Phytochemical Screening and Antimicrobial Activity of Limonia

acidissima Ethanol Extract against Microbes from Clinical Isolates

Ahmad Shobrun Jamil

, Siti Rofida, Dian Priyani, Wahidatin Nabila, Evi Wulandari

Department of Pharmacy, Faculty of Health Sciences, University of Muhammadiyah Malang Jalan Bendungan Sutami 188

A Malang 65145, East Java, Indonesia

Keywords: Antimicrobial, Ethanol fraction, Limonia acidissima, Phytochemical screening

Abstract: The aim of this study is to determine the content of secondary metabolites ethanol fraction of fruit peel of

Limonia acidissima qualitatively and test the activity of antimicrobial on Escherichia coli, Staphylococcus

aureus, and Candida albicans. Phytochemical screening was carried out by running for separating the extract

in silica gel by eluent n-hexane: ethyl acetate. The Thin Layer Chromatography (TLC) plate results then

sprayed with a stain viewer; Alkaloid stain viewer: Dragendorff reagent; Terpenoids: Anisaldehyde-sulfuric

acid reagents. The plate is heated to a temperature of 100°C; Flavonoids: H2 SO4 10% reagents; Polyphenols

and tannins: FeCl 3 reagents 10%; Anthraquinone: 10% KOH reagent in methanol. The antimicrobial test was

carried out using disk diffusion methods with concentrations of 50 mg/ml, 75 mg/ml and 100 mg/ml with

control of nystatin and chloramphenicol. Phytochemical screening results of ethanol fraction Limonia

acidissima can be detected in the content of alkaloids, terpenoids, anthraquinones, and saponins.

Antimicrobial test results obtained data that the ethanol fraction Limonia acidissima has an increasing

inhibitory activity of 50 mg/ml, 75 mg/ml, and 100 mg/ml

1 INTRODUCTION

Infectious disease is a serious threat in the field of

medicine. Coupled with the development of

antimicrobial resistance is very high from year to year

(Gyles 2011). This is exacerbated by the high

nosocomial infections that occur in health

installations (Brusselaers, Vogelaers, and Blot 2011).

The rate of increase in antimicrobial resistance

increases significantly from year to year which causes

fewer antibiotic treatment options and more

expensive medical costs (Bingyun Li and Thomas J.

Webster 2018).

The use of antibiotics with new variants and

increasing the dose of antibiotic therapy are some of

the ways taken so far to deal with infectious diseases.

But the two ways above are of course not enough to

overcome this problem. One way that needs to be

taken is to explore various natural resources that are

potentially used as an antimicrobial alternative.

Several amounts of research show that there are many

potential sources from plants as antimicrobial agents.

Twelve plant extracts which are extracted using

acetone solvent has good antibacterial activity against

multidrug-resistant bacteria that cause diarrhea, one

of which is Staphylococcus aureus (Bisi-johnson et

al. 2017).

In another study the extract of the Myrtus

communis and Cinnamomum zeylanicum plant

extracted using ethanol solvent had antibacterial

activity against several multidrug-resistant bacteria

tested ( S. aureus, E. coli, P. aeruginosa and S.

enteric ). It is even known that M. communis extract

has better antibacterial activity than penicillin (Zandi

2015). Likewise in several plant extract tests on

Aeromonas hydrophila it was found that extracts from

several plant species namely Olea europea, Myrtus

communis, Thymus vulgaris, Rosmarinus officinalis,

and Achillea falcata, were known to have better

antibacterial activity than some antibiotics (Anwar et

al. 2014).

Microbiologists have several reasons for making

plants as hope as antimicrobial agents because

various research results on plant phytochemical

compounds are proven to have potential as

antimicrobials (Rios and Recio 2017), (Mostafa et al.

2018), (Tchinda et al. 2017). Plants have the potential

as an alternative source of natural antimicrobials

which have a different mechanism of action than

commercial antibiotics, some even have competitive

152

Jamil, A., Roﬁda, S., Priyani, D., Nabila, W. and Wulandari, E.

Phytochemical Screening and Antimicrobial Activity of Limonia acidissima Ethanol Extract Against Microbes from Clinical Isolates.

DOI: 10.5220/0009126001520156

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 152-156

ISBN: 978-989-758-462-6

effects compared to some commercial antibiotics that

exist today (Abdallah 2011).

Kaemferia pandurata extract (256

micrograms/ml) and Senna alata (512

micrograms/ml) are known to have inhibitory activity

against the growth of methicillin-resistant

Staphylococcus aureus (MRSA), extended spectrum

beta-lactamase and resistant-carbapenemase bacteria

(Wikaningtyas and Sukandar 2016). In addition, due

to increasing public awareness about the importance

of sources of natural remedies made from plants

(Cowan, 1999).

