3.1. DPPH free radical scavenging effects of the organic substances
When DPPH (α, α-Diphenyl-b-picrylhydrazyl) free radical effects of the organic substances were compared according to Quercetin, it was found that 1a coded substances have partial free radical scavenging effect in 10 µL group (p < 0.05). Whereas 1b, 1c, and 2a coded substances had lower activity compared to Quercetin (p < 0.001).
There were a significant increase in DPPH free radical scavenging activity of 1a and 2a coded substances in 20 µL and 30 µL concentration groups because of the increase in concentration (p < 0.001). Whereas there was a significant decrease in DPPH free radical scavenging activity of (1b-c) and (2b-c) coded substances compared to Quercetin due to the increase in concentration (p < 0.0001).
3.2. Antioxidant effect of the substance groups in vitro medium
When the effects of the substance groups on LPO in vitro medium were compared; the amount of LPO was significantly increased in all groups compared to the control in 10 µL group (p < 0.0001). Whereas in 50 µL group; the amount of LPO was significantly increased in FR, (1b-c) and 2c coded substances compared to the control (p < 0.0001), and the increase in resveratrol and 2b coded substances was lower (p < 0.001). The amount of LPO was decreased compared to the control in Quercetin, (1-2a) (p < 0.05) with a more significant decrease compared to FR group (p < 0.0001).
LPO was significantly increased in FR, (2b-c) and 1c coded substances compared to the control in 100 µL group (p < 0.0001), although the increase was lower in 1b and 2a coded substances (p < 0.001).
Whereas the amount of LPO was significantly decreased in Quercetin and Resveratrol groups and 1a coded substance compared to the control and FR groups (p < 0.0001).
3.3. Effects of the (1a-b, 2c) substance groups on glutathione (GSH), malondialdehyde (MDA) in S. Cerevisiae
Table 2
Effects of the (1a-b, 2c) coded substances on glutathione (µmol/g pellet) and Malondialdehyde (nmol/g pellet) (MDA) in S. Cerevisiae
Materials
|
Control
|
1b
|
1a
|
2c
|
GSH
|
0,73 ± 0,03
|
0,12 ± 0,05 d
|
0,08 ± 0,05 d
|
0,01 ± 0,01 d
|
MDA
|
2,98 ± 0,30 a
|
3,20 ± 0,30 b
|
3,27 ± 0,30 b
|
2,97 ± 0,17 a
|
a: p > 0,05, b: p < 0,05, c: p < 0.01, d: p < 0.001 |
When effects of (1a-b, 2c) coded substances on the amount of glutathione in S. cerevisiae were compared with the control group, the amount was significantly decreased in all three groups (p < 0.001). When effects of (1a-b, 2c) coded substances on the amount of MDA in S. cerevisiae were compared with the control group; (1a-b) coded substances partially increased the amount of MDA (p < 0.05), while 2c coded substance, showed no statistically significant difference (p < 0.05).
3.4. Effects of the (2a-b,1c) S-substance groups on glutathione (GSH), malondialdehyde (MDA) in S. Cerevisiae
Table 3
Effects of (2a-b,1c) coded substances on glutathione (µmol/g pellet) and Malondialdehyde (nmol/g pellet) (MDA) in S. Cerevisiae
Materials
|
Control
|
2a
|
2b
|
1c
|
GSH
|
0,36 ± 0,09 a
|
0,27 ± 0,03 b
|
0,37 ± 0,04 a
|
0,76 ± 0,04 b
|
MDA
|
3,70 ± 0,93 a
|
5,64 ± 1,00 d
|
3,71 ± 0,75 a
|
3,62 ± 0,42 a
|
a: p > 0,05, b: p < 0,05, c: p < 0.01, d: p < 0.001, cd: p < 0.0001 |
When effects of (2a-b,1c) coded substances on the amount of glutathione in S. cerevisiae cell were compared with the control group; 2b showed no statistically significant difference (p > 0.05), while 2a coded substance partially decreased and 1c coded substance partially increased the amount of GSH. When effects of (2a-b,1c) coded substances on the amount of MDA in S. cerevisiae cell were compared with the control group; 2a coded substance significantly increased the amount (p < 0.0001), while 1c and 2b coded substances showed no statistically significant difference (p > 0.05).
