J. Chem. Chem. Eng. 8 (2014) 516-523 D DAVID PUBLISHING

Chromatographic Analysis of Chemical Composition of the Genus 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 (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 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 There is another feature of this place: all the plants growing on the mountain top experience excessive South Yakutia is situated in the heart of North East UV (ultraviolet rays), which are contained in solar Asia, and occupies the territory of Olekma-Charsky radiation. and Aldan plateau and the Northern spurs of Stanovoy Best adapted to such conditions are plants of range. Climate features allow us to classify this region family due to their unique chemical as 2nd or 3rd zone of “USDA-zones” scale with compounds. Of the 11 species of this family, living at minimum air temperatures in winter from -34.4 °C the top of Evota mountain, our attention was attracted (-30 F) to -44 °C (-40 F). The relief contains mainly three unique species of Rhododendron genus—Rh. hills (their altitude is limited to 900 m above sea level) adamsii Rehd., Rh. aureum Georgi. and procumbent with rounded flat tops. The unique landscape features form of Rh. parvifolium Adams also called as Rh. are the mountains situated on the north of lapponicum subsp. parvifolium (Adams) T. Yamaz. Nerungrinski district and named as Evota mountains Rh. aureum is a rare protected species listed in the (from Evota river on south-east side from them). red book of Yakutia [1]. Its leaves and flowers are The altitude of Evota mountain is 1,601 m above very popular in folk medicine for the treatment of sea level. Here, there are all natural zones conditional heart diseases [2, 3]. The chemical composition of on high-altitude—from forest at the foot of the this , the most studied. According the mountain to tundra and stony waste plot at the top. investigations from Irkutsk region [2-10], the leaves and sprouts of Rh. Aureum from Baical region contain *Corresponding author: Zaytseva Natalia Vladimirovna, Ph.D., research field: technology of practical application of the next biologically active matters: rhododendrin plants in the Northern regions. E-mail: [email protected].

Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants 517 Growing on the Mountain of Evota (South Yakutia)

(betuloside), betuligenol, ericolin, hyperoside, tannins, Thereby, the plants of Rhododendron can be a arbutin, rutin, gallic acid, ursolic acid, poisonous source of valuable medicinal matters of the natural glycoside of andromedotoxin. There are more than 50 derivation. In our opinion, severe highland conditions compounds besides mono- and sesquiterpenes of of growing plants, which is indicative of essential oil contaned more than 20 compounds South-Yakutia region, promote them in the synthesis (pinenes, myrcen, betuligenol/rhododendrole, etc.) [11]. of components that provide the processes of The oil has the fine exquisite aroma with tone of the adaptation and increased resilience organisms under tea rose scent. the powerful electromagnetic fields, UV radiation, Rh. adamsii is valuable medicinal plant used in oxygen shortage. This can be of great practical medicine Buryatia, Mongolia and Tibet as a means of importance for medical purposes and some problems increasing resistance to the human organism to of the health of the population at high widths. adverse conditions of existence, weariness, fatigue The aims of this research were: to study the [2, 3, 12, 13]. In Chinese and Mongolian medicine chemical composition of Rh. adamsii, Rh. lapponicum preparations of this plant named “Sagan Dayli” or and Rh. aureum by using method of thin layer “White wing”. According to literary sources [2, 3, 9, chromatography; to detect some features of 11, 14, 15], the composition of Rh. adamsii includes: biochemical composition depended on mountain essential oil of the complex chemical composition (in conditions of their growing. At this stage, our studies that number: germacron (26.2%); ± -transnerolidol are the searching ones. For species of South Yakutia (18.4%); juniper-camphor and nerolidol), resinous such studies are organized for the first time. substances, ursolic and oleanolic acids, triterpenoids, 2. Experiments tannins, flavonic derivatives. All parts of Rh. adamsii have very strong, sharp, but pleasing scent, reminding 2.1 Characteristics of the Plant Material scent of the rose and resins simultaneously—it does The leaves and sprout tops growing at the mountain this plant as especial amongst representatives of of Evota were collected for analysis. The collection of Rhododendron genus. raw materials for studies was carried out at June 29, The chemical composition of Rh. lapponicum (Rh. 2013, in dry solar weather, in the afternoon. parvifolium) is studied least of all. There are few scientific publications devoted to this species though it 2.2 Technology of the Plant Material Fixation is worthy of rapt attention. According to studies of Collected plant material was sorted and reduced, Makarov et al. [16], it was discovered the next matters and then it was dried out in dryer at 60 оС to dry in this species: tannins both pyrogallic and condition. Dried plant material was sacked in paper pyrocatechinic groups, quercetin, avicularin, coumarin, packages for keeping. Before undertaking analyses, aesculetin, the simple phenol compounds. According plant material was again dried in dryer at 60 оС to to published edition “Plant resources of Russia” (2009) Rh. constant mass (“absolutely dry mass”). parvifolium contains ursolic and oleanic acids, 2.3 Technology of the Extract Preparation sitosterin, arbutin (up to 4%), and phenol carboxylic acids [9]. According to Ref. [11], the essential oil of For extraction 1 g (accurate to 0.01 g) of absolutely this rhododendron containes more than 30 dry raw material was moistened is about 10 mL 70% components and the main from which are ethanol and then infused for 24 h under periodic isoaromadendren epoxide (13.8%), patchoulol (4.9%), shaking. Then, the extracts were evaporated to 1 mL a-cadinol (4.7%) and t-cadinen (4.2%). under slow heating.

