2.1. In vitro experiments
Results of determination of total flavonoids in
M. melissophyllum leaves extracts are given in
Table 1.
Table 1.
Content of total flavonoids (mg/g) in extracts of leaves of M. melissophyllum.
Table 1.
Content of total flavonoids (mg/g) in extracts of leaves of M. melissophyllum.
Extract | Et2O | CHCl3 | EtOAc | n-BuOH | H2O |
---|
Leaf | 0.79 | 1.14 | 1.31 | 0.41 | 1.98 |
The amount of flavonoids in extracts plays an important role in their antioxidative behavior. The richest in flavonoids proved to be the water extract, while the n-BuOH extract contains the lowest amount of these active substances.
Antiradical activity was observed in the study of
M. melissophyllum leaves extracts in different solvents on the content of DPPH, O
2·− and NO radicals (
Table 2), whereby the H
2O extract exhibited the strongest inhibitory effect, as the IC
50 value was achieved with the lowest concentration.
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). All of the assessed extracts were able to reduce the stable, purple-colored radical DPPH into yellow-colored DPPH-H reaching 50% of reduction with an IC
50 as follows: 9.21 μg/mL for H
2O, 11.34 μg/mL for EtOAc, 11.92 μg/mL for CHCl
3, 17.21 μg/mL for
n-BuOH, and 18.09 μg/mL for Et
2O extract. Comparison of the DPPH scavenging activity of the investigated extracts with those expressed by BHT (14.31 μg/mL) showed that H
2O, EtOAc, and CHCl
3 extracts expressed stronger antioxidant effects. When investigating neutralization of O
2·− and NO radicals, water extract has also exhibited the greatest ability of their scavenging.
Table 2.
IC50 values (μg/mL) of the neutralization of DPPH, O2·− andNO radicals with M. melissophyllum extracts.
Table 2.
IC50 values (μg/mL) of the neutralization of DPPH, O2·− andNO radicals with M. melissophyllum extracts.
| | | IC50 (μg/mL) | | | |
---|
Extract | Et2O | CHCl3 | EtOAc | n-BuOH | H2O | BHT |
---|
DPPH radical | 18.09 | 11.92 | 11.34 | 17.21 | 9.21 | 14.31 |
O2°− radical | 8.11 | 7.91 | 14.42 | 13.26 | 6.38 | 10.46 |
NO radical | 8.04 | 7.42 | 8.91 | 9.12 | 6.17 | 8.63 |
For the neutralization of DPPH, O
2·− and NO radicals, the most responsible compounds were flavonoids and phenolic acids present in the leaves of
M. melissophyllum [
9,
10], so obtained results can be related to the experiments in which the total amount of flavonoids was determined (
Table 1.), which show that water extract of
M. melissophyllum leaf contains the largest amounts of total flavonoids. It is well known that some flavonoids and phenolic acids isolated from
M. melissophyllum possess certain biological and pharmacological activity [
11,
12]. For example, apigenin, one of the flavonoids present in
M. melissophyllum, was shown to express strong antioxidant effects, increasing the activities of antioxidant enzymes and, related to that, decreasing the oxidative damage to tissues [
10,
13]. Luteolin is also thought to play an important role in the human body as an antioxidant, a free radical scavenger, an agent in the prevention of inflammation, a promoter of carbohydrate metabolism, and an immune system modulator. These characteristics of luteolin are also believed to play an important part in the prevention of cancer. Multiple research experiments describe luteolin as a biochemical agent that can dramatically reduce inflammation and the symptoms of septic shock [
14]. Furthermore, it can be supposed that such antiradical activity is caused, besides flavonoids, also by triterpenoids acids (especially ursolic, oleanolic, and pomolic acid) since nonpolar solvents also exhibited high antiradical potential [
15,
16,
17].
The hydroxyl RSC of the examined
M. melissophyllum extracts (1%, 5% and 10%) measured by the deoxyribose assay is shown(
Figure 1) The protective effects of the extracts on 2-deoxy-D-ribose were assessed as their ability to remove hydroxyl radicals, formed in Fenton reaction, from the test solution and to prevent its degradation.
M. melissophyllum extracts have exhibited different behavior related to production of OH radicals. Ethyl acetate and water extracts at all investigated concentrations expressed OH radical inhibition. A stronger inhibition of OH radical production was expressed by the water extracts, especially the 10% solution (51.2 ± 1.7 nmol/mL) in comparison with the control (76.3 ± 2.1 nmol/mL).