3.5. Effects of the (1a-b, 2c) coded substance groups on the fatty acid profile of S. Cerevisiae yeast cell
Table 4
Effects of the (1a-b, 2c) Coded Substance Groups on Fatty Acid Profile of S. Cerevisiae Yeast Cell (µg/1 g)
Fatty acids
|
Control
|
1b
|
1a
|
2c
|
12:0
|
34,73 ± 1,83 a
|
35,79 ± 0,97 a
|
14,50 ± 2,41 c
|
27,96 ± 1,69 b
|
16:0
|
191,65 ± 11,57
|
230,82 ± 11,50 d
|
273,29 ± 11,49 cd
|
233,01 ± 11,58 d
|
16:1
|
223,98 ± 10,54
|
96,50 ± 10,23 cd
|
112,45 ± 10,66 d
|
128,78 ± 10,42 d
|
18:0
|
62,14 ± 1,93
|
76,06 ± 1,33 c
|
87,95 ± 1,74 d
|
66,63 ± 1,16 b
|
18:1n9t
|
86,56 ± 0,68
|
158,22 ± 0,78 d
|
160,47 ± 0,59 d
|
169,82 ± 0,81 cd
|
18:1n9
|
35,55 ± 10,7 a
|
35,35 ± 10,29 a
|
63,40 ± 10,72 d
|
32,32 ± 10,67 b
|
18:2n6c
|
10,74 ± 0,02 a
|
10,28 ± 0,07 a
|
12,79 ± 0,89 b
|
13,02 ± 0,14 b
|
a: p > 0,05, b: p < 0,05, c: p < 0.01, d: p < 0.001, cd: p < 0.0001 |
When effects of the (1a-b, 2c) coded substance groups on the fatty acid profile of S. cerevisiae yeast cell were compared with the control; the amount of lauric acid (12:0) was partially decreased in 1a and 2c coded substances (p < 0.05), and the decrease in amount was more significant in 1b coded substance (p < 0.01). The amount of palmitic acid (16:00) significantly increased in all three groups compared to the control (p < 0.001), and this increase was more significant in 1a coded substance (p < 0.0001). The amount of stearic acid (18:0) increased in all three groups compared to the control, while the most significant increase was found in 1a coded substance (p < 0.001).
The amount of arachidic acid (20:0) significantly increased in all three groups compared to the control (p < 0.001), and the most significant increase was found in 1b coded substance (0.0001). The amount of palmitoleic acid (16:1). Significantly decreased in all three groups compared to the control (p < 0.001), and the most significant decrease was found in 1b coded substance (p < 0.0001). The amount of elaidic acid ((18:1) n9t) increased in all three groups compared to the control (p < 0.01), and the most significant increase was found in 2c coded substance (p < 0.0001). The amount of oleic acid ((18:1) n9) significantly increased in 1a coded substance (p < 0.01). Although the amount partially decreased in 2c coded substance (p < 0.05), and there was no statistically significant difference in 1b coded substance (p > 0.05). The amount of linoleic acid ((18:2) 6nc)) partially increased in 1a and 2c coded substances (p < 0.05), while no statistically significant difference was found in 1b coded substance (p > 0.05).
