Phytochemical Study of Endemic Species Helleborus Caucasicus and Helleborus Abchasicus

Abstract

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.

Full text

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; Accepted 23 February 2020
Abstract
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.
Keywords: Phytochemistry; UPLC-MS; Helleborus Caucasicus; Helleborus Abchasicus.
1. 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].
*
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.

HighTech and Innovation Journal Vol. 1, No. 1, March, 2020
29
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 [6-8].
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 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 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 Heleborus caucasicus, Helleborus abchasicus are presented in Table 2.
Table 2. Steroidal composition of Heleborus caucasicus, Helleborus abchasicus
#
Species name:
Heleborus caucasicus, Helleborus abchasicus
Heleborus Caucasicus
Heleborus 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].
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
533.22 852.201004.361057.97
1244.10
Peak #4 - 5.407 - QDa 1: MS Scan
Apex
313.7
423.2 468.1493.0
290.89354.62
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. 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.
6. References
[1] IUCN. (2006). Guidelines for using the IUCN Red List categories and criteria. Version 6.2. Retrieved from
www.redlist.org/info/categories criteria.html.
[2] Manvelidze, Z. K., Memiadze, N. V., Charazishvili, D. S., & Varshanidze, N. I. (2008). Diversity of a Floral Area of Adjara
(List of Wild Grown Plant Species). Annals of Agrarian Science, 6(2), 91-93.
[3] Varshanidze, N., Turmanidze, N., Dolidze, K., Zarnadze, N., Diasamidze, I., Epitashvili, T., & Katcharava, T. (2018).
Biodiversity of Medicinal Plants Containing Essential Oil and Their Spreading in Adjara. Universal Journal of Agricultural
Research, 6(3), 99–104. doi:10.13189/ujar.2018.060301.
[4] Memiadze, N. (2004). Botanical and geographical survey of the endemics of Ajara-Lazeti flora. Bull. Georg. Acad. Sci. 169 (2),
341-343.
[5] Dmitrieva, A. (1990). Key to flora of Adjara. Vol. II. Tbilisi: Metsniereba [in Russian].
[6] Jakeli, E., Varshanidze, N., Diasamidze I., Dolidze K., Zarnadze N. (2018). Biodiversity of medical plants of wild flora in
Ajara-South Colchis and their usage in folk medicine. 3-rd International Science Symposium “New Horizons in Science”,
Proceeding Book. At Pristina, Cosovo. 80-96.
[7] Novotny, L., Ghuloom, H. A. A. A., & Al-Hasawi, N. A. (2019). Structural Features and Biological Activities of
Bufadienolides. Research Journal of Pharmaceutical Biological and Chemical Sciences, 10(1), 1147-1157.
[8] Tomczyk, M., Gudej, J., & Sochacki, M. (2002). Flavonoids from Ficaria verna Huds. Zeitschrift Für Naturforschung C, Journal
of Biosciences, 57(5-6), 440–444. doi:10.1515/znc-2002-5-606.
[9] Guijas, C., Montenegro-Burke, J. R., Domingo-Almenara, X., Palermo, A., Warth, B., Hermann, G., … Siuzdak, G. (2018).
METLIN: A Technology Platform for Identifying Knowns and Unknowns. Analytical Chemistry, 90(5), 3156–3164.
doi:10.1021/acs.analchem.7b04424.