This behaviour of the water extract can be explained by the presence of phenolic acids and flavonoid glycosides [
18]. It is known from the literature [
19,
20] that bastard balm, and especially its leaves, is characterised by a high content of flavonoid glucosides. Luteolin 7-glucoside has been isolated from the
M. melissophyllum flower and leaf, whereas of phenolic compounds were detected rosmarinic, caffeic and chlorogenic acid in leaves and ursolic acid in the plant root and leaf [
20].
Figure 1.
Inhibition of degradation of 2-deoxyribose by different extracts of M. melissophyllum in the deoxyribose assay.
Figure 1.
Inhibition of degradation of 2-deoxyribose by different extracts of M. melissophyllum in the deoxyribose assay.
On the basis of this it can be supposed that the very pronounced protective effect of the H
2O and EtOAc extracts of
M. melissophyllum is due to the presence of flavonoids and glucosides, namely of luteolin, either being present as free or in the form of its glucosides. The suggested mechanism of flavonoid antioxidative action is as follows: the double bond in position 2,3 is conjugated with the C
4-carbonyl group, and free OH groups (C
5, C
3 and C
7) can form chelates with ions of d-elements. Once formed, complex with Fe
2+ ion prevents formation of OH
· radicals in Fenton’s reaction [
21]. It was determined that rosmarinic acid has stronger antioxidant effect than vitamin E. Rosmarinic acid prevents cell damage caused by free radicals and reduces the risk of cancer and atherosclerosis. In contrast to the histamines, rosmarinic acid prevents activation of the immune system cells that cause swelling and fluid collection [
22]. n-BuOH extract has prooxidative effects, when other two extracts (etheric and chloroformic) don`t show neither ani-, neither pro-oxidative effect.
The protective effects on lipid peroxidation (LP) of
M. melissophyllum extracts have been evaluated using the Fe
2+/ascorbate system of induction, by the TBA-assay (
Figure 2). Inhibition of LP was determined by measuring the formation of secondary components (mainly MDA) of the oxidative stress, using liposomes as an oxidizable substrate. In general, all of the examined extracts (except n-BuOH extract) expressed strong antioxidant capacity. Protective activity can be explained by present of phenolic acids and their influence on antioxidative capacity of ascorbic acid, which doesn`t show a strong antioxidative effect in lipid phase, but different phenolic compounds can result increase of its antioxidant activity [
23]. The largest inhibitory activity, again, was exhibited by water extract. The antioxidant activities of all extracts of
M. melissophyllum leaves were dose dependent.
Figure 2.
Inhibition of LP in Fe2+/ascorbate system of induction by different extracts of M. melissophyllum in the TBA assay.
Figure 2.
Inhibition of LP in Fe2+/ascorbate system of induction by different extracts of M. melissophyllum in the TBA assay.
The
n-BuOH extract shows a prooxidative effect that is increased by increasing concentration of added extract. It can be supposed that compounds with polar groups were extracted by
n-BuOH, and are present in high concentration in the extract. It is notable that molecules which show antioxidant activity, when they are present in high concentration, might behave as prooxidants [
24], so
n-BuOHextract of
Melittis melissophyllum L. leaves probably have this kind of activity.
2.2. In vivo experiments
The represented antioxidant activity results show that extracts of examined plant species, specially H
2O extract, are efficient in the protection of tissues and cells from oxidative stress. Anyway, according to variations in regard to antoxidant activity of tested by different
in vitro models, there are also requiste
in vivo tests that would confirm antioxidant activity.
In vivo tests are also necessary because a lot of plant phenols are biotransformed during their active metabolism. 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
M. melissophyllum 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 GSHR, GSHPx, Px, XOD and CAT (
Table 3 and
Table 5). In
Table 4 and
Table 6 the results of the same parameters obtained after pretreatment of experimental animals with the examined
M. melissophyllum extracts, followed by a single dose of carbon tetrachloride (CCl
4) as a well-known radical generator are presented.
