2.1. Determination of Total Phenolic and Flavonoid Content
Numerous investigations of qualitative composition of plant extracts have revealed the presence of high concentration of phenols in the extracts obtained using polar solvents [
21]. The extracts that display the highest antioxidant activity have the highest concentration of phenols. Phenols are very important plant constituents because they are acting as scavengers of intermediate peroxyl and alkoxyl radicals, myocardial infarction, arteriosclerosis, processes of aging, and cancer, and may prevent them, and chelating agents for metal ions which are of major importance for the initiation stage of radical reactions [
22]. Furthermore, phenols have therapeutic properties on different diseases like: Alzheimer’s and Parkinson’s disease, ischemic damage, arthritisdue to their antiradical property towards ROS. One of the very productive sources of polyphenols is food and food supplements of plant origin [
23]. In addition, successive extractions of
T. pratense were carried out, and for further work six different concentrations of extracts are prepared. Successive extraction was performed as the extraction of antioxidant substances of different chemical structure, was achieved using solvents of different polarity. Results of the amount of total phenolic contents and content of total flavonoids in
T. pratense extracts are given in
Table 1.
Table 1.
The amount of total phenolic contents andcontent of total flavonoids in T. pratense extracts.
Table 1.
The amount of total phenolic contents andcontent of total flavonoids in T. pratense extracts.
Extract | Et2O | CHCl3 | EtOAc | n-BuOH | H2O |
---|
Total phenolic content | 0.22 ± 0.03 | 0.16 ± 0.02 | 0.43 ± 0.01 | 0.21 ± 0.03 | 0.34 ± 0.03 |
Total flavonoids | 11.78 ± 0.04 | 9.24 ± 0.03 | 15.23 ± 0.01 | 11.87 ± 0.03 | 15.13 ± 0.05 |
The amount of total phenolics in T. pratense extracts ranged from 0.16 ± 0.02 mg GAE/g d.e. (CHCl3 extract) to 0.43 ± 0.01 mg GAE/g d.e. (EtOAc extract). A significant amount of these compounds has also been observed in the H2O extract (0.34 ± 0.03 mg GAE/g d.e.). Furthermore, a considerable total flavonoids content was determined in the H2O and EtOAc extracts. A little less amount of total flavonoids was determined in the n-BuOH extracts, while the smallest quantity of these compounds was found in the Et2O and CHCl3 extracts. HPLC-DAD analysis indicates a significant presence of flavonoids and phenolic in the EtOAc and H2O extracts. Quercetin glycosides and flavonoids (kaempferol-3-O-Glc, quercetin-3-O-Glc, luteolin-7-O-Glc and apigenin-7-O-Glc) were detected in EtOAc extract, while the presence of phenolic acids was proven in the H2O extract (such as caffeic acid) and flavonoids (luteolin, apigenin, naringenin and kaempferol. In addition, two phytoestrogens (daizdein and genistein) were detected in the H2O extract.
2.2. In Vitro Experiments
The antioxidant activity of
T. pratense extracts has been evaluated in a series of
in vitro tests (
Table 2). The 1,1-diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH) molecule is characterised as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so the molecules do not dimerise, as would be the case with most other free radicals. The delocalisation also gives rise to the deep violet colour, characterised by an absorption band in methanol solution centered at about 520 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present). In the DPPH assay, the ability of the investigated extracts to act as donors of hydrogen atoms or electrons in transformation of DPPH into its reduced form DPPH-H was investigated.
Table 2.
IC50 values (μg/mL) of extracts of T. pratense and standards (BHT and BHA) for different antioxidant assays.
Table 2.
IC50 values (μg/mL) of extracts of T. pratense and standards (BHT and BHA) for different antioxidant assays.