This research will be tested the anti-microbial

activity of ethanol extract of Limonia acidissima fruit

peel. Limonia acidissima is a plant that has various

potentials. Among other things, this plant has a high

antioxidant activity, has a protective activity against

body tissues (Balamuruganvelu et al. 2015). This

plant also has antitumor activity. Tests using male

model mice with ascitic lymphoma obtained

information that Limonia acidissima fruit extracts

could increase the life span of experimental animals,

significantly suppressing tumor growth compared to

the control group (Eluru, Taranalli, and Kawatra

2015).

The fruit also has wound healing activity, speed

up the process of epithelialization of the wound,

accelerating wound closure. It is monitored to

increase the activity of antioxidant enzymes such as

Superoxide Dismutase (SOD) and catalase enzymes

(Ilango and Chitra 2010). Another potential of this

plant is as an antimicrobial, it is known that Limonia

acidissima has antibacterial activity. Some research

records show the antimicrobial activity of Limonia

acidissima, among others, petroleum ether extract of

L acidissima leaf inhibits the growth of E. coli and S.

aureus (Harshali et al., 2015). The ethanolic extract

of Limonia acidissima leaves has the potential to

inhibit the growth of Gram-positive and negative

bacteria (Neelamadhab et al., 2013). The ethanol

extract of L. acidissima leaf has anti dermatophyte

activity in fungi such as Trichophyton

mentagrophytes, Microsporum canis and

Epidermatophyton floccosum (Buvanaratchagan,

2016).

Various types of Indonesian plant extracts are

known to have antimicrobial activity with various

levels (Pratiwi et al., 2015). Therefore, research on

antimicrobial activity on various plant extracts

always needs to be intensified in order to find

alternative solutions to the problem of resistance to

microorganisms in various infectious diseases.

Medicinal plants need to be explored antimicrobial

activity because the content of chemical compounds

in various extracts, has a variety of physiological

potential for humans. In addition, few compounds in

plants are toxic to humans (Zubaır et l., 2011). In this

study, the antimicrobial activity of Limonia

acidissima fruit peel is tested against Staphylococcus

aureus, Escherichia coli, and Candida albicans from

clinical samples and the chemical contents from

extracts and fractions of some of these plants are

observed. The three microbes were chosen because

they were general infectious agents and represented a

class of Gram-positive, Gram-negative bacteria and

fungal pathogens.

2 METHODS

2.1 Plant Simplisia Extraction

Plants Limonia acidissima L. obtained from the

district. Rasanae Barat Kota Bima, NTB. Plant

identification was carried out at UPT Materia Medika

Batu. Fruit flesh is washed with water until clean and

carried out drying at a temperature of 40 ° C. After

drying, then crushed with a grinding machine, so that

obtained fine powder. The fine powder is then sieved

using a SieveShaker with a certain degree of fineness.

Limonia acidissima fruit meat powder was

extracted stratified using three types of solvents (n-

hexane which is non-polar, ethyl acetate which is

semi-polar, and ethanol which is polar). 1000 grams

of powdered fruit peel of Limonia acidissima is

macerated using the first (n-hexane) at a ratio of 1: 4

(mass/volume), after 24 hours residue screening

results maceration first macerated with a second

solvent (ethyl acetate) in comparison with the

similarly, 24 then a filter is made and the residue is

macerated again using a third solvent (ethanol). The

filtrate from the third maceration (using ethanol

solvent) was concentrated with a rotary evaporator

until it was concentrated and the weight was stable.

2.2 Phytochemical Screening

Phytochemical screening was carried out according to

the method carried out by [21] by removing the

extract in silica gel with the eluent n-hexane: ethyl

acetate. TLC plate results then sprayed with stain

viewer to detect the compound, the Alkaloid:

Dragendorff Reagent (produce staining orange);

Terpenoids: Anisaldehyde-sulfuric acid reagents. The

plate is heated to 100°C (produces a purplestain);

Flavonoids: H

10% reagent (produce yellow

stain); Polyphenols and tannins: Reactionof 10%

FeCl

(produces a black stain); Anthraquinone: 10%

Phytochemical Screening and Antimicrobial Activity of Limonia acidissima Ethanol Extract Against Microbes from Clinical Isolates

153

KOH reagent in methanol (produces yellow or

brownish-yellow stains).

2.3 Antimicrobial Activity Test

The bacteria Escherichia coli, Staphylococcus

aureus, and the fungus Candida albicans are medical

samples obtained from the Biomedical Laboratory of

the Faculty of Medicine, University of

Muhammadiyah Malang. The antimicrobial activity

test is carried out by the disc diffusion method. The

ethanol fraction of Limonia acidissima fruit with a

concentration of 5 mg/ml, 7.5 mg/ml, and 10 mg/ml

was absorbed on paper discs. Then tested on

microbial samples Escherichia coli, Staphylococcus

aureus, and Candida albicans. The preparation was

incubated in an incubator for 24 hours, at 37 ° C then

the clear zone that appeared was measured.