As can be seen from
Table 3. the EtOAc extract decreased the GSHPx activity; Et
2O extract caused its increase, while the other three extracts caused no essential changes of this parameter. The EtOAc extract decreased the GSH content compared with control. All permanent extracts increased the index of GSH, but only the increase caused by treatment with H
2O extract are statistically remarkable. Since about 95% of glutathione in liver is reduced and only 5% is oxidized [
25], this increase in the GSH index by applying by
M. m
elissophyllum L. leaves extracts (except EtOAc extract) probably derives from some compounds that contains a free SH-group, regardless if these compounds are present in extract or secondary biomolecules from these extracts have influenced an increase in the synthesis of GSH or cysteine. Treatment with the Et
2O, CHCl
3, and H
2O extracts yielded an increase in GSHR activity, whereas the EtOAc extract caused a statistically significant decrease of this enzyme, which was in agreement with the action of this extract on GSH.
Table 3.
Effect of extracts of M. melissophyllum leaves on the biochemical parameters in the liver homogenate.
Table 3.
Effect of extracts of M. melissophyllum leaves on the biochemical parameters in the liver homogenate.
Parameter | Control | Extract |
---|
Et2O | CHCl3 | EtOAc | n-BuOH | H2O |
---|
GSHPx | 3.43 ± 0.17 | 3.96 ± 0.16a | 3.32 ± 0.18 | 2.84 ± 0.21a | 3.37 ± 0.15 | 3.46 ± 0.18 |
GSH | 2.61 ± 0.13 | 2.76 ± 0.11 | 2.86 ± 0.14 | 2.12 ± 0.17a | 2.87 ± 0.22 | 3.49 ± 0.13a |
GSHR | 3.96 ± 0.18 | 4.82 ± 0.21a | 5.36 ± 0.25a | 2.98 ± 0.14 a | 3.96 ± 0.19 | 5.52 ± 0.23a |
Px | 4.38 ± 0.13 | 4.81 ± 0.17 a | 4.93 ± 0.21 a | 3.89 ± 0.13 a | 5.11 ± 0.25 a | 4.87 ± 0.15 a |
LPx | 7.19 ± 0.23 | 7.36 ± 0.21 | 7.91 ± 0.19a | 6.71 ± 0.16 a | 7.12 ± 0.23 | 6.19 ± 0.27 a |
CAT | 4.41 ± 0.16 | 3.83 ± 0.17 a | 5.03± 0.19a | 3.20 ± 0.15 a | 4.52 ± 0.11 | 5.49 ± 0.13a |
XOD | 5.27 ± 0.17 | 6.17 ± 0.23a | 6.02 ± 0.16a | 4.23 ± 0.16 a | 5.11 ± 0.22 | 4.17 ± 0.19 a |
On the other hand, all the extracts (except the EtOAc 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 [
26]. Having in mind the results presented in
Table 1 and
Figure 1 it might be interesting to test the aqueous extract of
M. melissophyllum as a biological marker. As compared with control, intensity of LPx is statistically saignificant reduced during the treatment with ethylacetate and water extracts of
M. melissophyllum leaves. The result derived by treatment with ethylacetate and water extracts is in according with amounts got
in vitro experiment (
Figure 2). Using CHCl
3 extract leads to a significant increase of LPx intensity, whereas the other two extracts had no effect on this parameter. The CAT increased in the treatments with CHCl
3 and H
2O extracts, and decreased in the treatment with the Et
2O and EtOAc extract.
n-BuOH extract caused no essential changes of CAT with respect to control. An increased XOD value was observed only in the treatments with the Et
2O and CHCl
3 extracts.
Table 4 presents the values of biochemical parameters obtained for the liver homogenate of animals treated with extracts of
M. melissophyllum leaves and CCl
4. In comparison with the control (animals received only physiological solution), treatment with CCl
4 yielded a significant decrease in activities of all the enzymes (GSHPx, GSHR, Px, CAT, XOD), as well as of GSH content, the only increase being in the intensity of LPx. In combination with CCl
4 the extracts exhibited different effects on GSHPx: while the CHCl
3 and H
2O extracts showed a statistically significant increase, the EtOAc extract decreased the activity of this enzyme. The Et
2O and
n-BuOH extracts in combination with CCl
4 had no effect on the GSHPx activity.
Table 4.
Effect of M. melissophyllum leaves extracts and CCl4 on the liver homogenate biochemical parameters.
Table 4.
Effect of M. melissophyllum leaves extracts and CCl4 on the liver homogenate biochemical parameters.
