Chemical composition and antibacterial activities of essential oils from fruits of melicope pteleifolia (Champ. Ex Benth.) hartley grown in Lam Dong province, Viet Nam

ACADEMIA JOURNAL OF BIOLOGY 2020, 42(3): 89–94 DOI: 10.15625/2615-9023/v42n3.13916 89 CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITIES OF ESSENTIAL OILS FROM FRUITS OF Melicope pteleifolia (Champ. Ex Benth.) T.G. Hartley GROWN IN LAM DONG PROVINCE, VIETNAM Hoang Thi Binh1,*, Tran Thi Bao Tram1, Do Ngoc Dai2, Vuong Thuy Tien3, Le Minh Tam4, Nguyen Van Ngoc1 1Faculty of Biology, Dalat University, Vietnam 2Faculty of Agriculture, Forestry and Fisheries, Nghe An Economic University, Vietnam 3Thang Long Highschool for the gifted, Lam Dong Province, Vietnam 4Vaccine Company Limited of Dalat Pasteur Received 6 July 2019, accepted 20 July 2020 ABSTRACT In the present study, chemical composition and antibacterial properties of essential oil obtained from the aerial parts of the Melicope pteleifolia (Champ. ex Benth.) T.G Hartley in Dalat were evaluated. Essential oil was isolated through hydro-distillation. Twenty-nine constituents comprising 100% of the essential oil were characterized by gas chromatography/mass spectrometry (GC-MS) techniques. The major compounds in the essential oil were (+)-Sabinene (34.73%), Cis-α-bergamotene (13.15%), Z-α-trans-bergamotol (5.28%), β-mycrene (4.98%), and 1,3,6-octatriene, 3,7-dimethyl-(4.71%). Antibacterial activities of Melicope pteleifolia essential oil were investigated against Gram-positive and-negative bacteria. Results showed significant activities against Streptococcus pyogenes and Escherichia coli using an agar well diffusion method. The application of this essential oil in preventing and eliminating bacteria could be useful in fields as medicine and cosmetics. Keywords: Melicope pteleifolia, antibacterial activity, essential oil. Citation: Hoang Thi Binh, Tran Thi Bao Tram, Do Ngoc Dai, Vuong Thuy Tien, Le Minh Tam, Nguyen Van Ngoc, 2020. Chemical composition and antibacterial activities of essential oils from fruits of Melicope pteleifolia (Champ. Ex Benth.) T.G. Hartley grown in Lam Dong Province, Vietnam. Academia Journal of Biology, 42(3): 89–94. https://doi.org/10.15625/2615-9023/v42n3.13916. *Corresponding author email: binhht@dlu.edu.vn ©2020 Vietnam Academy of Science and Technology (VAST) Hoang Thi Binh et al. 90 INTRODUCTION Melicope J.R. Forst. & G. Forst., one of the largest genera in the family Rutaceae (Kubitzki et al., 2011) with 235 species, is widely distributed in the Hawaiian Islands, tropical Asia, Australia and New Zealand (Hartley, 2001). It has been divided into four sections comprising Lepta (with 102 species), Melicope (with 38 species), Pelea (with 85 species), and Vitiflorae (with 8 species) (Hartley, 2001). Euodia J.R. Forst. & G. Forst., the genus with the fewest number of species among the Rutaceae family, was merged with the genus Melicope by Hartley, 2001. According to Ho (2003), Euodia species were recorded in Vietnam including E. bodinieri (Dode.), E. calophylla (Guill.), E. crassifolia (Merr.), E. lepta (Spreng.) Merr., E. meliaefolia (Benrth.), E. oreophila (Guill.), E. pasteuriana (A. Chev. Ex Guill.), E. rutaecarpa (A. Juss.) Benth., E. simplicifolia (Ridl.), and E. sutchuenensis (Dode.), in which E. lepta is widely distributed in evergreen forests throughout Vietnam (Ho, 2003). The Plant List (2013) currently treated Euodia lepta (Spreng.) Merr. as a synonym of Melicope pteleifolia (Champ. ex Benth.) T.G. Hartley. Recently, we recorded several populations