Chemical composition and antimicrobial activity of the essential oils from the leaves and stems of Atalantia buxifolia from Vietnam

BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM - HỘI NGHỊ KHOA HỌC QUỐC GIA LẦN THỨ 5
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.

1. 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
8 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.
1

Hong Duc University, Thanh Hoa, Vietnam
Far Eastern Federal University, Vladivostok, Russia
3
Phan Chau Trinh High School, Quang Nam, Vietnam
4
Botanical Garden-Institute, FEB RAS, Vladivostok, Russia
*Email: ;
2


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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.
2. 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 4 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 4 °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 4 °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.


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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 1. NGHIÊN CỨU CƠ BẢN TRONG SINH HỌC

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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 2. 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
8
E. coli
500
500
16
C. albicans
250
500
8

In general, results obtained from MIC determination in Table 2 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).
4. 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


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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.

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THÀNH PHẦN HÓA HỌC VÀ HOẠT TÍNH KHÁNG VI SINH VẬT CỦA
TINH DẦU TỪ LÁ VÀ THÂN Atalantia buxifolia TỪ VIỆT NAM
Bùi Bảo Thịnh1,2,*, Nguyễn Văn Tân3,
Đậu Bá Thìn1, Roman V. Doudkin2,4
Tóm tắt. Tinh dầu từ lá và thân của Atalantia buxifolia từ Việt Nam thu được
bằng phương pháp chưng cất lôi cuốn hơi nước và phân tích bằng sắc ký khí (GC)
và sắc ký khí khối phổ (GC-MS). Tổng số 46 và 42 thành phần lần lượt chiếm
98,80% và 97,65% trong dầu thân và lá đã được xác định. Các thành phần chính
trong dầu lá là α-pinene (23,16%), β-caryophyllene (19,36%), limonene
(16,18%), caryophyllene oxide (10.45%) và sabinene (7,42%), trong khi dầu
thân chủ yếu chứa sabinene (32,14%), limonene (14,92%), β-caryophyllene
(10,72%), caryophyllene oxide (6,80%) và α-pinene (4,29%). Các loại tinh dầu
thu được đã được đánh giá về hoạt tính kháng vi sinh vật chống lại
Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, và nấm men
Candida albicans ATCC 10231 sử dụng xét nghiệm vi pha loãng. Cả tinh dầu lá và
thân đều thể hiện hoạt tính kháng vi sinh vật từ trung bình đến yếu đối với tất cả
các vi sinh vật được thử nghiệm với giá trị nồng độ ức chế tối thiểu (MIC) từ 125
− 500 μg/mL. Kết quả của nghiên cứu này đã cung cấp những dữ liệu mới về
thành phần hóa học và hoạt tính kháng vi sinh vật của tinh dầu A. buxifolia từ

Việt Nam.
Từ khóa: Hoạt tính kháng vi sinh vật, Atalantia buxifolia, tinh dầu, Rutaceae,
sabinene, α-pinene.

1

Trường Đại học Hồng Đức, Thanh Hóa, Việt Nam
Đại học Liên bang Viễn Đông, Vladivostok, Nga
3
Trường THPT Phan Châu Trinh, Quảng Nam, Việt Nam
4
Viện-Vườn Thực vật, FEB RAS, Vladivostok, Nga
*Email: ;
2