Protective properties of resveratrol in biological systems containing ethanol

Native plant-derived polyphenols are widely represented in nature and are used by humans. Resveratrol is such a substance. Its antimicrobial, antioxidant and other useful functions are being actively studied, which creates the prerequisites for the discovery of its new properties and the explanation of existing ones. The proposed study provides data on the interaction of resveratrol phytoalexin with a biological object - radish seeds. Plant seeds are biological objects that are very susceptible to external stress factors. In these studies, ethanol in concentrations from 0.5% to 12% was used as such a negative factor. Resveratrol in concentrations from 0.1% to 0.3% was used as a protective factor. The exposure of the germinated material was 64 hours. The criterion for the effectiveness of resveratol was the fact of seed germination. The data obtained prove a significant protective function of resveratrol. Thus, in environments with an alcohol concentration of 12% and a resveratrol concentration of 0.3% a significant increase in the germination of the studied seeds was noted. The data obtained allow us to simulate the behavior of resveratrol on biological systems, which may be useful for future studies with other biological objects. In recent years, there has been an actual increase in the need to examine the exact composition of plant raw materials and their tissues in order to obtain new preparations containing biologically active substances.


Introduction
The study of various plant raw materials makes it possible to obtain a group of valuable chemical compounds known as polyphenols [1,2,3]. Polyfinols include phytoalexins as subclass. They are a natural antibiotic that plants produce as a protection mean. Conducting the research into variety of subspecies of phytoalexins we can identify the most important: quercetin, resveratrol, luteolin, myricetin [4,5].
Natural phytoalexin such as resveratrol, which is released by some plants to protect against parasites. It is found in the skin and seeds of red grapes in the highest concentrations [6]. It is a very powerful its activity is much higher than that of vitamin E [7]. Resveratrol exhibits estrogenic and antioxidant activity, connecting to the same receptors as estradiol, one of the main estrogens of humans [8]. Even considering the fact that the hormonal activity of resveratrol is much lower than the  [9,10]. Even considering the fact that the hormonal activity of resveratrol is much lower than the estradiol (3-10 μM resveratrol corresponds to 0.1 nM estradiol). It may be present in the body in concentration that could affect even more strongly than estradiol in physiological concentrations [11,12].
Unfortunately, reliable experimental data concerning phytoalexin and stilbene groups remain poorly understood. This is about the degree of study of their ability to both absorb and generally interact with the human body. Research in this field is very important because it will help to create a reliable experimental base for the introduction of resveratrol and its derivatives into pharmaceutical and food products [12].
Various unicellular and multicellular objects, such as plant seeds, can be a basic medium for studying the effects of physiologically active components. They are very susceptible to external factors of exposure: lighting, stimulants and germination inhibitors, toxic compounds [13,14,15].
During the first germination phase, the cell wall of plants is particularly susceptible to foreign substances [16]. Some groups of substances are able to suppress cell growth, showing destructive effects on the metabolism of the whole cell [7].
It is known that ethanol is used as a solvent and antimicrobial agent. However, most vitamins and mineral species are suppressed by its disinfectant properties. In particular, vitamin C reduces its antioxidant activity to 73% in the presence of ethanol [17].
The question of the impact of resveratrol on the different functions of biological objects was interesting. In this paper, we investigated the effect of this substance on the germination function of agricultural seeds [18]. Each seed implies the presence of a complex structure, and the mechanism of its germination is due to many factors: the presence of moisture, temperature, illumination, etc. It is known that some compounds can serve as stimulators or growth inhibitors [19].
This paper also provides data on the protective properties of resveratrol in an unfavourable seed germination environment.

Materials and methods
The inhibitory agent was ethanol, which inhibits growth processes until complete processes [20]. It is also possible to determine the behavior of resveratrol in alcohol-containing products such as wine and beer [20]. This aspect is interesting in view of the fact that the skin of grapes of different varieties contains this compound in significant quantities. A substantial part of it will be transferred to the finished product.