Extract | Et2O | CHCl3 | EtOAc | n-BuOH | H2O | BHT | BHA |
---|
DPPH radical | 20.36 | 34.19 | 17.81 | 29.47 | 17.47 | 14.31 | 11.08 |
O2•− radical | 55.80 | 92.37 | 20.91 | 28.48 | 10.77 | 10.46 | 8.41 |
NO radical | 26.66 | 58.46 | 15.67 | 30.64 | 13.33 | 8.63 | 6.31 |
OH radical | 41.66 | 69.30 | 19.79 | 39.21 | 18.44 | 24.12 | 22.17 |
All of the assessed extracts of
T. pratense were able to reduce the stable, purple-colored radical DPPH to the yellow-colored DPPH-H form with IC
50 (50% of reduction) values as follows: 17.47 μg/mL for H
2O, 17.81 μg/mL for EtOAc, 20.36 μg/mL for Et
2O, 29.47 μg/mL for
n-BuOH, and 34.19 μg/mL for CHCl
3extract. Comparison of the DPPH scavenging activity of the investigated
T. pratense extracts with those expressed by BHT (14.31 μg/mL) and BHA (11.08 μg/mL) showed that neither of the extracts showed better antioxidant properties than the synthetic antioxidants. A high degree of correlation is observed between total phenol content and the ability of EtOAc and H
2O extracts to neutralize DPPH radicals. This is indicated by the fact that phenolic compounds play a key role in neutralizing free radical species which occurs by the mechanism of electron transfer. A lower degree of correlation defined between the content of total flavonoids in the two extracts and neutralization of DPPH radical, indicating that the content of flavonoids affect the level of free radical neutralization, but that is not directly correlated. These results are consistent with the data that is in
T. pratense are other types of phenolic compounds such as caffeic acid, which, in addition to flavonoids, may also participate in this type of reaction and contribute to the total antioxidant activity. When investigating neutralization of O
2•− and NO radicals, H
2O extract demonstrated highest activity. However, these activities were less than that of standards BHT and BHA. The lowest antioxidant activity was expressed by the CHCl
3 (IC
50 = 92.37 μg/mL for O
2•− and IC
50 = 58.46 μg/mL for NO radical) extract. The cellular damage resulting from hydroxyl radical (OH
•) is strongest among free radicals. Hydroxyl radical can be generated by biochemical reaction. Superoxide radical is converted by SOD (superoxide dismutase) to H
2O
2, which can subsequently produce extremely reactive OH
• radicals in the presence of transition metal ions such as iron and cooper [
24]. A good antioxidant potential of neutralization OH radical were shown by the H
2O (IC
50 = 18.44 μg/mL) and EtOAc (IC
50 = 19.79 μg/mL) extracts, which showed even better ability to neutralize OH radicals than BHT and BHA (IC
50 = 22.17 μg/mL). The worst effect on the neutralization of all the radicals studied
in vitro was shown by the
n-BuOH extract and this is probably due to the absence of any known “scavenger” of free radicals in that extract. GC-MS analysis showed that the
n-BuOH extract contains flavonoids in the form of glycosides and diglycosides. From the literature it is known that additional glycosylation reduces the antioxidant activity [
25]. Furthermore, it can be supposed that such antioxidant activity is caused, besides flavonoids and fenols, also by terpenoids since nonpolar solvents such as Et
2O also exhibited high antioxidant potential for neutralization of DPPH and NO radicals.
Lipid peroxidation is an established mechanism of cellular injury and is used as an indicator of oxidative stress. Polyunsaturated fatty acids peroxides generate malondialdehyde (MDA) and 4-hydroxyalkanals upon decomposition [
26]. Inhibition of LP was determined by measuring the formation of secondary components (mainly MDA) of the oxidative stress, using liposomes as an oxidizable substrate.The lipid peroxidation suppressing activity of
T. pratense leaves extracts is shown in
Figure 1, with BHT and BHA as control. In general, the examined extracts of
T. pratense leaves expressed strong antioxidant capacity. The largest inhibitory activity, again, was exhibited by H
2O extract. Solutions of all concentations (1%, 5% and 10%) have exhibited a stronger protective effect (from 40.36 to 43.67% of inhibition of LP) than BHT (26.15%) and BHA (36.88%). 5% and 10% solutions of EtOAc extracts demonstrate better protective effect of BHT, but only the most concentrated solution (10%) shows better inhibitory properties than BHA. The other two extracts (Et
2O and
n-BuOH), at the highest concentration (10%), have also exibited more intense protect effect than BHT.
