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Study of the Essential Oil of Limnophila Rugosa (Roth.)
Merr. in the South of Vietnam
a

Nguyen Truc Linh & Le Ngoc Thach

b

a

Faculty of Chemical Technology, University of Cantho , 3/2 Street, Ninhkieu Dist., Cantho
City , Vietnam
b

Department of Organic Chemistry, Faculty of Chemistry , University of Science, Vietnam
National University-HCM City , 227 Nguyen Van Cu Street, Dist 5., Hochiminh City , Vietnam
Published online: 12 Mar 2013.

To cite this article: Nguyen Truc Linh & Le Ngoc Thach (2011) Study of the Essential Oil of Limnophila Rugosa (Roth.) Merr. in
the South of Vietnam, Journal of Essential Oil Bearing Plants, 14:3, 366-372, DOI: 10.1080/0972060X.2011.10643947
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Jeobp 14 (3) 2011 pp 366 - 372

366

Journal of Essential Oil Bearing Plants
ISSN Print: 0972-060X Online: 0976-5026
www.jeobp.com

Study of the Essential Oil of Limnophila Rugosa
(Roth.) Merr. in the South of Vietnam
Nguyen Truc Linh 1, Le Ngoc Thach 2*
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1

Faculty of Chemical Technology, University of Cantho,
3/2 Street, Ninhkieu Dist., Cantho City, Vietnam
2
Department of Organic Chemistry, Faculty of Chemistry,
University of Science, Vietnam National University-HCM City,
227 Nguyen Van Cu Street, Dist 5., Hochiminh City, Vietnam
Received 14 February 2010; accepted in revised form 10 December 2010

Abstract: The essential oils of Limnophila rugosa (Roth.) Merr. (Scrophulariaceae) in the south of
Vietnam obtained by hydrodistillation under conventional heating or microwave irradiation showed a slight
difference in chemical compositions. The main constituents of these oils are trans-anethole (24.96-27.12 %)
and methyl chavicol (70.79-71.00 %). In addition, microwave method gave a higher extraction yield in a
considerably shorter time than conventional heating protocol. The physical, chemical and antimicrobial
properties of the oils were also determined.
Key words: Limnophila rugosa, Scrophulariaceae, chemical composition, trans-anethole, methyl
chavicol, microwave hydrodistillation.
Introduction: Limnophila rugosa (Roth.) Merr., an annual herb native to wetlands in Vietnam,
belongs to the Scrophulariaceae family 1,2. It is widely cultivated and used as food flavoring, seasoning
or culinary herb. In addition, the plant is also employed as a traditional medicine for colds, cough,
laxative, diarrhoea, gonorrhea and anaprodisia 3,18. The chemical composition of Limnophila rugosa
(Roth.) Merr. essential oil, obtained by hydrodistillation (steam distillation) with conventional heating,
from several regions including the north of Vietnam, 4,5 China, 6,7 Hungary, 8 and India 9,10,11,18 had been
studied. However, only some of these reports mention the antimicrobial activity of the oils. Moreover,
the essential oil of Limnophila rugosa (Roth.) Merr. from the south of Vietnam has not been studied
yet. Therefore, we decided to investigate this oil in term of both chemical and antimicrobial properties.
Recently, microwave-assisted extraction technique has been applied as a new and efficient method
to isolate essential oils from plant materials. 12 In 1996, we reported that a modified domestic microwave
oven could be used as a heating source for fast hydrodistillation of essential oils from plant materials.13
Unfortunately, this system was subsequently proved to be not applicable for long hydrodistillation
because water is quickly evaporated out of the distillation flask after several minutes.

In this paper, we report the first systematic study of essential oils from the aerial parts of
Limnophila rugosa in the south of Vietnam obtained by hydrodistillation using both conventional
*Corresponding author (Le Ngoc Thach)
E-mail: < >

© 2011, Har Krishan Bhalla & Sons


Le Ngoc Thach et al. / Jeobp 14 (3) 2011 366 - 372

367

heating (HDCH) and microwave irradiation (HDMW). We also introduce a newly-modified Clevenger
type apparatus 14 designed for long hydrodistillation. Oil contents in different parts of the plant and
versus age of material were also studied. The chemical compositions of oil products were analyzed by
GC-MS. The physical, chemical and antimicrobial properties of oils were also investigated.