2.4 Data Analysis

Anti-microbial test data in the form of inhibitory

zones were analyzed for the significance of

differences between treatments using ANOVA

followed by the Tukey test with a significance of P <

0.05 using SPSS program ver 23.

3 RESULTS AND DISCUSSION

Phytochemical screening results of ethanol fraction

Limonia acidissima can be detected in the content of

alkaloids, terpenoids, anthraquinones, and saponins

qualitatively as shown in Table 1.

While the results of the antimicrobial test of the

fraction gave a positive inhibitory growth on

Staphylococcus aureus and Escherichia coli but not

on Candida albicans molds from clinical samples as

listed in Table 2.

Table 1. Phytochemical screening results show Alkaloid, Terpenoid, Anthraquinone and Saponin content in the ethanol

fraction of Limonia acidissima

No Compound Stain Viewer Results Rf value Information

1 Alkaloids Dragendorff (+) 0.225

Orange color is

formed

2 Flavonoids H2SO4 10% - - -

3 Polyphenols and Tannins FeCl3 10% - - -

4 Terpenoids anisaldehyde-sulfuric acid (+) 0.0625; Purple color

0.1125;

0.175;

0.325;

0.4625

5 Anthraquinone KOH 10% in methanol (+) 0.05 Yellow formed

6 Saponin Froth test (+)

Formed stable froth

more than 3 cm

above the liquid

surface.

Salkowski Test (+)

The formation of a

red ring in the test

solution given

concentrated

H2SO4.

HSIC 2019 - The Health Science International Conference

154

Table 2. Test results for the antimicrobial activity of the ethanol fraction Limonia acidissima

The concentration of test material

Inhibitory zone diameter (mm)

Candida albicans Staphylococcus aureus Eschericia coli

5 mg / ml - 19.50 ± 2.50 13, 77 ± 1.72

7.5 mg / ml - 23.63 ± 1.89 15.47 ± 2.08

10 mg / ml - 26.76 ± 1.79 16.63 ± 2.11

Nystatin (Control +) 17.13 ± 1.35 - -

Chloramphenicol 30 µg / ml

(control +) - 15.30 ± 1.44 16.67 ± 1.05

Phytochemical screening results that show

positive on alkaloid compounds, terpenoids,

anthraquinone, and saponins that are in the polar

fraction. This means that only the more polar active

compounds are extracted in the ethanol fraction

according to the nature of the ethanol solvent

compared to the other two solvents that have been

used to extract Limonia acidissima powder, n-hexane

and ethyl acetate. The content of secondary

metabolites from plants have levels in different

polarity. It is possible in a group of compounds such

as flavonoids, saponins, phenol hydroquinone,

alkaloids, tannins, steroids can be extracted with

solvents that have different polarities. Polar

flavonoids will be extracted with polar solvents such

as ethanol, semi-polar flavonoids will be extracted

with semi-polar solvents such as ethyl acetate, and

non-polar flavonoids will be extracted with non-polar

solvents such as n-hexane, as well as other secondary

metabolite compounds (Widyawati et al. 2014).

Based on the antimicrobial activity test of marigolds

(Tagetes erecta) with various solvents (non-polar,

semi-polar and polar) it is known that antimicrobial

activity is best obtained in marigold extract with polar

solvents (Padalia and Chanda 2015).

It was found that the antimicrobial activity of

ethanol fraction of L. acidissima fruit against E. coli

in this study was higher than all parts of the fruit

extracted using ethanol in other studies (Audia Anda

Rini, Supriatno 2017). This fact shows the activity of

L. acidissima fruit flesh metabolite compounds works

better when extracted stratified as this research

method compared with the results of other studies that

use one-time maceration (not multilevel maceration).

Based on research exploration (Vijayvargia and

Vijayvergia 2014), (Vijayvargia, Choudhary, and

Vijayvergia 2014) [25] [26] overall L. Acidissima

plant extracts contain Alkaloids, flavonoids, phenols,

terpenoids, tannins, fats steroids, saponins,

glycosides, gum, mucilage, and essential oils.

The results of the detection of the presence of L.

acidissima metabolite compounds in this study are

known to be minus the presence of flavonoids,

polyphenols, and tannins. It turns out that the absence

of flavonoid compounds, polyphenols, and tannins in

this fraction increases the anti- mic r o ba activity of

E. coli.

The results of antimicrobial activity against S.

aureus, Limonia acidissima fruit extract of this study

looked the same as the study (Vijayvargia et al. 2014).

These results appear that fractionation with n-hexane,

ethyl acetate and ethanol did not affect the strength of

the antimicrobial activity of L. acidisima fruit peel.

4 CONCLUSIONS

Ethanol fraction from three factions (n-hexane, ethyl

acetate, and ethanol) Limonia acidisima showed

antimicrobial activity against E. Coli and S. aureus

but not Candida albicans. And the content in the

fraction contained alkaloids, terpenoids,

anthraquinone, and saponins.

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