Compositional characteristics of red clover (<i>Trifolium pratense</i>) seeds and supercritical CO<sub>2</sub> extracted seed oil as potential sources of bioactive compounds

Ying Zhou; Ye Tian; Priscilla Ollennu-Chuasam; Maaria Kortesniemi; Katri Selander; Kalervo Väänänen; Baoru Yang; Ying Zhou; Ye Tian; Priscilla Ollennu-Chuasam; Maaria Kortesniemi; Katri Selander; Kalervo Väänänen; Baoru Yang

doi:10.48130/fia-0024-0002

Composition	Content
Dry matter (%)	88.6 ± 0.1
Proteins (g/100 g DM)	6.9 ± 0.1
Lipids (g/100 g DM)	7.0 ± 1.1
Sugars (mg/100 g DM)	3,702.8 ± 632.7
Arabinose	40.9 ± 2.8
Xylose	13.1 ± 2.1
Xylitol	26.1 ± 4.0
Fructose	534.1 ± 96.3
Glucose	114.9 ± 19.3
Mannose	11.4 ± 1.5
Mannitol	29.4 ± 4.2
Inositol	38.6 ± 5.5
Sucrose	2,856.2 ±496.5
Maltose	31.4 ± 4.3
Galacturonic acid	6.7 ± 0.4
Arabinose to xylose ratio	3.2 ± 0.6
Organic acids (mg/100 g DM)	50.0 ± 4.5
Malic acid	14.0 ± 2.7
Citric acid	36.1 ± 1.9

Table 1.

Content of proteins, lipids, sugars, and organic acids in RCS.

No.	Identification	Content (mg/100 g DM)	UV λ_max (nm)	[M+Na]⁺/[M+H]⁺/[M-H]⁻ (m/z)	MS² (m/z)
Total phenolic compounds		228.4 ± 51.0
Flavanonols		102.2 ± 22.0
1	Taxifolin hexoside	60.0 ± 13.3	288	489.1321/467.1505/465.1069	467.1505 → 305.0989, 260.0501, 139.0380 465.1069 → 303.0518, 285.0411
2	Taxifolin	42.2 ± 8.7	289	327.0443/305.0623/303.0533	305.0623 → 287.0522, 259.0577, 231.0629, 195.0270, 153.0171, 149.0221 303.0533 → 285.0416, 275.0568, 259.0618, 241.0515, 217.0515, 178.9993, 153.0199, 125.0257
Flavanols		101.6 ± 24.2
4	Quercetin hexoside 1	2.7 ± 0.6	255, 371	487.0793/465.0975/463.0895	465.0975→ 303.0467 463.0895→ 301.0365, 151.0048
5	Quercetin 3-O-galactoside	1.1 ± 0.2	255, 353	−/465.0978/463.0893	465.0978 → 303.0465 463.0893 → 300.0287, 271.0258, 255.0309, 243.0309
6	Quercetin 3-O-glucoside	11.7 ± 3.0	255, 353	487.0801/465.0974/463.0903	465.0974 → 303.0469 463.0903 → 300.0286, 271.0257, 255.0307, 243.0309
7	Quercetin hexoside 2	19.2 ± 4.5	252, 365	487.0799/465.0977/463.0887	465.0977 → 303.0471 463.0887 → 301.0355
9	Quercetin hexoside 3	1.3 ± 0.2	270, 369	487.0792/465.0978/463.0888	465.0978 → 303.0465 463.0888 → 301.0367
10	Quercetin	63.1 ± 15.2	255, 370	325.0286/303.0471/301.0377	303.0471 → 229.0473, 153.0169 301.0377 → 273.0402, 178.9992, 151.0044
12	Kaempferol	1.2 ± 0.2	270, 365	−/287.0523/285.0418	−
13	Isorhamnetin	1.4 ± 0.3	260, 371	−/317.0624/315.0521	−
Isoflavones		1.6 ± 0.4
11	Formononetin malonyl- hexoside	1.6 ± 0.4	265, 310	−/517.1283/515.1208	517.1283 → 269.0786 1,031.2513 ([2M-H]⁻) → 515.1230, 267.0677
Unknown compounds		23.0 ± 4.5
3	Unknown compound 1	5.2 ± 0.9	270	427.1527/405.1711/403.1626	427.1527 → 265.1018, 203.0510 403.1626 → 223.0989, 179.1088
8	Unknown compound 2	17.8 ± 4.2	275, 320	643.2288/−/619.2421	643.2288 → 449.1734 619.2421 → 193.0513, 178.0277, 149.0612
* Quantitative results are shown as means ± standard deviation of triplicate analyses. '−' means the MSMS spectrum was not provided.

Table 2.

