The Caucasian flora: a still-to-be-discovered rich source of antioxidants

Cellular redox homeostasis is a state of balance between the formation of Usually Reactive Oxygen and / or Nitrogen Species (ROS/RNS), endogenous antioxidant defence systems, and exogenous dietary antioxidants. The disturbance of redox homeostasis, by the overproduction of endogenous ROS/RNS, may increase the risk of development of so-called civilisation diseases. The solution seems to be either the increased production of endogenous or consumption of exogenous antioxidants. Plant-borne antioxidants act via different chemical and molecular mechanisms, such as decreasing the level of oxidative damage in cells directly by reacting with ROS/ RNS or indirectly – by inhibition of the activity and expression of free radical generating enzymes or by enhancing the activity or expression of intracellular antioxidant defence enzymes. Despite the fact that the Caucasian flora is rich of health promoting edible/medicinal plants, recent studies concerning the biological activity of these plants are very scarce. This review is summarising the state-of-art on the health-promoting potential of plants representing the Caucasian flora, whose antioxidant capacity have been investigated in various in vitro models


The significance of redox homeostasis
In 19 th century, the French physiologist Claude Bernard (1813-1878) pointed out the relevance of maintenance of "the milieu interior" or "the interior environment," as important for good health of every living organism.The maintenance of intracellular homeostasis may be a way to prevent or even cure some diseases [1].This concerns also redox homeostasis, which can be disrupted easily by reactive species derived from oxygen (Reactive oxygen species -ROS), Nitrogen (Reactive Nitrogen Species -RNS), Sulphur (Reactive Sulfur Species -RSS), or Selenium (Reactive Selenium Species-RSeS) [2][3][4][5].
Reactive species are produced in different parts of the cell (e.g.plasma membranes, chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum, apoplasts, and cell walls) under both normal and stress conditions (e.g.microbial infections, extensive exercise, or the influence of pollutants/toxins, different types of radiation) [6].Mitochondria represent one of the major endogenous sources of oxidising agents with their mitochondrial electron transport chain and NADPH oxidase (NOx) reaction.Nonmitochondrial sources of free radicals include microsomal cytochrome P 450 enzymes, the Fenton reaction, peroxisomal beta-oxidation and the phagocytic cell respiratory burst [7].In some cases, cellular "redox cascades" are also triggered, resulting, for instance, in the formation of RSS [2,3].Overproduction of reactive species evokes the stressful status of cells commonly referred to as "Oxidative Stress."(OS).
However, ROS play a dual role in cell metabolism, depending on their concentration.At low or moderate concentrations, they serve as secondary messengers in intracellular signalling cascades, whereas at high concentrations, they may cause different kinds of damage to biomolecules [8].Not surprisingly, healthy cells maintain an equilibrium between the formation and utilisation of ROS/RNS which can be disturbed by the formation of reactive species, which in time results in enhanced of cell damage [9].
Redox homeostasis in the organism can be re-established by the action of intracellular antioxidant molecules, proteins and enzymes, and also by the intake of dietary antioxidants [10].In tissues, both cells and the extracellular matrix respond to an insult of ROS by activating multiple additional internal enzymatic defence mechanisms, which assist in the quenching of ROS and their derivatives.The non-enzymatic antioxidants are represented by molecules, which possess the ability to inactivate different reactive species.Together with small molecule antioxidants, metal scavengers, and redox active proteins, they provide a first line of defence against ROS [11].
Mammalian antioxidant defence systems are not limited to endogenous antioxidants [12].Dietary antioxidants such as vitamins (vitamin E and vitamin C) carotenoids, polyphenols (flavonoids, phenolic acids, lignans and stilbenes), and some minerals (Zn, Se, Mn and Cu) can affect the activity of endogenous antioxidants.Endogenous and exogenous antioxidants may act synergistically in order to maintain or re-establish the redox homeostasis of the organism [13].These considerations lead us to the question, if a balanced diet composed of the "best ingredients" found around the globe may be helpful to prevent civilisation diseases.

