Optimum Conditions for Extraction of Antibacterial Compounds

from Citrus Aurantifolia Fruit Peel Waste

Rima Munawaroh
Faculty of Pharmacy, Universitas Muhammadiyah Surakarta
Jl. A. Yani Tromol Pos I Pabelan Kartasura 57102
e-mail: rima.munawaroh@ums.ac.id

Abstract
Citrus fruit peel is a major waste in citrus fruit processing industry. The research on extraction active
compounds of Citrus aurantifolia (lime) fruit peel waste and antibacterial activity assay has been done. The
aim of research was to get optimum condition to extract their active compounds which have antibacterial
activity. The dried lime fruit peel was extracted by maceration method using ethanol 48%, 72%, and 96%.
The dried and fresh lime fruit peel were also extracted using ethyl acetate. Antibacterial assay was done by
diffusion agar against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. The result
showed that optimal condition to extract antibacterial compound using fresh sample with ethyl acetate as
solvent. The ethyl acetate extract of fresh sample was more active against S. aureus than E. coli.

Keywords: extraction, antibacterial, Citrus aurantifolia, fruit peel

Introduction different sugar groups attached to the C-7 than

Citrus aurantifolia (lime) cultivated hesperidin, have antibacterial activity against
throughout the world, especially in hot S. aureus and E. coli with MIC more than 1
subtropical or tropical area (Morton, 1987). mg/ml (Mandalari et al., 2007).
The main commercial products are lime juice Ethanol 56% is the best solvent for
and essential oils (Chamblee et al., 1997) but extracting total flavonoids from dried lime peel
the essential oil has economic value is much powder count as hesperidin (total flavonoid
higher than the juice (Bates et al., 2001). Pectin content 3.46%) (Mujahid, 2011). Ethanolic
from citrus fruit peel has also been produced extract 40%, 96% of whole dried fruit lime
and used as a gelling agent in food products has antibacterial activity against S. aureus
and stabilizer in the beverage product (May, with MIC 32-64 mg/ml and E. coli with MIC
1990). 64-256 mg/ml (Aibinu et al., 2007).
Citrus fruit peel is a major waste in juice The aim of research was to get optimum
and citrus essential oil industry because fruit condition to extract antibacterial compounds
peel consisting of flavedo, albedo, membranes, from lime fruit peel. The dried lime fruit peel
and pulp occupies between 50-65% of total was extracted by maceration method using
weight of citrus. If no further processing it will ethanol 48%, 72%, and 96%. The dried and
be waste and causes serious environmental fresh lime fruit peel were also extracted using
pollution. Therefore industry is interested in ethyl acetate. Antibacterial assay was done by
improving commercial value of this waste diffusion agar.
(Bocco et al., 1998).
In addition to essential oils and pectins, Research Methodology
citrus fruit peel also contains carotenoids, 1. Materials
coumarins, furanocoumarins, and typical Materials used are lime fruits were taken
citrus flavonoids (flavanone, flavanone from Temanggung, Central Java (harvested in
glycosides, polimethoxyflavone) (Agocs et January 2011); ethanol, ethyl acetate, distilled
al., 2007; Dugo et al., 1999; Li et al., 2006). water (technical); Escherichia coli ATCC
Lime peel (the flavedo and albedo) contains (American Type Culture Collection) 25922
the main flavonoid hesperidin (258 mg/100 dan Staphylococcus aureus ATCC 25923
g fresh fruit peel) (Nogata et al., 2006). (collection of Laboratory of Microbiology,
Neohesperidin, flavanones glycosides are just Faculty of Pharmacy UMS); BHI medium