Parameter | Control + CCl4 | Extract + CCl4 |
---|
Et2O | CHCl3 | EtOAc | n-BuOH | H2O |
---|
GSHPx | 2.12 ± 0.17 | 2.31 ± 0.15 | 2.61 ± 0.18a | 1.62 ± 0.23a | 2.18 ± 0.24 | 2.58 ± 0.18a |
GSH | 2.26 ± 0.17 | 2.18 ± 0.12 | 2.35 ± 0.19 | 1.86 ± 0.12 a | 1.92 ± 0.15 a | 2.49 ± 0.25 |
GSHR | 2.53 ± 0.21 | 3.46 ± 0.28a | 4.25 ± 0.24a | 2.08 ± 0.15a | 2.26 ± 0.26 | 3.07 ± 0.26a |
Px | 3.47 ± 0.18 | 3.05 ± 0.17a | 2.97 ± 0.25a | 2.84 ± 0.21a | 3.06 ± 0.23 | 3.01 ± 0.24a |
LPx | 8.91 ± 0.29 | 7.12 ± 0.21a | 7.06 ± 0.24 a | 6.92 ± 0.17a | 6.98 ± 0.24 a | 6.81 ± 0.24a |
CAT | 2.08 ± 0.17 | 2.17 ± 0.22 | 2.47 ± 0.25 | 1.02 ± 0.12a | 2.10 ± 0.14 | 1.44 ± 0.18a |
XOD | 4.61 ± 0.25 | 3.69 ± 0.23a | 5.38 ± 0.21a | 3.02 ± 0.19a | 3.98 ± 0.17 a | 2.39 ± 0.14 a |
Treatment with CCl
4 caused no significant reduction of GSH content compared with that seen in untreated animals. Combined treatment with the extracts and CCl
4 had a different effect on the GSH content in the liver homogenate. While the EtOAc and
n-BuOH extracts caused a decrease, the Et
2O, CHCl
3 and H
2O extracts showed no effect on this parameter. The Et
2O, CHCl
3 and H
2O extracts in combination with CCl
4 increased the GSHR activity in the liver homogenate and the EtOAc extract reduced it. On the other hand, the
n-BuOH extract did not influence this parameter. Bearing in mind the fact that the CCl
4-induced oxidative injuries of the liver require a high consumption of GSH, which is regenerated via the GSHR activity, it can be concluded that the extracts showed no protective effect, as they did not cancel out the effect of treatment with CCl
4. All the extracts (except
n-BuOH one) in combination with CCl
4 yielded a statistically significant decrease in Px, the EtOAc extract exhibiting the strongest effect. All extracts of
M. melissophyllum leaves combine with CCl
4 have showed a statistically significant decrease of LPx intensity, and this behavior of the extract probably results from the presence of secondary biomolecules like flavonoids and phenolic acids. Handa
et al. [
27] determined that secondary biomolecules such as flavonoids, xanthones and tannins in combination with CCl
4 have protective effects on liver. Phenolic components present in
M. melissophyllum leaves (rutin, luteolin, kvercetin) are known as strong inhibitors of CCl
4-induced LP [
28]. Flavonoids could affect the initiation phase of lipid peroxidation, where they influence the metabolism of CCl
4, they scavenge the free radicals, or they decrease the microsomal enzyme systems that are claimed for CCl
4 metabolism [
29]. In continuation of this process, flavonoids can scavenge lipoperoxides and their radicals or they can act as chelating agents for Fe
2+ ion, and in this way can stop Fenton reactions [
30]. On the basis of these results, we can conclude that all of extracts of
M. melissophyllum leaves showed protection effect in relation to the CCl
4-induced lipid peroxidation. The EtOAc and H
2O extracts in combination with CCl
4 yielded a statistically significant decrease in CAT activity. Administration of CCl
4 significantly decreased the activity of XOD (4.61 ± 0.25 nmol/mg of protein x min
-1) compared with the untreated animals (5.27 ± 0.17 nmol/mg of protein x min
-1). Further, the Et
2O, EtOAc,
n-BuOH and H
2O extracts in combination with CCl
4 significantly lowered the activity of XOD. On the contrary, CHCl
3 extract significantly increased XOD activity. Some recent studies point to the relationship between elevated XOD activity and oxidative stress in hypertension and the production of oxygen radicals in diabetes [
31]. However, allopurinol, a XOD inhibitor known in clinical practice, reduces oxidative stress in diabetes [
32], interacting with some peroxy radical species, such as Cl
3OO
•. It can be supposed that the active constituents present in Et
2O, EtOAc,
n-BuOH and H
2O extracts act similarly, reducing the activity of this enzyme.
