The essential oils from the leaves and stems of Atalantia buxifolia in Vietnam were obtained by hydrodistillation and analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS).
BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM - HỘI NGHỊ KHOA HỌC QUỐC GIA LẦN THỨ DOI: 10.15625/vap.2022.0002 CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF THE ESSENTIAL OILS FROM THE LEAVES AND STEMS OF Atalantia buxifolia FROM VIETNAM Bui Bao Thinh1,2,*, Nguyen Van Tan3, Dau Ba Thin1, Roman V Doudkin2,4 Abstract The essential oils from the leaves and stems of Atalantia buxifolia in Vietnam were obtained by hydrodistillation and analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) A total of forty-six and forty-two components accounting for 98.80 % and 97.65 % of the leaf and stem oil, respectively, were identified The major constituents in leaf oil were α-pinene (23.16 %), β-caryophyllene (19.36 %), limonene (16.18%), caryophyllene oxide (10.45 %), and sabinene (7.42 %), while the stem oil contained predominantly sabinene (32.14 %), limonene (14.92 %), β-caryophyllene (10.72 %), caryophyllene oxide (6.80 %), and α-pinene (4.29 %) The essential oils obtained were evaluated for antimicrobial activity against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and the yeast Candida albicans ATCC 10231, using the broth microdilution assay Both leaf and stem oil exhibited moderate to weak antimicrobial activity against all tested microorganisms with the minimum inhibitory concentration (MIC) value from 125 to 500 μg/mL The results of this study have provided new data on the chemical composition and antimicrobial activity of A buxifolia essential oil from Vietnam Keywords: Antimicrobial activity, Atalantia buxifolia, essential oil, Rutaceae, sabinene, α-pinene INTRODUCTION The genus Atalantia (Rutaceae) includes about 17 species that grow mainly in tropical and subtropical regions of Southeast Asia and China As of 2021, there are about Atalantia species found in Vietnam Several compounds such as acridone alkaloids, coumarins, limonoids, sesquiterpenoids, tyramines, and oximes have been isolated from plants of this genus (Chang et al., 2018; Posri et al., 2019; Vijayakumar et al., 2020) These compounds showed antiallergic, antibacterial, antifeedant, cytotoxic, expectorant, and antimalarial activities (Chang et al., 2018; Vijayakumar et al., 2020) Essential oils and their various components have received much attention in medicine and the food industry (Herman et al., 2019) Many essential oils have been demonstrated to have significant antibacterial, antifungal antimycotic, antihelmintic, and antiseptic properties against a wide range of microorganisms as well as antispasmodic and antioxidant properties Hong Duc University, Thanh Hoa, Vietnam Far Eastern Federal University, Vladivostok, Russia Phan Chau Trinh High School, Quang Nam, Vietnam Botanical Garden-Institute, FEB RAS, Vladivostok, Russia *Email: bui.ba@dvfu.ru; buibaothinh9595@gmail.com PHẦN NGHIÊN CỨU CƠ BẢN TRONG SINH HỌC Recent investigations into the chemical composition and bioactivity of essential oils from several Atalantia species have been reported (Thirugnanasampandan et al., 2015; Yang et al., 2015; Baskar et al., 2018; Pang et al., 2021) Atalantia buxifolia (the synonym name is Severinia buxifolia) which belongs to the genus Atalantia, is an evergreen citrus plant native to China (Swingle, 1916) This plant grows wild in the northern and central provinces of Vietnam Various parts of this plant have been used for folk medicine for several purposes such as the treatment of cough, snakebites, malaria, paralysis, traumatic swelling, chronic rheumatism, and pain (Loi, 2015; Chang et al., 2018) The chemical compositions of essential oil from A buxifolia of American, Cuba, China, and Egypt origin have been investigated in several studies (Scora, 1966; Scora and Ahmed, 1994; Pino et al., 2006; Safaa et al., 2016; Pang et al., 2020) To the best of our knowledge, there is no information on the chemical composition of the essential oils of A buxifolia from Vietnam Furthermore, the environment may affect the chemical composition and possibly the biological activity of different oils (Bailen et al., 2013) Thus, the present research reports (1) the chemical composition of the essential oil of A buxifolia growing in the wild in Vietnam, and (2) the antimicrobial activity profiles of the essential oil from this plant using the broth microdilution assay MATERIALS AND METHODS 2.1 Plant material The leaves and stems of A buxifolia were collected from Tien Phuoc, Quang Nam province, Vietnam in June 2020 The plant sample was authenticated by Assoc Prof Dau Ba Thin of Hong Duc University The plant materials were air-dried under laboratory shade for one week to reduce moisture content Afterward, the dried materials of each sample were ground using a small electric mill before the extraction 2.2 Isolation of essential oil The essential oil samples from the leaves and stems of A buxifolia were isolated using the hydrodistillation method The plant materials were introduced into a 5L flask and distilled water was added until it covered the sample Hydrodistillation was carried out for hours using a Clevenger-type apparatus under normal pressure according to the Vietnamese Pharmacopoeia (2009), as described previously (Thin et al., 2021) The extracted oil was collected, measured, and dried using anhydrous sodium sulfate, and stored at °C for further analysis 2.3 Gas chromatography Gas chromatography (GC) analyses were carried out on an Agilent GC 7890A gas chromatograph equipped with an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm), and FID detector, as described previously (Thinh et al., 2022) Helium was used as the carrier gas (flow rate: 1.0 mL/min) The oven temperature was initiated at 60 °C, then increased from 60 to 220 °C at a rate of °C/min and kept for 10 min at 220 °C Detector and injector temperatures were 260 °C and 250 °C, respectively The split ratio was adjusted to 10:1 10 