34 PHARMACON, Volume 18, Juni 2017

(CONDA Pronadisa), MH Agar (Oxoid); Table 1. Lime fruit peel and solvent used in ex-
chloramphenicol 30 μg/disc, paper discs with traction
diameter of 6 mm (Oxoid), DMSO (dimethyl Dried peel Solvent
sulfoxide) (Merck); McFarland Equivalence 20 g Ethanol 48% 150 ml
Turbidity Standards 0.5 (Remel), 0.9% NaCl (ethanol 96%:aquadest = 1:1v/v)
(Merck). 20 g Etanol 72% 150 ml
(ethanol 96%:aquadest = 3:1v/v)
2. Equipments 20 g Ethanol 96% 150 ml
Equipments used are rotary evaporator 1122,3 g Ethanol 96% 12900 ml
(Heidolph efficient Laborota 4000), 300 g Ethyl acetate 3400 ml
waterbath (Memmert), analytical scales, Fresh peel Solvent
oven (Memmert), vortex, autoclave (Portable 590,26 g Ethyl acetate 2950 ml
Pressure Steam Sterilizer, China), oven
(Memmert), LAF (Laminar Air Flow), Diffusion test (Kirby Bauer disc
shaker incubator (Excella 24 New Brunswick diffusion).Preparation of S. aureus and E.coli
scientific), incubator, ruler. inoculum with growth method (Wanger,
2007): At least 3-5 well-isolated colonies with
3. Stages of Research the same morphological type were selected
Identification of citrus fruit. Identification from an overnight culture on MH agar plate.
of fruit was carried out in Pharmaceutical Top of each colony was touched with a sterile
Biology Division, Faculty of Pharmacy, loop and transferred into a tube containing 4-5
Gadjah Mada University, Yogyakarta based on ml of BHI medium and incubated in a shaker
morphologic characteristics in determination incubator at 37°C, 200 rpm until its turbidity
literature. The species of citrus fruit is Citrus the same as or exceeded turbidity of 0.5 Mc
aurantifolia (Christm. & Panz. Swingle), Farland (usually 2-3 hours) and then bacterial
familia Rutaceae. turbidity was adjusted to turbidity of 0.5 Mc
Preparation of fresh and dried lime fruit Farland standard using sterile 0.9% NaCl (1-2
peel. Lime peel fruit that were used include x 108 CFU/ml bacteria population).
flavedo, albedo, segment, and pulp or whole Inoculation on agar plate (Wanger,
fruit after juices and seeds were taken. Citrus 2007): Inoculum suspension was used within
fruit washed, cut, squeezed the juice by hand; 15 minutes after turbidity adjustment. Two
fresh peel dipped into the water one time, hundred micro liter of bacterial suspension
drained, cut into small pieces and blended. ] was inoculated on MH Agar plate by spreader
Fresh lime fruit peel dried in oven at 41-430C glass (thickness of Agar ± 4 mm).
until easily broken, blended, and sieved 10/40 Extract test solution made ​​with
mesh. concentrations of 50, 100, 150, 200, 400 mg/
Extraction of lime fruit peel. Dried fruit ml DMSO; loading test solution 10 μl/disc or
peel extracted with ethanol 48%, 72%, 96% equal to 500, 1000, 1500, 2000, 4000 μg/disc.
and ethyl acetate; fresh fruit peel extracted Discs application on inoculated agar
with ethyl acetate (table 1). These fruit peel plates (Wanger, 2007): Paper discs were
macerated for 24 hours 3 times (each time with impregnated with a series concentration of
new solvent) and stirred occasionally. Liquid test solutions (extract) and allowed to dry for
extract was filtered, collected, and evaporated ± 30 minutes and then each disc (including
with a vacuum rotary evaporator at 45-50°C positive control disc/antibiotic) was placed
and then continued by waterbath at 60°C until on inoculated plates. Plates were left for 15
a thick extract obtained. minutes so that substances in paper discs