Like those given in
Table 3, the results in
Table 5 show that all the extracts caused a statistically significant decrease in blood GSHPx activity. All extracts reduced the GSH content in the hemolysate too, the decrease being statistically significant. The activity of GSHR decreased after treatment with Et
2O, EtOAc and
n-BuOH extracts.
Table 5.
Effect of extracts of M. melissophyllum leaves on the biochemical parameters in blood hemolysate.
Table 5.
Effect of extracts of M. melissophyllum leaves on the biochemical parameters in blood hemolysate.
Parameter | Control | Extract |
---|
Et2O | CHCl3 | EtOAc | n-BuOH | H2O |
---|
GSHPx | 5.94 ± 0.22 | 4.81 ± 0.16a | 4.95 ± 0.19a | 2.77 ± 0.23a | 4.79 ± 0.16a | 3.89 ± 0.16a |
GSH | 6.78 ± 0.12 | 6.22 ± 0.14a | 6.13 ± 0.17a | 5.56 ± 0.21a | 3.96 ± 0.21a | 4.37 ± 0.19 a |
GSHR | 7.67 ± 0.24 | 6.17 ± 0.24a | 7.87 ± 0.27 | 5.56 ± 0.17a | 6.81 ± 0.19a | 7.72 ± 0.28 |
Px | 3.72 ± 0.17 | 3.39 ± 0.21 | 3.69 ± 0.25 | 2.94 ± 0.19a | 3.46 ± 0.27 | 3.65 ± 0.18 |
LPx | 4.81 ± 0.24 | 4.59 ± 0.28 | 3.78 ± 0.17a | 2.96 ± 0.13a | 4.74 ± 0.19 | 4.07 ± 0.24a |
CAT | 4.28 ± 0.26 | 4.77 ± 0.18a | 3.75 ± 0.19a | 3.18 ± 0.16a | 4.11 ± 0.18 | 3.86 ± 0.23 |
XOD | 4.76 ± 0.29 | 5.92 ± 0.31a | 5.44 ± 0.26a | 5.58 ± 0.19a | 5.41 ± 0.27a | 5.48 ± 0.24a |
As for the Px activity, all extracts reduced the activity of this enzyme, the difference being statistically significant only with the EtOAc extract. Three extracts, CHCl
3, EtOAc and H
2O, induced a significant decrease of LPx intensity, while the Et
2O and
n-BuOH ones decreased the level of this enzyme insignificantly. A statistically significant decrease in CAT activity was produced by the CHCl
3 and EtOAc extracts. On the other hand, all extracts caused a statistically significant increase in XOD activity. XOD is an enzyme which is present in measurable amounts only in liver and jejunum. Otherwise, because of different irregularities in liver, this enzyme should be cleared by the circulation. Consequently, an increase of XOD in the blood could be an indicator of liver damage [
33]. Nowadays, increase of XOD activity is related to amplification of oxidative stress and production of free radicals [
34]. From the results obtained during this research, we can conclude that the high level of XOD in the experimental animals blood-hemolysate that were treated by
M. melissophyllum leave extracts is probably consequent of liver damage, so these extracts showed hepatotoxic effects. High production of oxygen radicals (priority O
2−·) and H
2O
2 probably influenced the increased production of XOD. We must bear in mind that only one of two forms of XOD is related to high production of oxygen radicals, while the other form has a completely different metabolic way, a mechanism proposed by Kisher
et al. [
35]. According to this mechanism, results obtained from this research could be interpreted otherwise. Particulary, the other form of XOD leads to the formation of NADH+H
+, which could be included in the respiratory chain with a view to synthesize ATP, that could be interpreted as the ultimate eventual protective effect of
M. melissophyllum leave extracts.
In
Table 6 the results of biochemical parameters measured in the blood-hemolysate of animals treated with extracts of
M. melissophyllum leaves and CCl
4 are presented. The activity of glutathione peroxidase (GSHPx) in the blood of animals treated with CCl
4 (
Table 6) was increased compared with the control (
Table 5). However, only the administration of EtOAc and
n-BuOH extracts significantly decreased the activity of GSHPx. Other administered extracts did not cause notable changes.
Table 6.
