Free radical scavenging actions of three Trifolium species in the protection of blood plasma antioxidant capacity in vitro

Figures & data

Table 1. Phytochemical composition of the examined plant extracts, derived from aerial parts of three Trifolium species (Kolodziejczyk-Czepas et al., 2013a,b; a compilation of data).

	Phenolic acids (equivalent of chlorogenic acid)	Isoflavones (equivalent of daidzin)	Other flavonoids (equivalent of quercetin glucoside)	Clovamides (equivalent of chlorogenic acid)	The total content of phenolics
Trifolium species	Concentration (mg/g of dry mass) ± SD
T. pratense crude extract	5.92 ± 0.14	10.50 ± 0.33	3.89 ± 0.18	–	20.31
T. pratense phenolic fraction	5.92 ± 0.10	5.01 ± 0.15	3.89 ± 0.20	–	14.82
T. pallidum phenolic fraction	14.20 ± 0.55	7.31 ± 0.52	0.61 ± 0.00	12.94 ± 0.15	35.06
T. scabrum phenolic fraction	0.66 ± 0.00	72.76 ± 0.43	7.67 ± 0.17	–	81.09

Table 2. The free radical scavenging activities of the examined Trifolium extracts. The results are expressed as percent of radical scavenging ability (% RSA). The table includes mean values ± SD of measurements done in triplicate.

	ABTS^• radical scavenging activity (%)				DPPH^• radical scavenging activity (%)
Concentration of the extract (µg/ml)	T. pratense (crude extract)	T. pratense (phenolic fraction)	T. pallidum (phenolic fraction)	T. scabrum (phenolic fraction)	T. pratense (crude extract)	T. pratense (phenolic fraction)	T. pallidum (phenolic fraction)	T. scabrum (phenolic fraction)
1.5	10.595 ± 2.178	18.870 ± 9.042	16.375 ± 1.967	11.981 ± 7.692	2.43 ± 2.13	11.74 ± 0.96	4.79 ± 1.81	5.88 ± 1.37
3	13.387 ± 1.467	26.224 ± 7.129	23.938 ± 0.583	10.353 ± 0.087	5.42 ± 1.69	22.57 ± 1.23	8.66 ± 1.35	5.42 ± 1.75
6.25	18.209 ± 0.686	37.341 ± 3.920	28.575 ± 1.292	12.480 ± 0.055	9.51 ± 2.02	39.74 ± 1.95	15.20 ± 1.49	7.35 ± 1.71
12.5	24.447 ± 0.401	61.389 ± 3.455	33.658 ± 0.891	17.336 ± 1.075	13.87 ± 1.33	65.75 ± 1.43	25.15 ± 1.69	12.17 ± 3.69
25	28.689 ± 0.329	83.907 ± 2.145	49.265 ± 0.853	26.028 ± 4.356	23.54 ± 2.00	89.36 ± 0.60	45.24 ± 1.47	16.64 ± 2.24
50	41.060 ± 1.281	89.490 ± 4.429	73.375 ± 0.450	40.223 ± 4.483	42.22 ± 1.05	91.82 ± 0.14	79.37 ± 3.60	25.59 ± 1.41

Table 3. Comparison of the ferric reducing ability of the examined Trifolium extracts (expressed as mM of Fe²⁺). The table includes mean values ±SD of measurements done in triplicate.

	The ferric reducing ability (mM of Fe^2+)
Concentration of the extract (µg/ml)	T. pratense (crude extract)	T. pratense (phenolic fraction)	T. pallidum (phenolic fraction)	T. scabrum (phenolic fraction)
1.5	0.012 ± 0.004	0.106 ± 0.017	0.027 ± 0.003	0.005 ± 0.003
3	0.032 ± 0.007	0.171 ± 0.018	0.058 ± 0.000	0.014 ± 0.000
6.25	0.061 ± 0.009	0.346 ± 0.022	0.113 ± 0.002	0.032 ± 0.006
12.5	0.129 ± 0.009	0.671 ± 0.011	0.229 ± 0.008	0.062 ± 0.003
25	0.240 ± 0.024	1.266 ± 0.031	0.417 ± 0.026	0.111 ± 0.010
50	0.421 ± 0.041	2.606 ± 0.355	0.809 ± 0.018	0.197 ± 0.036

Table 4. The comparison of the free radical scavenging effects of 10 Trifolium plant-derived extracts. The table comprises a compilation of EC₅₀ values for either ABTS^• or DPPH^• scavenging measured during the present study (results for T. pratense, T. pallidum, and T. scabrum) and data from our previous work.

The Trifolium extract	EC50 (μg/ml) for ABTS^• scavenging	EC50 (μg/ml) for DPPH^• scavenging
T. pratense (crude extract)	51.32	56.43
T. pratense (phenolic fraction)	21.69	12.27
T. pallidum	29.77	30.06
T. scabrum	55.42	86.82
T. alexandrinum	5.53^a	2.02^a
T. fragiferum	54.65^a	37.93^a
T. hybridum	42.38^a	47.66^a
T. incarnatum	48.64^a	28.76^a
T. resupinatum var. majus	22.94^a	15.70^a
T. resupinatum var. resupinatum	20.27^a	14.39^a

^aEC₅₀ values calculated for measurements of free radical scavenging properties of T. alexandrinum, T. fragiferum, T. hybridum, T. incarnatum, T. resupinatum var. majus, T. resupinatum var. resupinatum; Kolodziejczyk-Czepas et al. (2014).

Figure 1. Comparison of the protective effects of Trifolium pratense extracts on the total antioxidant capacity of blood plasma under ONOO⁻-induced oxidative stress conditions. The TAC of human blood plasma was determined spectrophotometrically by the ABTS^• (Panel A) and DPPH^• (Panel B) radicals decolourization, as well as by measurements of the ferric reducing ability (the FRAP assay) (Panel C). Results are presented as means ± SD of six independent experiments (six different donors); #p < 0.0 and ##p < 0.01 for ONOO⁻-treated plasma (with or without extracts) versus control plasma; *p < 0.05 and **p < 0.01 for plasma treated with ONOO⁻ in the presence of extracts versus plasma treated with ONOO⁻ in the absence of extracts.

Figure 2. Evaluation of the protective effects of Trifolium pallidum and Trifolium scabrum extracts on total antioxidant capacity of blood plasma under ONOO⁻-induced oxidative stress conditions. The total antioxidant capacity was estimated by the reduction of ABTS^• (Panel A) and DPPH^• (Panel B) radicals and by the measurements of the ferric reducing ability of plasma (Panel C). Results are presented as means ± SD of six independent experiments (six different donors); #p < 0.05 for ONOO⁻-treated plasma (with or without extracts) versus control plasma, and *p < 0.05 and **p < 0.01 for plasma treated with ONOO⁻ in the presence of extracts versus plasma treated with ONOO⁻ in the absence of extracts.

Kolodziejczyk-Czepas J, Olas B, Malinowska J, et al. (2013a). Trifolium pallidum and Trifolium scabrum extracts in the protection of human plasma components. J Thromb Thrombol 35:193–9

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