of M. pteleifolia in the evergreen forests of Bidoup-Nui Ba National Park and the nearby areas. In ethnomedicine, diseases such as arthritis, fever, chickenpox, epidemic influenza, meningitis and infective hepatitis have been treated using roots and leaves of M. pteleifolia (Duke & Ayensu, 1985). The vegetative parts as well as the flowers and fruits of M. pteleifolia are also extensively used in both the ethnic communities and the traditional health care system in Vietnam (Bich et al., 2004). Phytochemical studies on parts of M. pteleifolia reported several biologically active compounds including leptol A (Li et al., 2003), chromenes (Li et al., 1997a; Li et al., 1997b; Li & Zhu., 1998), chromans (Li et al., 1998) and benzopyrane derivatives (Thang et al., 2007). The chemical compositions of essential oil of leaves, stems and flowers of M. Lepta, obtained by hydrodistillation, were reported by Thang et al. (2015). According to their report, (E)-β- ocimene (24.4%), α-pinene (9.8%), (Z)-β- ocimene (6.3%) and δ-cadinene (5.2%) were the main compounds of leaves oil of M. lepta, while the stems oils contained spathulenol (26.0%), (E)-β- ocimene (9.9%) and (Z)-9- octadecenamide (7.7%) and the flowers oil comprised of cis-carane (19.2%), α-cadinol (10.8%), α-pinene (10.5%) and (E)-β-ocimene (9.0%) (Thang et al., 2015). However, until now, there has been no report on the volatile constituents of essential oil derived from fruits of M. pteleifolia and the antibacterial activities of the essential oil of this species. The purpose of this study is to identify the chemical compositions and evaluate the antibacterial activity of essential oil from fruits of M. pteleifolia. MATERIALS AND METHODS Plants materials The aerial parts of Melicope pteleifolia (Champ. ex Benth.) T.G. Hartley) were collected from Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam, between June and August 2018 at an altitude of 1,867 m. The plant was identified by Dr. Nguyen Van Ngoc, a researcher at the Faculty of Biology, Dalat University, Vietnam. The specimens were deposited in the herbaria DLU of Dalat University, Vietnam. Bacteria The two bacteria for testing, Streptococcus pyogenes (Streptococcus ß- hemolytic type A; gram (+); S. pyogenes) and Escherichia coli (gram (-); E. coli), were provided by the General Hospital of Lam Dong Province, Vietnam and were grown in nutrient agar (NA) at 30 oC for 24 hours. Isolation of the essential oil Essential oil was extracted from fruits of M. pteleifolia using hydro-distillation. 500 g Chemical composition and antibacterial activities 91 of fresh fruits were placed in a distillation apparatus with 10 L of water and hydro- distilled for 2.5 h. After that, sodium sulphate was used to dry the anhydrous essential oil, which was then kept at 4 oC until use for GC- MS analyses. Gas chromatography-Mass spectrometry (GC-MS) Essential oil from fruits of M. pteleifolia fruits was analyzed using GC-MS method on an Agilent Technologies HP 6890N Plus Chromatograph connected to a mass spectrometer HP 5973 MSD. The analytical conditions were; Column: Agilent DB-5MS: Length: 30 m, Film: 0.25 μm, diam: 0.25 mm; MS transfer line temperature: 220 oC; Ion source temperature: 200 oC; Injector temperature: 220 oC; Temperature program: from 70 oC (15 min) up to 250 oC with increments of 10 oC/min: Flow: 1.2 ml/min; Mass range (m/z): 50−450. Identification of the constituents The constituents of essential oil were identified based on the retention times (RT) of the co-injected standard terpenes. Further identification was