CBU INTERNATIONAL CONFERENCE ON INNOVATIONS IN SCIENCE AND EDUCATION 2020 (MEDICINE AND PHARMACY) MARCH 18-20, 2020, PRAGUE, CZECH REPUBLIC WWW.CBUIC.CZ PHYTOCHEMICAL STUDY OF ENDEMIC SPECIES HELLEBORUS CAUCASICUS, HELLEBORUS ABCHASICUS AND FICARIA POPOVII SPREAD IN SOUTHERN COLCHIS 1 Medea Beridze , Aleko Kalandia2, Indira Japaridze3, Maia Vanidze4, Natela Varshanidze5, Nazi Turmanidze6, Ketevan Dolidze7, Inga Diasamidze8, Eteri Jakeli9 Abstract: There are 176 endemic plants 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 objectiveis to study the phytochemical content of endemic species of Helleborus caucasicus, Helleborus abchasicus and Ficaria popovii spread in southern Colchis. The research method for the phytochemical content included separation analysis, whichwas performed byusing UPLC-MS (Waters Acquity QDa detector). Three Steroidal glycosideswere isolated from the MeOH extract of the plants of Helleborus caucasicus and Helleborus abchasicus: Hellebrigenin-D-glucose, 20 – Hydroxyecdysone and Hydroxyecdysone – 3 glucoside. Two saponins (Hederagenin 3-O -α-L-arabino pyranoside, Hederagenin28-O-[α-L-rhamno-pyranosyl(1→4)-β-D-glucopyranosyl-(1→6)]βD-lucopyranoside) and four flavonoids (kaempferol 3-O-β-ᴅ- (6ʺ-α-ʟ-rhamnopyranosyl)-glucopyranoside (nicotiflorin), apigenin 8-C-β-ᴅ-glucopyranoside (vitexin), luteolin 8-C-β-ᴅ-glucopyranoside (orientin), quercetin 3-O-rutinoside) were isolated from the tubers and flowers of Ficaria Popovii. Three Steroidal glycosides and Hydroxyecdysone -3 glucoside were isolated from the MeOH extract of Helleborus caucasicus.In addition, two saponins and four flavonoids were isolated from the tubers and leaves of FicariaPopovii. UDC Classification: 577, DOI: https://doi.org/10.12955/pmp.v1.89 Keywords:Phytochemistry, UPLC-MS,Ficaria Popovii, Helleborus caucasicus,Helleborus abchasicus 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 (IUCN. 2006). 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(Manvelidze et al., 2010). 1837 species of plants are common in southern Colchis, including 176 endemic ones (Varshanidze et al., 2018).Among the endemics, the following genera are distinguished by their decorative and medicinal properties: Helleborus and Ficaria, bothspecies of genusflower in winter-early spring. (Memiadze, 2004). 1 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of Biology, Batumi, Georgia, medeaberidze89@mail.ru 2 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of chemistry, Batumi, Georgia,a.kalandia@bsu.edu.ge 3 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of chemistry, Batumi, Georgia,indira.djafaridze@bsu.edu.ge 4 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of chemistry, Batumi, Georgia, maia.vanidze@bsu.edu.ge 5 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of Biology, Batumi, Georgia, natela.varshanidze@gmail.com 6 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of Biology, Batumi, Georgia,turmanidze.nazi@bsu.edu.ge 7 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of Biology, Batumi, Georgia, diasamidze.inga@bsu.edu.ge 8 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of Biology, Batumi, Georgia, ketodolidze@yahoo.com 9 Batumi Shota Rustaveli State University, Faculty of Natural Sciences and Health Care, Department of Pharmacy, Batumi, Georgia, eteri_jakeli@yahoo.com 1 CBU INTERNATIONAL CONFERENCE ON INNOVATIONS IN SCIENCE AND EDUCATION 2020 (MEDICINE AND PHARMACY) MARCH 18-20, 2020, PRAGUE, CZECH REPUBLIC WWW.CBUIC.CZ The genus Helleborus is represented by 2 species: Helleborus caucasicus and Helleborus abchasicus. Furthermore,the genus Ficaria is represented by 1 species -Ficaria popovii.