Figure 1.
Inhibition of LP in Fe2+/ascorbate system of induction by by five different extracts of T. pratense leaves, and BHT and BHA (as a positive control) in the TBA assay.
Figure 1.
Inhibition of LP in Fe2+/ascorbate system of induction by by five different extracts of T. pratense leaves, and BHT and BHA (as a positive control) in the TBA assay.
Such a good antioxidant activity of H
2O and EtOAc extracts is expected because it is known that the antioxidant activity of phenols is primarily a result of the ability of these compounds to act as donors of hydrogen atoms removing free radicals with the formation of less reactive phenoxyl radicals [
27]. The increased stability of the formed phenoxyl radicals primarily attributed to electron delocalization and the existence of multiple resonant forms. Researching dependence of activity on the structure was found to have three structural features important factors of radicals removal potential and/or antioxidant potential of flavonoids: (1)
o-dihydroxy function of ring B, which serves as the target of radicals; (2) 2,3-double bond in conjugation with 4-oxo function, which are responsible for electrons delocalization of the ring B; and (3) the additional presence of 3- and 5-hydroxyl groups for the maximum radical scavenging potential [
28]. The positive relationship between increased hydroxylation and increased antioxidant activity of flavonoids was found in different lipid systems, such as oil and liposomes systems. Also, for phenolic acids and coumarins has been shown that vicinal diol groups are important for radical scavenging capacity, and that methoxylation or glycosylation of o-hydroxy group in the coumarins and esterification of phenolic acids reduce the antioxidant activity of these compounds [
29]. Furthermore, the action of some flavonoids is based on their ability to chelate transition metal ions, thereby preventing the formation of radicals (initiators of LP), caught radicals initiators of LP (ROS), scavenge lipid-alkoxyl and lipid-peroxyl radicals and regenerate α-tocopherol by reduction of α-tocopheryl radicals. Different metals have different binding affinity of the flavonoids [
30]. Thus, for example, iron has the highest binding affinity for 3-OH group of ring C, then catechol group ring B and at the end of 5-OH group of ring A, while the copper ions bind to the first ring catechol group B [
31]. Recently, many scientists study the antioxidant properties from other
Trifolium species. For example, Kolodziejczyk
et al. [
32] investigated the antioxidative effects of the clovamide-rich fraction, obtained from aerial parts of
Trifolium pallidum, in the protection of blood platelets and plasma against the nitrative and oxidative damage, caused by peroxynitrite (ONOO
−) and established that the presence of clovamide-rich
T. pallidum extract partly inhibited ONOO
−-mediated protein carbonylation and nitration. In addition, they determined that the
T. pallidum extract reduced lipid peroxidation in plasma but, the antioxidative action of the tested extract in the protection of blood platelet lipids was less effective. Malinowska
et al. [
33] investigated
in vitro oxidative changes in human plasma induced by the model of hyperhomocysteinemia in the presence of the phenolic fractions from selected clovers (
T. pallidum and
T. scabrum) and established that the tested phenolic fractions significantly inhibited the oxidative stress in plasma treated with homocysteine or homocysteine thiolactone. The phenolic fractions from
T. pallidum and
T. scabrum also caused a distinct reduction of plasma lipid peroxidation (measured by the level of thiobarbituric acid reactive substance) induced by the model of hyperhomocysteinemia.
2.3. In Vivo Experiments
The presented antioxidant activity results show that the EtOAc and H
2O extracts of
T. pratense leaves are efficient in protection of tissues and cells from oxidative stress. However, studies on their antioxidant status in animal model are needed to evaluate their potential health benefits. In addition, examinations of the
in vivo activity of extracts of
T. pratense leaves were conducted. The experimental animals were given 1 mL/kg of 2% of Et
2O, CHCl
3, EtOAc,
n-BuOH or H
2O extract (i.p.) of
T. pratense leaves for 7 days. After 7 days, the animals were sacrificed. In the liver homogenate and blood-hemolysate of sacrificed animals the following biochemical parameters were determined: LPx intensity, content of GSH and activities of GSHPx, GSHR, Px, CAT and XOD (
Table 3 and
Table 4). In
Table 5 and
Table 6 the results of the same parameters obtained after pretreatment of experimental animals with the examined
T. pratense extracts, followed by a single dose of carbon tetrachloride (CCl
4) as a well-known radical generator are presented.
