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J. Chem. Chem. Eng. 8 (2014) 516-523
Chromatographic Analysis of Chemical Composition of
the Genus Rhododendron Plants Growing on the
Mountain of Evota (South Yakutia)
Zaytseva Natalia Vladimirovna* and Pogulyaeva Irina Alexandrovna
Nerungri Technical Institute, The Branch of North-East Federal University named by M.K. Ammosov, South Yakutia 678960, Russia
Received: March 29, 2014 / Accepted: April 30, 2014 / Published: May 25, 2014. Abstract: The data on chemical compounds of three Rododendron species (Rh. adamsii Rehd., Rh. aureum Georgi. and Rh. lapponicum subsp. parvifolium (Adams) T. Yamaz.) from the mountain of Evota top in South Yakutia are shown. Extracts of these plants was analyzed by method of planar chromatography in thin layers using different specific detectors to exposure of some groups of biologically active matters. During the researches, it was established that polyphenol compounds of different degree of condensment prevail in rhododendron composition; catechines, flavonoids, coumarins, saponins, essential oils, phenol carboxylic acids and arbutin are present too. Due to detected features, all the researched species of rhododendrons are perspective for practical using in food industry and as adaptogenes for maintenance of people health in the conditions of north. Key words: South Yakutia, the chemical composition of Rhododendron, adaptation to high-altitude conditions, planar chromatography.
1. Introduction
South Yakutia is situated in the heart of North East
Asia, and occupies the territory of Olekma-Charsky
and Aldan plateau and the Northern spurs of Stanovoy
range. Climate features allow us to classify this region
as 2nd or 3rd zone of “USDA-zones” scale with
minimum air temperatures in winter from -34.4 °C
(-30 F) to -44 °C (-40 F). The relief contains mainly
hills (their altitude is limited to 900 m above sea level)
with rounded flat tops. The unique landscape features
are the mountains situated on the north of
Nerungrinski district and named as Evota mountains
(from Evota river on south-east side from them).
The altitude of Evota mountain is 1,601 m above
sea level. Here, there are all natural zones conditional
on high-altitude—from forest at the foot of the
mountain to tundra and stony waste plot at the top.
*Corresponding author: Zaytseva Natalia Vladimirovna,
Ph.D., research field: technology of practical application of plants in the Northern regions. E-mail: nz_demetra@mail.ru.
There is another feature of this place: all the plants
growing on the mountain top experience excessive
UV (ultraviolet rays), which are contained in solar
radiation.
Best adapted to such conditions are plants of
Ericaceae family due to their unique chemical
compounds. Of the 11 species of this family, living at
the top of Evota mountain, our attention was attracted
three unique species of Rhododendron genus—Rh.
adamsii Rehd., Rh. aureum Georgi. and procumbent
form of Rh. parvifolium Adams also called as Rh.
lapponicum subsp. parvifolium (Adams) T. Yamaz.
Rh. aureum is a rare protected species listed in the
red book of Yakutia [1]. Its leaves and flowers are
very popular in folk medicine for the treatment of
heart diseases [2, 3]. The chemical composition of
this plant, the most studied. According the
investigations from Irkutsk region [2-10], the leaves
and sprouts of Rh. Aureum from Baical region contain
the next biologically active matters: rhododendrin
D DAVID PUBLISHING
Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)
517
(betuloside), betuligenol, ericolin, hyperoside, tannins,
arbutin, rutin, gallic acid, ursolic acid, poisonous
glycoside of andromedotoxin. There are more than 50
compounds besides mono- and sesquiterpenes of
essential oil contaned more than 20 compounds
(pinenes, myrcen, betuligenol/rhododendrole, etc.) [11].
The oil has the fine exquisite aroma with tone of the
tea rose scent.
