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Xhmhü PACTHTEJiBHoro cbipbü. 2016. №4. C. 83-88. DOI: 10.1425 8/j cprm.2016041340
UDC 615.322:547.913
COMPONENT COMPOSITION AND BIOLOGICAL ACTIVITY OF ESSENTIAL OILS OF GENUS DRACOCEPHALUM L.
© Ye.M. Suleimen1*, A.B. Myrzagaliyeva2, Zh.A. Ibatayev1, Zh.B. Iskakova1, T.N. Samarkhanov2, B.Z. Medeubayeva2
1The Institute of Applied Chemistry, L.N. Gumilyov Eurasian National University, K. Satbayevstr., 2, Astana, 010008 (Republic of Kazakstan), e-mail: suleimen_em@enu.kz
2S. Amanzholov East Kazakstan State University, 30-th Guards division str., 34, Oskemen, 070019 (Republic of Kazakstan), e-mail: anara_vkgu@mail.ru
This article presents data on the study of the chemical composition, antiradical and cytotoxic activity of three species of the genus Dracocephalum L., which grow in East Kazakstan. Essential oils are obtained from aerial parts of plants D. nutans, D. ruyschiana, D. thymiflorum and the average yield of oil was 0,2%. The chemical composition of essential oils were determined by the GC/MS.
Cytotoxic activity of essential oils was determined by the survival of Artemia salina aquatic crustaceans. Essential oils of D. nutans, D. ruyschiana and D. thymiflorum in all concentrations (1-10 mg/mL) tested exhibit acute lethal toxicity - all larvae were died. Antiradical activity was determined by the method based on colorimetry of free radicals (DPPH). The experimental results showed that essential oils from D. nutans, D. ruyschiana and D. thymiflorum at all tested concentrations (0,1-1,0 mg/mL) exhibited low antiradical activity compared with the standard drug - butylhydroxyanisole.
Keywords: Dracocephalum L., essential oils, chemical composition, GC/MS, cytotoxic activity, antiradical activity.
Introduction
The nature of East Kazakstan land is diverse and in many respects unique. The unique geographical position of the East Kazakstan region is that it is located in the depths of the largest continent of Eurasia within its central part, on the border of the Great Plains - Western Siberia, Central Asia and Kazakstan. In the territory of the region there is continental pole of the planet and the geographical center of Eurasia. East Kazakstan occupies the southwestern part of the Altai (Kazak Altai), Zaysan hollow, Kalba highlands, Saur-Tarbagatay ridges, Irtysh valley and eastern Kazak hills, it covers an area of 283,3 thousand km2. It is bordered on the North by Russia, on the East by China, on the South the border pass with Almaty, on the West - with the Pavlodar and Karaganda regions. On the
- territory of East Kazakstan is the law clear vertical
Suleimen Yerlan Melsuly - Candidate ofChem. Science, PhD, Director of the Institute of Applied Chemistry, Associate Professor of Chemistry Department of L.N. Gumilev, e-mail: suleimen_em@enu.kz Myrzagaliyeva Anar Bazarovna - Doctor of biological sciences, Professor, Vice-rector for academic and educational work, e-mail: anara_vkgu@mail.ru Ibatayev Zharkyn Abykenovich - Candidate of Chem. Science, Leading Researcher of the Institute of Applied Chemistry, e-mail: ZharynAstana@gmail.com Iskakova Zhanar Baktybaevna - Candidate ofChem. Science, Leading Researcher of the Institute of Applied Chemistry, e-mail: zhanariskakova@mail.ru Samarkhanov Talant Nurzhakypovich - Master of geography, Senior Researcher, e-mail: talant.68@mail.ru Medeubayeva Balzhan Zaruetovna - Master of chemistry. Senior teacher of department of chemistry, e-mail: medeubayeva_bz@mail.ru
zones of climate, vegetation and soils [1].
Dracocephalum L. (dragonhead) are widely distributed around the world. The genus includes about 70 species. In flora of East Kazakstan among the genuses of the family Lamiaceae the genus Dracocephalum occupies a leading position, including 9 species, representing 12% of the total number of species of the family Lamiaceae. Dracocephalum grow across all territory of East Kazakstan in various ecological conditions, rising up to 2500 m above sea level. They are part of the various phytocenoses [2]. The genus dragonhead (Dracocephalum) is widely distributed in the flora of East Kazakstan and is used in folk medicine. The aerial
Corresponding author.
part of dragonhead contains large amounts of essential oils, tannins, flavonoids, vitamins [3].