carried out by comparing their mass spectra with values from NIST 08 and Wiley 275 libraries or with mass spectra from the literature (Adams, 2007). Measuring antibacterial activities using the agar well diffusion method The agar well diffusion method (Devillers et al., 1989; Valgas et al., 2007) was used to test for antibacterial activity of the essential oil from fruits of M. pteleifolia . A broth culture (1%, containing 106–108 CFU/ml) of the respective bacterial strains was poured over base plates containing 7 ml nutrient agar at 4 oC in sterile 9 cm Petri dishes. Different concentrations of the essential oil, including undiluted and four dilutions (50, 25, 12.5 and 5%), were used to evaluate antimicrobial activity. Sterile dimethyl sulphoxide (DMSO) was used to dilute the essential oil of M. pteleifolia. DMSO was used as a negative control while chloramphenicol 250 mg (Vidipha Central Pharmaceutical Joint Stock Company, Vietnam) was used as a positive control. Plates were incubated at 30 oC for 24 hours. After that, the growth inhibition zones were measured in millimeters. Each test was performed in triplicate, from which the size of the growth inhibition zone was averaged. Statistical analysis All data analyses were performed using Microsoft excel 2017. Mean ± standard deviation was used to present data calculated from triplicate determinations. Statistical differences were considered significant at P < 0.05. RESULTS AND DISCUSSION Chemical compositions of the essential oil from fruits of of M. pteleifolia The analysis of hydro-distilled essential oil from M. pteleifolia fruits using GC-MS method identified 29 compounds (Table 1). The main components identified were (+)-sabinene (34.7%), cis-α-bergamotene (13.2%), Z-α-trans-bergamotol (5.3%), β- mycrene (5.0%) and 1,3,6-octatriene,3,7- dimethyl- (4.7%). Among those, sabinene was the most abundant, although this compound was not found in the essential oils from leaves, stems and flowers of M. lepta (Thang et al. 2015). Moreover, Thang et al., (2015) reported that (E)-β-ocimene (24.4%), spathulenol (26.0%) and cis-carane (19.2%) were the dominant constituent of essential oils derived from leaves, stems and flowers of M. lepta. The different parts of this plant species contain quantitatively and qualitatively different compound compositions. In addition, geographic differences also affect the constituents of essential oils of plant species (Saei- Dehkordi et al., 2010). Hoang Thi Binh et al. 92 Table 1. Chemical composition of the essential oil of Melicope pteleifolia fruits No Compounds RSI RT Area (%) 1 2-Thujene 953 4.56 1.0 2 -Pinene 901 4.79 1.8 3 (+)-Sabinene 961 6.00 34.7 4 -Pinene 953 6.19 1.2 5 -Mycerene 945 6.57 5.0 6 -Terpinene 934 7.84 1.1 7 o-Cymene 928 8.20 1.4 8 D-Limonene 936 8.47 3.8 9 1,8-Cineole 916 8.62 0.3 10 Cis-ocimene 925 8.85 1.0 11 1,3,6-octatriene,3,7-dimethyl- 943 9.49 4.7 12 -Terpinene 954 10.22 2.2 13 p-Mentha-1,4(8)-diene 931 12.23 0.5 14 Linalool 955 13.64 3.7 15 Geijerene 939 16.78 2.1 16 6-octenal,3,7 dimethyl- 912 17.46 0.2 17 Levomenthol 924 18.40 0.3 18 Terpinen-4-ol 920 18.47 3.6 19 -Terpineol 931 18.98 0.9 20 -citral 936 20.23 0.5 21 Linalyl acetate 928 20.57 0.7 22 -Citral 922 20.95 0.8 23 Pregeijerene 955 21.48 2.3 24 Copaene 916 23.04 0.9 25 Cis--bergamotene 953 23.94 13.2 26 Cadina-1(10),4-diene 905 25.16 1.8 27 Bergamotol,Z--trans- 838 25.24 5.3 28 Benzene,5-allyl-1,2,3-trimethoxy- 922 25.46 1.6 29 (±)-trans-nerolidol 956 25.68 3.5 Notes: RSI: Reversed Search Index; RT: Retention times. Antibacterial activities