The Antibacterial Activity of Isolated Flavonoid Fractions from

Ethanol Ethanolic Peel of Citrus Sinensis (Valencia Orange) with

Citrus Limon (Lemon) against Staphylococcus Aureus and

Pseudomonas Aeruginosa

Nik Nur Shamiha N. D

1,2*

, Ghayatery Nagatamby

, Fadli Asmani

, Eddy Yusof

School of Pharmacy, Management and Science University, Selangor Darul Ehsan, Malaysia

ICHLAS, Management & Science University, Selangor, Malaysia

Keywords: Citrus sinensis; Citrus limon; Combination antibacterial activity; Flavonoid; Skin and soft tissue infections

(SSTIs)

Abstract: Background: Skin and soft tissue infections (SSTIs) are most common infections encountered by all

physicians. Eventhough pharmacological industries have produced a number of new antibiotics, resistance to

these drugs by microorganisms has increased. Objective: The aim of this study is to evaluate the antibacterial

activity of isolated flavonoid fractions from ethanolic peel extract of C.sinensis (Orange), C.limon (Lemon)

and its combination against S.aureus and P.aeruginosa. Methods: The ethanolic peel extract of both plant were

screened for phytochemical identification of flavonoid by lead acetate test and shinoda test. The extract of

both plants were evaluated for preliminary antibacterial activity using disk diffusion method. Thin-layer

chromatography and column chromatography was performed to isolate flavonoids. Isolated flavonoids were

subjected to determination of minimum inhibitory concentration and antibacterial assay by disk diffusion

method. Results: Isolated flavonoids of C.sinensis (17mm, 8mm), C.limon (20mm, 9mm) and its combination

(24mm, 14mm) produced antibacterial activity that is comparable to the Ciprofloxacin disc (30mm, 9mm)

against S.aureus and P.aeruginosa respectively. Thus, these results suggested that C.limon produced a better

antibacterial activity against both bacteria compared to C.sinensis. However, the combination of both plants

isolated flavonoid fractions produced much better antibacterial activity against S.aureus and P.aeruginosa in

comparison with individual flavonoid fractions of both plants. Conclusion: Therefore, Citrus fruits peels that

is being as primary waste in juicing industries can be further developed as marketable natural source of

antibiotic as a treatment of SSTIs.

INTRODUCTION

Skin and soft tissue infections (SSTIs) are reﬂect

inﬂammatory microbial invasion of the epidermis,

dermis and subcutaneous tissues (Matthew

S.Dryden, 2010). Some skin colonizing

microorganisms, in particular Staphylococcus

aureus (S.aureus) and β-haemolytic group A

streptococci but also Gram-negative bacteria such

as Pseudomonas aeruginosa (P.aeruginosa), viruses

and fungi, have the potential to cause infection,

particularly when the skin barrier is breached

(Lacey et al., 2015). SSTIs are treated by using

antibiotics. The problem arises as these bacteria are

developing resistance strains against antimicrobial

agents as a result, there is an urgent need to find an

alternative way to cure the bacterial infections.

Plant would be a better alternative because of their

antimicrobial traits, which are due to compounds

synthesized in the secondary metabolism and it has

lesser side effects compared with synthetic

antibiotics (Kailash D. Sonawane et al., 2011).

Citrus fruits have been of interest for extraction

of antimicrobial metabolites by large numbers of

researchers but the peels are less studied (Akhilesh

et al., 2012). Moreover, the peels part of citrus fruits

is abundance with secondary metabolites which

serves as plant defense mechanisms against

predation by microorganisms, insects, and

herbivores (Xinmiao Lv et al., 2015). Among the

secondary metabolites, flavonoids are known for its

antimicrobial properties. Since, they are known to

Shamiha N. D., N., Nagatamby, G., Khan, J., Asmani, F. and Yusuf, E.

The Antibacterial Activity of Isolated Flavonoid Fractions from Ethanol Ethanolic Peel of Citrus Sinensis (Valencia Orange) with Citrus Limon (Lemon) against Staphylococcus Aureus and

Pseudomonas Aeruginosa.

DOI: 10.5220/0009844400002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

 2022 by SCITEPRESS – Science and Technology Publications, Lda. All r ights reserved

be synthesized by plants in response to microbial

infection, it should not be surprising that they have

been found in vitro to be effective antimicrobial

substances against a wide array of microorganisms

(Xinmiao Lv et al., 2015).