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM 2.4 Gas chromatography-mass spectrometry Gas chromatography-mass spectrometry (GC-MS) analyses were executed on an Agilent GC 7890A gas chromatograph equipped with an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm), and a mass spectrometer (HP 5973 MSD), as described previously (Thinh et al., 2022) The oven temperature program was the same as the one used for GC The injector temperatures were 250 °C and helium was used as the carrier gas at a flow rate of 1.0 mL/min The mass spectra were recorded at 70 eV of ionization voltage and emission current of 40 mA The mass range was 35350 amu with a sampling rate of 1.0 scan/s 2.5 Identification of essential oil components The constituents of A buxifolia essential oils were identified on the basis of retention indices (RI) determined with reference to a homologous series of n-alkanes, under identical experimental conditions, co-injection with standards (Sigma-Aldrich, St Louis, MO, USA) or known essential oil constituents, MS library search (NIST, 2018) and by comparing with literature data (Adams, 2007; FFNSC, 2012; Thinh et al., 2022) 2.6 Antimicrobial screening The antimicrobial activity of the essential oils was tested against test microorganisms including Gram-positive bacteria (Staphylococcus aureus ATCC 6538), Gram-negative bacteria (Escherichia coli ATCC 8739), and yeast (Candida albicans ATCC 10231) The minimum inhibitory concentration (MIC) determination, designated as the lowest concentration of each essential oil that completely inhibited the growth of the microorganisms, was determined by a two-fold serial broth microdilution assay using sterile 96-well flat-bottom microtiter plates as described previously (Thinh et al., 2022) The testing media included Mueller-Hinton agar (MHA) for the bacteria and Sabouraud agar (SA) for fungi Each microorganism was inoculated in each microtiter plate well at an inoculum size of 1×106 CFU/mL and incubated at 37 °C for 24 h Positive controlcolumn; - Not identified A few reports on the essential oil of this species from different origins have been published previously The major constituents of A buxifolia leaf oil of American origin were α-santalene (24.36 %), trans-β-santalol (20.75 %), limonene (18.86 %), germacreneB (10.56 %) and β-caryophyllene (6.66 %) (Scora and Ahmed, 1994), while (E)-β-santalol (26.9 %) and α-santalene (22.2 %) were reported to be the main components of A buxifolia leaf oil of Cuba origin (Pino et al., 2006) Furthermore, Pang et al (2020) reported that the main compositions of the essential oil from A buxifolia leaves from PHẦN NGHIÊN CỨU CƠ BẢN TRONG SINH HỌC 13 China were β-caryophyllene (25.8 %), caryophyllene oxide (19.1 %), and epiglobulol (7.5 %), while Safaa et al (2016) analyzed the chemical compositions of A buxifolia leaf essential oil from different seasons at Egypt, and limonene (35.50 % - 19.17%), Z-αsantalol (29.19% - 13.73 %), α-santalene (20.87 % - 8.10 %), and γ-elemene (7.75 % 5.54 %) were determined to be the main compositions Our results were generally different according to literature findings as concerns the major compounds The observed differences may be probably due to genetic variability and/or environmental conditions (Bailen et al., 2013) In addition, to the best of our knowledge, this is the first time that a detailed composition of stem essential oil of A buxifolia is given 3.2 Antimicrobial activity of the essential oils The essential oils obtained from the leaves and stems of A buxifolia were evaluated for antimicrobial activity against S aureus, E coli, and C albicans, using the broth microdilution assay The essential oils demonstrated variable levels of antimicrobial activity against all examined microorganisms (Table 2) The leaf essential oil exhibited moderate antimicrobial action against the growth of S aureus, E coli, and C albicans with MIC values of 125 μg/mL, 500 μg/mL, and 250 μg/mL, respectively Meanwhile, the stem essential oil possessed activity against S aureus, E coli, and C albicans with MIC values of 250 μg/mL, 500 μg/mL, and 500 μg/mL, respectively Table Antimicrobial activity of the essential oils from the leaves and stems of Atalantia buxifolia Minimum inhibitory concentration (MIC), μg/mL Microorganisms Leaves Stems Gentamicin Amphotericin B S aureus 125 250 E coli 500 500 16 C albicans 250 500 In general, results obtained from MIC determination in Table indicated that the leaf essential oil exhibited better antimicrobial activity than the stem essential oil This difference may be attributed to the synergistic effects between the major and minor components of the oils (Chouhan et al., 2017) Furthermore, the essential oils from the leaves and stems of A buxifolia were most active against the gram-positive S aureus with a MIC value of 125 μg/mL and 250 μg/mL, respectively This could be due to cell wall lipopolysaccharides of the gram-negative bacteria, which can inhibit the diffusion of hydrophobic essential oil constituents into the cells (Cox et al., 2000) CONCLUSIONS In summary, the present study investigated the chemical composition and antimicrobial activity of essential oils obtained from the leaves and stems of A buxifolia from Vietnam The major components of leaf oil were α-pinene, β-caryophyllene, limonene, caryophyllene oxide, and sabinene, while stem oil consists mainly of sabinene, limonene, β-caryophyllene, caryophyllene oxide, and α-pinene Both essential oils exhibited activity against all examined microorganisms with the MIC value from 125 to 14 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM 500 μg/mL Thus, this study has provided new data on the chemical composition and antimicrobial activity of A buxifolia essential oil from Vietnam REFERENCES Adams, R P., 2007 Identification of essential oil components by gas chromatographymass spectrometry 4th ed Carol Stream (IL): Allured Publishing Corporation Bailen, M., Julio, L F., Diaz, C E., 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