Chemistry of Natural Compounds, Vol. 37, No. 4, 2001 ESSENTIAL OIL AND ANTIMICROBIAL EVALUATION OF THE Pistacia eurycarpa* F. Demirci,1 K. H. C. Baser,1 I. Calis,2 and E. Gokhan3 UDC 547.915 The compositions of microdistilled and hydrodistilled essential oils of the mastix of Pistacia eurycarpa Yalt. (Anacardiaceae) were compared. The essential oils were analyzed by GC/MS: α- and β-pinenes were found as the major constituents. The antimicrobial activity of the hydrodistilled oil was determined due to the ethnomedical uses of the oleo-gum resin on skin diseases. Key words: Pistacia eurycarpa, essential oil, microdistillation, GC/MS, antimicrobial activity. The genus Pistacia consists of small trees of the cashew nut family Anacardiaceae and is native to tropical and subtropical Asia where its members have long been cultivated for a variety of uses [1]. It is represented by six species in Turkey [2]. The trunk of Pistacia species produces a characteristic exudate called mastic gum (mastix) which is an oleo-gum resin, known and used since ancient times. P. lentiscus is the main source for this material which is widely cultivated and commercialized, whereas P. terebinthus and P. atlantica also produce similar exudates. The well known P. vera is mainly cultivated for its nuts known as “pistachio”. The first uses of the mastic gum and oil for medicinal purposes date back from the time of Dioscorides, Galenus, and Theophrastus against rabies, snake bites, baldness, and scabies, as well as in prescriptions for stomach, intestine, bladder, and liver inflammations. Another important application was against oral and dental diseases. Recent interest on the chemistry and biological activity of the different parts of Pistacia species support evidence of their ancient uses [3–9]. As part of our investigation, the oleo-gum resin was obtained by incisions of the P. eurycarpa tree. It has been used against jaundice and skin infections as well as for the treatment of burns and scalds in extreme situations with success in Batman by the peasants. The strong fragrance of the oelo-gum resin encouraged a preliminary evaluation of the volatiles using microdistillation [10–11], which were analyzed by GC/MS. Thereafter further hydrodistillation yielded an essential oil which was also analyzed by GC/MS using the same conditions and later compared with the microdistilled volatiles. This essential oil was subjected to an antimicrobial assay using various pathogenic microorganisms. Literature survey showed that the nuts (seeds) of Jordanian Pistacia eurycarpa were recently investigated for its fatty acid composition [12]. The essential oils and chemical compositions of various parts (esp. gum-oleo resin) of Pistacia species have been subject to several investigations [5, 8, 13–16 and cited references]. ______ *Presented at the 4th International Symposium on the Chemistry of Natural Compound (SCNC), 6-8 June 2001, Isparta, Turkey. 1) Medicinal and Aromatic Plant and Drug Research Centre (TBAM), Anadolu University, 26470, Eskisehir, Turkey. http://www.tbam.anadolu.edu.tr/; 2) Faculty of Pharmacy, Department of Pharmacognosy, Hacettepe University, 06100, Ankara, Turkey; 3) Aziziye Private High School, Yeni Mahalle, Ankara, Turkey. Published in Khimiya Prirodnykh Soedinenii, No. 4, pp. 282-284, May-June, 2001. Original article submitted June 19, 2001. 332 0009-3130/01/3704-0332$25.00 © 2001 Plenum Publishing Corporation TABLE 1. Composition of the Volatiles of Pistacia eurycarpa Obtained by Different Methods RRI Compound A, % B, % RRI Compound A, % B, % 1014 1032 1076 1118 1132 1174 Tricyclene α-Pinene 0.1 66.0 0.1 75.0 1586 1597 Pinocarvone Bornyl acetate 0.1 1.1 0.2 0.7 Camphene β-Pinene Sabinene Myrcene 0.7 4.9 0.4 0.1 0.9 5.6 0.5 0.2 1611 1648 1663 1664 Terpinen-4-ol Myrtenal cis-Verbenol trans-Pinocarveol 0.1 0.6 0.5 2.9 0.1 0.3 0.3 1.6 1203 1213 1218 1224 Limonene 1,8-Cineole β-Phellandrene o-Mentha-1(7),5,8-triene 1.3 0.3 0.2 1.6 0.2 0.1 0.1 1674 1683 1694 1697 1246 1280 1290 1439 (Z)-b-Ocimene p-Cymene Terpinolene γ-Campholene aldehyde α,p-Dimethylstyrene α-Campholene aldehyde Chrysanthenone Pinocamphone Linalool Isopinocamphone Methyl citronellate 0.1 1.0 0.1 0.3 0.1 2.3 0.1 0.2 0.3 0.1 - Tr. 0.8 Tr. 0.1 0.1 0.9 0.1 0.2 0.2 Tr. Tr. 1706 1709 1719 1725 1751 1797 1804 1845 1864 1882 Total p-Mentha-1,5-dien-8-ol trans-Verbenol