Rh. adamsii is valuable medicinal plant used in
medicine Buryatia, Mongolia and Tibet as a means of
increasing resistance to the human organism to
adverse conditions of existence, weariness, fatigue
[2, 3, 12, 13]. In Chinese and Mongolian medicine
preparations of this plant named “Sagan Dayli” or
“White wing”. According to literary sources [2, 3, 9,
11, 14, 15], the composition of Rh. adamsii includes:
essential oil of the complex chemical composition (in
that number: germacron (26.2%); ± -transnerolidol
(18.4%); juniper-camphor and nerolidol), resinous
substances, ursolic and oleanolic acids, triterpenoids,
tannins, flavonic derivatives. All parts of Rh. adamsii
have very strong, sharp, but pleasing scent, reminding
scent of the rose and resins simultaneously—it does
this plant as especial amongst representatives of
Rhododendron genus.
The chemical composition of Rh. lapponicum (Rh.
parvifolium) is studied least of all. There are few
scientific publications devoted to this species though it
is worthy of rapt attention. According to studies of
Makarov et al. [16], it was discovered the next matters
in this species: tannins both pyrogallic and
pyrocatechinic groups, quercetin, avicularin, coumarin,
aesculetin, the simple phenol compounds. According
to published edition “Plant resources of Russia” (2009) Rh.
parvifolium contains ursolic and oleanic acids,
sitosterin, arbutin (up to 4%), and phenol carboxylic
acids [9]. According to Ref. [11], the essential oil of
this rhododendron containes more than 30
components and the main from which are
isoaromadendren epoxide (13.8%), patchoulol (4.9%),
a-cadinol (4.7%) and t-cadinen (4.2%).
Thereby, the plants of Rhododendron can be a
source of valuable medicinal matters of the natural
derivation. In our opinion, severe highland conditions
of growing plants, which is indicative of
South-Yakutia region, promote them in the synthesis
of components that provide the processes of
adaptation and increased resilience organisms under
the powerful electromagnetic fields, UV radiation,
oxygen shortage. This can be of great practical
importance for medical purposes and some problems
of the health of the population at high widths.
The aims of this research were: to study the
chemical composition of Rh. adamsii, Rh. lapponicum
and Rh. aureum by using method of thin layer
chromatography; to detect some features of
biochemical composition depended on mountain
conditions of their growing. At this stage, our studies
are the searching ones. For species of South Yakutia
such studies are organized for the first time.
2. Experiments
2.1 Characteristics of the Plant Material
The leaves and sprout tops growing at the mountain
of Evota were collected for analysis. The collection of
raw materials for studies was carried out at June 29,
2013, in dry solar weather, in the afternoon.
2.2 Technology of the Plant Material Fixation
Collected plant material was sorted and reduced,
and then it was dried out in dryer at 60 оС to dry
condition. Dried plant material was sacked in paper
packages for keeping. Before undertaking analyses,
plant material was again dried in dryer at 60 оС to
constant mass (“absolutely dry mass”).
2.3 Technology of the Extract Preparation
For extraction 1 g (accurate to 0.01 g) of absolutely
dry raw material was moistened is about 10 mL 70%
ethanol and then infused for 24 h under periodic
shaking. Then, the extracts were evaporated to 1 mL
under slow heating.
Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)
518
2.4 Technology of the Chromatogram Preparing
We used the standard technology of the
chromatography analysis [17, 18]. Adsorbent is silica
gel precoated on plates for thin-layer chromatography
(the brand is “Sorbfil” (PTSH-AF-А-UF)). Solvent
system is n-butanol:acetic acid:water (4:1:5).
Detection was carried out with the next spray
reagents: (1) 10% ethanolic solution of KOH; (2) 5%
ethanolic solution of AlCl3; (3) VSR (vanilin-sulfuric
acid reagent) (1% sulfuric vanilin); (4) FeSO4
(iron-II-sulfate reagent) (1% aqueous solution); and (5)
DRG (dragendorff reagent) (solution of basic bismuth
nitrate in glacial acetic acid and water/water solution
of potassium iodide).
Each chromatogram was described with color,
brightness and form of the spots, features of their
distribution on the chromatographic track; the main
quantitative index was Rf-index.