Plant-derived antioxidants
Plants contain various antioxidant mechanisms to maintain their own redox homeostasis.Similar to animal cells, plant cells can produce both, enzymatic and nonenzymatic antioxidants.Catalases, superoxide dismutase (SOD), peroxidases, and some other enzymes, which are included in the ascorbate-glutathione cycle, such as ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, belong to the enzymatic antioxidants found in plants [8].These enzymes are supported by nonenzymatic plant antioxidants, such as ascorbate, glutathione (GSH), carotenoids, tocopherols, anthocyanins, and diverse phenolic compounds [14].In fact, plant-based antioxidants are highly effective in controlling the level of ROS/RNS as they can also modulate enzymatic activities [15].
The most abundant group of plant-derived substances with antioxidant properties are polyphenols [16,17].In order to evaluate their antioxidant activity, a range of experimental models are commonly employed, from simple chemical methods (e.g.ferric reducing antioxidant power (FRAP), 2,2 0 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) tests) through biologically more relevant cellular-based assays, such as the cellular antioxidant activity assay, to the most accurate animal models and clinical trials in humans.In vitro studies are common, because they are relatively simple, fast and inexpensive, still these studies do not consider metabolic, biochemical, and other physiological parameters [17,18].In vivo studies are performed mainly in prokaryotic and eukaryotic cells or laboratory animals and studies involving clinical trials remain scarce [18].
Simple chemical assays are most popular for the preliminary evaluation of antioxidant activity of plant extracts and their bioactive constituents.The majority of the in vitro tests represents redox-linked colourimetric assays.Polyphenols provide an ideal chemical structure for scavenging free radicals because of their phenolic hydroxyl groups, which can donate a hydrogen atom or an electron to a free radical.Furthermore, they can also count on an extended conjugated aromatic system to delocalise unpaired electrons [19].
Nonetheless, the in vitro and in vivo effectiveness of polyphenols as antioxidants is still a matter of debate, and, moreover, human studies on this topic are extraordinarily scarce, also when compared to those in animals.Another problem is the bioavailability of polyphenols.Their potential health benefits in humans and in animal models depend on absorption, distribution, metabolism, and elimination.The most common polyphenols present in the human diet are not necessarily the most active within the body, either because of their lower intrinsic activity or because they are poorly absorbed from the intestine, highly metabolised, or eliminated rapidly [20,21].
Despite these apparent drawbacks, diets rich in antioxidants are frequently the focus of epidemiological studies, which aim to correlate certain dietary habits and local peculiarities with the maintenance of health and prevention of certain diseases.Nowadays, these studies are often motivated by demographical changes or ageing of the population [22].The "NutRedOx" network (EU COST Action 16112) is one fine example of this approach of "healthy living and ageing via healthy lifestyle and healthy eating" [23,24].The factsand mythssurrounding the famous "Mediterranean Diet" is another story [25][26][27][28].Interestingly, the focus on such local diets often moves or follows behind the global market, which, in theory, enables the design of particularly attractive new diets with selected ingredients from across the world.It is therefore hardly surprising that culinary and scientific experts are nowadays increasingly joining forces to sampleand investigatediets and dietary ingredients from various places.

The botanical diversity of the Caucasian flora
Within this context, the Caucasus, and here regions in Armenia and Georgia appear to be particularly rich in such health promoting edible or medicinal plants.
Armenia is positioned at the junction of several biogeographical zones.These areas are characterised by amazing botanical diversity, with approximately 3600 plant species in Armenia alone.In Armenia, these biogeographical zones are closely linked, which results in relatively few endemic species of Armenian flora.Overall, 123 endemic plant species are described, a fraction which represents just 3% of the total Armenian flora, which overall is very rich in plants with pharmacological significance [29].These plants are commonly employed in traditional medicine for the prevention and treatment of various diseases from Amirdovlat Amasiatsi(XX-YY), the 15 th century Armenian physician, for instance, demonstrated a preference for wild-growing plants as a source of drugs.He recommended the use of saffron, mandrake and hashish as pain-relieving and sleepinducing drugs [30,31].Despite this promising botanical richness, there is only limited literature published in recent years related to the biological activity of plants belonging to the Armenian flora.

