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Chemical Composition of the Essential Oil of Phlomis linearis Boiss. & Bal., and

Biological Effects on the CAM-Assay: A Safety Evaluation

Betl Demircia,b,*, Mehmet Y. Dadandc, Dietrich H. Paperb, Gerhard Franzb, andKemal Hsn Can Basera

a

Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470 Eskis ehir,Turkey. E-mail: bdemirca@anadolu.edu.trb Department of Pharmaceutical Biology, Faculty of Chemistry and Pharmacy, University of

Regensburg, 93040 Regensburg, Germanyc Department of Biology, Faculty of Science and Letters, Erciyes University, 38039 Kayseri,

Turkey

* Author for correspondence and reprint requests

Z. Naturforsch. 58 c, 826829 (2003); received June 20/August 15, 2003

Phlomis linearis Boiss. & Bal. of the Lamiaceae family growing in central, east and south-east Anatolia is an endemic species for Turkey. The essential oil obtained from the aerialparts by hydro distillation was subsequently analyzed by GC/MS. The main components of

the oil were found as

-caryophyllene (24.2%), germacrene D (22.3%) and caryophylleneoxide (9.2%), among 49 identified compounds, representing 94.5% of the total essential oil.The overall biological activity of the essential oil (100 g/pellet) was tested on the chorioal-lantoic membrane (CAM) of the fertilized hens egg in order to examine the anti-angiogenicand anti-inflammatory activity. None of the tests showed pronounced activity, toxicity orirritation at the tested concentration.

Key words: Phlomis linearis, Essential Oil, CAM-Assay

Introduction

The genus Phlomis as perennial herbs or shrubsof Lamiaceae are represented by thirty four spe-cies of which twenty one are endemic for Turkey(Huber-Morath, 1982). The leaves and flowers ofPhlomis species are used as aromatic amara, car-minative, stimulant and are locally known asBallkotu, Calba, Calba, Salba in Turkish tradi-tional medicine (Baytop, 1997, 1999). The endemicspecies Phlomis linearis Boiss. & Bal. grows insteps, rocky igneous slopes, volcanic rubble at 1400to 2400 m and has a distribution area from centralto east and southeast Anatolia. This species is dis-tinguished by a wide tubular calyx with a leavesize 4 to 8 times longer than the width. The calyxand bracteoles also contain intense long hairs, thecorolla is two lipped, generally in yellow and theupper lip is occasionally brownish (Dadand, 2002)

Chemical constituents of Phlomis linearis have

been investigated by Cals and co-workers (1990,1991); new glycosides, iridoids, neolignans, andtheir derivatives have been isolated. Some Phlo-mis species have also been investigated for both

09395075/2003/11000826 $ 06.00 2003 Verlag der Zeitschrift fr Naturforschung, Tbingen http://www.znaturforsch.com D

their biological activities and essential oil composi-

tions by different groups (Mirza and Nik, 2003;Couladis et al., 2000; Sokovic et al., 2002a,b; Risticet al., 2000)

The in vivo application on the chorioallantoicmembrane of the fertilized egg (CAM-assay) is auseful and well established method to determineanti-angiogenic and anti-inflammatory activity ofindividual compounds or complex plant extracts.It can be used for testing natural compounds insmall amounts for revealing various modes of ac-

tion and the complex mechanisms related to an-giogenesis and inflammation. Furthermore, pos-sible side effects such as membrane irritation,toxic and anticoagulant properties of the investi-gated material in question can be revealed (Paper,1998; DArcy and Howard, 1967; Brgermeisteret al., 2002; and references cited herein).

In this study, we have isolated the volatiles bywater distillation from the aerial parts of P. linea-ris. The essential oil was then analyzed by gas

chromatography/mass spectroscopy (GC/MS). Toevaluate the essential oil and its potential anti-an-giogenic and anti-inflammatory activity, the CAM-assay was employed.