518 Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)

2.4 Technology of the Chromatogram Preparing yellow-green fluorescent in UV-365 nm (indication of flavonoids); with VSR—rich bright red-brown to We used the standard technology of the bright orange and golden (indication of catechins, chromatography analysis [17, 18]. Adsorbent is silica phlobaphens, anthocyanin and quercetin). gel precoated on plates for thin-layer chromatography Other general sign for all studied chromatograms is (the brand is “Sorbfil” (PTSH-AF-А-UF)). Solvent yellow or yellow-green zone with R 0.90-0.97, which system is n-butanol:acetic acid:water (4:1:5). f colouration increases after processing by KOH Detection was carried out with the next spray (indication of kaempferol). In UV-256 nm, this zone reagents: (1) 10% ethanolic solution of KOH; (2) 5% is blue fluorescent (indication of aurons). In VSR, this ethanolic solution of AlCl3; (3) VSR (vanilin-sulfuric zone is colored red or violet (indication of single-core acid reagent) (1% sulfuric vanilin); (4) FeSO4 phenolic acids (arbutin, benzoic acid), saponins and (iron-II-sulfate reagent) (1% aqueous solution); and (5) DRG (dragendorff reagent) (solution of basic bismuth essential oils). nitrate in glacial acetic acid and water/water solution The small green (vis.) and bright-rose fluorescent of potassium iodide). (in UV and KOH) zone with Rf 1.0 represents the Each chromatogram was described with color, chlorophyll. The white fluorescence in the manner of brightness and form of the spots, features of their fine bands in this part of tracks is due to caffeic and/or distribution on the chromatographic track; the main ferulic acids. The zone with Rf 0.5-0.6 in the middle of tracks has quantitative index was Rf-index. The developed chromatograms were photographed distinct bright-blue fluorescence in UV-365 nm. It on Panasonic DMC-FS62 in visible (vis.) and most probably is coumarin or its predecessor on ultraviolet light (UV-254 nm and UV-365 nm). biosyntesis—coumaric acid. The identification of matters was conducted in The analysis of the rhododendron chemical accordance with information presented in publishing composition gives the following results. [17-21]. 3.1 Rhododendron Aureum Georgi 3. Results and Discussion There is a large number of phenolic matters both The analysis of chromatograms is indicatives of hydrolysed and nonhydrolysed nature in composition significant chemical resemblance of studied of the extract of this species (Fig. 1). The derivatives rhododendron species. All the chromatographic tracks of pyrogallol dominate—it is distinguished of Rh. are colored dark-grey or grey-yellow in vis. and aureum from two other rhododendron species. At especially intensive—with FeSO4 (Figs. 1-3). This interaction with iron ions phenolic compounds are fact is evidenced of presence of great number of colored blue dominating along the whole length of phenolic compounds with different chromatographic track. Rh. aureum shows three major blue zones in the mobility degree in extracts. Rf 0.57-0.67; 0.82-0.88 and 0.91-0.97 that could All tracks show one major yellow-grey or satisfy the chromatographic descriptions of following grey-olive zone (vis.) in the Rf range 0.7-0.8 that is matters: gallic acid, hydrolysed tannins, pyrogallol. well colored with all detectors and gives specific Grey-olive zone at Rf 0.67-0.77 represents yellow-green fluorescence in UV-365 nm. With nonhydrolysed tannic matters (catechins). In VSR,

