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Evalution of antibacterial and antioxidant activity of methanolic and hydromethanolic extract of sweet orange peels

Recent Research in Science and Technology 2011, 3(11): 22-25
ISSN: 2076-5061
Evalution of Antibacterial and Antioxidant Activity of Methanolic and
Hydromethanolic Extract of Sweet Orange Peels
Dayanand Dubey1*, K. Balamurugan2, R.C. Agrawal1, Rahul Verma1 and Rahi Jain1
1Jawaharlal Nehru Cancer Hospital & Research Centre, Idgah Hills, Bhopal, India.
2Annamalai University, Dept. of Pharmacy, Chidambaram, Tamilnadu, India
Article Info Abstract
Article History The in vitro antibacterial and antioxidant properties of hydromethanolic extract of peel from
Citrus sinensis (Sweet orange) was investigated. In this study the antibacterial activity of
Citrus sinensis peel extract against different gram positive and gram negative bacteria by
disc diffusion method and antioxidant activity by Fenton reaction was undertaken. Citrus
sinensis peels had shown mild activity against gram positive and gram negative bacteria and
the MIC value was measured. In another experiment antioxidant activity was observed and
IC50 was shown at 600 µg/ml.
Received : 20-04-2011
: 2
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*Corresponding Author
Tel : +91-8085605968
©ScholarJournals, SSR Key Words: Orange peel, Antioxidant activity, Antibacterial activity
Citrus is one of the most important commercial fruit crops
grown in all countries of the world [1]. Antioxidants are the
substances, compounds or nutrients in our foods which can
prevent or slow oxidative damage to our bodies. These agents
are able to remove the deleterious effects of free radicals
within our body. Nowadays, considerable interest is focused on
the development and evaluation of natural antioxidants and
radical scavengers from plant materials which are rich in
polyphenolic compounds. It is well known that plant derived
polyphenols have remarkable antioxidant and free radical
scavenging activity resulting in multiple beneficial nutritional
physiological effects in the humans. The essential oil obtained
from citrus fruits is having excellent antimicrobial properties
and is used in cosmetic industry [2]. An antimicrobial is a
substance that kills or inhibits the growth of microorganism
such as bacteria, fungi or protozoans. Antimicrobial drugs
either kill microbes (microbicidal) or prevent the growth of
microbes (microbistatic). Disinfectants are antimicrobial
substances used on nonliving objects. Citrus fruit products are
known to potent antimicrobial agents against bacteria and
fungus. These citrus fruits are rich source of flavonones and
many polymethoxylated flavones which are very rare in other
plants [3]. Antimicrobial and antioxidant properties of peel and
pulps of some citrus fruits were investigated [4]. These
compounds, not only play an important and physiological role,
but are also of commercial interest because of their multitude
of applications in the food and pharmaceutical industries
activities of the world.
Materials and Methods
Plant material
The fresh sweet orange fruits were collected from local
shops of Bhopal (M.P), India.
Preparation of Extract
The whole peels of sweet orange were fresh dried and
were mechanically crushed into powder and extracted with
distilled water at room temperature up to 48 h. The extract was
filtered and concentrated in water bath under reduced pressure
to obtain semisolid material which was then powdered. The
powdered material was soaked separately in methanol solvent
and hydro-methanol (50 % v/v) separately to obtain the
extracts [5].
Test organism
The following gram negative and gram positive bacteria
viz. Pseudomonas aeruginosa, Staphylococcus aureus,
Staphylococcus epidermidis, Shigella flexineri, Bacillus subtilis
and Escherichia coli were used for antibacterial activities which
were from stock culture of our laboratory.
Antibacterial activity
Antibacterial activities of 50 % methanolic, extract from
fruit of Orange peels were investigated using Disk diffusion
method [6]. 20ml of sterile Nutient agar was added in the test
tube after that petri plates were prepared and cultures were
swabbed on the top of the solidified media and allowed to dry.
The test was conducted at four different concentrations of the
crude extract 25 %, 50%, 75% and 100% (100µl per disk), the
disk which was soaked with extract were placed on the surface
of the medium the placed on the surface of the medium the
Dayanand Dubey et al. /Rec Res Sci Tech 3 (2011) 22-25
plate and were incubated for 12-18 h at 37ºC and the zone of
inhibition was recorded.
Antioxidant activity
The hydroxyl radical attached deoxyribose and initiated a
series of reaction that eventually resulted in the formation of
Thiobarbituric acid reaction substance (TBARs). The
measurement of TBARs gives an index of free radical
scavenging activity. the reaction mixture consisted of a
deoxiribose (3mM, 100µl), ferric chloride (Fe3+, 0.2 mM 50µl),
EDTA (0.1 mM 50 µl), ascorbic acid (0.1 mM 100 µl), stock
solution of orange peel extract 20 mg/ml were prepared from
which 200-2000 µl were added in reaction mixture, the final
volume was made up to 1ml by adding adequate quantity of
phosphate buffer saline (pH 7.4) and incubated for 1 h at 37°C.