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Methods and Materials

Plant material

Aerial parts of the plant were collected froma step area in Kayseri, Erciyes Mountain, atabout 2000 m, on July 24, 2002, by one of us(M.Y.D.) and M. G. Halc. A voucher specimen(MYD1621) was kept at the Herbarium of ErciyesUniversity, Faculty of Science and Letters.

Isolation of the essential oil

The aerial parts were dried in shade at roomtemperature. The essential oil was obtained bywater distillation using a Clevenger type apparatusfor 3 h having a yield of 0.05% (w/v).

Gas chromatography mass spectrometry

The essential oil was analysed by gas chroma-tography/mass spectrometry using a Hewlett-Packard GCD system. Innowax FSC column(60 m 0.25 mm, 0.25 m film thickness) was usedwith helium as carrier gas. GC oven temperaturewas kept at 60 C for 10 min and programmed to220 C at a rate of 4 C/min, and then kept con-

stant at 220 C for 10 min and programmed to240 C at a rate of 1 C/min. Split flow was ad-justed at 50 ml/min. The injector temperature was250 C. Mass spectra were recorded at 70 eV. Massrange was from m/z 35 to 425. n-Alkanes wereused as reference points in the calculation of rela-tive retention indices (RRI). Relative percentagesof the characterized components were as listed inTable I. Library search was carried out using bothWiley GC/MS Library and Baser Library of

Essential Oil Constituents.

CAM-assay

Sample preparation: The essential oil (10 mg/ml)was dissolved in a 2.5% (w/v) agarose (Merck,Darmstadt) solution. For ease of application pel-lets of these solutions (10 l) were prepared andapplied drop wise on circular teflon supports of3 mm diameter, cooled to room temperature for

solidification and applied on to the chorioallantoicmembrane (CAM).

Preparation of the test medium: The fertilizedhens eggs were previously incubated for 6572 hat 37 C and a relative humidity of 80%. The eggswere positioned in a horizontal position and ro-

Table I. The composition of the essential oil of Phlomislinearis.

RRI Compound %

1479 -Elemene tr1497 -Copaene 0.31528 -Bourbonene tr1535 -Bourbonene 1.41553 Linalool 0.61589 -Ylangene 0.51600 -Elemene 0.71612 -Caryophyllene 24.21650 -Elemene 0.11668 (Z)--Farnesene 6.61687 -Humulene 3.51704 -Muurolene 0.41706 -Terpineol 0.21726 Germacrene D 22.3

1740 -Muurolene 1.41755 Bicyclogermacrene 1.11773 -Cadinene 1.01776 -Cadinene tr1808 Nerol tr1827 (E,E)-2,4-Decadienal 0.21838 (E)--Damascenone 0.31857 Geraniol 0.51868 (E)-Geranyl acetone 0.51900 Nonadecane 0.21958 (E)--Ionone 0.62001 Isocaryophyllene oxide 0.82008 Caryophyllene oxide 9.2

2045 Humulene epoxide-I tr2046 Norbourbonone 0.12071 Humulene epoxide-II 0.72095 Hexyl benzoate 0.22131 Hexahydrofarnesyl acetone 5.82179 3,4-Dimethyl-5-pentylidene-2(5H)-furanone 0.62198 Thymol 0.52209 T-Muurolol 0.32239 Carvacrol 1.22255 -Cadinol 1.12300 Tricosane 0.62324 Caryophylla-2(12),6(13)-dien-5-ol 0.4

(Caryophylladienol II)2384 Hexadecanol 0.92389 Caryophylla-2(12),6-dien-5-ol 0.1

(Caryophyllenol I)2392 Caryophylla-2(12),6-dien-5-ol 1.1

(Caryophyllenol II)2400 Tetracosane 0.12500 Pentacosane 1.32600 Hexacosane tr2622 Phytol 0.12655 Benzyl benzoate 0.22700 Heptacosane 1.72900 Nonacosane 0.8

Total 94.5

RRI: Relative retention indices calculated against n-al-kanes.

%: calculated from TIC.tr: Trace (< 0.1%).