There are previous studies on antimicrobial

activity of essential oil from peels of orange and

lemon against several microorganisms (Shalu

Hasija et al., 2015). However, there are lack of

studies on antibacterial activity of flavonoid

isolated from peels of orange and lemon. Moreover,

there are no studies on combined antibacterial

activity of flavonoid isolated from orange peel and

lemon peel. Hence, this study is mainly focused on

investigating the antibacterial activity of combined

flavonoid fractions from ethanolic peel extract of

C.sinensis with C.limon against a gram-positive

organism (S.aureus) and a gram-negative organism

(P.aeruginosa) that is linked with SSTIs.

2 METHODOLOGY

2.1 Collection and Preparation of

Plant Materials

The peels of lemon and oranges part was separated

from the fruits. The peels were air-dried at room

temperature for almost a week until constant

weight. Then, it was grinded into coarse powder by

using a mechanical blender (Sapna B.Shetty et al.,

2015).

Both peels’ powder was macerated for a week

with 96% ethanol. Filtered and evaporated the

solvent by using rotary evaporator to obtain a

concentrate extract of the peels. The extract were

stored in cold room until further use (Augustine

Ahmadu & Ufuoma Omonigho, 2013).

2.2 Preliminary Screening for

Flavonoid

Both of the plants extract were subjected for two

tests: Lead acetate and Shinoda test, for

identification of flavonoids (Sangha R. Bijeka et al.,

2015).

2.3 Preliminary Antibacterial Assay

Both of the plants extract was tested on its

antibacterial activity against S.aureus and

P.aeruginosa by disk diffusion method with three

different concentration (1mg/mL, 10mg/mL and

100mg/mL).

2.4 Thin-Layer Chromatography

(TLC)

TLC was conducted on both plants extract by using

five different solvent mixture (Oyvind M. Andersen

& Kenneth R. Markham, 2006) as described in

Table 1.

Table 1: Different solvent mixture for TLC.

Solvent mixture

Chloroform-Acetic acid 100:4

Chloroform-Methanol-Acetic acid 90:5:5

Chloroform-Methanol-Water 40:10:1

Chloroform-Methanol-Water 65:45:12

Ethyl acetate-Methanol-Water 50:3:10.

TLC was conducted to determine the number of

flavonoid components in both plants extract and

also to identify solvent mixture that provide best

fractionation or separation between those

components (Abe Rita Temidayo, 2013).

2.5 Column-Chromatography

(Fractionation/Isolation of

Flavonoid)

Best solvent mixture identified from TLC was used

for isolating flavonoid fractions from both plants

extract (Abe Rita Temidayo, 2013). Chloroform-

Acetic acid 100:4 solvent system was used for

C.Limon while Chloroform-Methanol-Water

40:10:1 was selected for C. Sinensis. Silica gel was

used as adsorbent. Fractions were collected in

several test tubes and tested on TLC to ensure all the

flavonoid components are collected. Moreover, the

collected fractions were also tested on lead acetate

test again to ensure it is flavonoid fractions.

2.6 Determination of Minimum

Inhibitory Concentration (MIC)

Stock solution of flavonoid fraction was

prepared for both plants and its combination:

100mg/ml. Then, from this stock solution

transferred 1ml into test tube that contains 1ml

ethanol (50mg/ml), these procedure were repeated

until several dilution from 25 mg/ml - 0.2mg/ml.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Each concentration was tested on selected bacteria

by disk diffusion method. The first lowest

concentration that produced zone of inhibition

represent the MIC.

2.7 Antibacterial Assay

The in- vitro antibacterial activities was carried out

using the disk diffusion method. Mueller Hinton

Agar (MHA) plate was inoculated with selected

bacteria; S.aureus and P.aeruginosa. The MHA was

prepared and stored at 4°C. Three different

concentration of flavonoid fraction were placed on

the plate (lemon, orange and its combination).

Positive control was Ciprofloxacin and negative

control was Ethanol. Plates were incubated at 37ºC

for 24 hours in the incubator. Zone of inhibition was

measured and repeated for three times.

3 RESULTS AND DISCUSSION

Both plants peel extracts showed positive result for

flavonoid screening by producing yellow

precipitate (Lead acetate test) and orange (Shinoda

test). The result of preliminary antibacterial assay

was, both plants peel extracts showed antibacterial

effect against S.aureus and P.aeruginosa except

C.limon extract does not show any antibacterial

effect against P.aeruginosa at lowest concentration

(1mg/mL). Upon TLC for both plants extract on

different solvent mixture (Table 1), solvent mixture

number 3 was selected for orange while solvent

mixture 1 was selected for lemon to isolate its

flavonoid components. Solvent mixture was chosen

based on two factors which are good retention factor

(0.25 – 0.35) and spot on TLC (considerable

distance between one spot to another). Table 2 and

Table 3 shows the result of isolating flavonoid

fractions from orange and lemon peel extract

respectively.