Table 3.
Effect of extracts of T. pratense leaves on the biochemical parameters in the liver homogenate.
Table 3.
Effect of extracts of T. pratense leaves on the biochemical parameters in the liver homogenate.
Parameter | Control | Et2O extract | CHCl3 extract | EtOAc extract | n-BuOH extract | H2O extract |
---|
GSH | 4.17 ± 0.21 | 3.54 ± 0.19 a | 3.17 ± 0.17 a | 4.41 ± 0.15 | 3.91 ± 0.26 | 3.81 ± 0.23 |
GSHPx | 5.18 ± 0.26 | 4.48 ± 0.19 a | 4.38 ± 0.21 a | 3.64 ± 0.25 a | 4.29 ± 0.16 a | 4.31 ± 0.25 a |
GSHR | 6.16 ± 0.28 | 5.67 ± 0.24 a | 5.41 ± 0.26 a | 6.78 ± 0.17 a | 5.93 ± 0.19 | 5.71 ± 0.22 |
Px | 5.81 ± 0.23 | 5.58 ± 0.18 | 5.89 ± 0.21 | 6.17 ± 0.16 | 6.02 ± 0.24 | 6.65 ± 0.25 a |
LPx | 7.24 ± 0.28 | 6.56 ± 0.91 a | 6.95 ± 0.16 | 5.34 ± 0.23 a | 6.41 ± 0.23 a | 6.19 ± 0.22 a |
CAT | 5.11 ± 0.19 | 4.78 ± 0.19 | 5.18 ± 0.11 | 6.08 ± 0.25 a | 5.78 ± 0.23 a | 5.86 ± 0.27 a |
XOD | 6.32 ± 0.27 | 6.03 ± 0.14 | 6.17 ± 0.26 | 5.38 ± 0.28 a | 5.43 ± 0.21 a | 5.12 ± 0.28 a |
Table 4.
Effect of extracts of T. pratense leaves on the biochemical parameters in blood hemolysate.
Table 4.
Effect of extracts of T. pratense leaves on the biochemical parameters in blood hemolysate.
Parameter | Control | Et2O extract | CHCl3 extract | EtOAc extract | n-BuOH extract | H2O extract |
---|
GSH | 6.17 ± 0.17 | 5.32 ± 0.19 a | 5.13 ± 0.16 a | 5.64 ± 0.28 a | 5.53 ± 0.23 a | 5.37 ± 0.17 a |
GSHPx | 8.57 ± 0.24 | 7.31 ± 0.26 a | 7.14 ± 0.19 a | 7.87 ± 0.21 a | 7.77 ± 0.25 a | 7.51 ± 0.23 a |
GSHR | 6.64 ± 0.18 | 6.81 ± 0.19 | 6.92 ± 0.25 | 7.51 ± 0.23 a | 7.34 ± 0.19 a | 7.15 ± 0.18 a |
Px | 3.27 ± 0.19 | 2.54 ± 0.23 a | 2.79 ± 0.28 a | 3.14 ± 0.19 | 2.48 ± 0.25 a | 2.63 ± 0.20 a |
LPx | 7.81 ± 0.29 | 7.46 ± 0.27 | 6.59 ± 0.31 a | 6.08 ± 0.23 a | 6.64 ± 0.33 a | 5.98 ± 0.26 a |
CAT | 5.37 ± 0.27 | 5.87 ± 0.24 | 5.66 ± 0.29 | 6.14 ± 0.18 a | 6.05 ± 0.11 a | 5.72 ± 0.22 |
XOD | 6.15 ± 0.29 | 6.62 ± 0.29 | 6.44 ± 0.17 | 5.13 ± 0.14 a | 5.21 ± 0.28 a | 5.88 ± 0.26 a |
Table 5.
