⁻, 289.0709 [M-H-C₁₅H₁₃O₆]⁻), 46 (425.0893 [M-H-C₈H₈O₃]⁻, 407.0782 [M-H-C₈H₈O₃-H₂O]⁻, 289.0721 [M-H-C₁₅H₁₃O₆]⁻) and 57 (425.0886 [M-H-C₈H₈O₃]⁻, 407.0789 [M-H-C₈H₈O₃-H₂O]⁻, 289.0734 [M-H-C₁₅H₁₃O₆]⁻) were detected, which could be identified as procyanidin B1 [41], procyanidin B3 [41], procyanidin B2 [41] and procyanidin B7 [41] by comparing the retention time of the reference standards and literatures. Peak 41 had a molecular weight of 289.0713 (C₁₅H₁₄O₆) with fragments of 245.0843 [M-H-CO₂]⁻, 203.0713 [M-H-H₂O-C₃O₂]⁻ and 151.0401 [M-H-C₈H₁₀O₂]⁻, therefore it was identified as catechin [23]. Based on the same fragment, peaks 44 (289.0735 [M-H-C₇H₂O₄]⁻, 151.0386 [M-H-C₇H₄O₄-C₈H₁₀O₂]⁻), 51 (203.0710 [M-H-H₂O-C₃O₂]⁻, 187.0396 [M-H-C₄H₆O₃]⁻ and 151.0401 [M-H-C₈H₁₀O₂]⁻), 58 (316.0225 [M-H-C₆H₁₁O₅]⁻, 271.0249 [M-H-C₆H₁₁O₅-CO_-H₂O]⁻ and 151.0034 [M-H-C₁₄H₁₆O₉]⁻), 64 (285.0413 [M-H-C₆H₁₁O₅]⁻ and 151.0041 [M-H-C₁₄H₈O₇]⁻), 66 (301.0352 [M-H-C₆H₁₀O₅]⁻, 255.0296 [M-H- C₆H₁₀O₅-C₂H₂O]⁻ and 151.0034 [M-H-C₁₄H₁₁O₉]⁻), 67 (330.0381 [M-H-C₆H₁₁O₅]⁻, 315.0155 [M-H-C₆H₁₁O₅-CH₃]⁻, 271.0249 [M-H-C₆H₁₁O₅-CH₃-CO-H₂O]⁻ and 151.0034 [M-H-C₁₅H₁₈O₉]⁻), 68 (151.0020 [M-H-C₁₄H₁₆O₇]⁻), 69 (285.0413 [M-H-C₆H₁₁O₅]⁻ and 151.0041 [M-H-C₁₄H₈O₇]⁻), 70 (301.0379 [M-H-C₆H₁₀O₅]⁻ and 151.0039 [M-H-C₆H₁₀O₅-C₇H₆O₂-CO]⁻), 72 (301.0372 [M-H-C₆H₁₀O₅]⁻, 255.0293 [M-H-C₆H₁₀O₅-C₂H₂O]⁻ and 151.0016 [M-H-C₁₄H₁₁O₉]⁻), 73 (245.0504 [M-H-H₂O-2CO]⁻ and 151.0035 [M-H-C₈H₆O₄]⁻), 75 (315.0530 [M-H-C₆H₁₀O₅]⁻, 271.0255 [M-H-C₈H₁₄O₆]⁻ and 151.0034 [M-H-C₁₅H₁₈O₈]⁻), 83 (151.0034 [M-H-C₈H₆O₃]⁻), 85 (316.0231 [M-H-CH₃]⁻ and 151.0016 [M-H-C₉H₈O₄]⁻), 86 (151.0034 [M-H-C₈H₈O]⁻), 88 (151.0034 [M-H-C₈H₆O₂]⁻) and 91 (300.0270 [M-H-CH₃]⁻ and 151.0016 [M-H-C₉H₈O₃]⁻) were identified as the type of ion of 151.0037 (C₇H₄O₄), which were the characteristic fragment of flavonoid components. Indeed, they were epicatechin gallate [23], epicatechin [23], myricetin-3-O-galactoside [23], quercitrin [28], quercetin-3-O-glucuronide [28], laricitrin-3-O-glucoside [37], luteolin-3’-glucoside [23], astilbin [38], isoquercitrin [22], astragalin [38], myricetin [21], isorhamnetin-3-O-glucoside [28], quercetin [22], laricitrin [37], naringenin [23], kaempferol [22] and isorhamnetin [28]. Peak 45 had the mass spectrum ion at 197.0454 (C₉H₁₀O₅) with MS/MS ions at 182.0256 [M-H-CH₃]⁻, 167.0014 [M-H-CH₃-CO₂]⁻, 138.0317 [M-H-CH₃-CO₂-CH₃]⁻ and 123.0090 [M-H-CH₃-2CO₂-CH₃]⁻, which can be identified as syringic acid [27]. Peak 47 produced [M−H]⁻ at m/z 325.0926 with the molecular formula C₁₅H₁₈O₈. A loss of C₆H₁₀O₅ in MS/MS was confirmed to be a hexose neutral loss. The other ions were 145.0294 [M-H-C₆H₁₀O₅-H₂O]⁻ and 117.0343 [M-H-C₆H₁₀O₅-CO]⁻. Peak 47 was tentatively identified as p-coumaric acid glucoside [28]. Peak 48 had the major ions at m/z 189.0769 (C₈H₁₄O₅) and the MS/MS fragment was 71.0503 [M-H-C₂H₅-CO₂-C₂H₅O]⁻. Therefore, it was identified as diethyl malate [24]. Peak 53 was identified as ethyl gallate by its characteristic fragmentation pattern, which MS/MS spectra ions m/z 169.0142 [C₉H₁₀O₅-C₂H₄]⁻ and 125.0239 [C₉H₁₀O₅-C₂H₄-CO₂]⁻ were observed [28]. Peak 54 had the MS ion of 493.1341 and the MS/MS spectra ions of m/z 331.0822 [M-H-C₆H₁₀O₅]⁻ and 316.0553 [M-H-C₆H₁₀O₅-CH₃]⁻. Hence, peak 54 was tentatively identified as malvidin-3-O-glucoside [41]. By the similar regular pattern, peaks 62 (303.0510 [M-H-C₆H₁₀O₅]⁻ and 316.0553 [M-H-C₆H₁₀O₅-2H₂O-CO]⁻), 65 (303.0510 [M-H-2C₆H₁₀O₅]⁻), 74 (317.0648 [M-H-C₆H₁₀O₅]⁻ and 302.0429 [M-H-C₆H₁₀O₅-CH₃]⁻), 78 (303.0767 [M-H-C₆H₁₀O₅-CO₂]⁻) and 80 (477.1196 [M-H-C₉H₁₀O₃]⁻ and 331.0874 [M-H-C₉H₁₀O₃-C₆H₁₀O₅]⁻) were displayed in MS/MS spectra, which could be identified as delphinidin-3-O-glucoside [42,43], delphinidin-3,5-O-diglucoside [44], petunidin-3-O-glucoside [41], delphindin-3-O-(6-O-acetylglucoside) [44] and malvidin-3-O-(6-O-coumaroylglucoside) [44]. Peak 59 gave the [M−H]⁻ ion at the m/z 389.1242 with MS/MS spectra ions m/z 227.0994 [M-H-C₆H₁₀O₅]⁻ and 143.0505 [M-H-C₆H₁₀O₅-C₂H₂O-C₂H₂O]⁻. Peak 59 was tentatively identified as trans-piceid [23]. By the similar regular pattern, peaks 72 (227.0994 [M-H-C₆H₁₀O₅]⁻ and 185.0606 [M-H-C₆H₁₀O₅-C₂H₂O]⁻), 76 (185.0587 [M-H-C₆H₁₀O₅-C₂H₂O]⁻ and 143.0501 [M-H-C₆H₁₀O₅-C₂H₂O-C₂H₂O]⁻) and 84 (143.0504 [M-H-C₆H₁₀O₅-C₂H₂O-C₂H₂O]⁻) were displayed in MS/MS spectra, which can be identified as cis-piceid [23], trans-resveratrol [23] and resveratrol [23] with their different retention times. Peak 63 showed the mass spectrum ion at 463.0884 (C₂₁H₂₀O₁₂). The product fragment ions were 301.0379 [M-H-C₆H₁₀O₅]⁻, 300.0277 [M-H-C₆H₁₁O₅]⁻, 271.0245 [M-H-C₆H₁₀O₅-CHO]⁻, 243.0291[M-H-C₆H₁₀O₅-CHO₂]⁻ and 151.0031 [M-H-C₆H₁₀O₅-C₇H₆O₂-CO]⁻. Therefore, it was identified as hyperoside [23]. Peak 77 gave a mass spectrum ion at 435.1300 (C₂₁H₂₄O₁₀) with MS/MS fragments of 273.0769 [M-H-C₆H₁₂O₆]⁻, 179.0349 [M-H-C₁₅H₁₄O₅]⁻ and 167.0352 [M-H-C₆H₁₂O₆-C₇H₆O]⁻, which was detected to be phloridzin [45]. Peak 81 had a molecular weight of 303.0497 (C₁₅H₁₀O₇) with fragments of 285.0398 [M+H-H₂O]⁺, 257.0456 [M+H-H₂O-CO]⁺, 229.0492 [M+H-H₂O-2CO]⁺ and 153.0188 [M+H-C₈H₅O₃]⁺, therefore it was identified as morin [46]. Peak 82 had the mass spectrum ion at 303.0497 and MS/MS experiment gave fragmentations information at 229.0492 [M-H-CO-2H₂O]⁻ and 153.0188 [M-H-C₈H₆O₃]⁻,which can be identified as delphinidin [28]. By the similar regular pattern, peaks 87 (213.0559 [M-H-2H₂O-CO] and 137.0233 [M-H-C₈H₆O₃]) and 90 (229.0498 [M-H-2H₂O-CO-CH₃] and 153.0188 [M-H-CH₃-C₈H₆O₃]) were showed in MS/MS spectra, which can be identified as cyanidin [28] and petunidin [28]. Peak 89 was identified as syringetin with MS/MS spectra ions m/z 330.0385 [M-H-CH₃]⁻ and 315.0153 [M-H-C₂H₆]⁻ [47]. Peak 92, the molecular weight was 193.1588 (C₁₃H₂₀O) and the fragment ion was observed at m/z 56.9419 [M+H-C₁₀H₁₅]⁺, which was identified as ionone [48]. Peak 93 had a molecular weight of 173.1536 (C₁₀H₂₀O₂) with fragment of 76.0221 [M+H-HCOOH]⁺, therefore it was identified as ethyl caprylate (C₁₀H₂₀O₂). The molecular weight of peak 94 was 195.1748. MS/MS ions of m/z 177.1634 [M+H-H₂O]⁺ and 163.0394 [M+H-H₂O-CH₂]⁺ were observed, which revealed that it was theaspirane. Moreover, the MS spectrums of all compounds were shown in Supplementary Materials (Figures S1–S94).