Table 2: Flavonoid fractions of C.sinensis peel extract

(orange)

Fractions (C.sinensis)

Flavonoid test

(Lead acetate test)

1-5 (Presence spot)

Yellow precipitate

(Component A)

11-15 (Presence spot)

Yellow precipitate

(Component B)

Table 3: Flavonoid fractions of C.limon peel extract

(Lemon).

Fractions (C.limon)

Flavonoid test

(Lead acetate test)

1-5 (Presence spot)

Yellow precipitate

(Component A + B)

12-14 (Presence spot)

Yellow precipitate

(Component C)

17-19 (Presence spot)

Yellow precipitate

(Component D)

21-23 (Presence spot)

Yellow precipitate

(Component E)

Table 4 shows the MIC of flavonoid fractions

from the peel extract of orange, lemon and its

combination respectively against the selected

bacteria. The result is suggestive that when the

flavonoid fractions are combined (lemon with

orange), a lesser concentration of flavonoid is

needed to inhibit the growth of both S.aureus and

P.aeruginosa than individual flavonoid fraction of

orange and lemon. Furthermore, it is noted that a

higher concentration of flavonoid is needed to

inhibit growth of P.aeruginosa compared to

S.aureus.

Table 4: MIC of flavonoid from orange, lemon and its

combination.

Figure 1 shows graph represent comparison of

flavonoid fractions antibacterial activity against

S.aureus and P.aeruginosa. All three flavonoid

fractions produced antibacterial effect against

S.aureus and P.aeruginosa. It is noted that

antibacterial effect of flavonoid against S.aureus is

more compared to P.aeruginosa. It is associated

with impermeability of gram-negative bacteria

(P.aeruginosa) towards antimicrobial agents, in this

case, it is flavonoids. The wall structure of Gram-

negative bacteria, and speciﬁcally the presence of

an outer envelope, is often responsible for the

impermeability of these micro-organisms to

antimicrobial agents (S.P. Denyer & J.Y. Maillard,

2002). When the antibacterial effect of C.sinensis

and C.limon is compared, C.limon produced a better

antibacterial effect against both bacteria. This can

be due to presence of more flavonoid components

in C.limon (Component A-E) than C.sinensis

(Component A & B). Moreover, the combination of

both plants flavonoid produced a further increase in

Microorganis

MIC (mg/mL)

C.sinensi

C.limo

Combinatio

S.aureus

12.5

0.78

0.2

P.aeruginosa

The Antibacterial Activity of Isolated Flavonoid Fractions from Ethanol Ethanolic Peel of Citrus Sinensis (Valencia Orange) with Citrus

Limon (Lemon) against Staphylococcus Aureus and Pseudomonas Aeruginosa

antibacterial effect against S.aureus and

P.aeruginosa. Combined flavonoid fractions

produced an antibacterial effect against S.aureus

(24mm) which was nearly equal to positive control,

Ciprofloxacin disc (30mm). Both flavonoid fraction

of C.sinensis (8mm) and C.limon (9mm) produced

effective antibacterial effect against P.aeruginosa

comparable to positive control (9mm). Also it was

found that combination of C.sinensis and C.limon

flavonoid fractions produced an antibacterial effect

against P.aeruginosa (14mm) better than the

positive control (9mm).

Figure 1: Graph that compare the antibacterial activity of

flavonoid fractions from orange, lemon and its

combination against S.aureus and P.aeruginosa.

4 CONCLUSIONS

This research has found that flavonoid fractions of

all three (C.sinensis, C.limon and its combination)

significantly (p<0.05) produced inhibitory effect

against S.aureus and P.aeruginosa.

Thus,C.sinensis, C.limon and its combination holds

promise as a potential to be developed into solely

herbal based antimicrobial agent to cure SSTIs.

ACKNOWLEDGEMENTS

The author is very thankful and grateful towards the

research committee and lecturers of Management

and Science University, Malaysia for providing all

the needed materials, equipment as well continuous

guidance and support throughout completing this

research project.

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The Antibacterial Activity of Isolated Flavonoid Fractions from Ethanol Ethanolic Peel of Citrus Sinensis (Valencia Orange) with Citrus

Limon (Lemon) against Staphylococcus Aureus and Pseudomonas Aeruginosa

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

The Antibacterial Activity of Isolated Flavonoid Fractions from Ethanol Ethanolic Peel of Citrus Sinensis (Valencia Orange) with Citrus

Limon (Lemon) against Staphylococcus Aureus and Pseudomonas Aeruginosa