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Antibacterial activity of Vitex trifolia methanol extract against pathogenic

bacteria
To cite this article: L Zulkifli et al 2021 J. Phys.: Conf. Ser. 1869 012060

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Annual Conference on Science and Technology (ANCOSET 2020) IOP Publishing
Journal of Physics: Conference Series 1869 (2021) 012060 doi:10.1088/1742-6596/1869/1/012060

Antibacterial activity of Vitex trifolia methanol extract against

pathogenic bacteria

L Zulkifli1,2,*, M H Basri1 and A Syukur1,2

1
Department of Sciences Education Postgraduates, Mataram University, 83125
Mataram, Indonesia
2
Department of Sciences Education, Faculty of Teacher Training and Education,
Mataram University, 83125 Mataram, Indonesia

*lalu_zulkifli@unram.ac.id

Abstract. Vitex trifolia (Legundi plants) is a plant that is widely used as an ingredient of
traditional medicine in Lombok Island for a variety of microbial infections. In this study, a
qualitative test of V. trifolia methanol extract was shown to contain secondary metabolites, such
as steroids, saponins, alkaloids, flavonoids, terpenoids, and tannins. The inhibitory test of
methanol extract of roots, stems and leaves of V. trifolia against pathogenic bacteria
(Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Klebsiella
pneumoniae) has been carried out. The experimental design used was a Completely Randomized
Design. The treatment factor was the concentration of methanol extract with 4 levels (C1 = 7.5%,
C2 = 15%, C3 = 30%, C4 = 60%). The extraction method used was methanol maceration, and
the well diffusion method was used in antibacterial activity assay. Data were analyzed by using
Anova followed by DMRT test. Anova analysis results showed that differences in the
concentration of methanol extract of leaf have a significant effect in inhibiting the growth of all
tested bacteria. The DMRT showed that the best treatment was with 60% methanol extract. In
this study, S. aureus was the most sensitive bacterium to V. trifolia methanol extract, compared
to other test bacteria. It can be concluded that the methanol extract of V. trifolia is an effective
antibacterial source, especially against S. aureus.

1. Introduction
Plants have been used as a source of medicine in traditional or folk medicine from time immemorial due
to their magical power to cure diseases [1]. Nowadays, researchers have given more attention to discover
new antimicrobial drugs of plant origin because most of the available synthetic antibiotics are losing
their capacity to inhibit the growth of microorganisms [2]. This is mainly due to the ability of
microorganisms to develop resistance against the continuously using antibiotics.
Vitex trifolia (legundi plant) is a plant that has long been known as a traditional medicinal ingredient.
Parts of plants that can be used as medicine are all parts of the leaves, stems, roots, flowers and seeds.
The leaves of Vitex trifolia were used as a remedy for rheumatism, gout, sinus, hydrocele and
hemorrhoids [3]. V. trifolia leaf extract also has anti-inflammatory activity in Wistar rats by inhibiting
prostaglandin synthesis and inhibiting increased vascular permeability [4]. The hydro-alcoholic extract
of Vitex trifolia leaves also showed significant results on anti-inflammatory activity in acute and sub-
acute inflammation which were tested on Wistar rats [5].

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Annual Conference on Science and Technology (ANCOSET 2020) IOP Publishing
Journal of Physics: Conference Series 1869 (2021) 012060 doi:10.1088/1742-6596/1869/1/012060