PHARMACON, Volume 18, Juni 2017 35

diffuse into plates and then incubated in peel ethyl acetate extract, and fresh peel ethyl
inverted position (upside down) either acetate extract. Lime fruit peel extracts more
aerobically (37°C for 16-18 hours. Diameter actively inhibit S. aureus than E.coli.
of clear zone around discs was measured using
a ruler to the nearest millimeter. Table 2. Antibacterial activity of lime fruit peel
against S. aureus
Inhibition zone diameter (mm)*
Results and Discussions Loading Ethanolic
Fresh lime fruit peel occupies 52.47% of extract Ethyl acetate extract
extract
the total weight of lime fruit, when all the fruit (μg/disc) Fresh
peel is used after the juices and seeds taken. Dried peel Dried peel
peel
Lime fruit peel contains essential oils so the 500 6,0 ± 0,0 6,0 ± 0,0 6,2 ± 0,3
fruit peels are dried in oven at 43-45oC and 1000 6,0 ± 0,0 6,8 ± 0,3 7,0 ± 0,0
obtained 14.41% dried lime fruit peel powder. 2000 6,3 ± 0,6 7,8 ± 0,3 8,0 ± 0,0
Dried lime fruit reduced in size in order 4000 8,5 ± 0,5 12,5 ± 0,0 14,3 ± 0,3
to increase the surface area and facilitate Chloram-
25,3 ± 0,6 25,3 ± 0,6 25,3 ± 0,6
solvent into plant cells thereby enhancing phenicol
the extraction process. Maceration chosen DMSO 6,0 ± 0,0 6,0 ± 0,0 6,0 ± 0,0
as the method of extraction because of the * included disc diameter (6 mm)
cold extraction method, so that less stable Table 3. Antibacterial activity of lime fruit peel
compounds in heating is no damage relatively. against E. coli
Saturation of the solvent avoided by stirring Inhibition zone diameter (mm)*
and remaceration with new solvent, so the Loading Ethanolic
active compound more extracted. Liquid Ethyl acetate extract
extract extract
extract is evaporated at temperature less than (μg/disc)
Dried peel Dried peel Fresh peel
or equal to 600C so thermolabile compounds
are not degraded. Yield of dried lime fruit 500 6,0 ± 0,0 6,0 ± 0,0 6,0 ± 0,0
peel ethanolic extract with ethanol 48%, 1000 6,0 ± 0,0 6,0 ± 0,0 6,8 ± 0,3
72%, and 96% are 25.10%, 24.10%, 20.60% 2000 6,5 ± 0,5 6,8 ± 0,8 7,7 ± 0,6
respectively. 4000 6,8 ± 0,8 8,2 ± 0,3 9,5 ± 0,5
Dried lime fruit peel ethanolic extract Chloram-
26,0 ± 0,0 25,3 ± 0,6 25,3 ± 0,6
48%, 72%, 96% dried on loading 500, 1000, phenicol
1500 μg/disc does not inhibit S.aureus. DMSO 6,0 ± 0,0 6,0 ± 0,0 6,0 ± 0,0
Improved loading of ethanolic extract 96% to * included disc diameter (6 mm)
2000 and 4000 μg/disc inhibit S.aureus with
inhibition zone diameter 6.3 mm and 8.5 mm These results are similar to results
respectively. Ethanolic extract 96% inhibit Chanthaphon et al. (2008) which states that
S.aureus in a large loading (4000 μg/disc), the ethyl acetate extract of fresh lime fruit peel
therefore we try using the ethyl acetate extract more active against Gram-positive bacteria
of dried and fresh lime fruit peel, because of than Gram-negative bacteria; antibacterial
essential oil and less polar compounds in fruit activity of ethyl acetate extract of fresh lime
peel.Yield of dried and fresh lime fruit peel fruit peel to S.aureus larger than dried peel
ethyl acetate extract are 8.11% and 1.84% extract. This indicates the loss of certain
respectively. antibacterial compounds during the drying
Table 2 and 3 shows that the three lime process, especially volatile compounds.
peel extract has antibacterial activity against S. Aibinu et al. (2007) have proven that lime
aureus and E. coli, the greater loading extracts fruit peel essential oil also has antibacterial
per disc, the greater diameter inhibition zones. activity.
Antibacterial activity from the lowest to the Ethyl acetate extract more active
highest is dried peel ethanolic extract, dried antibacterial than ethanolic extract because of

36 PHARMACON, Volume 18, Juni 2017

less polar compound, such as coumarins and lipopolysaccharide that limiting diffusion of
furanocoumarins which have antibacterial hydrophobic compounds so E. coli are less
activity. Meranzin hydrate (coumarin) and sensitive to plants antibacterial (Tajkarimi et
oxypeucedanin hydrate (furanocoumarin) al., 2010).
were isolated from ethyl acetate extract
of flavedo Citrus grandis Osbeck. fruit Conclusion
from Japan has antibacterial activity against The optimal condition to extract
S.aureus with MIC 0.75 mg/ml and 0.24 mg/ antibacterial compounds from lime fruit peel
ml (Mokbel et al., 2006). was using fresh peel with ethyl acetate as
Extracts are more active inhibit S.aureus solvent. Ethyl acetate extract of fresh peel was
than E. coli, possibly because E.coli (Gram more active against S.aureus than E. coli.
negative bacteria) have the outer membrane

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