(Dmitrieva, 1990). Helleborus caucasicusandHelleborus abchasicus(Ranunculaceae) are evergreen, blooming in autumnwinter-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. Ficaria popovii (Ranunculaceae) is endemic of the narrow-local ephemeroid nature of Adjara.Its vegetation begins in November, blooms in January-February, fruiting is in progress in March-April, and dries in early May.Among these species, Helleborus caucasicus and Helleborus Abchasicus are widely distributed. Helleboruscaucasicus is an important source of chemical compounds with a great medical potential for the treatment of some serious diseases such as cancer, ulcers, diabetes, as well as some medical problems such as toothache, eczema, low immunity and arthritis. Ficaria popovii tubers and leaf extracts are used as a diuretic, blood purifier and wound healer, as well as in salads made from leaves that cleanse the blood of pathogenic microbes (Jakeli et al. 2018). It is the first time that wehave studied the detailed phytochemical content of Ficaria popovii tubers and leaves,and Helleborus caucasicus and Helleborus abchasicus rootstocks in southern Colchis. Methods and materials Plant material: the leaves, tubers and rhizomes of three species-Helleborus caucasicus H. Abchasicus,Ficaria popoviithat were collected in Adjara(Tab. 1). Table 1: Information about test samples # 1 2 3 Test species Helleborus caucasicus Helleborus abchasicus Ficaria popovii Samples collected area v. 1 Maisi, Adjara s.Kutaisi, Imereti v. Tsikhisdziri, Adjara Samples data February 2020 February 2020 March 2020 Source: Authors Ultra Performance Liquid Chromatography (UPLC)- Preparation of a sample for chromatographic examination of saponins: Various parts of the plant Ficaria popovii were taken for analysis - Flower, leaves and tubers, and the rhizomes and leaves of Helleborus caucasicus and Helleborus abchasicusas well. 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 there was an aqueous residue.(In the case of concentrated leaf extract, the sample was further treated with chloroform to remove chlorophyll green pigments).In order to elute and concentrate saponins, we divided the concentrated water fraction by C18.In the initial stage, the sorbent was conditioned, in particular, the sorbent was activatedwith methanol and balancedby using water.In the first stage after sampling, we removed unwanted components with water.In the final stage, the research components wereeluted with methanol (100%).The resulting eluent waslater 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 into a 0.45 μm filter. The flavonoids were tentatively identified according to their retention time (Rt), the wavelength of maximum absorbance (λmax), pseudomolecular ion ([M−H]-) and compounds mass database METLIN (https://metlin.scripps.edu). Concentration of analytical samples:Ficaria popovii - flower - g/200 μl (2.5 g/500 μl), leaves - g/25 μl (20 g/500 μl) and tubers - g/50 μl (10 g/500 μl).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). Results and discussion The detected flavonoids of Ficaria popovii are presented in Table 2. Four flavonoids were found, the UPLC - flavonoids profile of the ethanolic extract of the sample Tuber (The substance 1, 2, 3 and 4) and flower (The substance 1 and 2) of Ficaria popovii). Flavonoids Composition of Ficaria popovii tubers and leaves. 