2.3. 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. The corresponding relationship between fraction number and peak number of the 77 potential active components.

Fraction Number	Peak Number	Identification	Antioxidant Activity	Thrombin Inhibitory Activity	Lipase Inhibitory Activity
F1	5, 6, 10, 12	citric acid, tartaric acid, L-malic acid, shikimic acid	yes	yes	yes
F2	12	shikimic acid		yes	yes
F3	15, 16	succinic acid, trans-4-hydroxycinnamic acid	yes	yes
F4	17	tyramine	yes	yes
F5	18	gallic acid	yes	yes
F6	18	gallic acid	yes	yes
F9	19	coumalic acid	yes	yes
F10	21	protocatechuic acid	yes
F11	21	protocatechuic acid			yes
F13	22	(+)-gallocatechin	yes
F14	23, 24	acetylsalicylic acid, caftaric acid		yes
F16	25, 26, 27, 28	gentisic acid, protocatechualdehyde, homovanillic acid, methyl gallate	yes		yes
F17	29	4-hydroxybenzoic acid	yes		yes
F18	30	chlorogenic acid	yes
F19	31	p-coutaric acid	yes		yes
F20	32, 33, 34	m-coumaric acid, coutaric acid, dimethyl succinate			yes
F21	35	benzoic acid			yes
F22	36, 37, 38	procyanidin B1, (−)-epigallocatechin, procyanidin B3	yes	yes	yes
F23	39, 40, 41, 42, 43	vanillic acid, 2-Hydroxycinnamic acid, catechin, fertaric acid, caffeic acid	yes		yes
F24	44	epicatechin gallate	yes		yes
F25	45	syringic acid	yes	yes	yes
F26	46, 47, 48	procyanidin B2, p-coumaric acid glucoside, diethyl malate	yes	yes	yes
F27	49, 50, 51	desaminotyrosine, 2-hydroxybenzoic acid, epicatechin	yes	yes	yes
F28	53, 54	ethyl gallate, malvidin-3-O-glucoside	yes	yes	yes
F29	55	p-coumaric acid	yes	yes	yes
F30	55	p-coumaric acid	yes	yes	yes
F31	56	homogentisic acid	yes	yes
F32	57	procyanidin B7	yes
F36	58	myricetin-3-O-galactoside	yes	yes
F37	59	trans-piceid	yes
F39	60, 61	ellagic acid, dihydrocaffeic acid	yes
F40	62, 63	delphinidin-3-O-glucoside, hyperoside	yes
F42	65, 66	delphinidin-3,5-O-diglucoside, quercetin-3-O-glucuronide	yes		yes
F43	67, 68, 69, 70	laricitrin-3-O-glucoside, luteolin -3-O-glucoside, astilbin, isoquercitrin	yes		yes
F44	70	isoquercitrin	yes		yes
F45	71	astragalin		yes
F46	72	cis-piceid	yes
F48	73, 74, 75	myricetin, petunidin-3-O-glucoside, isorhamnetin-3-O-glucoside			yes
F49	76, 77	trans-resveratrol, phloridzin			yes
F51	778	delphindin-3-O-(6-O-acetylglucoside)		yes
F54	79, 80	ethyl caffeate, malvidin-3-O-(6-O-coumaroylglucoside)	yes
F55	81, 82, 83, 84	morin, delphinidin, quercetin, resveratrol	yes		yes
F56	85	laricitrin	yes	yes	yes
F57	85	laricitrin	yes	yes	yes
F58	86	naringenin		yes	yes
F59	87	cyanidin	yes		yes
F60	88, 89, 90	kamepferol, syringetin, petunidin	yes		yes