Furthermore, V. trifolia in several studies showed inhibitory activity against pathogenic microbes.
The methanol extract of V. trifolia leaves gave significant results in inhibiting gram-positive bacteria
such as B. cereus and B. pumilus with the inhibition zones of 15.49 mm and 14.82 mm respectively and
gram-negative bacteria, Salmonella flexneri and Shigella sonnei with inhibition zones of 12, 25 mm and
12.93 mm, respectively [6]. Ethanol, methanol and ethyl acetate extracts from V. trifolia leaves at a
concentration of 25 mg/ml gave significant results on the antibacterial activity in E. coli, Shigella
flexneri, Proteus mirabilis, Pseudomonas diminuta, Enterobacter cloacae, and Staphylococcus aureus
[7]. Other studies report that acetone, ethanol and aqueous extracts of Vitex trifolia leaves show
antibacterial activity against Bacillus subtilis (0.5 cm, 0.75 cm and 0.1 cm inhibitory zones) and E. coli
(successive inhibition zones also 0.15 cm, 0.25 cm and 0.35 cm) [8].
In addition to being useful as a medicinal and antimicrobial ingredient, V. trifolia is also useful as
larvicide. The methanol extract of V. trifolia leaves showed the highest larvicidal activity (LC50 = 41.41
ppm) compared to V. peduncularis (LC50 = 76.22 ppm), V. altissima (LC50 = 128.04 ppm) [9]. The
ethanol extract of V. trifolia leaves also showed activity as a repellent against Aedes aegypty with an
ED50 value of 14.809% and an ED99 of 41.442%. [10].
The compound content in Vitex trifolia has been reported in several studies. The ethanol extract of
V. trifolia leaves contained secondary metabolite compounds including alkaloids, saponins, flavonoids,
and glycosides [11]. A qualitative analysis of the leaves of V. trifolia with standard methods using
petrollium ether, benzene, acetone, ethanol and water, indicating the presence of alkaloids, saponins,
tannins, phenols, terpenoids, flavonoids, and steroids [8].
In this study, we evaluate the presence of secondary metabolites of V. trifolia form Lombok Island,
qualitatively, and evaluate the antimicrobial activity against two spesies of Gram positive bacteria and
two spesies of Gram negative bacteria.

2. Material and methods

This study was an experimental laboratory study with treatment factors for variations in the
concentration of legundi plant leaves namely concentrations of 7.5%, 15%, 30% and 60%, against
clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Kleibsella
pneumoniae. The experiment was done in a completely randomized design (CRD) with one factor
consisting of four levels of concentration treatment (C1 = 7.5%, C2 = 15%, C3 = 30%, C4 = 60%). Each
treatment was repeated 3 times.

2.1. Preparation of V. trifolia methanol extract

Legundi plants includes the roots, stems and leaves of legundi plants. The location of the sampling of
legundi plants is in the rice field area of Lingsar Village, Narmada District, West Lombok Regency.
Methanol extraction by maceration method from roots, stems and legundi leaves was carried out in the
Chemistry Laboratory FKIP Unram.
Legundi plant extraction is done by maceration using methanol as a solvent. The extraction procedure
was carried out in accordance with the Natheer procedure [7]. Plant samples were dried aerated and
blended. Subsequently, 1 kg of each plant part, root, stem, and leaf were incubated at room temperature
for 48 hours in 3 liter of methanol. Filtering is carried out to obtain the filtrate. The filtrate is then
concentrated with a vacuum rotary evaporator, and the extracts were then weighed. Fifty percent of the
extract was used for phytochemical testing and the rest was used for antibacterial assay. Four variations
of the extract concentration (7.5%, 15%, 30%, and 60%) were made.

2.2. Phytochemical test

Phytochemical tests to determine the content of secondary metabolites produced by V. trifolia plant was
carried out using the standard procedure of Harborne [12]. a). Steroid test. The extract was added 10
drops of CH3COOH and 2 drops of H2SO4. The solution is shaken slowly and left for several minutes.
The change in color to blue or green indicates the presence of steroids; b). Saponin test. The extract is
boiled with 10 ml of water in a water bath, the filtrate is shaken and allowed to stand for 1 minute.