3.6. Effects of the (2a-b, 1c) coded substance groups on the fatty acid profile of S. Cerevisiae yeast cell
Table 5
Effects of the (2a-b, 1c) coded substance groups on the fatty acid profile of S. Cerevisiae yeast cell (µg/ 1 g)
Fatty acids
|
Control
|
2a
|
2b
|
1c
|
12:0
|
49,49 ± 2,5
|
28,16 ± 2,09 c
|
25,85 ± 2,01 c
|
0
|
14:0
|
95,39 ± 2,48
|
104,26 ± 2,84 b
|
106,51 ± 2,05 b
|
44,03 ± 2,42 d
|
15:0
|
71,15 ± 2,22
|
63,14 ± 1,04 c
|
69,99 ± 1,81 c
|
31,87 ± 1,09 d
|
16:0
|
458,04 ± 4,40
|
543,83 ± 4,53 cd
|
554,20 ± 4,55 cd
|
272,08 ± 4,43 cd
|
16:1
|
160,49 ± 4,41
|
280,23 ± 2,73 cd
|
286,99 ± 2,78 cd
|
215,52 ± 2,91 cd
|
17:1
|
138,34 ± 21,47
|
121,40 ± 21,32 b
|
129,88 ± 22,04 b
|
60,98 ± 21,06 cd
|
18:0
|
80,44 ± 12,77 a
|
86,92 ± 12,92 b
|
80,91 ± 12,81 a
|
43,24 ± 12,62 cd
|
18:1n9t
|
172,71 ± 11,65
|
264,24 ± 12,58 cd
|
259,32 ± 12,92 cd
|
121,42 ± 11,65 cd
|
18:1n9
|
72,49 ± 2,31 a
|
48,52 ± 2,60 d
|
75,39 ± 2,19 a
|
38,35 ± 2,45 cd
|
18:2n6c
|
140,14 ± 20,63
|
148,41 ± 21,33 b
|
166,30 ± 21,38 d
|
97,89 ± 22,03 cd
|
18:2n6t
|
40,32 ± 12,57 a
|
46,24 ± 9,77 a
|
47,27 ± 9,17 a
|
31,42 ± 9,70 a
|
a: p > 0,05, b: p < 0,05, c: p < 0.01, d: p < 0.001, cd: p < 0.0001 |
When effects of (2a-b, 1c) coded substance groups on the fatty acid profile of S. cerevisiae yeast cell were examined; the amount of lauric acid (12:0) decreased in (2a-b) coded substances compared to the control (p < 0.01).
The amount of myristic acid (14:0) partially increased in (2a-b) compared to the control (p < 0.05), while a significant decrease was found in 1c coded substance (p < 0.001).
The amount of pentadecanoic acid (15:0) partially decreased in all three groups compared with the control (p < 0.01), the most significant decrease was in 1c coded substance (p < 0.001). The amount of palmitic acid (16:0) significantly increased in (2a-b) coded substances (p < 0.0001), although the amount significantly decreased in 1c coded substance (p < 0.0001). The amount of stearic acid (18:0) partially increased in 2a coded substance (p < 0.005) compared to the control, a significant decrease was found in 1c coded substance (p < 0.0001). Whereas no statistically significant difference was found in 2b coded substances (p > 0.05). The amount of palmitoleic acid (16:1) significantly increased in all three groups compared to the control (p < 0.001) with the most significant increase was found in 2b coded substances (p < 0.0001). The amount of heptadecenoic acid (17:1) partially decreased in all groups compared to the control (p < 0.05), and the most significant decrease among the groups was in 1c coded substance (p < 0,0001). The amount of elaidic acid ((18:1) n9t) significantly increased in (2a-b) coded substances compared to the control (p < 0.0001), although it was significantly decreased in d6 coded substance (p < 0.0001). The amount of oleic acid ((18:1) n9c) significantly decreased in 1c and 2a coded substances (p < 0.001), although it was partially decreased in 2b coded substances (p < 0.05).
3.7. Effects of the (1a-b, 2c) coded substance groups on lipophilic vitamins and phytosterol profile of S. Cerevisiae yeast cell
Table 6
Effects of the (1a-b, 2c) coded substance groups on lipophilic vitamins and phytosterols of S. Cerevisiae
Vitamins
|
Control
|
1b
|
1a
|
2c
|
α-tocopherol
|
2,85 ± 1,51
|
5,32 ± 0,82 c
|
14,61 ± 1,33 cd
|
7,90 ± 1,36 d
|
δ- tocopherol
|
0,79 ± 0,33
|
0,89 ± 0,24 b
|
0,89 ± 0,19 b
|
1,02 ± 0,34 c
|
Vitamin D2
|
1,28 ± 0,78
|
1,81 ± 0,39 c
|
1,66 ± 0,60 b
|
0,25 ± 034 cd
|
Vitamin K1
|
0,16 ± 0,15
|
0,46 ± 0,34 c
|
0,60 ± 0,48 cd
|
0,47 ± 0,32 c
|
Vitamin K2
|
0,49 ± 0,08
|
0,28 ± 0,09 d
|
0,08 ± 0,07 cd
|
0,56 ± 0,07 b
|
Ergosterol
|
3,23 ± 0,88 a
|
3,25 ± 1,22 a
|
2,72 ± 0,44 d
|
5,68 ± 1,40 cd
|
Stigmasterol