. 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.

2.4. 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.

2.5. 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. Precision, stability, linearity, LODs and LOQs of 12 compounds from red wine.

Analytes	Precision				Stability	Linearity			LOQ (μg/mL)	LOD (μg/mL)
	Intra-Day, RSD (%), (n = 3)			Inter-Day RSD (%) (n = 9)	RSD (%) (n = 6)	Range (μg/mL)	Equation	r2
	Low	Middle	High
gallic acid	2.2	2.2	1.3	3.4	2.5	20.00~320.0	y = 0.3627x + 0.0831	0.9998	0.12	0.040
coumalic acid	3.2	1.5	1.9	4.4	1.8	6.030~96.48	y = 0.2339x − 0.0078	0.9994	2.0	0.75
protocatechuic acid	4.9	2.2	1.7	4.2	1.1	1.005~16.08	y = 0.1498x + 0.0290	0.9993	0.12	0.040
proanthocyanidin B1	3.6	2.1	1.8	4.6	2.2	6.000~96.0	y = 0.1156x + 0.0121	0.9996	0.50	0.12
catechin	5.0	1.3	2.0	3.3	2.5	6.030~96.5	y = 0.2149x + 0.0288	0.9997	0.12	0.040
caffeic acid	2.5	2.3	1.3	4.7	2.2	3.000~48.00	y = 0.2645x + 0.0428	0.9992	0.12	0.040
syringic acid	4.8	2.4	2.1	4.8	1.8	2.973~47.56	y = 0.5234x + 0.0300	0.9997	0.25	0.08
epicatechin	4.2	2.0	1.2	4.4	2.9	3.030~48.48	y = 0.3627x + 0.0831	0.9998	0.50	0.12
p-coumaric acid	4.4	1.7	1.4	4.1	2.1	3.015~48.24	y = 0.2339x + 0.0078	0.9994	0.25	0.08
isoquercitrin	4.6	1.9	1.3	4.6	1.8	0.2500~64.64	y = 0.1495x + 0.0431	0.9994	0.25	0.08
isorhamnetin-3-O-glucoside	5.0	2.1	1.7	3.3	2.3	0.990~15.84	y = 0.1156x + 0.0121	0.9996	0.50	0.12
quercetin	4.3	2.6	2.2	4.6	2.5	0.7500~31.46	y = 0.2166x − 0.0094	0.9995	0.75	0.25

). As can be seen from

Table 5. The recovery of 12 compounds from red wine (n = 3).

Analytes	Original (μg)	Spiked (μg)	Found (μg)	Recovery (%)	RSD (%)
gallic acid	29.34	20.00	45.81	99.8	1.8
		40.00	68.85	97.0	1.4
		60.00	89.14	99.8	1.0
coumalic acid	9.724	6.030	14.65	87.8	4.1
		12.06	21.19	95.1	2.2
		18.09	26.77	86.7	3.1
protocatechuic acid	3.298	1.005	3.720	83.4	4.3
		2.010	4.864	90.6	2.5
		3.015	6.338	98.4	1.4
proanthocyanidin B1	9.027	6.000	14.01	89.7	4.0
		12.00	21.58	103.6	2.3
		18.00	26.27	94.1	2.1
catechin	11.43	3.000	14.15	97.7	3.6
		6.000	17.50	100.1	0.7
		9.000	20.61	100.0	0.3
caffeic acid	6.539	2.972	9.33	96.3	3.4
		5.945	13.14	105.0	2.1
		8.918	15.26	98.9	1.0
syringic acid	6.204	2.973	8.97	97.7	3.4
		5.945	12.08	97.5	3.2
		8.918	14.95	101.7	1.8
epicatechin	3.922	3030	6.932	101.2	1.6
		6.060	10.04	101.9	0.7
		9.090	13.18	105.9	1.4
p-coumaric acid	4.473	3.015	7.590	105.6	3.6
		6.030	10.15	92.5	1.1
		9.045	13.78	100.6	0.6
isoquercitrin	8.802	4.040	12.75	98.1	3.2
		8.080	16.75	98.8	2.1
		12.12	20.64	98.8	1.9
isorhamnetin-3-O-glucoside	3.144	0.9900	4.053	98.1	1.1
		1.980	5.123	97.4	2.6
		2.970	6.019	102.1	2.4
quercetin	3.146	1.966	4.928	95.5	1.6
		3.933	6.950	96.7	1.3
		5.899	8.82	92.2	4.1

, 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.