FeSO4 reagent this zone is colored green-brown this zone is colored bright red or red-wine due to

(indication of catechins); with KOH and AlCl3 synthesis of red pigments in acid. Perhaps these are the reagents—bright yellow (vis.) and distinct products of acid condensation of pyrocatechin

Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants 519 Growing on the Mountain of Evota (South Yakutia) derivatives—anthocyanins, leucocyanidins and of coumarinic nature: scopoletin (at Rf 0.37-0.43) and phlofabens [21]. umbelliferone (at Rf 0.50-0.57). Oxycinnamonic acids The qualitative reactions on the other groups of such as chlorogenic and coumaric ones posses almost matters show that yellow (in vis.) and yellow-green the same chromatographic features. The bright-white fluorescent (in UV-365 nm) zone with Rf 0.67-0.77 fluorescent stripe at the solvent front is a sign of conformes to flavanoids. In KOH and AlCl3, the caffeic and ferulic acids. fluorescence increases. The most probable matter with Thereby, we detected the next compounds in the Rh. such characteristic could be the quercetin. aureum extract: hydrolysed and nonhydrolysed

The zone in the Rf range 0.82-0.88 is weak without tannins, catechins, flavonoids (kaempferol, quercetin), additional spraying, but weak green fluorescence simple phenolic matters (arbutin), oxycinnamonic becomes more visible after spraiyng with KOH and acids (chlorogenic, coumaric, gallic, caffeic and

AlCl3 (indication of kaempferol). ferulic), coumarins (scopoletin, umbelliferone),

The zone in the Rf range 0.90-0.97 is yellow (vis.) phenologlycosides. The phenolic matters of the but in VSR it is colored red-orange and in middle degree of condensment (catechins) prevail.

FeSO4—violet. It is a sign of the simple phenols 3.2 Rhododendron Adamsii Rehd. (arbutin, hydroquinone and phenolic acids). The dark-blue fluorescent band in lower part of The chromatographic analysis of Rh. adamsii track indicatives of “heavy” phenolic matters extract reveals nonhydrolised polyphenols in chemical with low index of chromatographic mobility because composition of this plant (Fig. 2). Tracks have weak of their relationship with milk sugars dark-blue fluorescent bands in the lower range of Rf (phenologlycosides). detected as phenologlycosides.