Identification of phenolic compounds in the RCS by UPLC-DAD-ESI-QTOF*.

Composition	Content
Lipids (g/100 g DM)	7.0 ± 1.1
Fatty acids (µg/g DM)	46,760.5 ± 2,686.1
Myristic acid (C14:0)	58.3 ± 6.7
Palmitic acid (C16:0)	6,369.6 ± 338.9
Stearic acid (C18:0)	2,993.1 ± 149.8
Arachidic acid (C20:0)	65.7 ± 3.2
Behenic acid (C22:0)	55.7 ± 3.6
Lignoceric acid (C24:0)	48.1 ± 21.6
Total SFA	9,590.5 ± 507.2
Palmitoleic acid (16:1 n-7)	45.8 ± 2.2
Oleic acid (C18:1 n-9)	178.9 ± 4.6
Vaccenic acid (18:1 n-7)	7.6 ± 2.7
Eicosenoic acid (20:1 n-9)	23.3 ± 4.4
Erucic acid (22:1 n-9)	43.0 ± 8.5
Nervonic acid (24:1 n-9)	8.0 ± 4.6
Total MUFA	310.5 ± 10.4
Linoleic acid (18:2 n-6)	33,769.6 ± 1,971.1
α-Linolenic acid (18:3 n-3)	2,863.8 ± 191.1
γ-Linolenic acid (18:3 n-6)	24.2 ± 5.5
Eicosadienoic acid (20:2 n-6)	2.9 ± 0.4
Dihomo-α-linolenic acid (20:3 n-3)	5.7 ± 1.4
Arachidonic acid (20:4 n-6)	5.7 ± 1.5
Eicosapentaenoic acid (20:5 n-3)	187.6 ± 37.0
Total PUFA	36,859.5 ± 2,188.1
Total n-3	3,057.1 ± 219.0
Total n-6	33,802.4 ± 1,969.7
Tocopherols, mg/100 g DM	94.9 ± 4.4
α-Tocopherol	91.9 ± 4.2
β-Tocopherol	1.3 ± 0.1
γ-Tocopherol	1.7 ± 0.1
δ-Tocopherol	−
* Results are shown as means ± standard deviation of triplicate analyses. '−' means the compound was not detected in the sample.

Table 3.

Composition of and content of lipids, fatty acids and tocopherols in RCS*.

Composition	Content (mg/100 g)	% in the fatty acids
Fatty acids	89,482.4 ± 2,515.0
Myristic acid (C14:0)	84.7 ± 1.0	0.09
Palmitic acid (C16:0)	9,445.0 ± 394.8	10.55
Stearic acid (C18:0)	4,955.4 ± 207.3	5.54
Arachidic acid (C20:0)	137.2 ± 13.0	0.15
Behenic acid (C22:0)	27.4 ± 4.7	0.03
Lignoceric acid (C24:0)	63.0 ± 7.6	0.07
Total SFA	14,712.7 ± 599.0	16.44
Palmitoleic acid (16:1 n-7)	81.2 ± 3.8	0.09
Oleic acid (C18:1 n-9)	14,036.8 ± 2,598.0	15.69
Eicosenoic acid (20:1 n-9)	59.6 ± 9.2	0.07
Erucic acid (22:1 n-9)	40.4 ± 4.7	0.05
Nervonic acid (24:1 n-9)	5.0 ± 1.7	0.01
Total MUFA	14,223.0 ± 2,607.4	15.89
Linoleic acid (18:2 n-6)	54,665.6 ± 2,159.7	61.09
α-Linolenic acid (18:3 n-3)	5,432.4 ± 81.8	6.07
γ-Linolenic acid (18:3 n-6)	31.2 ± 3.6	0.03
Eicosadienoic acid (20:2 n-6)	74.2 ± 51.4	0.08
Dihomo-α-linolenic acid (20:3 n-3)	5.4 ± 2.2	0.01
Arachidonic acid (20:4 n-6)	5.4 ± 0.8	0.01
Eicosapentaenoic acid (20:5 n-3)	332.3 ± 5.3	0.37
Total PUFA	60,546.6 ± 2,258.0	67.66
Total n-3	5,770.1 ± 88.3	6.45
Total n-6	54,776.5 ± 2,169.7	61.21
Tocopherols (mg/100 g)	40.0 ± 5.9	−
α-Tocopherol	38.9 ± 5.8	−
β-Tocopherol	0.7 ± 0.1	−
γ-Tocopherol	0.5 ± 0.1	−
δ-Tocopherol	−	−
* Results are shown as means ± standard deviation of triplicate analyses. '−' means the compound was not detected in the sample.

Table 4.

Composition and content of fatty acids and tocopherols in RCS oil*.