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tated several times. Then the eggs were openedon the snub side. Before the opening 1015 ml ofalbumin were aspirated from a hole on the pointedside. At two third of the height (from the pointedside) the eggs were traced with a scalpel and after

that the shells were removed with forceps. Thecavity was covered with film and the eggs wereincubated at 37 C at a relative humidity of 80%for further 75 h. If the formed CAM had approxi-mately a diameter of 2 cm one pellet (1 pellet/egg)was placed on it. The eggs were incubated for onefurther day and then evaluated under the stereo-microscope. For every test compound 1015 eggswere utilized.

Evaluation and scoring: For the evaluation of

the effects, a scoring system was used (see TablesII and III). In brief; score: 0.5, no anti-angiogeniceffect; score: 0.51, weak to medium anti-angio-genic effect; score: 1, medium to strong anti-angio-genic effect. As controls suramin (Merck, Darm-stadt) and sodium dodecyl sulphate (SDS) (Merck,Darmstadt) at the concentration of 50 g/pelletwere also tested. As blank, CAMs treated with so-lidified agarose-solution in pellet form (2.5%, w/v)were also included. Each experiment was per-

formed in triplicate (DArcy and Howard, 1967;Brgermeister et al., 2002).

Results and Discussion

The chemical compositions of various Phlomisspecies have been investigated. However, to thebest of our knowledge this is the first investigationof the volatile fraction of P. linearis. The hydrodis-tillation of the aerial parts yielded a yellowish

essential oil with a pleasant odour, however in apoor yield. Subsequent GC/MS analysis resultedin detection and identification of 49 components

Table II. Effects of Phlomis linearis essential oil in the CAM-assay.

Test Sample Score* Irritation (%)

Phlomis linearis oil 0.2 0.2 100 g/pelletAgarose 0.2 0.2 Blanc (2.5%, w/v)

Suramin 0.5 0.2 Positive control (50 g/pellet)Sodium dodecyl sulphate (SDS) 0 85 5 Negative control (50 g/pellet)

* Formula for scoring:

Average sco re =Number of eggs (score 2) 2 + number of eggs (score 1) 1

Total number of eggs (score 0, 1, 2)

representing 94.5% of the total oil composition(Table I). Sesquiterpene hydrocarbons were themajor volatiles with -caryophyllene (24.2%), ger-macrene D (22.3%), and (Z)--farnesene (6.6%),respectively. Oxygenated sesquiterpenes were pre-

sent mostly as epoxides like caryophyllene oxide(9.2%). Noteworthy was the presence of variouscaryophyllene derivatives in different amountsthat probably contribute to the characteristicodour of the oil. Oxygenated monoterpenes andmonoterpene hydrocarbons were the minor por-tion of the whole oil which certainly also have im-pacts to the odour.

Due to the ethno-botanical use and various bio-logical activities of Phlomis species we have also

subjected the essential oil for potential anti-angio-genic and anti-inflammatory activity evaluationusing the CAM-assay. At 100 g/pellet the essen-tial oil showed a very weak angiogenic effect com-pared to standard substances (Table II and III).When applied to the CAM the essential oilshowed at this concentration neither toxic, nor irri-tant effects.

The essential oil composition of Phlomis olivieriBenth. from Iran was very recently reported, ses-quiterpenes were among the major componentswith germacrene D (28.1%) and -caryophyllene(16.1%) of fifteen identified compounds (Mirzaand Nik, 2003). Phlomis lanata Willd. essential oilwas analyzed to reveal forty-eight compounds rep-resenting 97% of the oil where -pinene (25.4%),limonene (15.7%) and trans-caryophyllene (8.8%)were found as major components (Couladis et al.,2000). Another study was on the essential oils

from the leaves ofPhlomis fruticosa L. Forty com-ponents were identified, -caryophyllene (12%),(E)-methyl isoeugenol (15.3%) and -asarone

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Table III. Score values for the evaluation of the anti-angiogenic effect on the CAM.

Score Effect Observation/Interpretation

0 no Normal embryo formation.0.5 very weak No capillary free area.

Area with reduced density of capillaries around the pellet not larger than its own area.

