Phytochemical Study of Endemic Species Helleborus Caucasicus and Helleborus Abchasicus

The floristic region of Adjara represents “Hotpoint” of Caucasian which is distinguished with the uniqueness of its it relict Colchis flora.It represents one of the most powerful refuge in western Eurasia , which is not touched by the chilling because has the special geographical location. There are 176 endemic plants spread in southern Colchis, of which 45 ones can be used for some medical treatments. The bioecology and detailed phytochemical content of some medicinal plant populations have not been studied so far. The research objective is to study the phytochemical content of endemic species of Helleborus caucasicus and Helleborus abchasicus spread in southern Colchis. The research method for the phytochemical content included the separation analysis, which was performed by using UPLC-MS (Waters Acquity QDa detector). Three Steroidal glycosides were isolated from the MeOH extract of the plants of Helleborus caucasicus and Helleborus abchasicus: Hellebrigenin-D-glucose, 20 – Hydroxyecdysone and Hydroxyecdysone – 3 glucoside. Three Steroidal glycosides and Hydroxyecdysone -3 glucoside have been isolated from the MeOH extract of Helleborus caucasicus.


Introduction
The floristic region of South Kolkheti (Adjara) isthe part of the Caucasus Ecoregion, which is includedamong the 200 world-renowned ecoregions by the World Wildlife Fund (WWF). These ecoregions are characterized by plant diversity, high levels of endemism, taxonomic uniqueness and rarity of biomes globally [1].
Southern Kolkheti (Adjara), in the Caucasus ecoregion, is characterized by the special diversity and originality of the flora, which is present due to the flora complexes rich in plant clusters and relict, and endemic species formed in the third period [2].
1837 species of plants are common in southern Colchis, including 176 endemic ones [3]. Among the endemics, the following genera are distinguished by their decorative and medicinal properties: Helleborus caucasicus and Helleborus abchasicus flowers in winter-early spring [4]. The genus Helleborus is represented by 2 species: Helleborus caucasicus and Helleborus abchasicus [5].
Helleborus caucasicus and Helleborus abchasicus (Ranunculaceae) are evergreen, blooming in autumn-winter-spring seasons, rooted, herbaceous plants, growing on cliffs. Their vegetation begins at the end of November, blooming starts in December, fruiting is in progress in March-April. Among these species, Helleborus caucasicus and Helleborus Abchasicus are widely distributed. Helleborus caucasicus is an important source of chemical compounds with the great medical potential for the treatment of some serious diseases. Glycosides, buffadienolides, monocytes, biocides and steroid saponins are found in its roots and rhizomes. Among them 0.1 percent of colerborine P which has an affect similar to stroftineon the heart. Colerborine P is used for circulatory disorders of quality II and III, most often in chronic heart failure. This has a particularly long and fast effect. In folk medicine in Adjara, the decoction of the root and rhizomes of Helleborus is used taking into account the dosage due to its toxic properties (1/2 teaspoon of roots in 0.5 l of water) for the treatment of cancer, hemorrhoids, cough, pleurisy, tuberculosis, purulent wounds, dandruff, diseases of the joints, diabetes, urological diseases, diseases of the liver, nervous system and kidneys; it is also used to lose weight [6][7][8].
It is the first time that we have studied the detailed phytochemical content of Helleborus caucasicus and Helleborus abchasicus rootstocks in southern Colchis.

Methods and Materials
Plant material: the leaves and rhizomes of two species-Helleborus caucasicus, Helleborus Abchasicus that were collected in Adjara (Table 1). Ultra Performance Liquid Chromatography (UPLC)-Preparation of a sample for chromatographic examination of saponins: Various parts of the plant were taken for analysis -the rhizomes and leaves of Helleborus caucasicus and Helleborus abchasicusas. Raw material of the sample was taken for analysis; Extraction of the crushed sample (2.5 g) was performed with methanol (100% 50-50 ml) three times in an ultrasound bath. The next step intended to filter the extracts by using a vacuum pump. We concentrated methanolic extracts at a temperature of 400 0 C under vacuum conditions until aqueous residue. (In the case of concentrated leaf extract, the sample was further treated with chloroform to remove chlorophyll green pigments). We divided the concentrated water fraction by C18. In the initial stage, the sorbent was conditioned; in particular, the sorbent was activated with methanol and balanced by using water. In the first stage after sampling, we removed unwanted components with water. In the final stage, the research components were eluted with methanol (100%). The resulting eluent was later concentrated to a dry mass. For chromatographic analysis, dry mass extraction was performed by using the mobile phase (acetonitrile: a mixture of methanol). The sample for chromatography was filtered inèto a 0.45 μm filter.

Results and Their Review
The detected Steroidal composition of Heleborus caucasicus, Helleborus abchasicus are presented in Table 2.

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Using UPLC-MS/MS, the steroid composition of the plant Helleborus caucasicus and Helleborus abchasicus was studied. In particular, 4 substances have been identified, 2 of which are found in leaves -Ecdysterone and Furostan, and 4 in tubers -Ecdysterone, Bufadienolide, Furostan and Hellebrigenin-D-glucose. Based on the results obtained, it can be concluded that the steroid composition of leaves and tubers of Helleborus caucasicus and Helleborus abchasicus is similar.

Conclusion
Vegetation of Helleborus caucasicus and Helleborus abchasicus begins at the end of November, blooming starts in December, fruiting is in progress in March-April. Three Steroidal glycosides were isolated from the MeOH extract of the plants of Helleborus caucasicus and Helleborus abchasicus-Hellebrigenin-D-glucose, 20 -Hydroxyecdysone and Hydroxyecdysone -3 glucoside. On the basis of the conducted analysis, it is possible to make a conclusion that three Steroidal glycosides were isolated from the MeOH extract of the plants of Helleborus caucasicus and Helleborus abchasicus-Hellebrigenin-D-glucose, 20 -Hydroxyecdysone and Hydroxyecdysone -3 glucosides. Steroidal glycosides that contribute to the biological activity of the plants, were identified in the Helleborus caucasicus and Helleborus abchasicus.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.