The developed chromatograms were photographed
on Panasonic DMC-FS62 in visible (vis.) and
ultraviolet light (UV-254 nm and UV-365 nm).
The identification of matters was conducted in
accordance with information presented in publishing
[17-21].
3. Results and Discussion
The analysis of chromatograms is indicatives of
significant chemical resemblance of studied
rhododendron species. All the chromatographic tracks
are colored dark-grey or grey-yellow in vis. and
especially intensive—with FeSO4 (Figs. 1-3). This
fact is evidenced of presence of great number of
phenolic compounds with different chromatographic
mobility degree in extracts.
All tracks show one major yellow-grey or
grey-olive zone (vis.) in the Rf range 0.7-0.8 that is
well colored with all detectors and gives specific
yellow-green fluorescence in UV-365 nm. With
FeSO4 reagent this zone is colored green-brown
(indication of catechins); with KOH and AlCl3
reagents—bright yellow (vis.) and distinct
yellow-green fluorescent in UV-365 nm (indication of
flavonoids); with VSR—rich bright red-brown to
bright orange and golden (indication of catechins,
phlobaphens, anthocyanin and quercetin).
Other general sign for all studied chromatograms is
yellow or yellow-green zone with Rf 0.90-0.97, which
colouration increases after processing by KOH
(indication of kaempferol). In UV-256 nm, this zone
is blue fluorescent (indication of aurons). In VSR, this
zone is colored red or violet (indication of single-core
phenolic acids (arbutin, benzoic acid), saponins and
essential oils).
The small green (vis.) and bright-rose fluorescent
(in UV and KOH) zone with Rf 1.0 represents the
chlorophyll. The white fluorescence in the manner of
fine bands in this part of tracks is due to caffeic and/or
ferulic acids.
The zone with Rf 0.5-0.6 in the middle of tracks has
distinct bright-blue fluorescence in UV-365 nm. It
most probably is coumarin or its predecessor on
biosyntesis—coumaric acid.
The analysis of the rhododendron chemical
composition gives the following results.
3.1 Rhododendron Aureum Georgi
There is a large number of phenolic matters both
hydrolysed and nonhydrolysed nature in composition
of the extract of this species (Fig. 1). The derivatives
of pyrogallol dominate—it is distinguished of Rh.
aureum from two other rhododendron species. At
interaction with iron ions phenolic compounds are
colored blue dominating along the whole length of
track. Rh. aureum shows three major blue zones in the
Rf 0.57-0.67; 0.82-0.88 and 0.91-0.97 that could
satisfy the chromatographic descriptions of following
matters: gallic acid, hydrolysed tannins, pyrogallol.
Grey-olive zone at Rf 0.67-0.77 represents
nonhydrolysed tannic matters (catechins). In VSR,
this zone is colored bright red or red-wine due to
synthesis of red pigments in acid. Perhaps these are the
products of acid condensation of pyrocatechin
Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)
519
derivatives—anthocyanins, leucocyanidins and
phlofabens [21].
The qualitative reactions on the other groups of
matters show that yellow (in vis.) and yellow-green
fluorescent (in UV-365 nm) zone with Rf 0.67-0.77
conformes to flavanoids. In KOH and AlCl3, the
fluorescence increases. The most probable matter with
such characteristic could be the quercetin.
The zone in the Rf range 0.82-0.88 is weak without
additional spraying, but weak green fluorescence
becomes more visible after spraiyng with KOH and
AlCl3 (indication of kaempferol).
The zone in the Rf range 0.90-0.97 is yellow (vis.)
but in VSR it is colored red-orange and in
FeSO4—violet. It is a sign of the simple phenols
(arbutin, hydroquinone and phenolic acids).
The dark-blue fluorescent band in lower part of
track indicatives of “heavy” phenolic matters
with low index of chromatographic mobility because
of their relationship with milk sugars
(phenologlycosides).
