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,

40100 Shah Alam, Selangor Darul Ehsan, Malaysia

ICHLAS, Management & Science University,

40100 Shah Alam, 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.

1 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

194

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: 10.5220/0008359801940197

In Proceedings of BROMO Conference (BROMO 2018), pages 194-197

ISBN: 978-989-758-347-6

 2018 by SCITEPRESS – Science and Technology Publications, Lda. All r ights reserved

secondary metabolites, flavonoids are known for its

antimicrobial properties. Since, they are known to 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

1 Chlorofor

-Acetic acid 100:4

2 Chlorofor

-Methanol-Acetic acid 90:5:5

3 Chlorofor

-Methanol-Water 40:10:1

4 Chlorofor

-Methanol-Water 65:45:12

5 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. Each concentration was tested

on selected bacteria by disk diffusion method. The

first lowest concentration that produced zone of

inhibition represent the MIC.

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

195

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

(

Com

onent 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

)

Yellow precipitate

(

Com

onent C

)

17-19 (Presence

)

Yellow precipitate

(

Com

onent D

)

21-23 (Presence

)

Yellow precipitate

(

Com

onent 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.

Microor-

ganism

MIC (mg/mL)

C.sinensis C.limon Combi-

nation

S.aureus 12.5 0.78 0.2

P.aeru

inosa 50 50 25

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

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

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

196

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.

REFERENCES

Akhilesh Khushwaha, Raghvendra Pratap Singh, Vikas

Gupta, and Madhulika Singh. (2012). Antimicrobial

properties of peels of Citrus fruits. International

Journal of Universal Pharmacy and Life Sciences 2(2).

Augustine A Ahmadu and Ufuoma Omonigho (2013).

Flavonoids from the leaves of Physalis angulata Linn.

African Journal of Pharmaceutical Research &

Development, 5, 40-43.

Kailash D. Sonawane. Maruti J. Dhanavade, Chidamber B.

Jalkute, and Jai S. Ghosh (2011). Study Antimicrobial

Activity of Lemon (Citrus lemon L.) Peel Extract.

British Journal of Pharmacology and Toxicology 2(3):

119-122, 2011.

Lacey, K. A., Geoghegan, J. A., & McLoughlin, R. M.

(2016). The Role of Staphylococcus aureus Virulence

Factors in Skin Infection and Their Potential as

Vaccine Antigens. Pathogens (Basel, Switzerland),

5(1).

Matthew S.Dryden. (2010). Complicated skin and soft

tissue infection. J Antimicrob Chemother 2010; 65

Suppl 3: iii35–44.

Oyvind M. Andersen & Kenneth R. Markham. (2006).

Flavonoids: Chemistry, Biochemistry and applications.

S.P. Denyer and J.-Y. Maillard. (2002). Cellular

impermeability and uptake of biocides and antibiotics

in Gram-negative bacteria. Journal of Applied

Microbiology Symposium Supplement 2002, 92, 35S–

45S.

Sangha R Bijekar, M.C Gayatri, L Rajanna. (2015),

Antimicrobial Activity of Isolated Flavonoid Fractions

from Drypetes Roxburghii (Wall.) Huresawa and Its

Phytochemical Fingerprinting. International Journal of

Innovative Research in Science, Engineering and

Technology, 4, 6214-6224.

Sapna B. Shetty, Prabu Mahin-Syed-Ismail, Shaji

Varghese, Bibin Thomas-George, Pathinettam

KandathilThajuraj, Deepak Baby, Shaista Haleem,

Sreeja Sreedhar, Darshan Devang-Divakar. (2016).

Antimicrobial effects of Citrus sinensis peel extracts

against dental caries bacteria: An in vitro study.

Journal of clinical and experimental denstistry.

Shalu Hasija, Geeta Ibrahim,and Ashok Wadia. (2015).

Antimicrobial Activity of Citrus Sinensis (Orange),

Citrus Limetta (Sweet Lime) and Citrus Limon

(Lemon) Peel Oil on Selected Food Borne Pathogens.

International Journal of Life Sciences Research, 3, 35-

39.

Xinmiao Lv, Siyu Zhao, Zhangchi Ning, Honglian Zeng,

Yisong Shu, Ou Tao, Cheng Xiao, Cheng Lu, and

Yuanyan Liu (2015). Citrus fruits as a treasure trove

of active natural

metabolites that potentially provide benefits for human

health. Chemistry Central Journal, 9, 68.

S.aureus 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

197