of the essential oil derived from fruits of M. Pteleifolia Antibacterial activities of essential oil derived from fruits of M. pteleifolia against Escherichia coli and Streptococcus pyogenes were examined after 24 hours of culture. Diameter of inhibition were used to express the anti-bacteria levels (Table 2). Table 2. Antibacterial activities of essential oil derived from fruits of M. pteleifolia Antibacterial activity (mm) Chloramphenicol DMSO Concentration (% of essential oil and DMSO) 100% 50% 25% 12.5% 5% E. coli 22.3 ± 0.6 - 17.3 ± 2.3 19.3 ± 1.2 12.0 ± 1.7 11.7 ± 1.2 11.3 ± 0.6 S. pyogenes 24.7 ± 0.6 - 24.3 ±1.5 21.0 ± 1.0 16.0 ± 1.0 12.3 ± 0.6 14.3 ± 2.1 Notes: “-”: No antibacterial activity. Chemical composition and antibacterial activities 93 The essential oil of M. pteleifolia fruits showed growth inhibition against both E. coli and S. pyogenes in a dose-dependent manner. Against S. pyogenes, pure (100%) essential oil produced inhibition zones 24.33 mm in diameter, comparable to those of chloramphenicol, a positive control. Against E. coli, the essential oil from fruits M. pteleifolia produced the best inhibitory activity at 50% concentration with an inhibition zone diameter of 19.33 mm. According to the classification of De Billerbeck (2007) on the susceptibility of bacteria against antibiotics based on the diameters of inhibitory zones (resistant: D < 6 mm; intermediate: 13 mm > D > 6 mm; sensitive: D > 13 mm), both tested bacteria strains were moderately sensitive to the essntial oil from fruits of M. pteleifolia. In this study, the gram-negative bacteria (E. coli) are less susceptible than gram-positive bacteria (S. pyogenes) at every concentrations examined. This can be explained based on the outer membrane of bacteria. The outer membrane of gram-negative bacteria contains hydrophilic lipopolysaccharides (LPS) which creates a barrier against macromolecules and hydrophobic compounds, making gram- negative bacteria more tolerant to hydrophobic component of the essential oil (Trombetta et al., 2005). Recently many reports showed that the antimicrobial effects of many essential oils is depend on the individual components and the combination of components within. Major components of essential oils, such as monoterpene or sesquiterpene hydrocarbons and their oxygenated derivatives, have potential antimicrobial activities (Diao et al., 2014). Essential oils rich in phenolic compounds have been reported to have antimicrobial activity (Aligiannis et al., 2001; Panizi et al., 1993; Sivropoulou et al., 1996). CONCLUSION Results of this study show that the essential oil from fruits of M. pteleifolia contains 29 components with the major compounds being (+)-sabinene (34.73%), cis- α-bergamotene (13.15%), Z- α-trans- bergamotol (5.28%), β-mycrene (4.98%) and 1,3,6-octatriene, 3,7-dimethyl-(4.71%). The antibacterial properties of the essential oil from fruits of M. pteleifolia harvested in Dalat, Vietnam, have growth inhibiting activities against E. coli and S. pyogenes with different efficacy depending concentrations. Acknowledgements: We would like to thank the director and staffs of the General Hospital of Lam Dong Province, Vietnam for allowing us to use the bacteria in antibacterial tests. We also thank the staff of the Center of Analytical Services and experimentation HCHC for analysis of our essential oil samples by GC- MS method. REFERENCES Adams R. P., 2007. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectrometry. 