|
198,40 ± 11,22
|
240,86 ± 11,41 d
|
246,16 ± 11,12 d
|
320,22 ± 11,96 cd
|
β-sitosterol
|
0, 52 ± 0,37 a
|
0,51 ± 0,22 a
|
1,23 ± 0,87 d
|
0,83 ± 0,59 b
|
a: p > 0,05, b: p < 0,05, c: p < 0.01, d: p < 0.001, cd: p < 0.0001 |
When effects of ((1a-b, 2c) coded substances on lipophilic vitamins and phytosterol profile in S. Cerevisiae Yeast Cell were examined; the amounts of α-tocopherol, δ- tocopherol, vitamin K1, and Stigmasterol increased in all groups compared to the control. The most significant increase in the amount of α-tocopherol was found in 1a coded substance (p < 0.0001), this increase was lower in 2c coded substance (p < 0.001). The most significant increase in the amount of δ-tocopherol was found in 2c coded substances (p < 0.01), although relatively lower increases were found in (1a-b) coded substances (p < 0.05). The most significant increase in the amount of vitamin K1 was observed in 1a coded substance (p < 0.0001), although there were partial increases in 1b and 2c coded substances (p < 0.01). The amount of Stigmasterol increased in (1a-b) coded substances (p < 0.001), while the most significant increase was found in 2c coded substances (p < 0.0001). The amounts of ergosterol and β-sitosterol were not statistically significant in 1b coded substance compared to the control (p > 0.05), while these amounts significantly increased in 1a and 2c coded substances (p < 0.0001). The amount of vitamin D2 increased in (1a-b) coded substances compared to the control, and the most significant increase was found in 2c coded substance (p < 0.01). Whereas this amount significantly decreased in 2c coded substance compared to the control (p < 0.0001).
3.8. Effects of the (2a-b, 1c) coded substance groups on lipophilic vitamins and phytosterol profile of S. Cerevisiae Yeast Cell
Table 7
Effects of the (2a-b, 1c) coded substance groups on lipophilic vitamins and phytosterols of S. Cerevisiae
Vitamins
|
Control
|
2a
|
2b
|
1c
|
α- tocopherol
|
1,59 ± 0,26
|
6,53 ± 0,07 cd
|
3,17 ± 0,18 d
|
2,46 ± 0,14 d
|
δ- tocopherol
|
0,38 ± 0,08
|
0,15 ± 0,10 c
|
0,16 ± 0,20 c
|
0,10 ± 0,10 c
|
Vitamin D2
|
0,02 ± 0,04
|
0,05 ± 0,12 b
|
0,09 ± 0,13 b
|
0,01 ± 0,00 b
|
Vitamin K1
|
0,22 ± 0,15
|
0,37 ± 0,11 b
|
0,42 ± 0,16 c
|
0,36 ± 0,16 b
|
Vitamin K2
|
1,15 ± 0,45
|
1,30 ± 0,26 b
|
1,22 ± 0,25 b
|
0,56 ± 0,46 c
|
Ergosterol
|
10,25 ± 0,24
|
12,09 ± 1,17 b
|
15,75 ± 1,38 cd
|
8,58 ± 1,35 c
|
Stigmasterol
|
525,61 ± 11,71
|
917,93 ± 11,11 cd
|
849,76 ± 11,41 cd
|
535,51 ± 11,11 b
|
β-sitosterol
|
0,96 ± 0,21
|
0,31 ± 0,54 c
|
0,31 ± 0,29 c
|
0,49 ± 0, 34 c
|
a: p > 0,05, b: p < 0,05, c: p < 0.01, d: p < 0.001, cd: p < 0.0001 |
When effects of (2a-b, 1c) coded substances on lipophilic vitamins and phytosterol profile in S. Cerevisiae Yeast Cell were examined; the amounts of α- tocopherol, vitamin K1, and Stigmasterol increased compared to the control group. The most significant increases in amounts of α- tocopherol Stigmasterol were found in 2a coded substance (p < 0.0001), although the most significant increase in the amount of vitamin K1 was found in 2b coded substance (p < 0.0001). The amounts of ergosterol and vitamin K2 partially increased in (2a-b) coded substances (p < 0.05), although these amounts decreased in 1c substance group (p < 0.01). The most significant increase in the amount of ergosterol was observed in 2b coded substance (p < 0.0001), while the most significant increase in the amount of vitamin K2 was in 2a coded substance (p < 0.05). The amounts of δ- tocopherol and β-sitosterol decreased in all three groups compared to the control (p < 0.01). The amount of vitamin D2 partially increased in (2a-b) coded substances (p < 0.05), although this amount partially decreased in 1c coded substance (p < 0.05).
