Antimicrobial Activity of Grapefruit Seeds Extracts (In vitro Study)

Abstract

Aims: The purpose of this study is to determine the antimicrobial activity of aqueous and alcoholic extract of grapefruit (Citrus Paradisi Rutaceae) seeds. Materials and Methods: Aqueous and alcoholic (ethanol) extracts of grapefruit seeds "GSE" in 20% (w/v) concentration were investigated for activity against Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumonia, Candida albicans and a mixed oral flora. The level of antimicrobial effects was established using in vitro disc diffusion method. Their antibacterial and antifungal activity was compared to the activity of Chlorhexidine (CHX) solution in two concentrations (0.12 and 0.2%) as a control. Results: The aqueous GSE (20%) solution used in this study gave positive results with lethal effect on the tested organisms with zones of inhibition ranging from 10mm-18mm in diameter, which is comparable to that of chlorhexidine (7mm - 22mm) and (13mm - 20mm) for 0.12% and 0.2% solutions respectively. Ethanolic GSE did not show any antimicrobial activity. Conclusions: Aqueous GSE has a good antimicrobial effect, which makes it a good natural preparation for use as antiseptic or disinfectant.

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Waidulla N Al–Âni Department of Dental Basic Sciences
BSc, PhD (Asst. Prof.) College of Dentistry, University of Mosul
Nahla O M Tawfik Department of Dental Basic Sciences
MBChB, MSc, PhD (Lec.) College of Dentistry, University of Mosul
Enas Y Shehab Department of Dental Basic Sciences
BSc, MSc (Asst. Lec.) College of Dentistry, University of Mosul