According Budantsev A.L. the genus includes about 70 species [2]. In "Flora of Kazakstan" (1964) described 22 species of this genus found on the territory of the Republic of Kazakstan [4].
To investigate we have been selected and collected plant materials of the following species of dragonhead: D. nutans L., D. ruyschiana L. and D. thymiflorum L.
Previously it was defined that the essential oil D. nutans consists of about 25 monoterpenoids, such as pinocamphone (56,4%), (3-pinene (12,7%), isopinocamphone (4,3%), a-phellandrene (4,6%) and isopinocampheol (3,7%) [5].
The composition of essential oil of the Kazak species D. nutans is very different from the Indian species. It is indicated that the main components of the essential oil of the Kazakstan species D. nutans are decahydro-1,1,7-trimethyl-4-methylene-lH-cycloprop[e]azulen-7-ol, (14,2%), caryophyllene oxide (11,3%), 3,7-dimethyl-l,6-octadien-3-ol, (7,7%), myrtenyl acetate (5,7%), l,2,3,4,4A,5,6,8A-octahydro-7-methyl-4-methylene-naphthalene (5,3%), spathulenol (4,8%), l-methoxy-4-(l-propenyl)-benzene (4,7%), decahydro-l,l,4,7-tetra-[ethenyl]-4ah-cycloprop[e]azulen-4A-ol (4,7%), germacrene D (4,4%), 3(10)-caren-4-ol (4,2%), 4,ll,ll-trimethyl-8-methulene-bicyclo[7.2.0]undec-4-ene (3,7%), a-caryophyllene (3,6%), l,5,5,8-tetramethyl-12-oxabicyclo[9.1.0]dodeca-3,7-diene (2,1%) [6].
I), ruyschiana is cultivated in Baksa, Marosvasarhely/Tirgu Mures (Transylvania, Romania). The predominant compounds in the essential oil of D. ruyschiana (0,2%) are oxygenated bicyclic monoterpenes, such as pinocamphone (43,6%) and isopinocamphone (21,5%). Besides the main components, also identified monoterpenes: myrcene (3,1%), limonene (0,7%), p-cymene (1,5%) and (3-pinene (0,9%); sesquiterpenes: (3-caryophyllene (3,8%), caryophyllene oxide (1,6%), (3-cubebene (1,6%), germacrene-D (3,6%) and elemol (4,4%); and a phenylpropane: methyl chavicol (0,6%). Pinocamphone and isopinocamphone also have been identified as main constituents of essential oil of D. nutans [7, 8].
The chemical composition and biological activity of essential oil of D. thymiflorum or Moldavica thymiflora is poorly investigated, according to the database of Dr. Duke main components of the essential oil of D. thymiflorum are pulegone, caryophyllene, isomenthone, (3-elemene and 1,8-cineole [9].
Experimental
Plant material. For study the chemical composition and biological activity of essential oils, we have collected vegetable raw materials of three types of dragonhead: Dracocephalum nutans, D. ruyschiana, D. thymiflorum.
D. nutans - rhizomatous perennial. It grows in forests, shrubby thickets, on gravelly, sandy and rocky slopes, taluses and steppe pastures, often comes up to the timber line [4]. The plant for research was collected in the territory of East Kazakstan, Katon-Karagay district, the neighborhood of the village Enbek, the mountain Shagyl (N 49°12,200'; E 086°10,320' at the height of 990 m above sea level).
D. ruyschiana - a puberulent perennial plant with height 20-60 cm. It blossoms in the summer, in June-July, fruiting in July-August [4]. Samples of plants were also collected in the territory of East Kazakstan, in Katon-Karagay district, on the ridge Sarymsakty, tract Akimbay (N 49°12,407'; E 086°11,415' at the height of 1383 m above sea level).
D. thymiflorum - an annual plant 15-50 cm high. It grows in bushes, on edges of forests, often as thicket of weed on raw farm lands [4]. The plant was collected in the East Kazakstan region, in Katonkaragay District, on the rocky slopes of the ridge Sarymsakty, along the road to the pass Burhat (N 49°09,617'; E 086°02,198' at the height of 1012 m above sea level).
