Profile of phenolic compounds in Trifolium pratense L. extracts at different growth stages and their biological activities

Figures & data

Table 1. LC-MS/MS data for standard compounds.

	LC-MS/MS conditions for quantification of standard compounds
Compound	Retention time (min)	Precursor ion (m/z)	Product ion (m/z)	Collision energy (V)	Fragmentor voltage (V)
Hinic acid	0.52	191	85	20	150
Gallic acid	0.61	169	125	10	90
Catechin	0.75	289	245	10	150
Protocatechuic acid	0.80	153	109	9	105
Epigallocatechin gallate	0.80	457	169	16	165
Chlorogenic acid	0.84	353	191	10	100
Epicatechin	1.01	289	245	10	150
2,5-Dihydroxybenzoic acid	1.05	153	109	9	100
p-Hydroxybenzoic acid	1.11	137	93	10	80
Aesculetin	1.12	177	133	15	105
Caffeic acid	1.17	179	135	10	100
Vanillic acid	1.26	167	108	15	100
Syringic acid	1.32	197	182	7	90
p-Coumaric acid	1.68	163	119	9	90
Scopoletin	1.73	191	176	8	80
Umbelliferone	1.76	161	133	19	120
Ferulic acid	1.89	193	134	11	90
Sinapic acid	1.89	223	193	17	100
Vitexin	1.91	431	311	22	200
Luteolin 7-O-glucoside	2.15	447	285	30	230
Hyperoside	2.18	463	300	30	200
Rutin	2.21	609	300	42	135
Quercetin 3-O-glucoside	2.25	463	300	30	210
Apiin	2.61	563	269	36	250
o-Coumaric acid	2.63	163	119	5	100
Myricetin	2.70	317	179	20	150
Apigenin 7-O-glucoside	2.71	447	284	30	190
Quercitrin	2.78	447	300	27	190
Kaempferol 3-O-glucoside	2.85	431	268	41	135
Secoisolariciresinol	2.87	361	165	26	130
3,4-Dimethoxycinnamic acid	3.00	207	103	7	110
Baicalin	3.36	445	269	22	140
Daidzein	3.43	253	208	31	145
Matairesinol	3.66	357	122	24	130
Quercetin	3.68	301	151	15	130
Naringenin	3.86	271	151	16	130
Cinnamic acid	3.91	147	103	5	100
Luteolin	3.96	285	133	25	135
Genistein	4.13	269	133	32	145
Kaempferol	4.52	285	285	0	130
Apigenin	4.64	269	117	25	130
Isorhamnetin	4.79	315	300	21	160
Chrysoeriol	4.81	299	284	20	125
Baicalein	5.15	269	269	0	165
Amentoflavone	5.81	537	375	35	220

Table 2. Quantification of selected phenolics in examined T. pratense MeOH extracts.

	Content of selected phenolics in T. pratense extract (μg g^−1 of dw)
Compound	No 1	No 2	No 3
Phenolic acids
Hinic acid	nd^b	Nd	nd
Galic acid	nd	Nd	nd
Protocatechuic acid	0.197 ± 0.004^a	0.079 ± 0.003^b	nd
Chlorogenic acid	nd	Nd	nd
2,5-Dihydroxybenzoic acid	0.088 ± 0.001^a	0.087 ± 0.001^a	nd
p-Hydroxybenzoic acid	0.713 ± 0.034^a	0.180 ± 0.021^b	0.042 ± 0.004^c
Caffeic acid	1.041 ± 0.052^a	^b0.922 ± 0.043	0.086 ± 0.011^c
Vanillic acid	nd	Nd	nd
Syringic acid	nd	Nd	nd
p-Coumaric acid	0.246 ± 0.027^a	0.080 ± 0.013^b	nd
Ferulic acid	nd	Nd	nd
Sinapic acid	nd	Nd	nd
o-Coumaric acid	nd	Nd	nd
3,4-Dimethoxycinnamic acid	nd	Nd	nd
Cinnamic acid	nd	Nd	nd
Flavonoids
Apigenin	0.320 ± 0.037^a	0.144 ± 0.041^b	0.012 ± 0.001^c
Apigenin 7-O-glucoside	0.288 ± 0.052^a	0.034 ± 0.001^b	0.014 ± 0.002^c
Apigein	nd	Nd	nd
Vitexin	0.246 ± 0.001^a	0.080 ± 0.001^b	Nd
Amentoflavone	0.345 ± 0.001^a	0.018 ± 0.002 ^b	Nd
Baicalein	nd	Nd	Nd
Baicalin	nd	Nd	Nd
Daidzein	1.527 ± 0.077^a	0.878 ± 0.064^b	0.121 ± 0.031^c
Genistein	0.588 ± 0.056^a	0.561 ± 0.038^a	0.031 ± 0.004
Isorhamnetin	nd	Nd	Nd
Kaempferol	0.062 ± 0.001	0.015 ± 0.001	Nd
Kaempferol 3-O-glucoside	8.544 ± 0.095	1.344 ± 0.081	0.277 ± 0.049
Chrysoeriol	nd	Nd	Nd
Luteolin	0.229 ± 0.032^a	0.028 ± 0.003^b	0.011 ± 0.001^c
Luteolin 7-O-glucoside	nd	0.032 ± 0.004	Nd
Quercetin	0.128 ± 0.042^a	0.018 ± 0.002^b	0.017 ± 0.002^b
Quercetin 3-O-glucoside	24.243 ± 0.371^a	3.137 ± 0.109^b	0.817 ± 0.016^c
Quercitrin	nd	Nd	Nd
Hyperoside	45.259 ± 0.628^a	5.624 ± 0.212^b	1.540 ± 0.059^c
Rutin	0.157 ± 0.027^a	0.020 ± 0.001^b	Nd
Myricetin	nd	Nd	Nd
Naringenin	0.062 ± 0.005	0.014 ± 0.002	Nd
Epicatechin	nd	Nd	Nd
Catechin	nd	Nd	Nd
Epigallocatechin gallate	nd	Nd	Nd
Coumarins
Umbelliferon	nd	Nd	Nd
Aesculetin	0.606 ± 0.033^a	0.293 ± 0.028^b	0.076 ± 0.017^c
Scopoletin	nd	Nd	Nd
Lignans
Matairesinol	nd	Nd	Nd
Secoisolariciresinol	nd	Nd	Nd

