Investigation of biological activities and antioxidant effects of newly synthesized α-β substituted unsaturated ketone benzofuran derivatives

Effects of benzofuran-derived α - β unsaturated ketone-derived compounds, which are newly synthesized and not previously subjected to biological activity testing have been evaluated. The pour test compounds were determined for in vitro antioxidant activity using models namely 1,1-Dihenyl-2-picrylhydrazyl radical (DPPH * ) and Lipid Peroxidation (LPO) scavenging methods. Lipophilic vitamins and phytosterols analysis was carried out on an HPLC instrument. The fatty acids in the lipid extract were converted to methyl esters and then analyzed by gas chromatography (GC). Our results show that 1a, 2a, 2c coded substances used at 20 µL and 30 µL concentrations have more DPPH free radical scavenging activity than quercetin used as standard. Whereas the amount of LPO was signicantly decreased in Quercetin and Resveratrol groups and 1a coded substance compared to the control and FR groups (p < 0.0001). 1c coded substance partially increased the amount of GSH (p < 0.01). When effects of 1b, 2c coded substances on lipophilic vitamins and phytosterol prole in S. Cerevisiae yeast cell were examined; the amounts of α-tocopherol, δ- tocopherol, vitamin K 1 , and Stigmasterol increased in all groups compared to the control. When effects of 1c, 2a coded substances on lipophilic vitamins and phytosterol prole in S. Cerevisiae Yeast Cell were examined; the amounts of α- tocopherol, vitamin K 1 , and Stigmasterol increased compared to the control group.


Introduction
The benzofuran ring system itself is a common structural element that appears in a large number of medicinally important compounds [1]. Benzofuran neolignans and nor-neolignans, which are contained in most plants, have attracted much attention in medicinal chemistry for their wide range of various biological activities including insecticidal, fungicidal, antimicrobial, and antioxidant properties [2].
Chalcones and their derivatives show signi cant biological activities which are very important and helpful in drug designing due to the presence of double bonds in conjugation with the carbonyl group. Chalcones are obtained by the reaction of aromatic aldehydes with aromatic ketones. It has been demonstrated that chalcones have biological activity in a wide range of antibacterial, anticancer, antifungal, antiin ammatory, antituberculous, and antioxidant activity [3,4].
In this study, antioxidant effects of certain benzofuran substituted α -β unsaturated ketone derivative compounds which were synthesized newly and were not subjected to any biological activity test before was investigated in vitro with Saccharomyces cerevisiae, in the anaerobic culture medium. In the study lipid peroxidation (LPO), fatty acid levels, lipophilic vitamin values, protein and glutathione concentrations, and MDA levels were measured. For this purpose, Fenton's reagent (FR) and the organic substance-containing groups were used as the control.

Experimental study plan
The biological activity of in vitro newly synthesized organic substances that were not subjected to any experiment before was investigated. The organic substances have been synthesized in Fırat University, Science Faculty, Organic Laboratory of Chemistry Department. First, DPPH method was used to measure free radical scavenging activity of the organic substances. In later studies, yeast which is a single-cell eukaryotic organism (saccharomyces cerevisiae) was used. Yuva brand fresh yeast was supplied from the market, and stock cultures were formed to be used as sterile in the experiment in the laboratory setting. The organic substances were added into the liquid yeast culture medium, and the activity of these substances was studied with GSH, protein, MDA, fatty acid, and vitamin analysis.
The open formulas of benzofuran derivative organic substances investigated for biological activity in this study are as follows: 2.2. Free radical (DPPH) scavenging activity DPPH free radical scavenging activity was examined by the method described [8]. 25 mg/L α, α-Diphenylbpicrylhydrazyl (DPPH) prepared in methanol was used as a free radical. First, 0.002 g was weighed from each organic substance, dissolved in 1 mL DMSO, and taken to the Eppendorf tubes. We added 4 mL from the prepared DPPH solution was added to the glass tubes, we have taken 10 µL, 20 µL, 30 µL from each organic substance in the Eppendorf tubes, respectively, and added on DPPH solution. The mixture was left to incubation for 30 minutes in a dark environment at room temperature, and following the incubation, their absorbances were read against blank at 517 nm with a spectrophotometer [9].
Decreased absorbance, the remaining amount of DPPH was de ned as the free radical scavenging activity.
The results were calculated using the following formula:

Determination of MDA-TBA level
Five different groups were used to determine lipid peroxidation in vitro. The groups were divided as follows Lipid peroxides (TBARS) in tissue homogenate were estimated using thiobarbituric acid reactive substances by the method of Okhawa et al [25]. 0.5 mL of 8.1 % SDS, 1.0 mL of (20 % acetic acid/NaOH pH 3.5), 1.0 mL of 10 % TCA, 50 µL of 2 % BHT and 1.0 mL of 0.8 % TBA were added to 1.0 mL tissue homogenate. The mixture was heated in a water bath at 95 ℃ for 45 min. After cooling, 3 mL of nbutanol / pyridine mixture was added and shaken vigorously. After centrifugation at 5000 rpm for 7 min, the organic layer was taken and its absorbance was measured at 532 nm. 1.1.3.3-tetra methoxy propane was used as standard. The resulting nmol MDA/g tissue was calculated.

Determination of antioxidant and antiradical activities in growth media of Saccharomyces Cerevisiae
For this purpose; the rst YEDP (1 g yeast extract, 2 g bactopepton, 2 g glucose for 100 mL) medium was prepared for the development and growth of saccharomyces cerevisiae FMC 16 to be used in the experiment [10]. After the medium was prepared, it was divided into the following groups: After sonication, supernatant sections of samples centrifuged at + 4 ºC for 9 minutes at 9000 rpm were plucked to determine glutathione and protein contents. The amount of fatty acid in the medium was determined by adding hexane/isopropanol to the remaining pellet portion.
Reduced glutathione (GSH) was determined by the method of Ellman [11]. Brie y, 1 mL tissue homogenate was treated with 1 mL of 5 trichloroacetic acids (10 %) (Sigma, St. Louis, MO), the mixtures were centrifuged at 5000 rpm and the supernatant was taken. After deproteinization, the supernatant was allowed to react with 1 ml of Ellman's reagent (30 mM 5, 5'-dithiobisnitro benzoic acid in 100 mL of 0.1 % sodium citrate). The absorbance of the yellow product was read at 412 nm in a spectrophotometer. Pure GSH was used as the standard for establishing the calibration curve [12].
The total protein content of Saccharomyces cerevisiae was determined as described by Lowry. The procedure for measuring protein has followed the method by Lowry et al [13]. Using BSA (Bovine serum albumin) as standard. The absorbance was read at 750 nm using a spectrophotometer.
Lipid extraction of tissue samples made with hexane-isopropanol (3:2 v/v) by the method of Hara and Radin [14].

Preparation of oil acid methyl esters
To carry out the gas chromatographic analysis of the fatty acids present in the lipids, it is necessary to convert them to derivatives such as methyl esters which have a non-polar, volatile, and stable structure. Different methods have been used to convert fatty acids in lipids to derivatives such as methyl ester [15].
As stated, we used the acid-catalyzed esteri cation method, which is practical and highly e cient in its application and described below: To prepare the methyl esters, the lipid extract in the hexane/isopropanol phase was taken up in 30 ml of non-leaching test tubes. 5 ml of 2 % methanolic sulfuric acid was added and thoroughly mixed with vortex. The mixture was left to methylate at 50°C for 15 hours. After 15 hours, the tubes were removed from the oven and cooled to room temperature and 5 ml of 5 % sodium chloride was added and mixed well. The fatty acid methyl esters formed in the tubes were extracted with 5 ml of hexane and the hexane phase was pipetted upwards and treated with 5 ml of 2% KHCO3 and left for 4 hours to separate the phases. The mixture containing the methyl esters was then evaporated at 45 ° C and under a stream of nitrogen, dissolved in 1 ml of hexane, and analyzed in a gas chromatograph under 2 ml capped autoclaves.