Effect of T. pratense leaves extracts and CCl4 on the liver homogenate biochemical parameters.
Table 5.
Effect of T. pratense leaves extracts and CCl4 on the liver homogenate biochemical parameters.
Parameter | Control | Et2O extract + CCl4 | CHCl3 extract + CCl4 | EtOAc extract + CCl4 | n-BuOH extract + CCl4 | H2O extract + CCl4 |
---|
GSH | 3.81 ± 0.17 | 2.68 ± 0.18 a | 2.41 ± 0.19 a | 3.71 ± 0.23 | 3.02 ± 0.17 a | 3.59 ± 0.21 |
GSHPx | 4.41 ± 0.27 | 3.65 ± 0.21 a | 3.41 ± 0.18 a | 3.77 ± 0.30 a | 3.61 ± 0.22 a | 3.73 ± 0.18 a |
GSHR | 5.17 ± 0.21 | 5.21 ± 0.28 | 4.16 ± 0.22 a | 4.31 ± 0.24 a | 5.26 ± 0.17 | 4.67 ± 0.18 a |
Px | 4.47 ± 0.18 | 3.98 ± 0.16 a | 4.15 ± 0.15 | 4.86 ± 0.28 | 4.61 ± 0.23 | 4.78 ± 0.25 |
LPx | 8.48 ± 0.26 | 8.13 ± 0.24 | 7.87 ± 0.19 a | 6.46 ± 0.19 a | 6.93 ± 0.28 a | 6.97 ± 0.27 a |
CAT | 4.48 ± 0.17 | 3.76 ± 0.25 a | 3.77 ± 0.25 a | 4.68 ± 0.22 | 4.88 ± 0.21 a | 4.54 ± 0.18 |
XOD | 8.56 ± 0.28 | 8.89 ± 0.27 | 8.78 ± 0.25 | 8.04 ± 0.19 a | 7.95 ± 0.18 a | 8.03 ± 0.13 a |
Table 6.
Effect of extracts of T. pratense leaves and CCl4 on the biochemical parameters in blood hemolysate.
Table 6.
Effect of extracts of T. pratense leaves and CCl4 on the biochemical parameters in blood hemolysate.
Parameter | Control | Et2O extract + CCl4 | CHCl3 extract + CCl4 | EtOAc extract + CCl4 | n-BuOH extract + CCl4 | H2O extract + CCl4 |
---|
GSH | 4.84 ± 0.27 | 3.26 ± 0.23 a | 3.87 ± 0.20 a | 4.27 ± 0.22 a | 4.05 ± 0.16 a | 4.34 ± 0.21 a |
GSHPx | 7.10 ± 0.23 | 6.03 ± 0.21 a | 5.48 ± 0.29 a | 6.41 ± 0.25 a | 6.88 ± 0.27 | 6.11 ± 0.26 a |
GSHR | 5.31 ± 0.32 | 4.26 ± 0.28 a | 4.79 ± 0.34 a | 4.67 ± 0.17 a | 4.88 ± 0.34 | 4.69 ± 0.31 a |
Px | 2.19 ± 0.21 | 1.84 ± 0.26 | 1.70 ± 0.18 a | 1.36 ± 0.13 a | 1.66 ± 0.18 a | 1.90 ± 0.21 |
LPx | 9.23 ± 0.25 | 9.86 ± 0.27 a | 9.91 ± 0.30 a | 8.15 ± 0.26 a | 8.28 ± 0.20 a | 7.86 ± 0.19 a |
CAT | 4.82 ± 0.18 | 4.27 ± 0.23 a | 3.76 ± 0.25 a | 4.11 ± 0.16 a | 4.01 ± 0.14 a | 4.31 ± 0.24 a |
XOD | 8.21 ± 0.35 | 8.94 ± 0.27 a | 8.75 ± 0.28 a | 7.13 ± 0.18 a | 7.93 ± 0.17 | 7.38 ± 0.26 a |
As can be seen from
Table 3, all extracts decreased the GSH content compared with control. The Et
2O,
n-BuOH, H
2O extracts, and especially the CHCl
3 one, decreased the GSH content in the liver homogenate, whereas this value remained essentially unchanged in the treatment with EtOAc extract. The lowered GSH content in the case of treatment with the former four extracts suggests that the constituents of these extracts entered no reaction with GSH, either of the radical or conjunction type, which is they did not show a hepatoprotective effect. All the extracts produced a statistically significant decrease of GSHPx.