Neral Carvotanacetone α-Terpineol α-Terpinyl acetate Borneol Verbenone Carvone p-Methyl acetophenone Myrtenol trans-Carveol p-Cymen-8-ol cis-Carveol 1.2 3.5 0.1 1.1 0.1 0.1 1.0 0.1 0.1 1.1 1.0 0.3 0.1 93.7 0.6 2.6 Tr. 0.4 0.7 0.1 0.6 0.6 0.1 Tr. 95.6 1452 1499 1522 1536 1553 1562 1571 ______ A: Microdistillation; B: Hydrodistillation; RRI: Relative retention indices calculated against n-alkanes; %: Calculated from TIC data; Tr: Trace (<0.1 %). TABLE 2. Antimicrobial Activity of the Essential Oils of Pistacia eurycarpa (E. O.) Microdilution Broth Susceptibility Assay, MIC ( µg/mL) Pathogenic Microorganisms E. O. St. Escherichia coli Gr () anearobic Staphyloccocus aureus Gr (+) Pseudomonas aeruginosa Gr () Enterobacter aerogenes Gr () Proteus vulgaris Gr () anearobic Salmonella typhimurium Gr () Candida albicans (yeast) 62.5 125 250 62.5 15.62 125 125 62.5 15.62 250 125 31.25 62.5 125* ______ St: Chloramphenicol, * Ketoconazole This is the first report on the essential oil composition and biological activity of P. eurycarpa. The essential oils obtained by two different methods, namely microdistillation (A) and hydrodistillation (B), were compared. As seen in Table 1 both methods resulted in similar chemical compositions with comparable amounts. Microdistillation yielded thirty-eight volatile compounds representing 93.7% of the total with α- and β-pinene as the major components. Hydrodistillation yielded thirty-seven compounds but with quite higher amounts of the major components. High essential oil yield (14.2%) by hydrodistillation was also remarkable. 333 TABLE 3. Pathologies of Selected Microorgasims to Humans Microorganism Pathology Escerichia coli Wound infections, urinary tract infections, gastrointestinal system infections, dysentery, septicemia Food poisoning, respiratory system infections, wound infections Septicemia, eye, urinary tract and wound infections Gastro infestinal system infections, urinary tract infections Diarrhea, urinary tract infections, skin diseases, and wound infections Staphyloccocus aureus Pseudomonas aeruginosa Enterobacter aerogenes Proteus vulgaris Salmonella typhimurium Candida albicans Salmonellosis, Diarrhea, Food poisoning Candidiasis: infestinal system infections, urinary tract infections, wound and skin infections The essential oil of the oleo-gum resin of P. eurycarpa strongly inhibited microorganisms associated with common wound infections and skin diseases (Enterobacter aerogenes and Proteus vulgaris). The Gr (–) pathogen E. aerogenes was inhibited at a concentration of 62.5 mg/mL and P. vulgaris at 15.62 mg/mL more effectively by than the control drug chloramphenicol. The essential oil also showed inhibitory effects towards the other pathogens used in this study which are listed in Table 2 with the relevant MICs. More in-depth studies of the essential oils of Pistacia species against different pathologies are suggested. Bioassay guided fractionation and investigation of the bioactive components are subject to further studies. EXPERIMENTAL Plant Material. The mastix was collected by one of the authors (E. G.) from Batman, Sason, Yucebag koyu (Yucebag), on 27.07.2000. The voucher specimen of the identified plant material is deposited (00187) at the Herbarium of the Faculty of Science and Letters, Gazi University, Ankara. Microdistillation. The mastix was first subjected to microdistillation by Eppendorf MicroDistiller® to obtain the volatile compounds. 500 mg mastix was added to a sample vial containing 10 mL distilled water. NaCl (2.5 g) and water (0.5 mL) was placed in the collecting vial. n-Hexane (350 µL) was added into the collecting vial to trap the volatile components. The sample vial was heated to 108oC at a rate of 20oC/min, kept at the same temperature for 90 min, and then heated to 112o C at a rate of 20oC/min and kept at this temperature for 30 min. Finally the sample was subjected to post-run for 2 min under the same conditions. The collecting vial was cooled to –1oC during distillation. After the distillation was completed, the organic layer in the collection vial was separated and injected into the GC/MS system. Hydrodistillation. The mastix (50 g) was gently warmed up and later subjected to hydrodistillation using a Clevengertype apparatus for 3 h to yield a colorless essential oil (7.1 mL). Analysis of the Essential Oils. Both essential