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ABSTRACT
Aims: The purpose of this study is to determine the antimicrobial activity of aqueous and alcoholic
extract of grapefruit (Citrus Paradisi Rutaceae) seeds. Materials and Methods: Aqueous and alcoholic
(ethanol) extracts of grapefruit seeds "GSE" in 20% (w/v) concentration were investigated for activity
against Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumonia, Candida albicans and a mixed
oral flora. The level of antimicrobial effects was established using in vitro disc diffusion method. Their
antibacterial and antifungal activity was compared to the activity of Chlorhexidine (CHX) solution in
two concentrations (0.12 and 0.2%) as a control. Results: The aqueous GSE (20%) solution used in this
study gave positive results with lethal effect on the tested organisms with zones of inhibition ranging
from 10mm-18mm in diameter, which is comparable to that of chlorhexidine (7mm - 22mm) and
(13mm - 20mm) for 0.12% and 0.2% solutions respectively. Ethanolic GSE did not show any antimi-
crobial activity. Conclusions: Aqueous GSE has a good antimicrobial effect, which makes it a good
natural preparation for use as antiseptic or disinfectant.
Key Words: Grapefruit Seeds. chlorhexidine and aqueous extract.
Al–Âni WN, Tawfik NO, Shehab EY. Antimicrobial Activity of Grapefruit Seeds Extracts (In
vitro Study) Al–Rafidain Dent J. 2011 11(2): 341-345.
Received: 16/5/2010 Sent to Referees: 16/5/2010 Accepted for Publication: 28/6/2010
INTRODUCTION
The use of herbal medicine is widely
spread and growing (1). Plants have been
exploited for the treatment of many infec-
tions and diseases because those plants
readily contain substances for defense
against attacks by insects, herbivores and
microorganisms (2). The citrus fruits make
a group of plants of great medicinal im-
portance (3). The therapeutic efficacy of
citrus fruits such as grape fruits is support-
ed by the facts that they contain different
classes of polyphenolic flavenoids that
have been shown to exert antibacterial,
antifungal and antioxidant activities (4, 5).
Grapefruit seed extract (GSE) is a com-
mercial product derived from the seeds
and pulp of grapefruit (6, 7). The present
study contributes to the identification of
the antibacterial and antifungal effects of
the self – made aqueous and ethanolic ex-
tracts of GSs and to compare those effects
Antimicrobial Activity of Grapefruit Seeds
Extracts (In vitro Study)
ISSN: 1812–1217
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with an important orally used antiseptics
solution chlorhexidine gluconate (CHX) in
0.12% and 0.2% concentrations.
MATERIALS AND METHODS
1. Preparation seeds for extraction:
The seeds of the grape fruits were air-dried
for two weeks to prevent loss of active
components. Then were ground into pow-
der with electrical blender. Water and eth-
anol alcohol were used for the extraction
(8).
2. Preparation of aqueous extract:
Forty grams of dry seeds powder were
placed in 160ml of sterile distilled water
and left at room temperature for 24 hrs, the
mixture was filtered firstly by gauze and
secondly by filter paper (Whatman No. 1).
After filtration, it was placed into incuba-
tor at 37C°. The liquid was evaporated,
and the precipitated extract was left at the
base of the baker (9).
3. Sterilization of aqueous extract:
Five ml of distilled water was added to 1
gram of plant extract powder to produce
200mg/ml (20%w/v) solution, sterilization
was carried out by passing through a ster-
ile filter membrane 0.22µg (10).
4. Preparation of alcoholic extract:
Twenty grams of dry seeds powder was
added to 200ml of ethanol. The same pro-
cedure as in aqueous extract was ap-
pliedbut the solvent for extraction was
95% ethanol (9).
5. Sterilization of alcoholic extract:
Five ml of Dimethyl sulfoxide was added
to 1gm of seed extract powder and steri-
lized by pasteurization. The same concen-
tration of (20%w/v) is as aqueous extract
(10).
6. Sensitivity test (disc diffusion method):
Susceptibility testing was done on each
isolate (in triplicate) for each type of the
extract, CHX solution ( 0.12 and 0.2% )
were used as control using disc diffusion
method(11). Discs were prepared by adding
1ml of seed extract to 10 discs (12).
Four types of bacterial isolates were
used; Staphylococcus aureus, Proteus vul-
garis, Klebsiella pneumonia and a mixed
oral flora. The antifungal activity was test-
ed against one fungal isolates (Candida
albicans) by the disc diffusion method.
Colonies were streaked on Muller-
Hinton agar. Within 15 minutes after the
plates were inoculated, a prepared discs
were applied to the surface of the inoculat-
ed plates by sterile forceps, taking care
that each disc is 15mm apart from edge of
the plate. Then plates were placed in an
incubator at 37C for 24 hours for bacteria
and for 48 hours for Candida albicans(13).
After incubation, the plates were ex-
amined, the zone of inhibition of growth
were noted and measured. The antimicro-
bial activity of each extract was measured
from the diameters of zone of inhibition
for each organism and this was compared
with that of CHX solution (0.12% and
0.2%).
RESULTS
The antimicrobial effects of self made
20% (w/v) GSE (aqueous and ethanolic)
extracts and CHX solution (0.12% and
0.2%) are shown in Table (1).
The ethanolic extract showed no zone
of inhibition. The aqueous extract (20%)
was active against all gram negative bacte-
ria (Figure, 1). GSE produced the largest
zone of inhibition for normal flora (18mm)
(Table, 1), but exerted lower activity on
the growth of Staphylococcus aureus
(10mm). In comparison, CHX 0.2% (con-
trol I) showed the largest zone of inhibi-
tion against normal flora (20mm), while
exerted lower activity on the growth of S.
aureus, Proteus sp. and Klebsiella sp
(18mm). The zone of inhibition to CHX
0.12% (control II) is 7, 11, 18 and 22mm
for S. aureus, Proteus sp., Klebsiella sp,
and normal flora respectively. The aque-
ous extract (20%) was active against Can-
dida albicans with a zone of inhibition of
12mm in diameter (Table, 1).
Both CHX solutions (0.12% and
0.2% ) showed approximately the same
activity on the Candida albicans isolate
tested (13mm and 12mm respectively)
when compared with the aqueous GSE as
shown in Figure (1), Table (1).
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0
5
10
15
20
25
Inhibition zone ( mm )
Ca. Al. St.Au. Pr.sp. Kl.sp. Nor. Fl.
GSE (20%) CHX (0.2%) CHX (0.12%)
Figure (1): Comparison between the effect of 20% aqueous GSE and CHX solutions( 0.2% &
0.12%) on different types of microorganisms.
Table (1): Comparisons between the antimicrobial activity of the 20% aqueous GSE with
CHX solutions
Types of microorganism
Inhibition Zone ( mm )
GSE (20%)
CHX (0.2%)
CHX (0.12%)
Candida albicans
12
13
12
Staph. aureus
10
18
7
Proteus sp.
16
18
11
Klebsiella sp.
16
18
18
Normal flora
18
20
22
*Diameter of zone of inhibition in mm (Data represented as mean of three reading);**Disc diameter =
6mm
DISCUSSION
In this study, solutions of 20% con-
centrated GSE (aqueous and ethanolic)
were tested for antibacterial properties
against a number of gram negative and
gram positive organisms. The ethanolic
extract showed no activity against all test
microorganisms. This is in agreement with
Adedeji et al. where negative results were
also obtained. Kroum et al. found that
methanolic extract were more active than
ethanolic extract, but many studies showed
that ethanolic GSE possess good antibacte-
rial activities against many types of mi-
croorganisms (6, 15 & 16). The method of ex-
traction will influence the chemical com-
position, and thus, can have repercussion
regarding their biological properties (17).
Techniques of solubility of these substanc-
es are among other problems. Normaliza-
tion of methods and laboratory conditions,
in addition to the techniques used (extrac-
tion dissolution and dispersion, culture
medium) for testing biological and phar-
macological activities of solutions at the
definitive stage of laboratory screening are
desirable, in order to provide a common
basis for the comparison of results ob-
tained in various parts of the world in dif-
ferent organisms tested under similar la-
boratory conditions.
The aqueous GSE has consistently
good antimicrobial activity against all bio-
type tested. This is comparable to that of
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Antimicrobial effect of grapefruit seeds extracts