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Annual Conference on Science and Technology (ANCOSET 2020) IOP Publishing
Journal of Physics: Conference Series 1869 (2021) 012060 doi:10.1088/1742-6596/1869/1/012060

Added 2 drops of HCl 1 N. The formation of a stable foam indicates the presence saponins; c). Alkaloid
test. The extract was mixed with 2 ml of chloroform and 2 ml of ammonia then heated, shaken and
filtered. 3-5 drops of 2 N sulfuric acid are added to each filtrate, then shake and settle. The top of each
filtrate was taken and tested by Meyer, Wagner, and Dragendorf reagents. The formation of orange,
brown and white deposits indicates the presence of alkaloids; d). Flavonoid test. The extract was added
with hot water and boil for 5 minutes then filtered. To the 5 ml of filtrate, 0.05 g of Mg and 1 ml of
concentrated HCl were then added, and shaken vigorously. The formation of red indicates the presence
of flavonoids; e). Triterpenoid test. The extract was mixed with 2 ml of chloroform and 3 ml of
concentrated sulfuric acid. The formation of a brownish red color between the surfaces shows the
presence of triterpenoids. f). Tannin test. The extract is boiled with 20 ml of water and then filtered. A
few drops of 1% ferrochloride were added and the formation of a greenish-brown or black-blue color
indicates the presence of tannins.

2.3. Rejuvenation of tested bacteria

Test bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Kleibsella
pneumonia) were rejuvenated by inoculating as much as 1 ose on NA aseptic media aseptically and
incubated for 18-24 hours at 37oC. Test bacteria were taken from NA medium, then suspended into a
test tube containing 5 ml of 0.9% physiological salt. Addition of bacteria continued until the same
turbidity was obtained with the standard solution Mc. Farland on a scale of 0.5 [13].

2.4. Antibacterial activity assay

The Mueller Hinton Agar (MHA) plate was made for antibacterial testing. This media is made by means
of 38 grams of Mueller Hinton Agar (MHA) media suspended with 1 liter of distilled water. The solution
is then heated on a hot plate and stirred with a magnetic stirrer until it is homogeneous. Media were
sterilized by autoclaving at 121ºC for 15 minutes. Then the media were poured into petri dishes ± 10
mL each [14].
Inhibitory activity of extracts of leaves of Vitex plants was done by using the well diffusion method.
The culture suspension was spread evenly on the MHA media plate with a sterile cotton swab and
allowed to stand for 10 minutes in an incubator so that the microbial suspension permeated the culture
media. The wells are made by perforating the solid agar media which has been inoculated with a test
bacteria with a diameter of 6 mm. The size of the wellbore has been adjusted to the standard of wellbore
for sensitivity testing, which is between 6-8 mm. The pit is then injected with the extracts in 4 variations
of the concentration to be tested. The amount of extract injected is 50 µl. The media were then incubated
for 24 hours. Antimicrobial activity in the form of clear zone is then measured in mm [15].

2.5. Data analysis

Data were analyzed by using Anova, and DMRT at α = 0.05% using SPSS 23.

3. Results and discussion

3.1. Qualitative test of methanol extract

The results of extraction by methanol maceration showed that all groups of tested secondary metabolites
were detected, except for steroids was not detected in the roots and stems (Table 1).

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Annual Conference on Science and Technology (ANCOSET 2020) IOP Publishing
Journal of Physics: Conference Series 1869 (2021) 012060 doi:10.1088/1742-6596/1869/1/012060

Table 1. Phytochemical test results for several secondary metabolites of V. trifolia from roots, stems
and leaves.
Secondary metabolite Extract
No
group Root Stem Leaf
1 Steroid - - +
2 Saponin + + +
3 Alkaloid + + +
4 Flavonoid + + +
5 Triterpenoid + + +
6 Tannin + + +

3.2. Antibacterial activity

The average of the clear zone diameter data as a result of differences in concentration treatment on the
growth of test bacteria from 3 replications is presented in Figure 1.

Figure 1. The average diameter of inhibition zone of leaf methanol extract against S. aureus, S.
epidermidis, E.coli, and K. pneumonia.

The effect of these concentrations was analyzed by using ANOVA and showed significant different at
α = 0.05 (Table 2).