oils were analyzed using a Hewlett-Packard G1800A GCD system. HPInnowax FSC column (60 m × 0.25 mm L, with 0.25 µm film thickness). Helium (0.8 ml/min) was used as carrier gas. GC oven temperature was kept at 60oC for 10 min and programmed to 220o C at a rate of 4o C/min and then kept constant at 220o C for 10 min to 240oC at a rate of 1o C/min. Mass range was recorded from m/z 35 to 425. Injections were applied splitless. Injection port temperature was at 250oC. MS were recorded at 70 eV. Relative percentage amounts of the separated compounds were calculated automatically from peak areas of the total ion chromatogram (TIC). Alkanes were used as reference points in the calculation of relative retention indices (RRI). Library search was carried out using both “Wiley GC/MS Library” and “TBAM Library of Essential Oil Constituents”. The compounds identified are listed in Table 1. Bioassay. Microdilution broth susceptibility assay was used for the evaluation of the antimicrobial activity [17]. The stock solution of essential oil was prepared in DMSO. A dilution series of the essential oil was prepared in a sterile distilled water in a 96-well microtiter plate up to 1.94 µg/mL in sterile distilled water. Freshly grown bacterial suspensions in double strength Mueller-Hinton broth and a yeast suspension of Candida albicans in yeast medium were standardized to 108 CFU/mL. Sterile distilled water served as growth control. 100 µL of each microbial suspension was then added to each well. The last row 334 containing only the serial dilutions of the antimicrobial agent (chloramphenicol for bacteria and ketoconazole for C. albicans) without microorganism was used as negative control. After incubation at 37oC for 24 h the first well without turbidity was determined as the minimal inhibition concentration (MIC). Human pathogens Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Proteus vulgaris, and Salmonella typhimurium were obtained from the culture collection of the Microbiology Department in Anadolu University, and Candida albicans was obtained from the culture collection of Osmangazi University, Medical Faculty, Microbiology Department (Table 2). ACKNOWLEDGMENT The authors would like to thank Dr. Hayri Duman of the Faculty of Science and Letters, Gazi University, Ankara, for the identification of the plant material and Dr. Salih Kafkas for contributions of the poster presentation. It is also acknowledged that part of this work was presented (E. G.) at a high school project competition organized by TUBITAK. REFENENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. V. H. Heywood, Flowering Plants of the World, Oxford University Press, Oxford, 1979, 198. P. H. Davis, Flora of Turkey and the Aegean Islands, Edinburgh University Press, Edinburgh, 2, 544, 1966. T. Baytop, Therapy With Medicinal Plants in Turkey, 2nd Edition, Nobel Twp Kitapevleri, 1999, 322. J. Perikos, The Chios Gum Mastic, Print All Ltd. Graphic Arts, Athens, 1993. G. Tumen, K. H. C. Baser, and N. Kirimer, In Proceedings of the 11th Symposium on Plant Originated Crude Drugs, 122, 1997. L. Iauk, S. Ragusa, A. Rapisarda, S. Franco, and V. M. Nicolosi, J. Chemother., 8, 207 (1996). S. Nishimura, M. Taki, S. Takaishi, Y. Iijima, and T. Akiyama, Chem. Pharm. Bull., 48, 505 (2000). P. Magiatis, E. Melliou, A. L. Skaltsounis, I. B. Chinou, and S. Mitaku, Planta Med., 65, 749 (1999). M. S. Al-Said, A. M. Ageel, N. S. Parmar, and M. Tariq, J. Ethnopharmacol., 15, 271 (1986). C. Bicchi and P. Sandra, Microtechnigues in Essential Oil Analysis. In: P. Sandra and C. Bicchi (eds.), Capillary Cas Chromatography in Essential Oil Analysis, A. Huethig Verlag, Heidelberg, 1987, 85. R. Briechle, W. Dammertz, R. Guth, and W. Volmer, GIT Lab. Fachz., 41, 749 (1997). K. I. Ereifej, M. K. Hammouri, G. N. Al-Karaki, and N. Asilzada, Int. J. Chem., 7, 71 (1996). S. Kusmenoglu, K. H. C. Baser, and T. Ozek, J. Essent. Oil Res., 7, 441 (1995). V. Castola, A. Bighelli, and J. Casanova, Biochem. Syst. Ecol., 28, 79 (2000). M. H. Boelens, and R. Jimenez, Flav. Fragr. J., 6, 271 (1991). H. L. De Pooter, N. M. Schamp, E. A. Aboutabl, S. L., El Thoamy, and S. L. Doss, Flav. Fragr. J., 6, 229 (1991). E. W. Koneman, S. D. Allen, W. M. Janda, P. C. Schreckenberger, and W. C. Winn, Color Atlas and Textbook of Diagnostic Microbiology, Lippincott-Raven Pub., 1997, 785. 335