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DALAT UNIVERSITY JOURNAL OF SCIENCE Volume 10, Issue 2, 2020 3-13 ANTIBACTERIAL ACTIVITIES AND CHEMICAL COMPOSITION OF ESSENTIAL OIL OF BLUMEA BALSAMIFERA (L.) DC., DISTRIBUTED IN LAMDONG PROVINCE, VIETNAM Hoang Thi Binha*, Nguyen Minh Tria, Nguyen Huu Quana, Nguyen Van Ngoca a The Faculty of Biology, Dalat University, Lamdong, Vietnam * Corresponding author: Email: binhht@dlu.edu.vn Article history Received: August 25th, 2019 Received in revised form (1st): October 28th, 2019 | Received in revised form (2nd): December 19th, 2019 Accepted: December 23rd, 2019 Abstract In the present study, the chemical composition and the antibacterial properties of the essential oil obtained from fresh leaves of Blumea balsamifera (L.) DC. in Lamdong are reported. The hydrodistillation method was used to isolate essential oil from leaves of this species, and gas chromatography/mass spectrometry (GC-MS) techniques were used to analyze the chemical constituents of the essential oil. Thirty six chemical constituents of the essential oil derived from fresh leaves of B. balsamifera were identified, in which the major compounds of the essential oil were camphor, caryophyllene, caryophyllene oxide, β-eudesmol, thymol hydroquinone dimethyl ether, and t-eudesmol, accounting for 43.69%, 12.71%, 5.98%, 4.84%, 4.63%, and 3.32%, respectively. Moreover, by using the agar well diffusion method, the antibacterial effects of B. balsamifera essential oilagainst Staphylococcus aureus and Escherichia coli were tested by the inhibition zone diameter test to evaluate the antibacterial activity. Keywords: Antibacterial activity; Blumea balsamifera; Essential oil; Lamdong. DOI: http://dx.doi.org/10.37569/DalatUniversity.10.2.597(2020) Article type: (peer-reviewed) Full-length research article Copyright © 2020 The author(s). Licensing: This article is licensed under a CC BY-NC 4.0 3 DALAT UNIVERSITY JOURNAL OF SCIENCE [NATURAL SCIENCES AND TECHNOLOGY] THÀNH PHẦN HOÁ HỌC VÀ HOẠT TÍNH KHÁNG KHUẨN CỦA TINH DẦU BLUMEA BALSAMIFERA (L.) DC. PHÂN BỐ Ở LÂM ĐỒNG, VIỆT NAM Hoàng Thị Bìnha*, Nguyễn Minh Tría, Nguyễn Hữu Quâna, Nguyễn Văn Ngọca Khoa Sinh học, Trường Đại học Đà Lạt, Lâm Đồng, Việt Nam * Tác giả liên hệ: Email: binhht@dlu.edu.vn a Lịch sử bài báo Nhận ngày 25 tháng 8 năm 2019 Chỉnh sửa lần 01 ngày 28 tháng 10 năm 2019 | Chỉnh sửa lần 02 ngày 19 tháng 12 năm 2019 Chấp nhận đăng ngày 23 tháng 12 năm 2019 Tóm tắt Trong nghiên cứu này, thành phần hoá học và hoạt tính kháng khuẩn của tinh dầu thu từ lá tươi loài Blumea balsamifera (L.) DC. phân bố ở Lâm Đồng, Việt Nam đã được công bố. Tinh dầu lá tươi của loài B. balsamifera (L.) DC. được thu nhận bằng phương pháp cất kéo hơi nước và được làm khan bằng Na2SO4. Bằng phương pháp sắc ký khí ghép khối phổ (GC-MS) đã xác định được 36 thành phần hoá học có trong tinh dầu lá tươi loài B. balsamifera (L.) DC. ở Lâm Đồng, trong đó các hợp chất chính là camphor (43.69%), caryophyllene (12.71%), caryophyllene oxide (5.98%), β-eudesmol (4.84%), thymol hydroquinone dimethyl ether (4.63%), và t-eudesmol (3.32%). Bên cạnh đó, phương pháp khuếch tán giếng thạch cũng đã được sử dụng để đánh giá hoạt tính kháng khuẩn của tinh dầu này lên hai chủng vi sinh vật là Staphylococcus aureus và Escherichia coli, thông qua kích thước vòng kháng khuẩn cho thấy tinh dầu này có khả năng kháng cả hai chủng vi sinh vật thử nghiệm. Từ khóa: Blumea balsamifera; Hoạt tính kháng khuẩn; Lâm Đồng; Tinh dầu. DOI: http://dx.doi.org/10.37569/DalatUniversity.10.2.597(2020) Loại bài báo: Bài báo nghiên cứu gốc có bình duyệt Bản quyền © 2020 (Các) Tác giả. Cấp phép: Bài báo này được cấp phép theo CC BY-NC 4.0 4 Hoang Thi Binh, Nguyen Minh Tri, Nguyen Huu Quan, and Nguyen Van Ngoc 1. INTRODUCTION Blumea DC. (1833) is a genus belonging to the Asteraceae family with approximately 100 species distributed throughout the Old World tropics (Anderberg, 1994, pp. 273-291; Anderberg & Eldenäs, 