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Experimental
Materials: All Limnophila rugosa samples were collected in the morning in Trang Bang Dist.,
Tay Ninh Province. The plant was identified by the Department of Botany, School of Biology, University
of Science, Vietnam National University-Ho Chi Minh City.
Gas chromatography: GC analyses were performed on an Aglient 6890N GC equipped with
an FID and DB-5 capillary column (30 m x 0.32 mm x 0.25 μm). Nitrogen was used as the carrier at
a flow rate of 1.74 ml/min. The constant pressure mode at 9.32 psi was chosen on GC program. The
oven temperature was programmed from 60°C to 240°C at 3°C/min. The injector and detector
temperature was 250°C. The injection volume was 1.0 μl. The split ratio was 10:1. The percentage
composition of the constituents of the oils was determined by area normalization.
Gas chromatography-mass spectrometry (GC/MS): An Agilent 7890 GC coupled to a 5973A

Network Mass Selective Detector using HP-5MS capillary non-polar column (30.0 m x 0.25 mm x
0.25μm). Helium was employed as the carrier at a flow rate 1.20 mL/min. The constant pressure mode
at 10.604 psi was chosen on GC program. The injection temperature was 250°C; the injection volume
was 1.0 μl; the split ratio was 10:1, and the ionization voltage was 70 eV. The oven temperature was
programmed from 60°C to 240°C at 3°C/min. The retention indices (arithemetic indices) of the oil
components were calculated relative to the homologous series of C9-C17 n-alkanes by using the equation
propounded by van den Dool and Kratz in 1963. Identification of constituents was based on comparing
their mass spectra with those of standard compounds registered in the NIST 2008 library. Moreover,
the identification was also confirmed by the retention indices of each individual constituent relative to
those reported in the literature compiled by Adams 15.
Density of oil products was determined by using glass densitometer, and their [α] 27
were meaD
sured by A. Krüss Optronic polarimeter (Germany).16
Typical hydrodistillation procedure: Fresh aerial parts of the plant (300 g) were cut into small
pieces, loaded onto a Clevenger type apparatus with distilled water (600 ml), and hydrodistilled using
HDCH or HDMW. The oil yield was measured as a function of time. 14
Anti microorganism test: The oil is diluted in dimethyl sulphoxide (DMSO). The concentration
can be calculated as following equations:
C0 = 100 μl-1: concentration of original oil
C1 = 1μl C0 + 9μl DMSO = 10-1 μl-1
C2 = 1μl C1 + 9μl DMSO = 10-2 μl-1
C3 = 1μl C2 + 9μl DMSO = 10-3 μl-1
C4 = 1μl C3 + 9μl DMSO = 10-4 μl-1
The antimicrobial activity of Limnophila rugosa aerial part oil was tested by paper disc method
(standard diameter of 6 mm) on Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, and Candida albicans.


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Results and discussion: The extraction yields in correspondence with extraction methods are
shown in Figure 1. The conventional method requires longer time (5 hours) but provides lower yield
(0.1347 %) in comparison with the microwave method (0.1560 % yield in 9 minutes). This result is
consistent with the fact that microwave heating accelerates the evaporation of volatile compounds
from plant materials. Therefore, microwave-assisted extraction has been recently considered as a
“green” technique due to its time and energy efficiency. 17 It should be noted that our newly-designed
apparatus for microwave-assisted hydrodistillation allows water to return to the distillation flask,
thus, providing enough water for long distillation processes. 13
The distribution of essential oils in different parts of plant materials was also investigated. As
illustrated in Figure 2, the leaf of the plant contains the highest amount of essential oil (0.2740 %),
followed by the aerial part after flowering (AF) (0.1994 %), aerial part before flowering (BF) (0.1347
%) and stem (0.0151 %). It was also found that the oil content in the plant is dependent on the age of
plant (nearly peak value observed at four months and slightly leveled off afterwards).

Figure 1. Oil yield vs. the hydrodistillation time of HDMW (left) and HDCH (right)

Figure 2. The oil yields vs. the plant part (left), and changes in oil yield
vs. the age of the plant (right) investigated by HDCH method


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The chemical compositions of the aerial part oil were identified by both GC-FID and GC-MSD
technique as shown in Table 1. Generally, oil products isolated by the two techniques show a slight
difference in oil compositions. For example, the oil obtained by HDCH method indicates the presence
of limonene, coumarin, pentadecane… while these minor components were not found in the oil produced
by HDMW method. In addition, the percentages of major components, trans-anethole and methyl
chavicol, are also variable with the heating methods. On the other hand, these values are considerably
different with that of the oil from China and the north of Vietnam. For example, only 24.96 % of transanethole was found in the oil from the south of Vietnam while the figures for the oil from the north of
Vietnam and China are 89.40 % and 76.39 %, respectively. In contrast, methyl chavicol, the major
component in the oil from the south of Vietnam (70.79 %), occurs as the minor one in the oil from the
north of Vietnam (0.41%) (Table 2).
The chemical constituents of the aerial part after flowering oil (AF) were also identified by GCMS technique. The result indicates a transformation between two major components after flowering.
This can be easily observed by an increasing of trans-anethole (from 24.96 to 30.35 %) and decreasing
of methyl chavicol (from 70.79 to 64.20 %) while the total percentage of these two components remains
unchanged (Table 1). In general, oils isolated by using different techniques show a slight difference in
compositions and abundance. Besides, the oil constituents of the aerial part of Limnophila rugosa
(Roth.) Merr. vary with the geographic position of the herbs. The percentage of each component is
variable with the periods of plant growth.
Physical and chemical properties of aerial part oils isolated by both HDCH and HDMW methods
were also determined (Table 3). 15 In general, the difference between two oils is negligible. However, if
compared with the oils from China and India, the difference becomes significant (Table 4).
The anti-microbial activity of Limnophila rugosa aerial part oil was tested by paper disc method
(standard diameter of 6 mm) on Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, and Candida albicans. In addition, a typical fungi (Candida albicans) was
also used to test the antifungal activity of this essential oil. Interestingly, oil obtained from the microwave
method exhibits stronger antimicrobial activity than the one from conventional method (Table 5). The
antibacterial activity of the oil arises from the two main components. The higher antimicrobial activity
of the oil obtained from HDMW method can be attributed to the larger amounts of the two main
components. Previous studies also demonstrated significant anti-bacterial and anti-fungal activities of
this oil. 18,19 Infusion of leaves is used as diuretic and stomachic treatment in the Philippines and
India 18. Moreover, L. rugosa’s oil has antifungal activity toward Candida albicans, which cause