1 weak medium Small capillary free area or area with significantly reduced density of capillaries.Effects not larger than double the size of the pellet.

2 strong Capillary free area around the pellet at least double the size of the pellet.

(10.9%) as major compounds, respectively (So-kovic et al., 2002a). Phlomis fruticosa L. from twodifferent localities in Yugoslavia were analyzedand investigated for their antimicrobial activities.Here, -pinene (3957%) was found to be the

major constituent in both samples (Ristic et al.,2000).

Baytop T. (1997), Trkce Bitki Adlar Szlg (Dictio- Dadand M. Y. (2002), Trkiyenin Phlomis L. (La-nary of Turkish Plant Names), TDK Yay., No. 280, miaceae) Cinsi Revizyonu [The Revision of theAnkara, Turkey. Phlomis L. (Lamiaceae) of Turkey]. Ph. D. Thesis, In-

Baytop T. (1999), Trkiyede Bitkiler ile Tedavi, Gec- stitute of Science and Technology, Gazi University,

miste ve Bugn (Therapy with Medicinal Plants in Ankara, Turkey.Turkey, Past and Present). Nobel Tp Basmevi, Istan- Huber-Morath A. (1982), Phlomis L. In: Flora of Turkeybul, Turkey, 2nd ed. and East Aegean Islands Davis P. H., ed.). Edinburgh

Brgermeister J., Paper D. H., Vogl H., Linhardt R. J., Univ. Press, Edinburgh, Vol. 7, pp. 102126.and Franz G. (2002), LaPSvS1, a (153)--galactan Mirza M. and Nik Z. B. (2003), Volatile constituents ofsulfate and its effect on angiogenesis in vivo and Phlomis olivieri Benth. from Iran. Flavour Fragr. J.in vitro. Carbohydrate Res. 337, 14591466. 18, 131132.

Couladis M., Tanimanidis A., Tzakou O., Chinou I. B., Paper D. H. (1998), Natural Products as angiogenesis in-and Harvala C. (2000), Essential oil of Phlomis lanata hibitors. Planta Med. 64, 686695.growing in Greece: Chemical composition and antimi- Ristic M. D., Duletic-Lausevic S., Knezevic-Vukcevic J.,crobial activity. Planta Med. 66, 670672. Marin P. D., Simic D., Vukojevic J., Janackovic P., and

Cals I., Basaran A. A., Saracog lu I., Sticher O., and Vajs V. (2000), Antimicrobial activity of essential oilsRedi P. (1990), Phlinosides A, B and C, three phenyl- and ethanol extract of Phlomis fruticosa L. (La-

propanoid glycosides from Phlomis linearis. Phyto- miaceae). Phytother. Res. 14, 267271.chemistry 29, 12531257. Sokovic M. D., Marin P. D., Janackovic P., Vajs V., Milo-

Cals I., Basaran A. A., Saracog lu I., Sticher O., and savljevic S., Dokovic D., Tesevic V., and Petrovic S.Redi P. (1991), Phlinosides D and E, phenylpropa- (2002a), Composition of the essential oils of Phlomisnoid glycosides, and iridoids from Phlomis linearis. fruticosa L. (Lamiaceae). J. Essent. Oil Res. 14, 167Phytochemistry 30, 30733076. 168.

DArcy P. F. and Howard E. M. (1967), A new anti- Sokovic M. D., Marin P. D., Simic D., Knezevic-Vukcevicinflammatory test, utilizing the chorioallantoic mem- J., Vajs V., and Petrovic S. (2002b), Antimutagenic ac-brane of the chick embryo. Br. J. Pharmac. Chemo- tivity of essential oil and crude extract ofPhlomis fru-ther. 29, 378387. ticosa. Pharm. Biol. 40, 311314.

Phlomis species are widely distributed in Tur-key, however, composition of the essential oil hasnot been investigated thoroughly. This is also thecase for their biological activities. Using the CAM-assay other essential oils and major common com-

ponents will be also tested in further work. Ourstudies on medicinal and aromatic Phlomis speciesare ongoing.