The bright-blue fluorescent (in UV-365 nm) zones
(increasing in KOH and AlCl3) represent the matters
of coumarinic nature: scopoletin (at Rf 0.37-0.43) and
umbelliferone (at Rf 0.50-0.57). Oxycinnamonic acids
such as chlorogenic and coumaric ones posses almost
the same chromatographic features. The bright-white
fluorescent stripe at the solvent front is a sign of
caffeic and ferulic acids.
Thereby, we detected the next compounds in the Rh.
aureum extract: hydrolysed and nonhydrolysed
tannins, catechins, flavonoids (kaempferol, quercetin),
simple phenolic matters (arbutin), oxycinnamonic
acids (chlorogenic, coumaric, gallic, caffeic and
ferulic), coumarins (scopoletin, umbelliferone),
phenologlycosides. The phenolic matters of the
middle degree of condensment (catechins) prevail.
3.2 Rhododendron Adamsii Rehd.
The chromatographic analysis of Rh. adamsii
extract reveals nonhydrolised polyphenols in chemical
composition of this plant (Fig. 2). Tracks have weak
dark-blue fluorescent bands in the lower range of Rf
detected as phenologlycosides.
The distinct blue fluorescence at Rf 0.60-0.68 is due
to umbelliferone or coumaric acid.
Fig. 1 Photographs of chromatograms of Rh. aureum extracts (indicating possible components of their chemical composition): (a) image in vis.; (b) image in UV-365 nm.
Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)
520
Fig. 2 Photographs of chromatograms of Rh. adamsii extracts (indicating possible components of their chemical composition): (a)—image in vis; (b)—image in UV-365 nm.
Grey-olive zone in the range of Rf 0.68-0.80 is the
major. With KOH and AlCl3, this zone is colored
bright-yellow in vis. and green fluorescent in UV-365
nm (indication of flavonoids); with
VSR—yellow-orange (indication of flavondiols); with
FeSO4—dark-brown (indication of tannins). On the
basis of these signs, it is possible to confirm the
presens of quercetin and tannins in composition of Rh.
adamsii.
Treatment with FeSO4 generates some additional
dark-brown zones in the range of Rf 0.80-0.93. With
КОН and AlCl3, these zones show green and blue
fluorescence (indication of flavonoids).
At the solvent front (in the range of Rf 0.90-1.00),
there are some zones with indivual color reactions:
(1) Without spraying—white fluorescent zone in
UV-365 nm (caffeic acid); blue fluorescent zone in
UV-256 nm (aurons);
(2) In VSR—blue (in vis.) and dark-violet (in
UV-365 nm) zone (saponins, essential oils);
(3) In FeSO4—weak red zone (arbutin) and light
brown zone (aurons, lignans).
Thereby, there are the next compounds in the
chemical composition of Rh. adamsii: nonhydrolysed
tannins, some fractions of flavonoids (including
quercetin and kaempferol), coumarins (umbelliferone),
saponins, aurons, essential oil, arbutin, caffeic acid.
3.3 Rhododendron Lapponicum Subsp. Parvifolium
(Adams) T. Yamaz
Chromatographic tracks of Rh. lapponicum extract
are like tracks of Rh. adamsii, but the first ones are
more prominent and rich of the following components
(Fig. 3):
(1) “Heavy” polyphenols are characterized by
fluorescent dark-blue zone at Rf 0.00-0.025;
(2) Scopoletin (and/or chlorogenic acid) is found at
Rf 0.24 as fluorescent blue zone; KOH intensifies the
fluorescence;
(3) Romedotoxin is found at Rf 0.25 as dark zone;
(4) Hyperin is revealed in VSR and UV-365 nm as
brown fluorescent zone at Rf 0.33-0.36;
(5) Umbelliferone (and/or coumaric acid) is found at
Rf 0.45 as fluorescent bright-blue zone; KOH
intensifies the fluorescence;
(6) Rutin is revealed in VSR and UV-365 nm as
Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)
521
Fig. 3 Photographs of chromatograms of Rh. lapponicum extracts (indicating possible components of their chemical composition): (a)—image in vis; (b)—image in UV-365 nm.
brown fluorescent zone at Rf 0.47.