^bNd – not detected Data are means ±SD of three measurements. In each different letters mean significant differences (p<0.05)

Table 3. Total phenolics and flavonoid content, antioxidant potential of extracts of T. pratense extracts and standard compounds.

		T. pretense			Standards
		No1	No3	No2	BHA	BHT
Content	Total phenolics (mg gallic acid equivalents g^−1 of dw)	41.96 ± 0.93^§a	36.00 ± 0.55^b	30.99 ± 1.02^c	-	-
	Total flavonoids (mg quercetin equivalents g^−1 of dw)	7.32 ± 0.59^a	5.78 ± 0.30^b	3.87 ± 0.23^c	-	-
Antioxidant activity	IC50 values (μg/mL for extracts)
	DPPH^•	22.98 ± 0.14^c	35.09 ± 0.51^d	44.85 ± 1.32^e	11.07 ± 0.07^a	14.32 ± 024^b
	HO^•	188.42 ± 1.65^c	204.30 ± 1.84^d	213.06 ± 0.28^e	119.88 ± 0.2^b	108.73 ± 1.3^a
	O2^•-	25.73 ± 0.36^c	27.40 ± 6.25^c	30.77 ± 1.20^d	8.53 ± 0.49^a	10.12 ± 0.34^b
	NO^•	11.23 ± 0.45^c	33.34 ± 0.63^d	42.89 ± 1.05^e	62.33 ± 0.10^a	87.90 ± 0.18^b
	H2O2	32.43 ± 0.39^c	44.10 ± 1.61^d	55.08 ± 1.05^e	17.20 ± 0.60^a	20.67 ± 0.63^b
	LP	53.07 ± 1.72^c	68.30 ± 1.12^d	74.90 ± 1.86^e	34.76 ± 1.14^b	26.08 ± 0.98^a
	Reducing power (mg of ascorbic acid equivalents (AAE)/g of dw)
	FRAP	428 ± 2.60^c	411.40 ± 0.98^d	376.90 ± 2.35^e	587.70 ± 1.80 ^a	54.20 ± 1.79^b

^§Values are means ± SD of three measurements; a,b,c,d,e means in the same row not sharing the same superscript are significantly different (p<0.01).

Table 4. Evaluated antibacterial activity of T. pratense extracts.

	Disc diffusion assay (Inhibition zone measured in mm)
	Test microorganisms
Tested sample	Pseudomonas aeruginosa	Escherichia coli	Enterobacter aerogenes	Enterococcus faecalis	Staphylococcus aureus	Bacillus cereus
T. pratense No1	abs*	13.4 ± 0.25^§b	12.5 ± 0.68^b	8.6 ± 1.38^b	7.4 ± 0.64^b	9.30 ± 0.74^b
T. pratense No2	abs	5 ± 0.09^c	3.9 ± 1.04^c	2.3 ± 1.74^c	1.2 ± 0.46^c	2.59 ± 0.77^c
Streptomycin	13.2 ± 0.11	29 ± 0.55^a	18 ± 0.55^a	14 ± 0.00^a	31.2 ± 0.44^a	19.30 ± 2.20^a
Agar dilution assay (MIC; µg ml^−1)
T. pratense No1	nd^¥	25	25^b	50	50^b	100
T. pratense No2	Nd	nd	50^c	nd	100^c	nd
Streptomycin	16	4	8^a	16	2^a	8

^§Values are means ± SD of three measurements; ^a,b,c means in the same column not sharing the same superscript are significantly different (p<0.01)

*abs – absence of inhibition zone; ^¥nt – not determined.