The reaction was stopped by adding 0.5 ml of 5 % TCA and
0.5 ml of 1 % TBA the mixture was than incubated for 20 min in
a boiling water bath. The absorbance was measured at 532
nm. Ascorbic acid was used as positive control. The results are
expressed as the percentage inhibition of TBARS as reported
Results and Discussion
Antibacterial activity
Hydromethanolic extract of orange peel at the different
concentration i.e. 25%, 50%, 75%, 100% exhibits antibacterial
against Staphylococcus aureus (6-14mm), Bacillus subtilis (6-
9mm), Staphylococcus epidermis (5-10mm), E. coli (7-12 mm),
Shigella flexineri (9-12mm), Pseudomonas aeruginosa (6-9
mm). The results are recorded in Table 1 and Figure 1-6.
Antioxidant activity
The extract of orange peel showed good antioxidant
property in Fenton reaction model, the test drug was compared
with a low concentration of ascorbic acid. Table 2 showed the
percent TBARS inhibition of Hydromethanolic extract of orange
peel and Ascorbic acid. It is clear from the above data that the
Orange peels which are considered as waste material of the
fruit consist of good Antibacterial and Antioxidant activity.
Table 1: Antibacterial activity of orange peel extract against different bacterial strains
S.No Microorganism Concentration of Hydromethanolic
extract Zone of inhibition Concentration of Methanolic extract
Zone of inhibition
25% 50% 75% 100% 25% 50% 75% 100%
1. Staphylococcus aureus 8mm 12mm 14mm 14mm 9mm 13mm 16mm 16mm
2 Bacillus subtilis NA 8mm 9mm 9mm 6mm 8mm 9mm 10mm
3. Staphylococcus epidermis NA 7mm 8mm 10mm NA 10mm 10mm 13mm
4. E. coli 7mm 9mm 10mm 12mm 8mm 12mm 14mm 16mm
5. Shigella flexineri 6mm 9mm 10mm 12mm 8mm 9mm 10mm 14mm
6. Pseudomonas aeruginosa NA 6mm 8mm 9mm 7mm 8mm 9mm 13mm
Table 2: In vitro Antioxidant activity of Hydromethanolic Extract of orange peel
S. No. Concentration
(20mg in1ml ) % of
TBARS Concentration (20mg In 1ml ) Of Orange Peels Extract
Ascorbic acid Hydromethanolic % of TBARs Methanolic % of TBARs
1. 200 50.3 200 45.30 200 45.85
2. 400 51.6 400 45.85 400 46.96
3. 600 81.8 600 49.72 600 63.53
4. 800 85.2 800 55.24 800 113.25
5. 1000 95.3 1000 59.11 1000 74.58
6. 1200 69.7 1200 66.85 1200 82.87
7. 1400 122 1400 63.54 1400 85.63
8. 1600 110.7 1600 67.95 1600 109.39
9. 1800 139.5 1800 72.38 1800 110.01
10. 2000 142.2 2000 74.59 2000 151.9
Methanolic extract of Orange peels consist of good
antioxidant and antibacterial activity as compared to
Hydromethanolic extract. Among many other bacterial species,
S. aureus showed good antibacterial activity at 100 %
methanolic extract concentration. Both (Hydromethanolic and
Methanolic extracts) showed good antioxidant activity when
done by Fenton reaction. Orange peel showed IC50 at
600µg/ml and standard Ascorbic Acid showed IC50 at 200
µg/ml. The results of antimicrobial and antioxidant activity are
found to be correlated with some previous work [4].
Dayanand Dubey et al. /Rec Res Sci Tech 3 (2011) 22-25
Figure 1: Inhibition of Staphylococcus aureus in a different
concentration of Hydromethanolic and Methanolic extract of Orange
peel Extract
Figure 2: Inhibition of Bacillus subtilis in a different concentration of
Hydromethanolic and Methanolic extract of Orange peel Extract
Figure 3: Inhibition of Staphylococcus epidermis in a different
concentration of Hydromethanolic and Methanolic extract of Orange
peel Extract
Figure 4: Inhibition of E coli in a different concentration of
Hydromethanolic and Methanolic extract of Orange peel Extract.
Figure 5: Inhibition of Shigella flexineri in a different concentration of
Hydromethanolic or Methanolic extract of Orange peel Extract
Figure 6: Inhibition of Pseudomonas aeruginosa in a different
concentration of Hydromethanolic and Methanolic extract of Orange
peel Extract
From the present investigation it may be concluded that
hydromethanolic extract of sweet orange peel has potent
antibacterial activity against gram positive and gram negative
bacteria. Hydromethanolic extract of Citrus sinensis showed
good antioxidant activity and IC50 was found at 600 µg/ml
concentration. Further studies are however needed to isolate
the active molecule responsible for both antimicrobial and
antioxidant activity. The studies thus may lead to the
formulation of a potent antimicrobial and antioxidant agent.
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Mathur, V. Gupta, V.K. Dua and S. Singh. 2011.
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[5] Alade P.I. and O.N. Irobi. 1993. Antimicrobial activity of
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[7] Kamran G., G. Yousuf and M.A. Ebrahimzadehb. 2009.
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Faculty of Agriculture. 22 (3): 277-281.
... Table 2 shows the antibacterial activity of petroleum ether extract of citrus peel against gram-positive bacteria. For Bacillus cereus (gram-positive), the zone of inhibition (mm) of the test extracts So, Sat, Ja, Kh, Le, Ada, Ba, Chi, Ma and Ka were 20, 13, 16, 20, 10, 15, 10, 16, 11 and 11 mm respectively while for Streptococcus aureus, the values for the above mentioned extracts were 20,15,18,20,11,16,11,17,12 and 10 mm respectively. The zones of inhibition of standard kanamycin for the two organisms were noted as 22 and 17 mm respectively. ...
... Table 2 shows the antibacterial activity of petroleum ether extract of citrus peel against gram-positive bacteria. For Bacillus cereus (gram-positive), the zone of inhibition (mm) of the test extracts So, Sat, Ja, Kh, Le, Ada, Ba, Chi, Ma and Ka were 20, 13, 16, 20, 10, 15, 10, 16, 11 and 11 mm respectively while for Streptococcus aureus, the values for the above mentioned extracts were 20,15,18,20,11,16,11,17,12 and 10 mm respectively. The zones of inhibition of standard kanamycin for the two organisms were noted as 22 and 17 mm respectively. ...
... The zones of inhibition of the standard kanamycin for these two organisms were 18 and 20 mm respectively. For Shigella sonnei, the zones of inhibition (mm) of the test extract were 25,16,20,22,12,17,15,18,15,13 mm respectively while for the control, the value was recorded as 24 mm. The highest zone of inhibition (ZOI, 18 mm) for So (C. ...
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... Antibacterial properties have also been reported for orange peel [152]. Dubey et al. demonstrated the robust antibacterial properties of orange peel extracts against different bacteria using the disk diffusion method [153]. The effectiveness of orange peel extract against Klebsiella pneumoniae has been demonstrated by Jabuk et al. [154]. ...
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... Antibacterial effects of various citrus peels have been demonstrated in the literature (Lawal D et al., 2013, Ramachandra YL et al., 2013, Dhanavade MJ et al., 2011). Dubey D et al. (2011 showed potent antibacterial activity (against Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Shigella flexineri, Bacillus subtilis and Escherichia coli) of extract from fruit of Orange peels. Cuishnie TP and Lamb AJ concludes that the phenolic compounds in citrus peels are responsible for antimicrobial activi ...
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... As reported previously, Cit. sinensis essential oil showed a variable antimicrobial effect against microorganisms (Fisher and Phillips, 2006;Dubey et al., 2011). Singh et al. (2010) described antimicrobial action of citrus species including Cit. sinensis, and the principal components were limonene, limelool, and citral. ...
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... The present study also corroborated several earlier studies where citrus peels proved its bactericidal activities against Bacillus cereus [26] and other pathogenic organisms [27]. In concordance with earlier studies; the phytochemical analysis [qualitative and quantitative] of the components of the citrus [26,[28][29][30][31] ...
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Background At the present time skin problems need to address seriously, antibiotic resistance development is very fast in skin infectious bacteria. For the prevention of all types of skin infections, natural plant extracts can provide suitable defense line because they contain active compounds. Wrinkles, sunburn, itching, black heads, white heads and uneven tone of the skin are the collective issues related to skin. Extensive use of antibiotics for skin diseases is restricted due to the development of worse antibiotic resistance. Objective In this study the antibacterial potential of plant extracts was assessed against skin infectious bacteria to get alternative cure of skin diseases. Method Staphylococcus aureus, Staphylococcus epidermidis strains were isolated from clinical samples and E.coli was used as non-pathogenic control strain from Jinnah University for Women strain repository. Their antibacterial activity was performed against eight plant extracts through well diffusion method. Result Among eight plant extracts, Neem (Azadirachta indica), Turmeric (Curcuma longa), Sandalwood (Santalum album), Shahtara (Fumaria parviflora) Chirayata (Swertia chirayita) and commercially available mixed Citrus peel showed good inhibition of bacterial growth. Discussion Natural sources such as fruits, herbs, minerals, leaves, flowers, roots, honey and rose water are now becoming part of skin care and treatment products because they possess antimicrobial activities. Skin products with herbal ingredients are now evolving as suitable solutions to the existing skin problems.