344
proven topical antibacterial solutions
(CHX). These results are also in agree-
ment with several scientific studies which
showed that GSE possess good antibacte-
rial activities against many types of mi-
croorganisms (5, 6 & 15). Chemical research
revealed the presence of flavonoids,
ascorbic acid, tocopherols, citric acid, li-
monoids, sterols and minerals in grapefruit
seeds and pulp extracts (4, 5 & 16). This bene-
ficial antibacterial effect of GSE has been
attributed to the antioxidant activity of
citrus flavonoids, such as naringenin and
hesperidine (4). The presences of those
compounds were confirmed by thin layer
chromatography (5). Heggers et al. with aid
of scanning transmission electron micros-
copy revealed GSE's antibacterial activity.
It was evident that GSE disrupts the bacte-
rial membrane and liberate the cytoplas-
mic contents with in 15 minutes after con-
tact even at more diluted concentration (7).
Fungi or yeast constitute a small pro-
portion of the usual oral microbiota with
Candida species being the most common
of the fungi present in healthy (30 – 45%)
individual. Aqueous CHX solution has a
wide spectrum of antimicrobial activity at
low concentrations and is especially effec-
tive against Candida albicans (11) and this
is in agreement of our result were both
CHX preparations had more or less same
antifungal activity and this was compara-
ble with that of our preparations.
Krajewska et al. found that 33%
(m/V) GSE concentration exert a potent
antifungal activity against the yeast like
fungi strains and had low activity against
germatophytes and moulds (18), while
Cvetnić and Vladimir – Kneźević re-
ported that GSE exhibit antifungal activity
against all tested strains of Candida albi-
cans in concentrations from 8.25% to
16.50% (m/V) (inhibition zones ranging
from 9mm to 11mm) (5) and this is in
agreement of our result.
The inhibitory action of these natural
products on mould could involve cyto-
plasmic membrane granulation, rupture
and inactivation and / or inhibition of in-
tercellular and extracellular enzymes. The-
se biological events could take place sepa-
rately or concomitantly culminating with
mycelium germination inhibition (19).
Brull and Coote reported that plant lytic
enzymes act on fungal cell wall causing
breakage of β-1,3 glycan, β-1,6 glycan and
chitin polymers which could be the cause
of death in those fungal strains (20).
Despite the fact that CHX were found
to be superior to the tested aqueous ex-
tract, the latter showed slight, but constant
activity against all the tested bacteria and
yeast. CONCLUSIONS
The use of 20% concentration of GSE
aqueous solution has effective anti bacteri-
al activities against Staphylococcus aure-
us, Proteus vulgaris, Klebsiella pneumo-
nia, Candida albicans and normal mouth
flora. The antibacterial activity of some of
that extracts was of such a level that it
would probably be therapeutically useful,
because its activity was comparable with
one of most widely used antiseptic solu-
tion.
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Antimicrobial effect of grapefruit seeds extracts