The distribution of compounds in upper part of the
tracks looks like tracks of Rh. adamsii practically in
everything. There are some main zones.
The first zone in the range of Rf 0.69-0.77 is
correspond to flavonoids, quercetin, nonhydrolysed
tannins.
The second zone in the range of Rf 0.80-0.95
consists of several subzones:
(1) Yellow-green in vis. and dark-green fluorescent
(in UV-365 nm) zone (in KOH the fluorescence in
intensified and colored yellow);
(2) Yellow-brown in vis. and white fluorescent (in
UV-365 nm) zone;
(3) Weak green in vis. and bright-rose fluorescent
(in UV-365 nm) zone.
This zone is blue fluorescent in UV-264 nm, in
VSR it is colored blue. In FeSO4 detected subzones
are accordingly colored dark-brown, red and
yellow-brown.
Thereby, the next compounds were detected in the
chemical composition of Rh. lapponicum:
phenologlycosides, alkaloids (andromedotoxin),
flavonoids (hyperin, rutin, quercetin, kaempferol),
coumarins (scopoletin, umbelliferone),
oxycinnamonic acids (coumaric, caffeic and ferulic),
saponins (ursolic and oleanic acids), nonhydrolysed
tannins, arbutin, aurons, lignans.
Thus, our results correspond to information
presented in publications [3-16]. However, it is
possible to draw a conclusion about tannins as the
major compound of rhododendron representatives.
Due to tannins these plants adapt to conditions of
highlands and UV radiation, and at the same time
tannins are products of intensive biosynthetic
processes in plants growing in conditions of intensive
solar radiation themselves. Fluorescent
compounds—coumarins (blue fluorescence UV-365
nm), oxycinnamonic acids (white fluorescence
UV-365 nm), flavonoids (yellow fluorescence in
UV-365 nm), and aurons (blue fluorescence in
UV-256 nm)—also reduce unfavourable influence of
UV radiation and present in extracts of studied species
of rhododendrons in relatively large amount.
Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)
522
The analysis of the chemical composition of
rhododendron species of Evota Mountain directs on
the following discourses:
(1) Rh. adamsii and Rh. lapponicum are near related
species, so they have practically the same features of
the extract chemical compositions.
(2) On presence and distribution of compounds in
plant extracts, it is possible to detect if the plant is in
favourable conditions for its growing and
development or feels the stress and deperssion. So,
according our researches Rh. adamsii does not realize
its biosynthetic potential but for Rh. aureum and Rh.
lapponicum the conditions of the Evota Mountain are
favourable. Therefore, these species give the whole
spectrum of compounds of different chemical groups.
4. Conclusions
The processes of the phenolic matters synthesis
proceed in plants of Rhododendron genus growing on
Evota Mountain. In studied extracts, both the matters
of starting processes of phenol biosynthesis
(monophenols, oxycinnamonic acids, coumarins) and
the products of phenol condensation, the final stage of
which are tannins. Catechins and polyphenols (an
average degree of phenolic matters condensation)
dominate.
Approximite generalised chemical composition of
studied rhododendron species includes the next
compounds: polyphenologlycosides, coumarins
(umbelliferone and scopoletin), flavonoids (quercetin,
kaempferol, rutin, hyperin), catechins and their
derivatives, tannins of different degree of
condensment, anthocyanins, essential oils,
monophenols (including arbutin), saponins, aurons
and oxycinnamonic acids (chlorogenic, coumaric,
caffeic, and/or ferulic).
The content of the alkaloid andromedotoxin is not
significant; on results of our studies its presence
carries the residual nature.
The obtained data on chemical composition of
studied plants can be used not only as information base
for the development of technology of practical
application in future but also as a reason for
estimation of phylogenetic species relationship and
their conditions with standpoint of the ecological
adaptation to conditions of growing.
Acknowledgments
The studies are executed on order of Ministry of
Education and Sciences of Russian Federation on the
basis of the project “Floristic studies in South Yakutia;
Discovery of resourse potential of plant
communities”.
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