The bright-blue fluorescent (in UV-365 nm) zones The distinct blue fluorescence at Rf 0.60-0.68 is due

(increasing in KOH and AlCl3) represent the matters 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 Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)

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 chemical composition of Rh. adamsii: nonhydrolysed major. With KOH and AlCl3, this zone is colored tannins, some fractions of flavonoids (including bright-yellow in vis. and green fluorescent in UV-365 quercetin and kaempferol), coumarins (umbelliferone), nm (indication of flavonoids); with saponins, aurons, essential oil, arbutin, caffeic acid. VSR—yellow-orange (indication of flavondiols); with 3.3 Rhododendron Lapponicum Subsp. Parvifolium FeSO4—dark-brown (indication of tannins). On the (Adams) T. Yamaz basis of these signs, it is possible to confirm the presens of quercetin and tannins in composition of Rh. Chromatographic tracks of Rh. lapponicum extract adamsii. are like tracks of Rh. adamsii, but the first ones are

Treatment with FeSO4 generates some additional more prominent and rich of the following components dark-brown zones in the range of Rf 0.80-0.93. With (Fig. 3):

КОН and AlCl3, these zones show green and blue (1) “Heavy” polyphenols are characterized by fluorescence (indication of flavonoids). fluorescent dark-blue zone at Rf 0.00-0.025;

At the solvent front (in the range of Rf 0.90-1.00), (2) Scopoletin (and/or chlorogenic acid) is found at there are some zones with indivual color reactions: Rf 0.24 as fluorescent blue zone; KOH intensifies the (1) Without spraying—white fluorescent zone in fluorescence;

UV-365 nm (caffeic acid); blue fluorescent zone in (3) Romedotoxin is found at Rf 0.25 as dark zone; UV-256 nm (aurons); (4) Hyperin is revealed in VSR and UV-365 nm as

(2) In VSR—blue (in vis.) and dark-violet (in brown fluorescent zone at Rf 0.33-0.36; UV-365 nm) zone (saponins, essential oils); (5) Umbelliferone (and/or coumaric acid) is found at

(3) In FeSO4—weak red zone (arbutin) and light Rf 0.45 as fluorescent bright-blue zone; KOH brown zone (aurons, lignans). intensifies the fluorescence; Thereby, there are the next compounds in the (6) Rutin is revealed in VSR and UV-365 nm as

Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants 521 Growing on the Mountain of Evota (South Yakutia)

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. phenologlycosides, alkaloids (andromedotoxin), The distribution of compounds in upper part of the flavonoids (hyperin, rutin, quercetin, kaempferol), tracks looks like tracks of Rh. adamsii practically in coumarins (scopoletin, umbelliferone), everything. There are some main zones. oxycinnamonic acids (coumaric, caffeic and ferulic),

The first zone in the range of Rf 0.69-0.77 is saponins (ursolic and oleanic acids), nonhydrolysed correspond to flavonoids, quercetin, nonhydrolysed tannins, arbutin, aurons, lignans. tannins. Thus, our results correspond to information

The second zone in the range of Rf 0.80-0.95 presented in publications [3-16]. However, it is consists of several subzones: possible to draw a conclusion about tannins as the (1) Yellow-green in vis. and dark-green fluorescent major compound of rhododendron representatives. (in UV-365 nm) zone (in KOH the fluorescence in Due to tannins these plants adapt to conditions of intensified and colored yellow); highlands and UV radiation, and at the same time (2) Yellow-brown in vis. and white fluorescent (in tannins are products of intensive biosynthetic UV-365 nm) zone; processes in plants growing in conditions of intensive (3) Weak green in vis. and bright-rose fluorescent solar radiation themselves. Fluorescent (in UV-365 nm) zone. compounds—coumarins (blue fluorescence UV-365 This zone is blue fluorescent in UV-264 nm, in nm), oxycinnamonic acids (white fluorescence

VSR it is colored blue. In FeSO4 detected subzones UV-365 nm), flavonoids (yellow fluorescence in are accordingly colored dark-brown, red and UV-365 nm), and aurons (blue fluorescence in yellow-brown. UV-256 nm)—also reduce unfavourable influence of Thereby, the next compounds were detected in the UV radiation and present in extracts of studied species chemical composition of Rh. lapponicum: of rhododendrons in relatively large amount.