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Citrus fruits are rich source of vitamin-C (Ascorbic acid). In the present study the antimicrobial and antioxidant activities of the peel and pulp of some of the citrus fruits has been investigated. The results indicate that the aqueous extracts from citrus fruit peel and pulp contains significant antimicrobial activity. In vitro antioxidant activity was determined by various procedures and it has been determined that aqueous and ethanolic fraction of peels and pulps of citrus fruits possessed maximum antioxidant activity in reference to standard antioxidant. Total polyphenolic content was found to be maximum in ethanolic extract of peels and chloroform extract of pulps, thus these have remarkable antioxidant property. The study thus revealed that peel and pulp of citrus fruits are useful for consumption and are beneficial for health. This study may thus lead to the formulation of an antimicrobial drug and can be used as a potent natural antioxidant additive or food products and as a dietary supplement.
Methanolic extracts from different parts (leaves, flowers, stem and pods) of Cassia surattensis and C. fistula were evaluated for potential antimicrobial activity against medically important bacterial, yeast and fungal strains. The antimicrobial activity was determined in methanol extracts using agar disc diffusion method. Methanol extracts of C. surattensis and C. fistula were effective on tested microorganisms. The methanolic extract with the greatest antimicrobial activity was that from stem of C. surattensis and C. fistula. Micrococcus sp, S. typhi, E. coli, A. lipoferum, K. pneumoniae, S. aureus, E. aerogenes, B. subtilis, A. niger and C. albicans were the most susceptible strains while the most resistant strain was S. cerevisiae. Hence, these plants can be used to discover bioactive natural products that may serve as leads in the development of new pharmaceuticals that address not fulfilled therapeutic needs.
This study examined the effect of volatile components of citrus fruit essential oils on P. digitatum and P. italicum growth. The hydrodistilled essential oils of orange (Citrus sinensis cvv. “Washington navel”, “Sanguinello”, “Tarocco”, “Moro”, “Valencia late”, and “Ovale”), bitter (sour) orange (C. aurantium), mandarin (C. deliciosa cv. “Avana”), grapefruit (C. paradisi cvv. “Marsh seedless” and “Red Blush”), citrange (C. sinensis x Poncirus trifoliata cvv. “Carrizo” and “Troyer”), and lemon (C. limon cv. “Femminello”, collected in three periods), were characterized by a combination of GC and GC/MS analyses. The antifungal efficacy of the oils was then examined at progressively reduced rates. Findings showed a positive correlation between monoterpenes other than limonene and sesquiterpene content of the oils and the pathogen fungi inhibition. The best results were shown by the citrange oils, whose chemical composition is reported for the first time, and lemon. Furthermore P. digitatum was found to be more sensitive to the inhibitory action of the oils.
The chemical composition of the essential oil obtained from the peel of Satsuma mandarin was analyzed by GC/MS analysis. As a result, 28 components were isolated. Among the 25 components identified, the monoterpenes hydrocarbons group was predominant, accounting for 86.62% (w/w) of the total oil. Of which, limonene was the most abundant (67.44%), followed by β-myrcene (7.15%), 3-carene (4.4%), α-pinene (2.52%), p-cymene (2.43%), β-pinene (1.46%), sabinene (0.77%), terpinolene (0.47%) and α-thujene (0.45%). Oxygenated monoterpenes (6.33%) and sesquiterpene (3.43%) were also present in the oil.
Water, ethanol, chloroform and hexane extracts of Acalypha wilkesiana leaves were investigated for in vitro antimicrobial activities by agar-diffusion and tube-dilution techniques. The water and ethanol extracts inhibited the growth of standard and local strains of bacteria and fungi including Staphylococcus aureus, Trichophyton rubrum, Trichophyton mentagrophytes, Candida albicans and Aspergillus flavus. The aqueous extract did not exert any inhibitory action on Klebsiella pneumoniae and Proteus mirabilis while the ethanol extract was active. The minimum inhibitory concentrations of the extracts ranged between 0.25 and 32 mg/ml, while the minimum cidal concentrations were between 1.0 and 64 mg/ml. The aqueous extract was found to be static in action while the ethanolic extract was uniformly cidal in effect.
Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Dept. of horticulture
  • G Kamran
  • G Yousuf
  • M A Ebrahimzadehb
Kamran G., G. Yousuf and M.A. Ebrahimzadehb. 2009. Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Dept. of horticulture, Faculty of Agriculture. 22 (3): 277-281.