Effect of extracts of M. melissophyllum leaves and CCl4 on the biochemical parameters in blood hemolysate.
Table 6.
Effect of extracts of M. melissophyllum leaves and CCl4 on the biochemical parameters in blood hemolysate.
Parameter | Control + CCl4 | Extract + CCl4 |
---|
Et2O | CHCl3 | EtOAc | n-BuOH | H2O |
---|
GSHPx | 6.08 ± 0.17 | 5.83 ± 0.21 | 5.77 ± 0.24 | 4.69 ± 0.21a | 4.94 ± 0.27a | 5.91 ± 0.33 |
GSH | 5.21 ± 0.13 | 4.84 ± 0.19a | 3.97 ± 0.24a | 4.08 ± 0.17a | 3.03 ± 0.18a | 3.86 ± 0.24a |
GSHR | 6.12 ± 0.29 | 5.76 ± 0.28 | 5.87 ± 0.22 | 4.12 ± 0.18a | 6.17 ± 0.29 | 6.58 ± 0.32 |
Px | 4.08 ± 0.22 | 3.27 ± 0.16a | 4.86 ± 0.24a | 3.15 ± 0.23 a | 3.04 ± 0.18 a | 4.11 ± 0.29 |
LPx | 5.11 ± 0.24 | 5.31 ± 0.17 | 4.92 ± 0.21 | 3.02 ± 0.24a | 5.17 ± 0.25 | 2.98 ± 0.12a |
CAT | 3.74 ± 0.24 | 3.19 ± 0.23a | 3.24 ± 0.19a | 3.07 ± 0.25a | 2.98 ± 0.18a | 3.13 ± 0.28a |
XOD | 6.19 ± 0.31 | 6.08 ± 0.26 | 4.96 ± 0.24a | 5.11 ± 0.17a | 6.97 ± 0.25a | 5.23 ± 0.21a |
The amount of GSH from the blood of non-CCl
4 treated animals was compered with CCl
4 treated animals (5.21 ± 0.13
vs. 6.78 ± 0.12), and it was observed that the amount of GSH is one of the important secondary biomolecules, that take a part in different detoxification processes in organism. Not only “radical“ GSH already in conjugation with toxic metabolites protects organism. From the results presented it is obvious that CCl
4 decreased the values of GSHR (6.12 ± 0.29 nmol/mL erythrocytes x min
−1), compared with the levels in the control group (7.67 ± 0.24 nmol/mL erythrocytes x min
−1). The results of GSHR activity were significantly lower only in the combination of CCl
4 with EtOAc extract (4.12 ± 0.18 nmol/mL erythrocytes x min
−1). Also, treatment with Et
2O and CHCl
3 extracts caused a decrease of GSHR activity, but not notably. Treatment with CCl
4, compared with the control animals (
Table 5 and
Table 6) did not cause notable differences in the activity of Px. Application of three extracts (Et
2O, EtOAc or
n-BuOH) with CCl
4, caused a statistically significant decrease of Px. On the other hand, the CHCl
3 extract significantly increased the activity of Px in combination with CCl
4. Similar to the values of Px, values of LPx showed an insignificant increase of activity in the blood of animals treated with CCl
4 (
Table 6), compared with the control (
Table 5). A clear ‘protective’ effect was seen in experimental animals administered H
2O extract and CCl
4 compared with untreated animals. Furthermore, EtOAc extract also significantly decreased the activity of LPx, while Et
2O, CHCl
3 and
n-BuOH extracts did not change notably the levels of lipid peroxidation. In all previous research works (
Figure 2,
Table 3,
Table 4,
Table 5), H
2O extract showed the best protective effect on LPx. Already it was noticed that these protective properties probably come from secondrary biomolecules such as the flavonoids and phenolic acids are. Administration of CCl
4 caused a decrease in the values of CAT activity in blood of experimental animals compared with the control. Combined treatment of experimental animals with CCl
4 and all of examined extracts reduced the activity of this enzyme, especially in the case of the
n-BuOH extract. The level of activity of xanthine oxidase (XOD) was increased in the blood of experimental animals after administration of CCl
4 (
Table 6) with respect to the untreated ones (
Table 5). On the contrary, lower activities of XOD were obtained after combined treatment of CCl
4 with CHCl
3, EtOAc or H
2O extracts. However, only the
n-BuOH extract exhibited a significant increase of XOD activity.