2007, pp. 374-390; Randeria, 1960). Almost all the species of the Blumea genus are widely distributed in tropical Asia with a few species in Australia and Africa (Anderberg, 1994, pp. 273-291; Anderberg & Eldenäs, 2007, pp. 374-390; Randeria, 1960). The genus is characterized by herbs, shrubs, or small trees; Stems not winged, with resin canals; Leaves alternate, simple, sessile or shortly petiolate, and mucronate-toothed to laciniate or sometimes pinnately lobed; Capitula heterogamous, disciform, solitary, or paniculate; Involucre campanulateglobose; Phyllaries numerous, imbricate, or reflexed in four or five series, outer series shortest; Marginal female florets in several rows, corolla yellow, filiform, and minutely 2- or 3-toothed (Shi et al., 2011). According to Pham (2003) and Vo (2003), a total of 32 Blumea species are recorded and distributed throughout Vietnam, of which 10 species are reported from Lamdong province, including Blumea adenophora, B. alata, B. balsamifera, B. densiflora var. hookeri, B. chevalierii, B. clarkei, B. fistulosa, B. hieracifolia var. hamiltonii, B. lacera, and B. virens (Pham, 2003). In particular, Blumea balsamifera is widely distributed in many areas of Vietnam. In ethnomedicine, B. balsamifera has anti-inflammatory, anticatarrhal, and expectorant effects, and has been used to treat asthmatic bronchitis and respiratory tract disorders (Chu, Du, & Liu, 2012). In traditional Vietnamese medicine, B. balsamifera leaves have been used to treat various diseases, such as fever, arthritis, and infective hepatitis (Do et al., 2004; Vo, 2003). To date, there have been many studies and reports about the chemical components in Blumea balsamifera, such as flavonoid compounds from leaves (Bui et al., 2017; Saewan, Koysomboon, & Chantrapromma, 2011) and components of essential oil from leaves of B. balsamifera grown in Bangladesh, China and Vietnam (Bhuiyan, Chowdhury, & Begum, 2009; Chu et al., 2012; Nguyen, Le, Nguyen, & Nguyen, 2004; Tran, Le, & Le, 2014). In Bangladesh, the chemical compositions of the essential oil from leaves of B. balsamifera were extracted with diethyl ether and the dominant components in the oil were borneol (33.22%), caryophyllene (8.24%), ledol (7.12%), tetracyclo[6,3,2,0,(2.5).0(1,8)]tridecan-9-ol, 4,4dimethyl (5.18%), phytol (4.63%), caryophyllene oxide (4.07%), guaiol (3.44%), thujopsene-13 (4.42%), dimethoxy-durene (3.59%), and γ-eudesmol (3.18%) (Bhuiyan et al., 2009). In China, the main components of the essential oil of B. balsamifera obtained by hydrodistillation were 1,8-cineole (20.98%), borneol (11.99%), β-caryophyllene (10.38%), 4-terpineol (6.49%), α-terpineol (5.91%), and caryophyllene oxide (5.35%) (Chu et al., 2012). In Vietnam, chemical compositions of the essential oilfrom the leaves of B. balsamifera grown in Thuathienhue province obtained by hydrodistillation were reported by Tran et al. (2014). According to this report, borneol (40.33%), β-caryophyllene (26.51%), and thujopsene-13 (5.56%) were the dominant components in the essential oil. Chemical compositions of the essential oil of B. balsamifera leaves have been reported by Bhuiyan, Chowdhury, and Begum (2009); 5 DALAT UNIVERSITY JOURNAL OF SCIENCE [NATURAL SCIENCES AND TECHNOLOGY] Chu et al. (2012); Nguyen et al. (2004); and Tran et al. (2014). However, the essential oil compositions may be affected by the choice of extraction methods. In addition, geographical variation also affects the composition of essential oils of plant species (Saei, Tajik, Moradi, & Khalighi, 2010). Moreover, until now there has been no report on the volatile constituents of essential oil composition as well as antibacterial activity of essential oil derived from the leaves of B. balsamifera distributed in Lamdong province. Thus, in this study, we report the chemical composition and the evaluation of antibacterial activity of the essential oil from the leaves of B. balsamifera distributed in Lamdong province. 