Gas chromatographic analysis of the fatty acid methyl esters
The fatty acids in the lipid extract were converted to methyl esters and then analyzed by SHIMADZU GC 17 gas chromatography. For this analysis, 25 m in length, 0.25 µm. Machery-Nagel (Germany) capillary column with an inner diameter of 25 microns and PERMABOND 25-micron lm thickness was used.
During the analysis, the column temperature was 120-220 ºC, the injection temperature was 240 ºC and the detector temperature was 280 ºC. The column temperature program was set from 120 ºC to 220 ºC.
The temperature increase was 5 ºC / min for 200 ºC and 4 ºC / min for 200 ºC to 220 ºC. 8 minutes were kept at 220 ºC and the total duration was determined as 35 minutes. Nitrogen gas was used as carrier gas. Before the analysis of the fatty acid methyl esters of the samples, the retention times of each fatty acid were determined by injecting mixtures of standard fatty acid methyl esters. After this process, necessary programming was performed to analyze mixtures of fatty acid methyl esters of the samples 2.7. Analysis of the amounts of vitamins ADEK and Cholesterol in the Yeast Cell 5 mL supernatant was placed in 25 mL lid-capped tubes and 5 % KOH solution was added. After vortexing, it was held at 85°C for 15 min. The tubes were removed, cooled to room temperature, and 5 mL of puri ed water was added and mixed.
The non-saponi ed lipophilic molecules were extracted with 2 x 5 ml of hexane. The hexane phase was evaporated with a nitrogen stream. The molecules were then dissolved in 1 mL (50 % + 50 %, v/v) acetonitrile/methanol mixture and taken into autosamplers and analyzed.

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 signi cant 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 signi cant 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).

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 signi cantly 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 signi cantly 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 signi cant decrease compared to FR group (p < 0.0001).
LPO was signi cantly 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 signi cantly 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 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 signi cantly 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 signi cant 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 ( 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 signi cant 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 signi cantly increased the amount (p < 0.0001), while 1c and 2b coded substances showed no statistically signi cant difference (p > 0.05).
3.5. Effects of the (1a-b, 2c) coded substance groups on the fatty acid pro le of S. Cerevisiae yeast cell When effects of the (1a-b, 2c) coded substance groups on the fatty acid pro le 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 signi cant in 1b coded substance (p < 0.01). The amount of palmitic acid (16:00) signi cantly increased in all three groups compared to the control (p < 0.001), and this increase was more signi cant 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 signi cant increase was found in 1a coded substance (p < 0.001).
The amount of arachidic acid (20:0) signi cantly increased in all three groups compared to the control (p < 0.001), and the most signi cant increase was found in 1b coded substance (0.0001). The amount of palmitoleic acid (16:1). Signi cantly decreased in all three groups compared to the control (p < 0.001), and the most signi cant 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 signi cant increase was found in 2c coded substance (p < 0.0001). The amount of oleic acid ((18:1) n9) signi cantly 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 signi cant 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 signi cant 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 pro le of S. Cerevisiae yeast cell When effects of (2a-b, 1c) coded substance groups on the fatty acid pro le 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 signi cant 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 signi cant decrease was in 1c coded substance (p < 0.001). The amount of palmitic acid (16:0) signi cantly increased in (2a-b) coded substances (p < 0.0001), although the amount signi cantly 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 signi cant decrease was found in 1c coded substance (p < 0.0001). Whereas no statistically signi cant difference was found in 2b coded substances (p > 0.05). The amount of palmitoleic acid (16:1) signi cantly increased in all three groups compared to the control (p < 0.001) with the most signi cant 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 signi cant decrease among the groups was in 1c coded substance (p < 0,0001). The amount of elaidic acid ((18:1) n9t) signi cantly increased in (2a-b) coded substances compared to the control (p < 0.0001), although it was signi cantly decreased in d6 coded substance (p < 0.0001). The amount of oleic acid ((18:1) n9c) signi cantly 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 pro le of S. Cerevisiae yeast cell When effects of ((1a-b, 2c) coded substances on lipophilic vitamins and phytosterol pro le in S. Cerevisiae Yeast Cell were examined; the amounts of α-tocopherol, δ-tocopherol, vitamin K 1 , and Stigmasterol increased in all groups compared to the control. The most signi cant 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 signi cant 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 signi cant increase in the amount of vitamin K 1 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 signi cant increase was found in 2c coded substances (p < 0.0001). The amounts of ergosterol and βsitosterol were not statistically signi cant in 1b coded substance compared to the control (p > 0.05), while these amounts signi cantly increased in 1a and 2c coded substances (p < 0.0001). The amount of vitamin D 2 increased in (1a-b) coded substances compared to the control, and the most signi cant increase was found in 2c coded substance (p < 0.01). Whereas this amount signi cantly 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 pro le of S. Cerevisiae Yeast Cell When effects of (2a-b, 1c) coded substances on lipophilic vitamins and phytosterol pro le in S. Cerevisiae Yeast Cell were examined; the amounts of α-tocopherol, vitamin K 1 , and Stigmasterol increased compared to the control group. The most signi cant increases in amounts of α-tocopherol Stigmasterol were found in 2a coded substance (p < 0.0001), although the most signi cant increase in the amount of vitamin K 1 was found in 2b coded substance (p < 0.0001). The amounts of ergosterol and vitamin K 2 partially increased in (2a-b) coded substances (p < 0.05), although these amounts decreased in 1c substance group (p < 0.01). The most signi cant increase in the amount of ergosterol was observed in 2b coded substance (p < 0.0001), while the most signi cant increase in the amount of vitamin K 2 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 D 2 partially increased in (2a-b) coded substances (p < 0.05), although this amount partially decreased in 1c coded substance (p < 0.05).