4th ed. Allured Publishing Corporation, Carol Stream IL, USA. Aligiannis N., Kalpoutzakis E., Mitaku S., Chinou I. B., 2001. Composition and antimicrobial activity of the essential oils two Origanum species. Journal of Agriculture and Food Chemistry, 49: 4168–4170. Bich D. H., Chung D. Q., Chuong B. X., Dong N. T., Dam D. T., Hien P. V., Lo V. N., Mai P. D., Man P. K., Nhu D. T., Tap N., Toan T., 2004. Medicinal plants and medicinal animals in Vietnam, Vol. 1. Science and Technology Publishers, Ha Noi (in Vietnamese). De Billerbeck V. G., 2007. Huiles essentielles et bactéries résistantes auxantibiotiques. Phytothérapie, 5: 249–253. Diao W. R., Hu Q. P., Zhang H., Xu J. G., 2014. Chemical composition, antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill.). Food Control., 35: 109–116. Duke J. A., Ayensu E. S., 1985. Medicinal Plants of China. Reference Publications, Inc, Taiwan. Hoang Thi Binh et al. 94 Hartley T. G., 2001. On the taxonomy and biogeography of Euodia and Melicope (Rutaceae). Allertonia, 8: 1–341. Ho P. H., 2003. An Illustrated Flora of Vietnam, Vol. 2. Youth Publishing House, Ho Chi Minh City (in Vietnamese). Kubitzki K., Kallunki J. A., Duretto M., Wilson P. G., 2011. Rutaceae. In: Kubitzki, K. (Ed.). The Families and Genera of Vascular Plants. Springer Verlag, Berlin, vol. 10: 276–356. Li G. L., Zeng J. F., Zhu D. Y., 1997a. The isolation and identification of four new 2,2-dimethyl chromenes. Acta Pharm Sin., 55: 1123–1126. Li G. L., Zeng J. F., Song C. Q., Zhu D. Y., 1997b. Chromenes from Evodia lepta. Phytochemistry, 44: 1175–1177. Li G. L., Zhu D. Y., 1998. Two chromenes from Evodia lepta. Phytochemistry, 48: 1051–1054. Li G. L., Zeng J. F., Zhu D. Y., 1998. Chromans from Evodia lepta. Phytochemistry, 47: 101–104. Li G. L., Zhu D. Y., Pandey R. K., 2003. Phytochemical and biological studies on Evodia lepta. In ACS Symposium, 859: 247–257. Magaldi S., Mata-Essayag S., De Capriles C. H., Perez C., Colella M. T., Olaizola C., Ontiveros Y., 2004. Well diffusion for antifungal susceptibility testing. International Journal of Infectious Diseases, 8(1): 39–45. Panizi L., Flamini G., Cioni P. L., Morelli I., 1993. Composition and antimicrobial properties of essential oils of four Mediterranean Lamiaceae. Journal of Ethnopharmacology, 39: 167–170. Saei-Dehkordi S. S., Tajik H., Moradi M., Khalighi-Sigaroodi F., 2010. Chemical composition of essential oils in Zataria multiflora Boiss. from different parts of Iran and their radical scavenging and antimicrobial activity. Food and Chemical Toxicology, 48(6): 1562–1567. Sivropoulou A., Papanikolaou E., Nikolaou C., Kokkini S., Lanaras T., Arsenakis M., 1996. Antimicrobial and cytotoxic activities of Origanum essential oils. Journal of Agricultural and Food Chemistry, 44: 1202–1205. Thang T. D., Thach T. D., Luong N. X., Hac L. V., Dung N. X., 2007. Structural determination of several chemical compounds from Evodia lepta (Spreng) Merr. Rogls. J. Anal Sci., 12: 25–28. Thang, T. D., Hoi, T. M., Do Ngoc, D. A. I., & Ogunwande, I. A., 2015. Composition of essential oils from Euodia lepta (Spreng.) Merr and Euodia calophylla Guill., grown in Vietnam. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 14(1): 60–66. Trombetta D., Castelli F., Sarpietro M. G., Venuti V., Cristani M., Daniele C., Saija A., Mazzanti G., Bisignano G., 2005. Mechanisms of antibacterial action of three monoterpenes. Antimicrobial agents and chemotherapy, 49(6): 2474–2478. Valgas C., Souza S. M., Smânia E. F., Smânia Jr A., 2007. Screening methods to determine antibacterial activity of natural products. Brazilian journal of microbiology, 38(2): 369–80.