2. MATERIALS AND METHODS 2.1. Plant materials Fresh leaves of Blumea balsamifera L. (DC.) were collected in June to August, 2019 at altitudes of 1,800 m in Lan Tranh commune inside the protected area of Bidoup-Nui Ba National Park, Lamdong province, Vietnam. The specimens (voucher specimens: LD04) were deposited at the DLU Herbaria of Dalat University and the plant was identified based on type specimens, original descriptions, digitized plant specimen images available on the web at JSTOR Global Plants, and diagnostic traits described in the taxonomic literature (Pham, 2003; Vo, 2003). 2.2. Isolation of the essential oil In the present study, the hydrodistillation method was used to extract essential oil from B. balsamifera leaves in Lamdong province. After the volatile essential oil was collected, sodium sulphate was used to dry the anhydrous essential oil of B. balsamifera and then the oil was kept at 4 oC until used for GC-MS analysis. 2.3. Gaschromatography-mass spectrometry (GC-MS) and identification of the constituents The components of the essential oil derived from leaves of B. balsamifera were separated and identified using the gas chromatography-mass spectrometry (GC-MS) method. GC-MS analyses were conducted using a Thermo Scientific ISQ Single Quadrupole MS with the following specifications: Column: Agilent DB-5MS; Length: 30 m; Film: 0.25 μm; Diameter: 0.25 mm; MS transfer line temperature: 220 oC; Ion source temperature: 200 oC; Injector temperature: 220 oC; Temperature programmed: 70 oC (15 min) increase 10 oC/min up to 250 oC; Flow: 1.2 ml/min; and Mass range (m/z): 50-450. Most of the constituents of the essential oil were identified on the basis of retention times (RT). Further identification was carried out by comparison of their mass spectra with those stored in the NIST 08 and Wiley 275 libraries or with mass spectra from the literature (Adams, 2007). 6 Hoang Thi Binh, Nguyen Minh Tri, Nguyen Huu Quan, and Nguyen Van Ngoc 2.4. Evaluation of antibacterial activity by the agar well diffusion method In this study, two bacterial strains were identified and obtained from the Institute of Drug Quality Control in Hochiminh City, Vietnam. The bacterial strains used in this study were Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739. Nutrient agar (NA) was used to grow the two bacteria strains at 30 oC for 24 hours and the bacteria were then maintained on nutrient agar at 4 oC. The antibacterial activity of the essential oil derived from fresh leaves of B. balsamifera was carried out by the agar well diffusion method (Devillers, Steiman, & Seigle, 1989; Valgas, Souza, Smania, & Smania, 2007). The bacteria were inoculated by the spread plate method on base plates containing 7 ml nutrient agar in sterile 9 cm Petri dishes (containing about 106-108 CFU/ml of the microorganisms). In the center of each dish, wells of approximately 6 mm diameter were created and 40 μL of essential oil solution, dimethyl sulphoxide (DMSO), and chloramphenicol were added to the wells. The sterile DMSO was used to the dilute essential oil of B. balsamifera to obtain four dilutions of 75%, 50%, 25%, and 12.5% respectively. The chloramphenicol 250 mg (Vidipha Central Pharmaceutical Joint Stock Company, Vietnam) and the DMSO were used as a positive control and a negative control, respectively. The dishes were incubated at 4 oC for two hours for sample diffusion and then incubated at 30 oC for 24 hours. After that, the growth inhibition zones were measured and analyzed. In this study, each test was performed in triplicate. 