Available online at www.HighTechJournal.org HighTech and Innovation Journal Vol. 1, No. 1, March, 2020 28 Phytochemical Study of Endemic Species Helleborus Caucasicus and Helleborus Abchasicus Medea Beridze a*, Aleko Kalandia b, Indira Japaridze b, Maia Vanidze b, Natela Varshanidze a, Nazi Turmanidze a, Ketevan Dolidze a, Inga Diasamidze a, Eteri Jakeli c a Department of Biology, Faculty of Natural Sciences and Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia. b Department of Chemistry, Faculty of Natural Sciences and Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia. c Department of Pharmacy, Faculty of Natural Sciences and Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia. Received 14 January 2020; Revised 20 February 2020; Accepted 23 February 2020; Published 01 March 2020 Abstract The floristic region of Adjara represents the "Hotpoint" of Caucasians, which is distinguished by the uniqueness of its relict Colchis flora. It represents one of the most powerful refuges in western Eurasia, which is not touched by the chill because of its special geographical location. 176 endemic plants are spread in southern Colchis, of which 45 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 that have spread in southern Colchis. The research method for the phytochemical content included the separation analysis, which was performed 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. Keywords: Phytochemistry; Bioecology; UPLC-MS; Helleborus Caucasicus; Helleborus Abchasicus. 1. Introduction The floristic region of South Kolkheti (Adjara, Georgia) is part of the Caucasus Ecoregion, which is included among 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 the 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, relics, and endemic species formed in the third period [2]. There are 1837 species of plants 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 flower in winter-early spring [4]. The genus Helleborus is represented by 2 species: Helleborus Caucasicus and Helleborus Abchasicus [5]. * Corresponding author: medeaberidze89@mail.ru http://dx.doi.org/10.28991/HIJ-2020-01-01-04  This is an open access article under the CC-BY license (https://creativecommons.org/licenses/by/4.0/). © Authors retain all copyrights. https://creativecommons.org/licenses/by/4.0/ HighTech and Innovation Journal Vol. 1, No. 1, March, 2020 29 Helleborus Caucasicus and Helleborus Abchasicus (Ranunculaceae) are evergreen, blooming in the autumn- winter-spring seasons, rooted, herbaceous plants, growing on cliffs. Their vegetation begins at the end of November, blooming starts in December, and 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 great medical potential for the treatment of some serious diseases. Glycosides, bufadienolides, monocytes, biocides, and steroid saponins are found in its roots and rhizomes. Among them is 0.1 percent of colerborine P, which has an effect similar to that of stroftineon in 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 [6-8]. It is the first time that we have studied the detailed phytochemical content of Helleborus caucasicus and Helleborus abchasicus rootstocks in southern Colchis. 2. Methods and Materials Plant material: the leaves and rhizomes of two species-Helleborus caucasicus, Helleborus Abchasicus that were collected in Adjara (Table 1). Table 1. Information about test samples # Test species Samples collected area Samples data 1 Helleborus caucasicus v. 1 Maisi, Adjara February 2020 2 Helleborus abchasicus s. Kutaisi, Imereti February 2020 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 abchasicus as. 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 4000C 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. Concentration of analytical samples: Helleborus caucasicus rhizomes - g/80 μl (15 g / 1200 μl) and leaves - g/4 ml (15 g/60 ml) and Helleborus abchasicus rootstock - g/200 μl (10 g/2000 μl) and leaves - g/4 ml (10 g/40 ml). 3. Results and Their Review The detected Steroidal composition of Helleborus caucasicus, Helleborus abchasicus are presented in Table 2. Table 2. Steroidal composition of Helleborus caucasicus, Helleborus abchasicus # Species name: Heleborus caucasicus, Helleborus abchasicus Helleborus Caucasicus Helleborus abchasicus Mass ESI-MS m/z Tubers Flowers Tubers Flowers 1 20 - Hydroxyecdysone (Ecdysterone)C27H44O7 480.3087 + + + + 2 Bufadienolide C24H34O2 354.2558 503.2[M +Na]+ + + 3 Furostan C27H46O 386.3548 355.2 [M + H]+ + + + + 4 Hellebrigenin-D-glucoseC30H42O11 578.2726 431.32 [M+2Na-H]+ + + Four steroidal compounds were isolated from the MeOH extract (the tubers and leaves ) of Helleborus caucasicus and Helleborus abchasicus: 20- Hydroxyecdysone (Ecdysterone), Bufadienolide, Furostan and Hellebrigenin-D- glucose. All four substances are identified in the extract of the rhizomes, while in the flowers 2 - Ecdysterone and Furostan. HighTech and Innovation Journal Vol. 1, No. 1, March, 2020 30 Figure 1. MS scan ESI-MS m/z: 503 [M+H] + The substance 1 - (Figure 1) is retention time 3.446 min, ʎ max324 nm (Table 2); In positive ionization mode, substance 1 mainly showed molecular ions ESI-MS m/z: 503.2 [M +Na]+; according to the obtained results and compounds mass database METLIN (https://metlin.scripps.edu) the substance 1 was identified as 20- Hydroxyecdysone (Ecdysterone) [9-11]. Figure 2. MS scan ESI-MS m/z: 355 [M+H]+ The substance 2 - (Figure 2) is retention time 5.407 min, ʎ max 313.7 nm (Table 2); In positive ionization mode, substance 2 mainly showed molecular ions ESI-MS m/z: 355.26 [M + H]+; according to the obtained results and compounds mass database METLIN (https://metlin.scripps.edu) the substance 2 was identified as – Bufadienolide. Peak #1 - 3.446 - QDa 18: MS Scan Apex 210.7 324.9 423.2 468.1493.0 370.41 502.51 503.24 533.22 852.20 1004.36 1057.97 1244.10 Peak #4 - 5.407 - QDa 1: MS Scan Apex 313.7 423.2 468.1493.0 290.89 354.62 514.73 844.65 1229.31 HighTech and Innovation Journal Vol. 1, No. 1, March, 2020 31 Figure 3. MS scan ESI-MS m/z: 431 [M+H] + The substance 3 - (Figure 3) is retention time 6.164 min; In positive ionization mode, substance 3 mainly showed molecular ions ESI-MS m/z: 431.32 [M+2Na-H]+; according to the obtained results and compounds mass database METLIN (https://metlin.scripps.edu) the substance 3 was identified as Furostan. Figure 4. MS scan ESI-MS m/z: 579+ [M+H] The substance 4 - (Figure 4) is retention time 6.164 min; In positive ionization mode, substance 4 mainly showed molecular ions ESI-MS m/z: 579+ [M+H]+; according to the obtained results and compounds mass database METLIN (https://metlin.scripps.edu) the substance 4 was identified as Hellebrigenin-D-glucose. Four steroidal compounds, were isolated from the MeOH extract of Helleborus caucasicus and Helleborus abchasicus: 20- Hydroxyecdysone (Ecdysterone), Bufadienolide, Furostan and Hellebrigenin-D-glucose. All four substances are identified in the extract of the rhizomes, while in the flowers 2 - Ecdysterone and Furostan. Peak #1 - 6.164 - QDa 1: MS Scan Apex 431.19 533.83 Peak #2 - 6.846 - QDa 18: MS Scan Apex 579.23 763.18 764.13 869.09 HighTech and Innovation Journal Vol. 1, No. 1, March, 2020 32 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. 4. 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. 5. Institutional Review Board Statement Not applicable. 6. 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