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 signi cantly 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.
[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 signi cantly 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 -OCH 3 bind to phenol ring.
It can be said that the radical formed when electron releasing groups (like -OCH 3 ) 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 (-OCH 3 ) 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

Effect of substance groups in anaerobic culture medium
When effects of the organic substances on biochemical parameters of Saccharomyces cerevisiae were studied, rst 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 Δ 9 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 in uenced 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 signi cantly 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 signi cant 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 H 2 O 2 .
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 Stransferase, and SOD. When the expression of these enzymes is elevated, protein amount is also indirectly raised [28].

Conclusions
In summary, biological activities of the newly synthesized organic substances were investigated both in vitro and in anaerobic culture medium containing Saccharomyces cerevisiae yeast and the obtained results were summarized below: DPPH free radical scavenging effect was investigated in vitro, and it was found that 1a and 2a coded substances showed an effective antioxidant effect, while 1c coded substance had more limited antioxidant effects compared to 1a and 2a coded substances.
Comparing effects of the substances on LPO in vitro medium; 1a and 2a signi cantly decreased the amount of MDA. These decreases can be explained by that 1a and 2a coded substances showed an antioxidant effect, decreasing lipid peroxidation and supporting that 1a and 2a coded substances showed strong antioxidant effects in parallel with the results of the study conducted on DPPH free radical scavenging effects.
When effects of the organic substances on biochemical parameters of Saccharomyces cerevisiae yeast were investigated; rst we aimed to examine the effects of these substances on lipid peroxidation in this microorganism. However, in the experiment performed, levels of lipid peroxidation products were found to be much low in the yeast. We concluded that Saccharomyces cerevisiae yeast might create a high resistance against the radicals we used.
In our study, we observed that substances coded (1a-b) and (2a-c) shown a remarkable level of increase in the amount of ergosterol compared to the controls (p < 0001) which may assist the studies of increasing the synthesis of ergosterol from yeast, supporting the importance of our study for biotechnology.

Figure 1
Chemical reaction mechanism of benzofuran derivative organic synthesis Antioxidant effects of substance groups on lipid peroxidation in vitro medium