All plants were collected in the second decade of July, 2014. Plant samples are stored in the herbarium of the Department of Biology of the S. Amanzholov East Kazakstan State University (Oskemen).
Extraction of essential oil. Essential oils were obtained from dried, crushed aerial parts of plants (-100 g) by steam distillation in a Clevenger apparatus for 3 h according to the method by the Pharmacopoeia [10]. During receiving essential oil used hexane as a trap. The essential oil was collected by decantation, dried over Na2S04, weighed and stored in sealed dark glass vials at 4-5 °C until analysis.
The yields for all samples averaged 0,1%.
Analytical GC. The constituent composition of essential oils were determined on a Clarus-SQ 8 GC/MS (PerkinElmer) with a mass-spectrometric detector. An essential oil sample (25 mg) was placed into a 25-mL volumetric flask, dissolved in hexane (15 mL), adjusted to the mark, and stirred until the oil was fully mixed.
Chromatographic conditions: capillary column Restek Rxi®-lms (0,25 mm x 30 m x 0,25 |im): sample volume 1,0 |iL: He carrier gas at 1 mL/min; flow division 1 : 25; column temperature 45 °C increasing at 1,5 °C/min to 200 °C, than at 15 °C/min to 280 °C and isotherm at 280 °C for 10 min; vaporizer temperature 280 °C; mass spectrometric detector at 240 °C with EI+ 70 eV; scan time 4-120 min; scan range 39-500 m/z.
The percent contents of constituents were calculated automatically from peak areas in the total ion chroma-togram. Constituents were identified from mass spectra and retention times, using the NIST library. Retention indices were calculated relative to «-alkanes.
Cytotoxic activity. Separating funnel filled with 55 mL of artificial sea water and 200 mg of Artemia salina eggs. Allowed standing for 3 days at the air supply until soft crustaceans gave the egg. One side of the tube covered with aluminum foil, and 5 minutes later, the larvae that are going on the bright side of the funnel, removed with Pasteur pipette.
20-40 larvae were placed in 990 |iL of seawater into each of the 24 micro titer plates. Dead larvae were counted using a microscope. Added 10 |iL of dimethylsulfoxide solution of 10 mg/mL sample. As a comparison, the drug actinomycin D or staurosporine was used. For a negative control 10 |iL was added only DMSO. After 24 h of incubation and further maintaining micro titer plates for 24 hours (to ensure immobility) counts the dead larvae by the microscope [11].
Mortality P determined by the following formula:
A-N-B
P =---100% (!)
P = A N B l00%Where A - amount of dead larvae after 24 h; N - amount of larvae died before the test; B - the
z
average amount of larvae died in a negative control; Z - the total amount of larvae.
Antiradical activity. Determination of antiradical activity of essential oil was carried out by the known technique of the colorimetry of free radicals based on reaction of the radical a 2,2-diphenyl-l-picrylhydrazil (DPPH) with standard of antioxidant [12, 13].
For determination of inhibition of DPPH to 0,1 mL of the test sample in the range of concentration of 0,25; 0,5; 0,75; 1 mg/mL added 3 ml of 6x 10"5 M solution of radical. Centrifuge test tubes were in a support, wrapped in black polyethylene. After intensive mixing, solutions were left in the dark and after 30 minutes were measured absorbance of solutions at 520 nm.
The values of antiradical activity (ARA) were calculated using the formula shown below:
ARA(%) = 04o - At)/A0 * 100% (2)
Where A0 - absorbance of control; At - absorbance of the working sample.
The optical density of the investigated samples measured on a spectrophotometer Cary 60 UV-Vis. Antiradical activity of essential oil, we compared with butylhydroxyanisole (BHA).
Antiradical activity is defined in relation to standard - of butylhydroxyanisole (BHA).
Results and Discussion
Chemical composition . As shown in the Table 1, in essential oil of D. nutans mail components are: cis, cis-nepetalactone - 35,0%, germacrene D - 6,3%, (3-cyclocitral - 4,0%, (3-bourbonene - 3,1% and cis, trans-nepetalactone - 2,9%. The main components of the essential oil of D. ruyschiana found a-pinene - 3,8%, 3-carene - 3,2%, (3-pinene - 2,7% and 3-octanol acetate - 2,5%, and in essential oil of D. thymiflorum - (3-caryophyllene oxide - 12,2%, spathulenol - 9,4%, palustrol - 3,9%, 1,8-cineol - 3,7% and humulene-l,2-epoxide - 3,7%.