522 Chromatographic Analysis of Chemical Composition of the Genus Rhododendron Plants Growing on the Mountain of Evota (South Yakutia)

The analysis of the chemical composition of for the development of technology of practical rhododendron species of Evota Mountain directs on application in future but also as a reason for the following discourses: estimation of phylogenetic species relationship and (1) Rh. adamsii and Rh. lapponicum are near related their conditions with standpoint of the ecological species, so they have practically the same features of adaptation to conditions of growing. the extract chemical compositions. Acknowledgments (2) On presence and distribution of compounds in plant extracts, it is possible to detect if the plant is in The studies are executed on order of Ministry of favourable conditions for its growing and Education and Sciences of Russian Federation on the development or feels the stress and deperssion. So, basis of the project “Floristic studies in South Yakutia; according our researches Rh. adamsii does not realize Discovery of resourse potential of plant its biosynthetic potential but for Rh. aureum and Rh. communities”. lapponicum the conditions of the Evota Mountain are References favourable. Therefore, these species give the whole spectrum of compounds of different chemical groups. [1] Rare and Threatened Species of Plants and Mushrooms. In The Red Book of the Sakha Republic (Yakutia); 4. Conclusions Publishing House “Sakhapoligraphizdat”: Yakutsk, 2000; p 256. The processes of the phenolic matters synthesis [2] Teljatev, V. V. Healing Treasures; Irkutsk the proceed in plants of Rhododendron genus growing on Eastern-Siberian Book Publishing House, 1991; p 400. [3] Medicinal Plants of Siberia for the Treatment of Evota Mountain. In studied extracts, both the matters Cardiovascular Diseases; Novosibirsk, 1991; p 240. of starting processes of phenol biosynthesis [4] Zapesochnay, G. G.; Benkovski, A. I. Polyphenols of (monophenols, oxycinnamonic acids, coumarins) and Plants: The Study of the Chemical Composition in the products of phenol condensation, the final stage of Leaves of Rhododendron Aureum Georgi. Chemistry of Natural Compounds 1965, 24, 289-292. which are tannins. Catechins and polyphenols (an [5] Mirovich, V. M.; Makarenko, S. P.; Paisova, O. I. Study average degree of phenolic matters condensation) of Chemical Composition of Overground Organs of dominate. Adams Rhododendron by High Performance Liquid Approximite generalised chemical composition of Chromatography. Bulletin of the East-Siberian Scientific Centre of Siberian Branch of Russian Academy of studied rhododendron species includes the next Medical Sciences 2005, 7, 164-166. compounds: polyphenologlycosides, coumarins [6] Murashkin, I. A. Phytochemical Study of Rhododendron (umbelliferone and scopoletin), flavonoids (quercetin, Aureum and Obtaining on Its Basis Medicines. Ph.D. kaempferol, rutin, hyperin), catechins and their Thesis, Ulan-Ude, 2003. [7] Potrazi, K. A.; Kopnin, A. A.; Dagaeva, S. O.; Margaryan, derivatives, tannins of different degree of A. A.; Sokolskaya, T. A. Quantitative Determination of condensment, anthocyanins, essential oils, an Arbutin in Raw Materials of Rhododendron Plants monophenols (including arbutin), saponins, aurons (Rhododendron Aureum Georgi) Using High-Performance Liquid Chromatography. Siberian and oxycinnamonic acids (chlorogenic, coumaric, Medical Journal 2009, 8(91), 134-138. caffeic, and/or ferulic). [8] Potrazi, K. A.; Margaryan, A. A.; Dagaeva, S. O.; The content of the alkaloid andromedotoxin is not Sokolskaya, T. A. Rhododendron Aureum is a Promising significant; on results of our studies its presence Source of New Medicines Drugs. Problems of Biological, Medical and Pharmaceutical Chemistry 2009, 6, 9-13. carries the residual nature. [9] Plant Resources of Russia: Wild Flowering Plants, Their The obtained data on chemical composition of Composition and Biological Activity. In Families studied plants can be used not only as information base Actinidiaceae-Malvaceae, Euphorbiaceae-Haloragaceae;

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