Armenian grapes and wine production
Among the Armenian plants described as being rich in antioxidants, grape (Vitis vinifera) features prominently.Armenia is considered a homeland of viticulture and earliest "wine culture".Recent archaeological excavations in the Areni-1 cave complex (South-Eastern Armenia) (Latitude: 39.730361 N Longitude:

45
.203722 E) have revealed important artefacts and installations, which suggest wine production dating back to around 4000 BC [32].Armenian grape cultivars were selected traditionally for thousands of years and the resulting variability of hybrids was extended further by cross-breeding.Currently, viticulture is one of the most important branches of the Armenian agriculture and the production of wine and brandy provides an important contribution to the country's economy.In three ampelographic collections, 140 grape cultivars are preserved, among them 125 are local accessions and 15 are varieties of foreign origin.Around 70 local varieties are old autochthonous cultivars.The areas of distribution of wild grapevine (Vitis vinifera ssp.sylvestris) in Armenia have recently declined sharply.Still, many wild populations are still growing in Southern Armenia [33].

Antioxidant activity of grape cultivars
In recent years, polyphenols present in such grapes have been of scientific and applied interest.The grape skin and seeds both are rich in phenolic compounds and flavonoids.Their content in grapes depends on the variety of grapevine and is also influenced by viticultural and environmental factors, such as altitude and soil conditions; Aroutiounian, et al. for example, evaluated the total phenolic content and antioxidant properties of a large number of Armenian aboriginal varieties, interspecific and intraspecific hybrids and wild species with different genetic background and geographic origin (Table 1) [33].
The results on Armenian grapes are also reflected by grapes grown in neighbouring Georgia.Here, 14 samples of dry grapes, titrated acidity, and active acidity of five species of white grapes, i.e.Tsolikouri, Tsitska, Klarjula, Krakhuna, and Kutaturi, were studied, spread across three regions of Western Georgia -Adjara, Imereti, and Samegrelo [34].Different spectrophotometric assays were employed to determine the total phenolic and total flavonoid content and antioxidant activity (Table 2).Due to the variety of grapes, differences between the content of biologically active compounds on the one side and the activities in the antioxidant activities on the other were observed.These parameters were directly proportional and the studies were therefore able to identify specific varieties of grapes, such as the Tsolikouri and the Tsitska, grown in Opcha (Imereti) and Keda (Adjara), as particularly high in antioxidant activity [35].

Antioxidant activity of wine
The antioxidant activities of grapes are reflected in corresponding activities of wine.The content of phenolic compounds and antioxidant properties of wines produced of five varieties of white grapes were studied in Adjara, Imereti, and Samegrelo.A range of phytochemicals, including (-)epicatechin, quercetin-3-glucoside, quercetin-3-ramnoside, quercitrin-3-glucuronide, and procyanidin B2 were identified by ultra performance liquid chromatographymass spectrometry (UPLC-MS).With the help of the spectral methods, the total amount of phenols, catechins, and flavonoids was defined in the wines of all five varieties of grapes and their antioxidant properties were established (0.01 mM DPPH 50% inhibition).It was observed that wines produced from Tsolikouri and Tsitska grapes contain high amount of phenolic compounds and are characterised by antioxidant activity [35,36].
Together, the research conducted on wines and grapes grown and produced in Armenia and Georgia revealed apparent differences among the cultivated varieties and wild species with respect to their total phenolic content and antioxidant activity [37,38].These differences imply that biological activity, and not simply taste, may represent an important "seal of quality" for these natural products and produce.Notably, the high antioxidant activity in grapes is not limited to juice.Skin and seeds are also rich in antioxidant components, and rather than "going to waste" by vermicomposting, could be turned into valuable new products for the food industry, for instance in form of additives and baking [39,40].Additionally, medical and also agricultural and cosmetic applications may be considered [41,42].