3.9. Effects of benzofurane derived organic substances in vitro medium
In line with the data obtained by investigating in vitro free radical scavenging effect, in vitro effect on MDA, and effect on the amount of GSH, MDA, and total protein in Saccharomyces cerevisiae yeast cell, and effect on vitamin and fatty acid synthesis in synthesized benzofuran derived compounds.
According to our results, when free radical scavenging effects of organic DPPH substances were compared; 2b coded substances had significantly higher scavenging effects compared to the other substances. When the effectiveness of these groups was compared according to increasing concentration, an increase was found in DPPH free radical scavenging effects of 1a and 2a coded substances. Many studies have supported the antioxidant effects of benzofuran-derived compounds. Grisar et al. [18] and Bindoli et al. [19] reported that interact with the oxidants formed as a result of lipid peroxidation of benzofuran-derived compounds and sulfhydryl group oxidants, destructing these oxidants, and thus they are antioxidants. The antioxidant feature of the organic substances is closely associated with the number and localization of OH· groups. A high number of OH· groups is the main factor increasing antiradical activity. Because hydrogen in OH· group functions as a donor, making radicals, especially hydroxyl, peroxyl, and peroxynitrite stable.
We found that DPPH free radical scavenging effect of (1b-c) and (2b-c) coded substances significantly decreased with increasing concentrations.
As is evident from its name, free radical inhibitors inhibit the radical reaction. Sometimes, a free radical inhibitor is referred to free radical trap. A free radical inhibitor reacts with active radicals, forming relatively stable and inactive radicals.
In the present study, looking at the interaction of various chalcones and pyrazoline with DPPH 1a and 2a coded substances showed a good effect. Examining the structures of these substances, it was found that electron-releasing groups bind to pyrazoline substituent phenol and the phenyl ring. As is known, phenol radicals have given good results since they are stable, namely inactive radicals. Moreover, it has been found that they show a better effect when electron releasing groups such as –OCH3 bind to phenol ring.
It can be said that the radical formed when electron releasing groups (like -OCH3) bind to phenol ring gives better results because it is more stable compared to phenol radicals. That is –OH group in the phenyl ring reacts with radical (DPPH) which is highly active, forming more unstable phenol radicals. In a reaction, it is always the stable product which forms. Unstable, namely highly active products are not likely to be found in the medium. In the above reaction also phenol radical is much more likely to be found in the medium because it is more stable than DPHH* radical. Furthermore, the presence of electron releasing groups (-OCH3) gives the structure extra stability. It has been found that phenol ring substitute chalcone derivates give no good results with DPPH* radical (except for 2b). This may be attributed to conjugation in these structures. Namely, conjugation in the structure causes a decrease in electron intensity in the phenol ring. Stability of phenol radical decreases as electron intensity decreases. This will result in the emergence of more active radicals, and in turn, this will negatively affect the result.
The reason for better results with pyrazoline substituent phenol derivatives than chalcone substituent phenol derivatives is that conjugation is not scattered through all the structures in pyrazoline substituent phenol derivatives. Furthermore, -NH group is an electron releasing group. Thus, electron intensity is higher compared to chalcone substituent phenol derivatives. The result of our study also supports that pyrazoline substituent phenyl radicals are more stable than chalcone substituent phenyl radicals.
We also investigated in vitro effect of organic substances of MDA in addition to their free radical scavenging effect. Covalent binding of free radicals to the membrane receptors, changes polyunsaturated fatty acid/protein ratio, initiating lipid peroxidation. Lipid peroxides formed by this way are easily broken down, forming reactive carbon compounds with the most active being MDA. Therefore, measurement of the MDA amount reflects lipid peroxidation degree in the tissues. MDA leads to decreased molecular oxygen, causing the formation of superoxide anion and hydrogen peroxide, that have damaging effects on cell and tissues.