Treatment with the EtOAc extract yielded an increase in GSHR activity, whereas the other four extracts caused a statistically significant decrease of this enzyme, which was in agreement with the action of this enzyme on GSH. Furthermore, only H
2O extract produced a statistically significant increase in Px activity. In addition to the very important role of peroxidase in the oxidative stress there are literature data on some other actions of peroxidases. Thus, some plant peroxidases oxidize phenols to phenoxy radicals to form polymers and enable their removal from industrial wastewaters [
34]. Having in mind the results presented in
Table 2, it might be interesting to test the aqueous extract of
T. pratense as a biological marker. The LPx intensity was lowered in the liver homogenate of animals treated with all extracts of
T. pratense leaves. The decrease was statistically significant in the case of Et
2O, EtOAc,
n-BuOH and H
2O extracts, which indicates the existence of a protective effect in the
in vivo experiments too. The CAT increased in the treatments with EtOAc,
n-BuOH and H
2O extracts, the other extracts caused no essential changes of CAT with respect to control. The results obtained in this assay showed a statistically significant decrease of XOD activity in the experimental animals treated with last three extracts (EtOAc,
n-BuOH and especially H
2O).
Summarizing the results obtained for all mice liver biochemical parameters, it can be concluded that the CCl
4 had hepatotoxic effects, because the decreased content of GSH and also activity of GSHPx, GSHR, Px and CAT, which significantly weakened antioxidant defense system of the body (
Table 5). On the other hand, it has led to an increase in the intensity of lipid peroxidation and the activity of the enzyme xanthine oxidase, which participate in the production of free radicals. It is assumed that CCl
4 toxicity stems from the possibility of its transformation into free radicals CCl
3• and CCl
3COO
•, the latter of which initiates the process LP higher polyunsaturated fatty acids, which eventually leads to cell death [
35]. Treatment of experimental animals with extracts and CCl
4 generally decreased the content of GSH, probably causing the appearance of its prooxidative metabolites. Application of three
T. pratense leaves extracts (Et
2O, CHCl
3 and
n-BuOH) in combination with CCl
4 resulted in a greater decrease of GSH content.
The administration of all examined extracts together with CCl
4 decreased the activity of GSHPx in the liver, particularly CHCl
3 and
n-BuOH extracts. A combination of Et
2O and
n-BuOH extracts and CCl
4 in the treatment of animals did not effect the essential change in GSHR activity. The application of CHCl
3, EtOAc and H
2O extracts exhibited a significant decrease of activity of this enzyme. In combination with CCl
4 the extracts exhibited different effects on Px: while the Et
2O extract showed a statistically significant decrease, the other four extracts had no statistically significant effect on the Px activity. All tested extracts showed inhibitory effects on LP in the liver of experimental animals, and the most active extract was EtOAc. It is assumed that the phenolic compounds present in the extract, effecting on CCl
4-induced LP in the following ways: removing CCl
3• radicals, inhibiting microsomal Cyt P450 system (whose increased activity accelerates the transformation of CCl
4 to free radical), removing peroxy- and lipoperoxy- radicals and complexing Fe
2+ ions [
36]. Combined treatment with the extracts and CCl
4 had a different effect on the activity of CAT in the liver homogenate. While the Et
2O and CHCl
3 extracts, caused a decrease, and the
n-BuOH extract an increase, the EtOAc and H
2O extracts showed no effect on this parameter. The application of EtOAc,
n-BuOH and H
2O extracts in combination with CCl
4 significantly lowered the activity of XOD. On the contrary, Et
2O or CHCl
3 extracts exhibited increasing activity on values of XOD, but statistically insignificant. Some recent studies point to the relationship between elevated XOD activity and oxidative stress in hypertension and the production of oxygen radicals in diabetes [
37]. However, allopurinol, a XOD inhibitor known in clinical practice, reduces oxidative stress in diabetes [
38], interacting with some peroxy radical species, such as CCl
3OO
•. It can be supposed that the active constituents present in EtOAc,
n-BuOH and H
2O extracts extracts act similarly, reducing the activity of this enzyme.