2 CBU INTERNATIONAL CONFERENCE ON INNOVATIONS IN SCIENCE AND EDUCATION 2020 (MEDICINE AND PHARMACY) MARCH 18-20, 2020, PRAGUE, CZECH REPUBLIC WWW.CBUIC.CZ Table 2: Flavonoids Composition of Ficaria popovii tubers and leaves Species name:Ficaria popovii m/z Retention Time flavonoids quercetin 3-O-rutinoside(rutin) 609 [M-H]kaempferol 3-O-β-ᴅ- (6ʺ-α-ʟ-rhamnopyranosyl)593 [M-H]glucopyranoside (nicotiflorin) luteolin 8-C-β-ᴅ-glucopyranoside (orientin) 447 [M-H]apigenin 8-C-β-ᴅ-glucopyranoside (vitexin) 431[M-H]- leaves tubers 5.653 6.237 + + + + 7.119 10.339 _ _ + + Source: Authors 5.653 - 609.03 250000 5.653 Peak 1 - QDa 18: MS Scan 18: QDa Negative(-) Scan (50.00 609.03 200000 Intensity Intensity 300000 200000 7.071 - 447.07 7.478 - 608.87 Figure 1: MS scan ESI-MS m/z: 609 [M+H]- 150000 100000 50000 100000 0 0 200.00 400.00 600.00 m/z 800.00 1000.00 1200.00 4.00 5.00 6.00 7.00 8.00 Minutes 9.00 10.00 Source: Authors Figure 2: MS scan ESI-MS m/z: 593 [M+H]6.237 Peak 1 - QDa 18: MS Scan 18: QDa Negative(-) Scan (50.00 300000 200000 100000 400000 Intensity 6.237 - 593.01 Intensity 400000 593.01 7.892 - 593.04 7.552 - 593.11 500000 300000 200000 100000 0 0 5.50 6.00 6.50 7.00 7.50 Minutes 8.00 8.50 9.00 200.00 400.00 600.00 800.00 m/z 1000.00 1200.00 Source: Authors Figure 3: MS scan ESI-MS m/z: 447 [M+H]7.119 Peak 2 - QDa 18: MS Scan 18: QDa Negative(-) Scan (50.00 100000 Intensity 150000 7.700 - 447.01 3.668 - 446.99 Intensity 200000 7.119 - 447.01 447.01 200000 100000 50000 50000 0 0 2.00 150000 3.00 4.00 5.00 6.00 Minutes 7.00 8.00 9.00 200.00 400.00 600.00 m/z 800.00 1000.00 1200.00 Source: Authors Substance 1 - (Fig.1) retention time is 5.653 min, ʎ max 254 and 354 nm (Table 2); according to the obtained results and compounds mass database METLIN (https://metlin.scripps.edu) substance 1 is quercetin 3-O-rutinoside(rutin) C27H30O16, MW 610,15; Substance 2 - (Fig.2) retention time is 6.237 min, ʎ max 266 and 346 nm (Table 2); according to the obtained results and compounds mass database METLIN, substance 2 is kaempferol 3-O-β-ᴅ- (6ʺα-ʟ-rhamnopyranosyl)- glucopyranoside (nicotiflorin) C27H30O15,MW 594.15 3 CBU INTERNATIONAL CONFERENCE ON INNOVATIONS IN SCIENCE AND EDUCATION 2020 (MEDICINE AND PHARMACY) MARCH 18-20, 2020, PRAGUE, CZECH REPUBLIC WWW.CBUIC.CZ Substance 3 - (Fig.3) retention time is 7.071 min, ʎ max 254 and 267 nm (Table 2); according to the obtained results and compounds mass database METLIN, substance 3 is luteolin 8-C-β-ᴅglucopyranoside (orientin) C21H20O11, MW 448.1; Figure 4: MS scan ESI-MS m/z: 431 [M+H]- 6.0x105 10.339 Peak 1 - QDa 18: MS Scan 18: QDa Negative(-) Scan (50 512.32 431.06 100000.0 Intensity 8.0x105 Intensity 120000.0 10.339 - 510.92 1.0x106 4.0x105 80000.0 60000.0 40000.0 2.0x105 20000.0 0.0 8.00 8.50 9.00 9.50 10.00 Minutes 10.50 11.00 400.00 11.50 450.00 500.00 m/z 550.00 Source: Authors Substance 4 - (Fig.4) retention time is 10.339 min, ʎ max 268 and 334 nm (Table 2); according to the obtained results and compounds mass database METLIN, substance 4 is apigenin 8-C-β-ᴅglucopyranoside (vitexin) C21H20O10, MW 432.105; Two known saponins, glycosides of oleanolic acid, have been isolated from the tubers of Ranunculus ficaria PopoviiL. (Ranunculaceae): 28-Glucosyloleanolic acid 3-arabinoside and 28-[Glucosyl-(1->6)glucosyl]oleanolic acid 3-arabinoside. Table 3: Saponins composition of Ficaria popovii tubers # Species name:Ficaria popovii Saponin Tubers 1 2 Mss 28-Glucosyloleanolic acid 3-arabinoside C41H66O12 28-[Glucosyl-(1->6)-glucosyl]oleanolic acid 3-arabinosideC47H76O17 ESI-MS m/z 912.50 769.45[M+F] 750.45 971.52[M+CH3COO] Source: Authors Figure 5:MS scan ESI-MS m/z: 769 [M+H]200000 8.583 - 768.86 8.716 - 768.89 100000 768.86 250000 200000 Intensity 150000 Intensity 8.583 Peak 1 - QDa 18: MS Scan 18: QDa Negative(-) Scan (50.00 150000 100000 50000 50000 0 0 7.00 7.50 8.00 8.50 9.00 Minutes 9.50 10.00 10.50 200.00 400.00 600.00 m/z 800.00 1000.00 1200.00 Source: Authors Substance 5 - (Fig.5) retention time is 8.583 min, (Table 3); according to the obtained results and compounds mass database METLIN (https://metlin.scripps.edu), substance 5 is 28Glucosyloleanolic acid 3-arabinoside. C41H66O12 MW 912.50; ESI-MS m/z [M+F]; Substance 6 - (Fig.6) retention time is 14.226 min, (Table 3); according to the obtained results and compounds mass database METLIN (https://metlin.scripps.edu), substance 6 is 28-[Glucosyl-(1>6)-glucosyl] oleanolic acid 3-arabinoside C47H76O17 MW 750.45; ESI-MS m/z 971.52 [M+CH3COO]; Using UPLC-MS / MS, 4 flavonoids and 2 saponins have been identified in the plant Ficaria popovii extract. In particular, in leaves identified 2 substances (quercetin 3-O-rutinoside and kaempferol 3-Oβ-ᴅ- (6ʺ-α---rhamnopyranosyl) - glucopyranoside), and in tubers 4 flavonoids (quercetin 3-Orutinoside, kaempferol). 3-O-β-ᴅ- (6ʺ-α-ʟ-rhamnopyranosyl) - glucopyranoside, luteolin 8-C-β-ᴅ- 4 CBU INTERNATIONAL CONFERENCE ON INNOVATIONS IN SCIENCE AND EDUCATION 2020 (MEDICINE AND PHARMACY) MARCH 18-20, 2020, PRAGUE, CZECH REPUBLIC WWW.CBUIC.CZ glucopyranoside and apigenin 8-C-β-ᴅ-glucopyranoside). 3-arabinoside and 28- [Glucosyl- (1-> 6) glucosyl] oleanolic acid 3-arabinoside.These are the first studies of the Ficaria popoviiflavonoid complex and saponins using UPLC-MS / MS. Figure 6: MS scan ESI-MS m/z: 971 [M+H]- 40000.0 30000.0 971.39 800000 Intensity 50000.0 Intensity 14.226 Peak 1 - QDa 1: MS Scan 1: QDa N 14.186 - 971.29 60000.0 20000.0 600000 400000 200000 10000.0 0.0 0 12.00 14.00 Minutes 16.00 200.00400.00600.00800.001000.00 1200.00 m/z Source: Authors Table 4: Steroidal composition of Heleborus caucasicus, Helleborus abchasicus # 1 2 3 4 Species name: Heleborus caucasicus, Helleborus abchasicus Mass ESI-MS m/z 20 - Hydroxyecdysone 480.3087 (Ecdysterone)C27H44O7 503.2[M Bufadienolide C24H34O2 354.2558 +Na]+ 355.2 [M + Furostan C27H46O 386.3548 H]+ Hellebrigenin-D431.32 578.2726 glucoseC30H42O11 [M+2Na-H]+ Heleborus Caucasicus Tubers Flowers + + + Heleborus abchasicus Tubers Flowers + + + + + + + + + Source: Authors Figure 7: MS scan ESI-MS m/z: 503 [M+H] + Peak #1 - 3.446 - QDa 18: MS Scan 210.7 324.9 423.2 468.1493.0 1244.10 503.24 502.51 370.41 533.22 1057.97 1004.36 852.20 Apex Source: Authors Four steroidal compounds, were isolated from the MeOH extract (the tubers and leaves ) of Helleborus caucasicus and Helleborusabchasicus: 20- Hydroxyecdysone (Ecdysterone), Bufadienolide, Furostan and Hellebrigenin-D-glucose. All four substances are identified in the extract of the rhizomes, while in the flowers2 - Ecdysterone and Furostan. Substance 7 - (Fig. 7) retention time is 3.446 min, ʎ max324 nm (Table 4); In positive ionization mode, substance 7 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), substance 7 was identified as 20- Hydroxyecdysone (Ecdysterone); Substance 8 - (Fig. 8) retention time is 5.407 min, ʎ max 313.7 nm (Table 4); In positive ionization mode, substance 8 mainly showed molecular ions ESI-MS m/z: 355.26 [M + H]+; according to the 5 CBU INTERNATIONAL CONFERENCE ON INNOVATIONS IN SCIENCE AND EDUCATION 2020 (MEDICINE AND PHARMACY) MARCH 18-20, 2020, PRAGUE, CZECH REPUBLIC WWW.CBUIC.CZ obtained results and compounds mass database METLIN (https://metlin.scripps.edu), substance 8 was identified as – Bufadienolide Figure 8: MS scan ESI-MS m/z: 355 [M+H]+ Peak #4 - 5.407 - QDa 1: MS Scan 313.7 423.2 468.1493.0 354.62 290.89 514.73 844.65 1229.31 Apex Source: Authors Figure 9: MS scan ESI-MS m/z: 431 [M+H] + Peak #1 - 6.164 - QDa 1: MS Scan 431.19 533.83 Apex Source: Authors Substance 9 - (Fig.9) retention time is 6.164 min; In positive ionization mode, substance 9 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), substance 9 was identified as Furostan Figure 10: MS scan ESI-MS m/z: 579+ [M+H] Peak #2 - 6.846 - QDa 18: MS Scan 763.18 869.09 579.23 764.13 Apex Source: Authors Substance 10 - (Fig.10) retention time is 6.164 min; In positive ionization mode, substance 10 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), substance 10 was identified as Hellebrigenin-Dglucose. Four steroidal compounds, were isolated from the MeOH extract of Helleborus caucasicus and Helleborus abchasicus: 20- Hydroxyecdysone (Ecdysterone), Bufadienolide, Furostan and Hellebrigenin-D-glucose(Table 4). All four substances are identified in the extract of the rhizomes, while in the flowers2 - Ecdysterone and Furostan. Using UPLC-MS / MS, the steroid composition of the plant Helleborus caucasicus and Helleborus abchasicus was studied. In particular, 4 substances were identified, 2 of which are found in leaves - 6 CBU INTERNATIONAL CONFERENCE ON INNOVATIONS IN SCIENCE AND EDUCATION 2020 (MEDICINE AND PHARMACY) MARCH 18-20, 2020, PRAGUE, CZECH REPUBLIC WWW.CBUIC.CZ Ecdysterone and Furostan, and 4 in tubers - Ecdysterone, Bufadienolide, Furostan and HellebrigeninD-glucose. Based on the obtained results, 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, and fruiting is in progress in March-April. Ficaria popovii (Ranunculaceae) blooms in January-February, fruiting is in progress in March-April, and dries in early May. 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. Two saponins (Hederagenin 3-O -α-L-arabino pyranoside, Hederagenin28-O-[α-L-rhamno-pyranosyl(1→4)-β-D-glucopyranosyl-(1→6)]β-D-lucopyranoside) and four flavonoids (kaempferol 3-O-β-ᴅ- (6ʺ-α-ʟ-rhamnopyranosyl)-glucopyranoside (nicotiflorin), apigenin 8-C-β-ᴅ-glucopyranoside (vitexin), luteolin 8-C-β-ᴅ-glucopyranoside (orientin), quercetin 3O-rutinoside) have been isolated from the tubers and flowers of Ficaria Popovii. 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. While, two saponins and four flavonoids were isolated from the tubers and flowers of Ficaria Popovii. Steroidal glycosides, saponinsand flavonoids, that contribute to the biological activity of the plants, were identified in the leaves and tubers of Ficaria Popovii, Helleborus caucasicus and Helleborus abchasicus. References Dmitrieva, A. (1990). [in Russian], Key to flora of Adjara. Vol. II. Tbilisi: Metsniereba. IUCN. (2006). Guidelines for using the IUCN Red List categories and criteria. Version 6.2. Retrieved from www.redlist.org/info/categories criteria.html. Jakeli, E., Varshanidze, N., Diasamidze I., Dolidze K., Zarnadze N. (2018). 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