When in vitro effects of substance groups on LPO were compared, the amount of LPO significantly increased in all groups compared to the control in 10 µL study group. Although the amount of LPO significantly increased in parallel with increasing concentrations in FR, (1b-c) and 2c coded substances compared to the control, this increase was lower in reveretrol and 2b coded substances. 1a and 2a coded substances significantly decrease the amount of MDA. According to our results, we thought that high MDA value in the groups containing phenol reactive in vitro medium proves that FeCl2 and H2O2 substances show radical effect, increasing lipid peroxidation. The same effect may be thought to be effective in the groups with a high amount of MDA. 1a and 2a coded substances significantly decreased the amount of MDA, which can be explained by that organic substances show antioxidant activity, decreasing lipid peroxidation.
3.10. Effect of substance groups in anaerobic culture medium
When effects of the organic substances on biochemical parameters of Saccharomyces cerevisiae were studied, first we aimed to investigate the effects of these substances on lipid peroxidation in this microorganism. However, at the end of the experiment we conducted, it was found that lipid peroxidation products were of very low levels in yeast. It was concluded that this may have resulted from a high resistance of Saccharomyces cerevisiae against the radicals we used.
Martin et al. [20] reported that monounsaturated fatty acids in Saccharomyces cerevisiae and other yeasts consist of acyl CoA precursors that are saturated through Δ9 desaturase. In the same study, the authors found that desaturase gene OLE1 in Saccharomyces cerevisiae created a response to a part of different stimuli containing different carbon sources, metal ions, and oxygen levels. Because the composition of the membrane and fatty acids is influenced by environmental factors. In addition, the fatty acid composition of the cell may also be affected by the carbon source of the growth medium [21, 22].
Also, studies have stated that the fatty acid content of a cell may also show variability depending on the lipid content of the growth medium [23, 24].
In our study, we demonstrated that the substance groups used affected lipophilic vitamins and phytosterol synthesis in Saccharomyces Cerevisiae yeast at different ratios. Comparing the substance groups, phytosterols more significantly increased in 1a and 2c coded substances.
Studies have shown that different carbon, nitrogen sources, and the other compounds in the growth medium of Saccharomyces cerevisiae yeast affect cell development, and ergosterol synthesis [25, 26]. As a result of our study, we concluded that the significant increase in the amount of ergosterol in (2a-c), 1a substances compared to the control may be helpful for the studies conducted to increase ergosterol synthesis and thus we demonstrated that the present study is important in terms of biotechnology.
We observed that 1c coded substance decreased the level of ergosterol compared to the control. This decrease might have resulted from the substances added into the medium, and based on the same mechanism of action in imidazole and pyrazole containing antifungal drugs, it can be proposed that 1c coded substance may show antifungal effects.
Numerous new synthetics aimed to have antibacterial and antifungal effects have been synthesized in a laboratory setting and they were tested whether to have the desired properties in our country and worldwide [25].
In the present study, when effects of the organic substances on the amount of glutathione synthesized from Saccharomyces cerevisiae yeast; it was found that 1c coded substance increased the amount of glutathione, while (1a-b) and (2a-c) coded substances increased the amount of synthesized glutathione. We think the increase in 1c coded substances was resulted from that the yeast develops a defense mechanism against radicals, increasing the amount of glutathione and thus may provide adaptation against oxidative damages. This opinion is also supported by a study conducted by Izawa et al [26]. In their study, these authors reported that increased intracellular glutathione may be one of the adaptations created against H2O2.
Besides, Penninckx [27] found that Saccharomyces cerevisiae yeast synthesized glutathione as a response to different food sources and oxidative stress.
Decreases in the amount of glutathione in (1a-b) and (2a-c) coded substances may be attributed to the destruction of the radicals in an early stage.
When total protein amounts of Saccharomyces cerevisiae yeast in the media containing different substance groups were compared; (1a-b) and 2b coded substances decreased the amount of total protein, although (2a-c) and 1c coded substances increased the amount of total protein. In parallel to the increase of GSH levels in some groups, antioxidant enzyme concentrations also increase. These antioxidant enzymes are the enzymes such as catalase, GSH-Px, glutathione reductase, glutathione S-transferase, and SOD. When the expression of these enzymes is elevated, protein amount is also indirectly raised [28].