Antioxidant activity of plant extracts
Another medicinally important family of Armenian plants on which there is some scientific literature concerning antioxidant activity is represented by the Lamiaceae family.Hayrapetyan et al. demonstrated that the antioxidant activity of Thymus serpyllum L. depends on the altitude of cultivation [43].The highest amount of antioxidants was found in ethyl acetate extracts of thyme growing in the Vorotan Gorge (14% w/w, 650 m a.s.l.), while the same activity of T. serpyllum growing in other places, such as the old city of Goris (2% w/w, 1250 m a.s.l.), the village Verishen (5% w/w, 1600 m a.s.l.), the gorge in the village Brun (7% w/w, 1700 m a.s.l.), the gorge in the village Chndzoresk (13% w/w, 1350 m a.s.l.), the gorge in the village Tegh (4% w/w, 1200 m a.s.l.), and the village Gorayk (3% w/w, 2400 m a.s.l.) was lower [43].These findings are particularly stimulating as they demonstrate that antioxidant content is not simply a property of a given plant species, it also depends decisively on a number of environmental factors, from the temperature, humidity, and soil to altitude.In this particular case, the plants grown at higher altitude were frequently lower in antioxidants (Figure 1).Such a correlation may be due to the fact that the conditions of growth at higher altitude are generally harsher and plants therefore produce less secondary metabolites, including antioxidants.Then again, these plants are also exposed to higher stress levels and this, in turn, may also increase the production of protective substances, explaining that certain plants found at higher altitude are actually more stress resistant and also richer in certain phytochemical than their counterparts found at lower altitudes.
Similarly, in a study conducted in neighbouring Georgia, several plants plants including Elaeagnus L., Prunus spinosa L., Iwasaki, Mukoiyama and Tiaxara Unshiu were chosen to determine the content of the phenols in the Georgian flora.It turned out that the highest amount of phenols and antioxidant activity was recorded at 120 m a.s.l., and was relatively low at 80 m a.s.l.The amount of phenols in silverberry (Elaeagnus L.) differed with the altitude from the sea level between 1863.3 mg/100 g to 2336.59 mg/100 g (fresh berry).Antioxidant activity was observed to vary around 16.4 mg (50% inhibition of 0.1 mM DPPH solution is produced).In another plant endemic to Georgia, P. spinosa L., the amount of phenols again differed according to the area of cultivation and ranged from 331.0 mg to 114.9 mg/100 g.Antioxidant activity was somewhere around 25.11 mg.As for Georgian tangerines, the total amount of phenols varied between 0.364 mg and 0.474 mg/ml (in juice).The highest value radical scavenging activity among the samples was found in the tangerines Tiaxara Unshiu (80.05 mg) and Iwasaki (74.45 mg), with lower activities in samples of Mukoiyama and Clemenules, that is, 79.9-191.72 mg (Figure 2) [44,45].
Once again, these findings underline the impact of environmental factors on the composition and activity of such plants.Armeniaand its neighbour Georgiaare Caucasian countries with varying altitude, and height clearly matters.
Similarly, plants of the genus Artemisia possess excellent biological activities, such as antioxidant, antimicrobial, anticancer and are employed widely in medicine, cosmetics and food production.Around 300 species of this genus have been reported and 16 are described as part of the Armenian flora [48].Thus, Babayan et al. were able to demonstrate that Artemisia vulgaris L., A. fragrans Willd., A. absinthium L., and A. splendens Willd.collected from the Aragatsotn region (1500-1600 m a.s.l.) during the flowering period displayed high antioxidant potential, although the radical scavenging activity depended on the specific conditions of the extraction method [49].Vardapetyan et al. presented a comparative analysis of phenolic component and radical scavenging activity of extracts of Crataegus laevigata, Plantago major, and Artemisia absinthum plants growing in Armenia.The authors demonstrated a direct relationship between the content of quercetin and the antiradical activity of selected ethanol extracts obtained from these plants [50].
Climatic and environmental changes also increase stress levels in plants, including the formation of a variety of free radicals, which plants have to deal with in order to survive.Since the chemical composition, that is, chemotype, and biological activity of substances obtained from the plants belonging to the same species may vary significantly, depending on the variety of cultivars, environment, elevation, and cultivation methods, it is essential to study examples of these plants growing in other regions.For instance, according to Avetisyan et al., the dominant constituent of Ocimum basilicum var.purpureum (purple basil) growing at high altitude Armenian landscape (1600 m a.s.l.) is methyl chavicol (estragole), whereas the major component for the other variety of the same species, O. basilicum var.thyrsiflora (Thai basil) is linalool [51].At the same time, the chemical composition of O. citriodorum hybrid plant differed from the first two basil varieties, grown at the same area.This variety contains a significant amount of the aldehyde citral, with another prevalent constituent being nerol, a monoterpene alcohol.The authors stated that essential oil from the O. basilicum var.thyrsiflora displayed the highest ability to neutralise free radicals.The relevant chemical structures of these important phytochemicals are provided in Figure 3.
To recapitulate, a major share of plants found within the Armenian flora exhibits considerable antioxidant potential.Nonetheless, these plantsand their antioxidant potentialstill have not been investigated fully.They should be considered as part of more comprehensive investigations to identify particularly promising plants with equally promising antioxidant activities and to identify the potential mechanisms and modes of actions associated with them.