2.5. Statistical analysis Data analysis was performed using Microsoft Excel 2017. Mean values ± one standard deviation were calculated from triplicate determinations and used in the data presentation. Differences were considered significant at P <0.05 in the statistical analysis of the data. 3. RESULTS AND DISCUSSION 3.1. Chemical constituents of the essential oil The steam distillation of Blumea balsamifera fresh leaves gave a yellowish essential oil with a very strong and pleasant odour. The results also showed that the yield of the essential oil of B. balsamifera fresh leaves was 0.16% (v/w) on a fresh weight basis. Thirty six compounds were identified in the essential oil of B. balsamifera leaves using GC-MS (Table 1 and Supplement 1). Table 1. Chemical constituents of the essential oil of Blumea balsamifera fresh leaves No. Name of chemical constituents RT % 1 2 3 4 -Pinene Camphene (+)-Sabinene 5.06 5.57 6.33 6.54 0.58 0.95 0.30 1.37 -Pinene 7 DALAT UNIVERSITY JOURNAL OF SCIENCE [NATURAL SCIENCES AND TECHNOLOGY] Table 1. Chemical constituents of the essential oil of Blumea balsamifera fresh leaves (cont.) No. Name of chemical constituents RT % 5 -Myrcene 6.94 0.20 6 o-Cymene 8.68 0.38 7 D-Limonene 8.97 2.60 8 5-Formal-4-nonene 9.11 0.15 9 Trans--ocimene 9.36 0.11 10 Cis-ocimene 10.04 0.93 11 -Terpinene 10.81 0.31 12 Filifolone 14.22 0.54 13 Linalool 14.42 0.51 14 2-Pinen-7-one 16.02 0.98 15 2-Methyl-2,4,6-octatriene 17.19 0.77 16 Camphor 17.56 43.69 17 Endo-borneol 18.46 2.14 18 -Citral 20.45 0.23 19 -Citral 21.15 0.58 20 Perillal 21.28 0.17 21 Silphiperfol-5-ene 22.34 0.41 22 7-Epi-silphiperfol-5-ene 22.69 0.74 23 Thymol hydroquinone dimethyl ether 23.80 4.63 24 Caryophyllene 23.99 12.71 25 Humulene 24.50 0.81 26 Aromadendrene 24.56 0.44 27 Elemol 25.74 0.66 28 Aristolene epoxide 25.80 0.29 29 (±)-trans-Nerolidol 25.86 0.84 30 -Longipinene 26.10 0.53 31 Caryophyllene oxide 26.24 5.98 32 Guaiol 26.38 2.10 33 10-Epi--eudesmol 26.73 2.24 34 -Eudesmol 26.83 3.32 35 Caryophylladienol II 26.88 1.98 36 -Eudesmol 27.13 4.84 Note: “RT” stands for Retention times. The results of the analysis showed that the essential oil of B. balsamifera fresh leaves mainly consisted of monoterpenes and sesquiterpenes. According to the results above (Table 1), the main components of the essential oil of B. balsamifera fresh leaves are camphor (43.69%), caryophyllene (12.71%), caryophyllene oxide (5.98%), 8 Hoang Thi Binh, Nguyen Minh Tri, Nguyen Huu Quan, and Nguyen Van Ngoc β-eudesmol (4.84%), thymol hydroquinone dimethyl ether (4.63%), and -eudesmol (3.32%). Of these, camphor was determined to be the predominant compound in this essential oil. Camphor is a monoterpene and is known as a waxy, flammable, and transparent solid with a strong odour (Mann, Davidson, Hobbs, Banthorpe, & Harborne, 1994). This compound is used for many fields in human life as a pest deterrent and preservative, a popular perfume ingredient, in pharmaceutical applications, and so on (Ahmed, 2016; Donkin, 1999; Ghosh, 2000; MacKinney, Soti, Shrestha, & Basnyat, 2015). In medicine, camphor has antispasmodic, antipruritic, anti-inflammatory, antiinfective, activities, and has been used as a rubefacient, contraceptive, mild expectorant, nasal decongestant, cough suppressant, etc. (Segal, Cohen, & Freeman, 1978; Zuccarini, 2009). In addition, camphor has also been used to prevent and cure serious, life threatening diseases as a significant antioxidant and anti-tumor agent (Edris, 2007; Ho, Wang, & Su, 2009). The present results also show that caryophyllene and caryophyllene oxide are the second and third most dominant, respectively, in the essential oil of B. balsamifera, and they are members of bicyclic sesquiterpene. Their biological effects include anti-inflammatory, anticarcinogenic, antimicrobial, antioxidative, and analgesic activities (Klauke et al., 2014; Langhasova et al., 2014; Medeiros et al., 2007; Sabulal et al., 2006; Singh, Marimuthu, de Heluani, & Catalan, 2006). These two compounds have applications as cosmetics and food additives, and they also have a strong potential for use in medical applications due to their anticancer and analgesic properties (Fidyt, Fiedorowicz, Strządała, & Szumny, 2016). Therefore, the results of the present study may explain the use of B. balsamifera to treat various diseases