Cytotoxic activity. Results of the study the cytotoxic activity of essential oils from areal parts of D. nutans, D. ruyschiana and D. thymiflorum are shown in Tables 2-4.
Based on this experiment it can be assumed that the essential oils of D. nutans, D. ruyschiana and D. thymiflorum in all concentrations tested exhibit acute lethal toxicity - all larvae are died.
86_
D. thy
tested
Table
RI
1
781
819
921
924
959
960
962
973
980
997
997
1001
1006
1012
1014
1014
1025
1035
1080
1081
1094
1111
1124
1133
1134
1138
1140
1148
1153
1159
1172
1191
1193
1194
1196
1209
1214
1229
1233
1257
1263
1295
1295
1305
1306
1319
1332
1350
1355
1359
1364
1373
1 ntiradical activity. Values of the antiradical activity of essential oils of D. nutans, D. ruyschiana and iflorum calculated by the formula (2) are given in Table 5.
"he experimental results showed that essential oils of D. nutans, D. ruyschiana and D. thymiflorum at all ancentrations exhibited low antiradical activity compared with the reference drug butylhydroxyanisole.
Chemical composition of the aerial parts essential oils of D. nutans, D. ruyschiana and D. thymiflorum
Compound
Composition (%)
D. nutans
D. ruyschiana
2
3
4
Hexanal
2-Hexenal
Benzaldehyde
a-Pinene
P-Pinene
1-Octen-3-ol 6-Methylheptan-3-on
2-Pentyl- furan P-Myrcene
3-Carene a-Ocimene Benzeneacetaldehyde o-Cymene 1,8-Cineol Limonene
3-Ethyl-4,5-dimethyl-1,4-hexadiene
cw-P-Ocimene
trans-P-Ocimene
Nonanal
Linalool
3.4-Diethylthiophene 3-Octanol acetate
Pinocarvone
Isopinocamphone
frawi-Pinocamphone
Cryptone
Naphthalene
Terpinen-4-ol
Myrtenal
a-Terpineol
P-Cyclocitral
/>-Ethylbenzyl alcohol
/>-Cumic aldehyde
5.5-Dimethyl-2-propyl-l,3-cyclopentadiene Pulegone
cw-3-Hexenyl-a-methylbutyrate cw-3-Hexenyl isovalerate Phellandral
cw-Chrysanthenyl acetate Isocarveol Dihydroedulan II n-Nonyl acetate Myrtenyl acetate p-Methoxy thiophenol cis, cis-Nepetalactone Eugenol
cis, fra«s-Nepetalactone
frawi-P-Damascenone
cw-Jasmone
a-Copaene
P-Bourbonene
P-Elemene
0,8 0,4 1,0
0,6 1,0
0,5
0,6 1,3 0,3
0,6 1,5
0,3
0,3
0,4
4,0
0,4
0,4
0,3
2,0
0,9 35,0
2,9
0,3 0,5 3,1
1,6 0,6 0,4
3.8 2,7
0,6
2,1 3,2
1,0 0,7
1.9 0,4
0,7 1,1 0,8 2,5
1,5
1,0
1,9 0,5 0,5 0,9
1,4
0,5
0,4
0,6 0,5 0,5 1,3 0,4
0,5
End of Table 1
1 2 3 4 5
1376 a-Dihydroionone 0,3 - -
1395 P-Caryophyllene 1,1 - 0,7
1396 Aristolene - 0,6 -
1406 P-Copaene 0,6 - -
1422 Geranyl acetone - 0,8 -
1440 trans-P-Farnesene 1,4 - -
1446 trans-P-Ionone 0,3 1,8 0,5
1450 Germacrene D 6,3 0,4 1,1
1455 a-Curcumene 0,6 - -
1460 y-Muurolen 0,7 - -
1464 Bicyclogermacrene - - 0,4
1464 S-Guaiene - - 0,4
1470 Unknown 1 - 0,5 -
1471 a-Cedrene 0,8 - -
1471 Cubebol - - 0,4
1479 y-Cadinene 0,5 - -
1480 P-Bisabolene 1,3 - -
1491 S-Cadinene 2,0 - -
1500 Pentadecane - - 0,4
1503 Norbourbonone - - 0,5
1505 Caryophyllene oxide isomer - - 0,7
1521 1,5-Epoxysalvial-4( 14)-ene - - 0,5
1528 Palustrol - - 3,9
1533 Spathulenol 0,3 - 9,4