Similar to the results presented in
Table 3, the results of biochemical parameters measured in blood hemolysates of animals treated with extracts of
T. pratense leaves are shown in
Table 4. Concentrations of GSH measured in blood hemolysate showed significantly lower levels after treatment of animals with all extracts. In addition, all examined extracts exhibited decreases of GSHPx values, compared with the control.
An unusual result was obtained by examining the impact on the value of GSHR extracts. All extracts increased the activity of this enzyme, and the EtOAc, n-BuOH and H2O extract statistically significantly show this increase. As for the Px activity, all extracts reduced the activity of this enzyme, the difference being statistically insignificant only with the EtOAc extract. Four extracts, CHCl3, EtOAc, n-BuOH and H2O, induced a significant decrease of LPx intensity, while the Et2O one decreased the level of this enzyme insignificantly. On the other hand, last three extracts (EtOAc, n-BuOH and H2O) caused a statistically significant increase in CAT activity. The same extracts decreased the activity of XOD, while the other extracts caused no essential changes.
In
Table 6 the results of biochemical parameters measured in the blood hemolysate of animals treated with extracts of
T. pratense leaves and CCl
4 are presented. As during testing of the effect of
T. pratense leaves extracts and CCl
4 on the liver homogenate biochemical parameters (
Table 5), application of CCl
4 caused a decrease of the GSH content and activities of GSHPx, GSHR, Px and CAT, and led to an increase in the intensity of lipid peroxidation and the activity of the enzyme xanthine oxidase. A comparasion of GSH values in
Table 4 and
Table 6 shows that the GSH value is significantly lower in the animals treated with CCl
4 (6.17 ± 0.17
vs. 4.84 ± 0.27). This indicates that GSH acts as one of the essential antioxidant systems. On the other hand, the extracts showed no protective effect; moreover, all of them produced a further decrease of the GSH value. The administration of all extracts, except
n-BuOH, significantly decreased the activity of GSHPx.
n-BuOH extract did not cause notable changes. From the results presentd in
Table 6, it is obvious that CCl
4 decreased the values of GSHR (5.31 ± 0.32 nmol/mL erythrocytes x min
−1), compared with the levels in the control group (6.64 ± 0.18 nmol/mL erythrocytes min
−1). The results of GSHR activity were significantly lower in the combination of CCl
4 with all extracts, except
n-BuOH. Also, treatment with
n-BuOH caused a decrease of GSHR activity, but not notably (4.88 ± 0.34 nmol/mL erythrocytes min
−1).
The LPx value showed a statistically significant increase with CCl
4-treated animals compared with the untreated ones, whereas the presence of EtOAc,
n-BuOH and H
2O extracts yielded a decrease of LPx. These results suggest that these three extracts had a protective effect. According to the literature data [
39], the reduction of the serum LPx might be the result of antioxidant activity of several classes of plant phenolic constituents, such as cinnamic acids (ferulic, caffeic, and chlorogenic), flavonoids and biflavonoids, 1,3,6,7-tetrahydroxyxanthones, and acylphoroglucinols such as hyperforin and adhyperforin. On the other hand, the Et
2O and CHCl
3 extracts significantly increased the activity of LPx in combination with CCl
4. Very indicative are the results obtained for the Px and CAT which are significantly decreased in combination of all extracts with CCl
4. A statistically significant decrease of XOD activity was observed in the case of treatment with the EtOAc and H
2O extracts, while
n-BuOH extract exhibited no influence on it. Application of other two extracts (Et
2O or CHCl
3) with CCl
4, expressed a statistically significant increase of XOD.