genital infection in human. 20 Further in vitro and in vivo tests are needed to confirm this finding. The
antifungal activity promises a broader usage of L. rugosa’s oil.
Conclusions: Two different techniques (HDMW and HDCH) were used to extract essential oil
from Limnophila rugosa (Roth.) Merr. HDMW method is more efficient to extract main components
(trans-anethole and methyl chavicol). Therefore, HDMW is a suitable technique for extracting essential
oil from plant material containing a large amount of water. HDCH method was applied to investigate
the change in composition of oil versus period of growth. The aerial parts can provide optimal oil yield
after 4 months. Interestingly, a transformation between trans-anethole and methyl chavicol was observed
in the oil of aerial part after flowering. In addition, the leaves were found to give the highest oil yield.
Besides, the essential oil from Limnophila rugosa (Roth.) Merr. indicates significant antibacterial
and fungal activities. Although further testing is needed, it is believed that these results would lead to
a wider application for Limnophila rugosa oil.

1.

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Table 1. Chemical composition (%) of the aerial parts
(BF and AF) of Limnophila rugosa (Roth.) Merr.

No.

Constituents

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

18
19
20

1-Octen-3-ol
3-Octanol
Limonene
Linalool
Methyl chavicol
cis-Anethole
p-Anisaldehyde
4-(1-Methylpropyl)phenol
trans-Anethole
1-(4-Methoxyphenyl)-2-propanol
Caryophylene
Coumarin
α-Caryophylene
Pentadecane
α-Farnesene
Nerolidol
2-(1-Methoxypropyl)phenol
Caryophylene oxide
γ-Gurzunene
Heptadecane
Total

% Oil composition
Before flowering
After flowering
HDCH

HDMW
HDCH
0.18
0.17
70.79
0.26
0.37
0.08
24.96
0.18
0.96
0.11
1.09
0.04
0.13
0.12
99.44

0.10
71.00
0.31
0.46
27.12
0.14
0.22
0.30
0.08
0.08
0.07
99.88

0.73
0.27
0.05
0.28
64.20
0.39
0.03
30.35
1.32
0.21
1.39
0.08
0.03
0.16
0.08
99.57


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372

Table 2. Comparison of the main constituents of aerial part
oils in the south of Vietnam and other regions
% Oil composition
Southern Vietnam
Northern Vietnam 5

Main constituents

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Linalool
Methyl chavicol
p-Anisaldehyde
trans-Anethole
Caryophylene
α-Caryophylene

0.17
70.79
0.37
24.96
0.96
1.09

China 7

0.10
0.41
0.09
89.4
0.74
0.85

0.08
21.94
0.05
76.39

0.08
0.15

Table 3. Physical and chemical properties of oil products
obtained by HDCH and HDMW methods

nD26 , 4

[α
α] 25
D

IA

IE

1.5226
1.5237

+ 0.307
+ 0.289

0.5745
0.5236

2.6552
2.5441

Method
HDCH

HDMW

0.9579
0.9585

IA : Index of Acide ; IE : Index of Ester
Table 4. Comparison of physical and chemical properties of aerial
part oils in the south of Vietnam and other regions
Properties

China7

Southern Vietnam

d
nD

0.9579 30
1.5226 26,4

[α] 25
D

+ 0.30725

- 0.4524

IA
IE

0.57
2.08

0.38
5.32

0.9756-0.9985
1.5283 22

India9
24

India10

0.986 10
1.530-1.53110

0.995922
1.522121

-

-

3.02
31.78

1.15
12.58

Table 5. Anti microorganism properties of Limnophila rugosa (Roth.) Merr. volatile oil

Microorganism
C* = 10 0
Bacillus subtilis
Escherichia coli, ATCC 25922
Staphylococcus aureus, ATCC 25923
Pseudomonas aeruginosa, ATCC 27853
Candida albicans
*C : oil’s concentration (μl-1)

22
16.5
27
13
37.5

Zone of inhibition (mm)
HDCH
HDMW
10 -1 10-2
10 -3 10 0 10 -1 10 -2
9
12
6
7
6

6
8

6
6
6

6
6
6
6
6

23
22
15
11
>60

6
11
6
6
6

6
6
6
6
6