1538 P-Caryophyllene oxide 1,0 0,6 12,2
1541 P-Spathulenol - - 0,8
1547 Isoaromadendrene epoxide 0,5 - 0,4
1547 Salvial-4( 14)-en-l -one 0,3 - -
1553 Aromadendrene oxide-(2) - - 0,4
1563 a-Guaiol 1,0 2,3 -
1564 Humulene-l,2-epoxide - - 3,7
1590 Isospathulenol - - 1,7
1600 Hexadecane - - 0,6
1618 5-Cadinol - - 0,5
1633 Bulnesol 0,9 2,4 -
1635 (lR,7S,E)-7-Isopropyl-4,10-dimethylenecyclodec-5-enol - - 2,1
1646 ent-Germacra-4( 15 ),5,10( 14)-trien-1 P-ol 0,6 - -
1649 Unknown 2 - 0,8 1,1
1824 Phthalic acid, isobutyl octyl ester - 0,7 0,5
1835 Perhydrofarnesyl acetone 0,3 0,6 0,6
1949 «-Hexadecanoic acid 0,5 1,2 0,7
2066 Biformen 0,3 - -
2097 frawi-Phytol 0,4 0,7 -
2346 Larixol 0,5 - -
2700 «-Heptacosane 0,4 - -
2900 «-Nonacosane - 0,9 0,5
Table 2. Cytotoxic activity of essential oil from areal parts of D. nutans
Parallel Number of larvae in control Number of larvae in sample The amount of surviving larvae in the control, % The amount of surviving larvae in sample, % Mortality, P,% The percentage of neurotoxicity, %
survived died survived died paralyzed
10 mg/mL
Medium 25 1 0 28 0 96 0 96 0
5 mg/mL
Medium 25 1 0 27 0 96 0 96 0
1 mg/mL
Medium 25 1 0 27 0 96 0 96 0
Table 3. Cytotoxic activity of essential oil from areal parts D. ruyschiana
Parallel Number of larvae in control Number of larvae in sample The amount of surviving larvae in the control, % The amount of surviving larvae in sample, % Mortality, P,% The percentage of neurotoxicity, %
survived died survived died paralyzed
10 mg/mL
Medium 25 1 0 23 0 96 0 96 0
5 mg/mL
Medium 25 1 0 24 0 96 0 96 0
1 mg/mL
Medium 25 1 0 25 0 96 0 96 0
Table 4. Cytotoxic activity of essential oil from areal parts D. thymiflorum
Parallel Number of larvae in control Number of larvae in sample The amount of surviving larvae in the control, % The amount of surviving larvae in sample, % Mortality, P,% The percentage of neurotoxicity, %
survived died survived died paralyzed
10 mg/mL
Medium 25 1 0 28 0 96 0 96 0
5 mg/mL
Medium 25 1 0 25 0 96 0 96 0
1 mg/mL
Medium 25 1 0 28 0 96 0 96 0
Table 5. Antiradical activity of various concentrations of essential oils from D. nutans, D. ruyschiana and D. thymiflorum, %
No Sample Essential oil concentration, mg/mL
0,1 0,25 0,5 0,75 1,0
1 Butylhydroxyanisole (BHA) 80,82 81,23 80,30 83,08 83,88
2 Dracocephalum nutans L. 5,53 6,46 6,59 7,79 7,32
3 Dracocephalum ruyschiana L. 3,51 4,57 3,89 4,97 4,26
4 Dracocephalum thymiflorum L. 5,65 7,57 7,83 8,64 8,70
Conclusion
Thus, during the researches the chemical composition, cytotoxic and antiradical activity of essential oils from D. nutans, D. ruyschiana and D. thymiflorum were determined.
The experimental data show that the essential oils of D. nutans, D. ruyschiana and D. thymiflorum showed high cytotoxic and low antiradical activities.
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Received June 10, 2016 Revised July 12, 2016