in traditional medicine in Vietnam. A comparison of the chemical composition between the essential oil of B. balsamifera leaves in this study and in previous studies is shown in Supplement 2. In the present study, camphor and caryophyllene are the dominant compounds in the essential oil of fresh B. balsamifera leaves, whereas 1,8-cineole and borneol were abundant compounds in the essential oil of B. balsamifera leaves reported in the previous studies of Bhuiyan et al. (2009); Chu et al. (2012); and Tran et al. (2014). According to the three previous reports, borneol was one of the main compounds of the essential oil of B. balsamifera leaves, but it was absent in this study. This issue is common because the quantity or quality of constituents in the essential oil of plant species may be influenced by the geographic variation (Saei et al., 2010; Sakee, Maneerat, Cushnie, & de Eknamkul, 2011). 3.2. Antibacterial activity of essential oil of B. balsamifera leaves The assays for antibacterial activity against bacteria in the essential oil of B. balsamifera showed antibacterial activity against both Gram-positive and Gramnegative bacterial strains used in this study and expressed by the diameter of inhibition (Table 2). 9 DALAT UNIVERSITY JOURNAL OF SCIENCE [NATURAL SCIENCES AND TECHNOLOGY] Table 2. Antibacterial activity of essential oil derived from leaves of B. balsamifera in Lamdong province Inhibition zone diameters (mm) Bacteria Concentration (% of essential oil in DMSO) Chloramphenicol DMSO 100% 75% 50% 25% E. coli 36.00 ± 4.00 - 12.00 ± 1.00 11.33 ± 0.57 8.33 ± 1.52 S. aureus 50.00 ± 2.00 - 12.70 ± 2.08 12.00 ± 1.73 11.50 ± 1.32 9.67 ± 2.08 12.5% 10.66 ± 1.52 7.00 ± 1.00 8.16 ± 0.76 Note: “-” is not active. The results revealed that the essential oil of B. balsamifera inhibited the growth of both S. aureus and E. coli at the different concentrations, in which the pure essential oil (the oil at 100% concentration) had the best inhibition zones, approximately 12mm in diameter, for both S. aureus and E. coli. However, in this assay, the Gram-negative bacteria (E. coli) were generally less susceptible than Gram-positive bacteria (S. aureus) at all concentrations of essential oil because the outer membrane of Gram-negative bacteria is composed of hydrophilic lipopolysaccharides, and this structure creates a barrier toward macromolecules and hydrophobic compounds, providing Gram-negative bacteria with higher tolerance toward hydrophobic essential oil components (Trombetta et al., 2005). Additionally, comparison between the inhibition zone diameters in Table 2 and the suggestion of de Billerbeck (2007) about the classification of antibiotics on the basis of their inhibition diameters (Resistant: D < 6 mm; Intermediate: 13 mm > D > 6 mm; Sensitive: D > 13 mm) showed that both tested bacteria strains were not sensitive (D > 13 mm) to the essential oil of B. balsamifera, but two strains (S. aureus and E. Coli) of bacteria were moderately sensitive to the essential oil. The activity against bacteria of the essential oil of B. balsamifera distributed in Lamdong Province is related to camphor (43.69%), the main component of the essential oil. Camphor is mainly responsible for the antibacterial activity of the plant oils which contain it, as it is known to have very efficient antibacterial properties (Jalsenjak, Peljnjak, & Kustrak, 1987; Sivropoulou et al., 1997). The result from this study may explain the use of B. balsamifera to treat various diseases in traditional medicine in Vietnam. 4. CONCLUSION In conclusion, analysis by GC-MS showed that thirty six compounds were identified in the essential oil obtained from fresh leaves of Blumea balsamifera in Lamdong, Vietnam. Camphor (43.69%), caryophyllene (12.71%), caryophyllene oxide (5.98%), β-eudesmol (4.84%), thymol hydroquinone dimethyl ether (4.63%), and -eudesmol (3.32%) were the main components. 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