Discussion of the results
The current experiment was conducted in the dark room conditions. All chemical glass equipment was subjected to high-grade heat treatment and had an exceptionally sterile surface. The seeds were placed in a Petrie dish on a filter paper substrate, after which the test (or control) solution was added to the sample.
Resveratrol was then added to each sample. The total amount of moisture per sample was 2 ml. The samples were then sealed and exposed for 64 hours. Every 4 hours of the process, the sprouted seeds in each sample were counted.
Although we had different approaches on creating the necessary combinations of samples and their percentage concentrations (distillated water with either liquid solutions of resveratrol or ethanol), studies should be limited to 36 samples (including one control sample), each containing 50 radish seeds. The studies produced a spectrum of data. It was found that the maximum and minimum germination times were substantially dependent on the component base of the solution. The sample with the highest alcohol content (12%) without resveratrol showed the longest time of 10% of seeds in the sample. At the same time, if resveratrol was present in the sample, this time decreased by 8 hours: resveratrol suppressed the inhibitory effect of alcohol (Figure 1).

Figure 1. The process of germination of all sample's combinations with resveratrol's and ethanol's concentration compound
It was also noted that resveratrol did not significantly affect the germination of seeds in the ethanolfree sample, hence it did not have intensive growth-stimulating properties, unlike protectors.
During the experiment, several photographs were taken that demonstrate the germination of radish seeds and the inhibitory effect of ethanol. Figure 2 shows photographs of the exposure process without adding resveratrol. The total exposure time was 64 hours. The most positive conclusion that we've got during the experiment was increasing the total germination at the all samples with addition resveratrol solution. With this result we can say about essential stimulation rendered for samples as we found into descriptions about native different resveratrol's properties.
In summary, if resveratrol shows its properties, is able to suppress to some extent the toxic effect of ethyl alcohol and allows seeds to germinate in greater quantities than in control samples (without resveratrol).
The total growth of the test seeds was stable, ranging from 16 to 100%. At the same time dependence between end-tracts of ethanol and resveratrol in the tested samples is established: effect of leakage properties of resveratrol appeared more strongly at higher concentration of ethanol. However, in order to talk about it with confidence, there were still a number of experiments to be put in and to understand most carefully in future research.
As a result of the experiments, we obtained a complex relationship consisting of combinations of ethanol-resveratrol to the percentage of germination. According to the data obtained, dependencies were formulated (Figure 4).  The analysis shows that with a maximum alcohol concentration of 12% and a maximum resveratrol concentration of 0.3%, the percentage of surviving grains is 16%, and in a similar medium without resveratrol only 3%. That is, seed survival increased 5.3 times.
The protective effect of resveratrol starts already at the concentration of ethyl alcohol 0.5%, which indicates its high biological activity in the medium. It is very sensitive to external stress factors.
It is noted that resveratrol at a concentration of 0.25% and 0.3% give similar results, this may indicate some limit value of concentration that has a pronounced protective effect. High concentration is not appropriate. This fact provides a prerequisite for justifying the use of resveratrol as a supplement to foods in technologically feasible concentrations.

Conclusion
As a result of the studies carried out, it was found that: 1. If we use resveratrol that obtained from the Sakhalin Highlander (Reynoutria sachalinensis) extract it confirms its antitoxic effect on radish seeds.
2. Phytoalexine resveratrol, in this study, reduced activity as a natural bioprotector and helped to reduce seed stress during germination by a factor of 5.3 at a total alcohol concentration of 12%.
3. The obtained data showed characteristic aspects of interaction of the system "seed-ethylenresveratrol." We determined resveratrol concentrations that stimulate seed germination better than others. This range can be shown with an interval of 0.25 to 0.3% resveratrol.
4. As a recommendation for further research, it would be necessary to continue studying resveratrol properties at other biological sites. This approach will help to obtain more information about this substance.