2.6. Contents of the Bioactive Components from Red Wine

UHPLC was applied to analyze the bioactive components in triplicates from red wine. The results showed that among the bioactive compounds, gallic acid was in the highest amounts of 46.94 μg/mL. The contents of coumalic acid (18.93 μg/mL), proanthocyanidin B1 (14.19 μg/mL), catechin (19.37 μg/mL), caffeic acid (12.05 μg/mL) and isoquercitrin (10.21 μg/mL) were in the range of 10.21–19.37 μg/mL. Protocatechin (4.768 μg/mL), syringic acid (9.87 μg/mL), epicatechin (7.148 μg/mL), p-coumaric acid (8.18 μg/mL), isorhamnetin-3-O-glucoside (6.460 μg/mL) and quercetin (5.353 μg/mL) were in low contents. Contents of 35 batches red wine were shown in (Figure 5). These components may be considered to be the main bioactive compounds from red wine and had the antioxidant activity, inhibitory activities of and lipase.

2.7. 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.

2.8. 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.

3. Materials and Methods

3.1. Reagents and Chemical

HPLC-grade Acetonitrile (ACN) and methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Trifluoroacetic acid (TFA) was obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China). Reference Standards including gallic acid (98.1%), catechin (99.2%), caffeic acid (99.7%), epicatechin (99.7%), isoquercitrin (98.0%) and quercetin (98.0%) were provided from National Institutes for Food and Drug Control (Beijing, China). Coumalic acid (98.0%), proanthocyanidin B1 (98.0%) and syringic acid (98.0%) were purchased from Chengdu Chroma-Biotechnology Co., Ltd. (Sichuan, China). P-coumaric acid (98.0%) and isorhamnetin-3-O-glucoside (98.0%) were offered by Shanghai Tauto Biotech Co., Ltd. (Shanghai, China). The water used in this study was purified by a Milli-Q water purification system (MA, USA). Total Antioxidant Capacity Assay Kit (ABTS) was supplied from Beyotime (Shanghai, China). Thrombin Inhibitor Screening Kit (Fluorometric) was obtained from Biovision (San Francisco, CA, USA). Lipase (TypeII, L3126) was purchased from Solarbio Science Co. Ltd. (Beijing, China) and 4-Methylumbelliferyl Oleate (4-MUO) was provided by Sigma-Aldrich (St. Louis, MO, USA). Red wine (2018 vintage, the central valley of Chile, alcoholic degree 13.0% (v/v)) was made of cabernet sauvignon, which was obtained from Ole’ Boutique Supermarket (Shenzhen, Guangdong, China).

3.2. 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.

3.3. 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 UHPLC 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).

3.4. UHPLC Analysis

The UHPLC analysis was performed using an Dionex ultra high performance liquid chromatograph (Dionex UltiMate 3000, Thermo, MA, USA) equipped with a Dionex UltiMate 3000 pump, a Dionex UltiMate 3000 autosampler and a Dionex UltiMate 3000 diode array detector. The chromatographic separation was achieved on an ACQUITY UPLC column (HSS T3, 1.8 μm, 2.1 mm× 100 mm, Waters, Ireland) [49] at 30 °C. The mobile phase was composed of water containing 0.1% TFA (A) [50] and methanol (B) at a flow rate of 0.3 mL/min. The elution program was conducted as follows: 0–15 min at 0–6% B; 15–20 min at 6–11% B; 20–30 min at 11–11% B; 30–45 min at 11–20% B; 45–55 min at 20–30% B; 55–60 min at 30–90% B. The injection volume was 2 μL. DAD wavelength was set as 220 nm. Method validation was accorded to the Chinese Pharmacopeia guidelines, which were included linearity, limits of detection (LOD), limits of quantification (LOQ), repeatability, precision, stability and recovery.

3.5. 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.

3.6. 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].

3.7. 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 (T₁ = 5 min, T₂ = 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 ΔRFU = 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].

3.8. 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].

3.9. 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.

4. 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.