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Annual Conference on Science and Technology (ANCOSET 2020) IOP Publishing
Journal of Physics: Conference Series 1869 (2021) 012060 doi:10.1088/1742-6596/1869/1/012060

Table 2. Anova test results regarding the effect of the concentration of methanol extract of V. trifolia
leaves on the growth of test bacteria.
Sum of
Tested bacteria df Mean Square F Sig.
Squares
S.aureus Between Groups 288.483 3 96.161 29.377 .000
Within Groups 26.187 8 3.273
Total 314.669 11
S.epidermidis Between Groups 53.476 3 17.825 14.906 .001
Within Groups 9.567 8 1.196
Total 63.043 11
E. coli Between Groups 58.189 3 19.396 10.011 .004
Within Groups 15.500 8 1.938
Total 73.689 11
K. pneumoniae Between Groups 68.543 3 22.848 11.813 .003
Within Groups 15.473 8 1.934
Total 84.017 11

ANOVA test results (Table 2) showed that the treatment of different extract concentrations on the
growth of the four test bacteria showed a significant difference (Sig. <0.05) based on the average
diameter of the inhibitory zone in the growth of the test bacteria.
Further tests were specifically conducted to know the significant differences of the average value
among the treatments in S. aureus. The DMRT results (not shown) showed that all treatments (7.5%,
15%, 30% and 60%) were different significantly from each other, where treatment with 60% of methanol
extract of V. trifolia leaves gave the best effect by being able to achieve an average inhibition zone
against S. aureus at the value of 15.1 mm. The content of secondary metabolites in the form of steroids,
saponins, alkaloids, flavonoids, triterpenoids and tannins detected as shown in Table 1, has more
inhibitory effect on Gram positive bacteria, in this case S. aureus and S. epidermidis, compared to Gram
negative bacteria, E. coli and K. pneumoniae. The use of crude extracts of plants parts and
phytochemicals of known antimicrobial properties can be of great significance in the therapeutic
treatments. In fact, many plants have been used due to their antimicrobial properties which are actually
the secondary metabolites synthesize by the plants.
The present study has shown that methanolic extract of V. trifolia leaf has promising antibacterial
activity. This is probably why the plant is widely used in traditional medicine. It is also used as anti-
inflammatory, antipyretic agent. They are also used as sedative for rheumatism, headache and common
cold in some countries. Vitex species used in traditional medicine to treat ailments like wounds,
allergies, asthma and body pains [16]. There were reports that methanol extract demonstrated inhibitory
effects to S. aureus but not E. coli [17]. Several research activities on antibacterial activities of crude
extracts have implicated the methanol extract for being more active than the other solvents extracts [18].
The activity of the plant against both Gram positive and Gram negative bacteria can be indicative of
the presence of broad spectrum antibiotic compounds or simply general metabolic toxins in the plant.
The antimicrobial activity of the extracts of V. trifolia may be due to the presence of flavonoids,
triterpenoids, and tannins in the plant extract [6-8].
In the present study Gram positive bacteria were found to be more susceptible to the plant extract
than Gram negative bacteria which corroborated the previous reports that plant extracts are more active
against Gram positive. Gram-negative bacteria are considered to be more resistant due to their outer
membrane which acting as a barrier to many environmental substances including antibiotics [19]. This
outer membrane includes the asymmetric distribution of the lipids with phospholipids and
lipopolysaccharide located in the inner and outer leaflets, respectively. This characteristic that is absent

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Annual Conference on Science and Technology (ANCOSET 2020) IOP Publishing
Journal of Physics: Conference Series 1869 (2021) 012060 doi:10.1088/1742-6596/1869/1/012060

in the Gram- positive bacteria might have acted as the additional barrier that hinders the movement of
foreign substance into the cell.

4. Conclusion
V. trifolia methanol extract contains 6 types of secondary metabolites, such as steroids, saponins,
alkaloids, flavonoids, triterpenoids and tannins. This study shows that the methanolic extract of Vitex
trifolia leaves exhibited appreciable antibacterial properties inhibiting growth of Gram positive bacteria,
especially to S. aureus. It could serve as useful source for new antimicrobial agents in the future.

Acknowledgment
The authors thank the Directorate of Research and Community Service, Ministry of Research and
Technology of the Republic of Indonesia, which has provided financial support through the Master's
Thesis Research scheme.

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Journal of Physics: Conference Series 1869 (2021) 012060 doi:10.1088/1742-6596/1869/1/012060

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