Chromatographic Analysis of Chemical Composition …...Chromatographic Analysis of Chemical...

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: [email protected].

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.


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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


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.


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


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.


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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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