Conclusions
Since the turn of the Millennium, a considerable amount of scientific data has been published concerning the antioxidant potential of plants.Some researchers suggest that approximately 60% of plant species investigated could possess medicinal value, including a large number of plants displaying antioxidant activity.
Plants are able to synthesise and accumulate enzymatic antioxidants during their metabolism.Among these substances, tocopherols, ascorbic acid, glutathione, poly-phenols, and other low molecular weight componentswhich act via different mechanisms on and in the human bodytake the centre stage.Almost all plants studied demonstrate some antioxidant activity in in vitro assays.Still, due to the complexity of biological systems, such activities may not translate directly into action within more complex organisms.In fact, antioxidants of plant origin may act via multiple mechanisms, there could be issues related to the limited bioavailability, and the presence or absence of transition metal ions and co-antioxidants may also interfere.As a consequence, information obtained in in vitro assays may not be extrapolated directly to in vivo studies as plant-originated antioxidants have to pass through a number of physiologicalmetabolic processes and interact with co-antioxidants and transition metal ions.
As the investigations of different varieties of grapes and thyme in Armenia and Georgia have demonstrated, the composition of antioxidants and activities associated with them may vary considerably, between different subspecies and as a result of environmental factors, including altitude.
Despite the considerable amount of literature available today, there isliterallya vast field to cultivate, in research, nutrition, agriculture, and medicine.Such studies need to focus on the detection of new plants with antioxidant potential, including those from the Caucasian landscapes.At the same time, the mechanisms, modes of action, and applications under both, in vitro and in vivo conditions, have to be considered.

Figure 1 .
Figure 1.The influence of altitude on antioxidant capacity of the plants from the Lamiaceae family (sketch was provided by prof.C. Jacob).

Figure 2 .
Figure 2. The influence of altitude on antioxidant capacity of the plants from the Lamiaceae family (sketch was provided by professor C. Jacob.

Table 1 .
Content of total phenols in Armenian grapes.

Table 2 .
Content of phenols in Georgian grapes.