See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/257932280

In vitro assessment of antioxidant, antibacterial and phytochemical analysis of

peel of Citrus sinensis

Article  in  Pakistan Journal of Pharmaceutical Sciences · January 2015

CITATIONS READS

17 2,625

7 authors, including:

Shaukat Ali Uzma Azeem Awan

Government College University, Lahore National University of Medical Sciences (NUMS), Rawalpindi, Pakistan
124 PUBLICATIONS   883 CITATIONS    15 PUBLICATIONS   74 CITATIONS   

SEE PROFILE SEE PROFILE

Tahseen Ghous Dr. Saiqa Andleeb

University of Azad Jammu and Kashmir University of Azad Jammu and Kashmir
13 PUBLICATIONS   184 CITATIONS    74 PUBLICATIONS   317 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Nano drug delivery systems View project

NA interference; A tool for treatment of human diseases View project

All content following this page was uploaded by Dr. Saiqa Andleeb on 05 November 2014.

The user has requested enhancement of the downloaded file.

SHORT COMMUNICATION

In vitro assessment of antioxidant, antibacterial and phytochemical

analysis of peel of Citrus sinensis

Basharat Mehmood1#, Kamran Khurshid Dar1#, Shaukat Ali1, Uzma Azeem Awan1,
Abdul Qayyum Nayyer1, Tahseen Ghous2 and Saiqa Andleeb1*
1
Microbial Biotechnology Laboratory, Department of Zoology, University of Azad Jammu and Kashmir, Muzzafarabad, Pakistan
2
Biochemistry Laboratory, Department of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan

Abstract: Antibacterial effect of Citrus sinensis peel extracts was evaluated against several pathogenic bacteria
associated with human and fish infections viz., Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia,
Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, Serratia marcesnces, Shigella flexneri,
Enterobacter amnigenus, Salmonella Typhimurium and Serratia odorifera. Methanol, ethanol, chloroform and diethyl
ether solvents were used for extraction. In vitro antibacterial activity was analyzed by agar well and agar disc diffusion
methods. It was found that ethanol extract showed highly significant inhibition of E. coli and K. pneumonia (12.6±0.94
mm and 11.6±1.2 mm) whereas methanol extract of C. sinensis also showed high zone of inhibition of S. odorifera
(10.0±2.16 mm). The potential activity of active extracts was assessed and also compared with standard antibiotics
through activity index formulation. The order of antioxidant activity through ABTS·+ and DPPH free radical scavenging
activity was ethanol>methanol>chloroform>diethyl ether. Phytochemical screening of all solvents had determined the
presence of terpenoids, alkaloids, steroids, glycosides and flavonoids. It was also found that Chloroform/Methanol (5:5)
and Butanol/Ethanol/Water (4:1:2.2) solvent systems showed significant separation of active phytochemical constituents.
These findings reveal the potential use of C. sinensis peel to treat infectious diseases, which are being caused by
microorganisms.

Keywords: Antibacterial, Antioxidant, Agar disc diffusion method, Bacterial pathogens, Citrus sinensis

INTRODUCTION bio-ethanol production (Kim et al., 2008; Kim et al.,

2007; Sharma et al., 2007). Likewise, essential oil of
Infectious diseases are serious health problem worldwide. citrus peel has been identified to exhibit antibacterial
The use of commercial anti-microbial drugs subjectively activity (Upadhyay et al., 2010; Palakawong et al., 2010;
in the treatment of many infectious diseases is being led Ayoola et al., 2008). Due to high anti-microbial effect it
to multiple drug resistance phenomenons in human is abundantly used in pharmaceutical products (Njoroge et
pathogenic microorganisms (Das et al., 2010; Sokmen et al., 2005). It has also been used as an anti-diabetic,
al., 2004). Because this problem is prevailing at very high larvicidal, anti-microbial, anti-fungal, hypotensive agent,
magnitude there is an unmet need to find and develop antioxidant, antiviral, insect repellent, antibacterial, anti-
alternative eco-friendly methods to treat bacterial or other mutagenic and anti-yeast agent (Ghasemi et al., 2009;
infectious pathogens without or with minimal toxicity. In Kanaze et al., 2008; Hamendra and Anand 2007;
the last few years, researchers had conducted studies on Proteggente et al., 2003; Caccioni et al., 1998; Han, 1998;
the use of medicinal plants as anti-microbial agents Stange et al. 1993; Kumamoto et al., 1986).
having bioactive compounds (Thenmozhi and Rajeshwari,
2010; Duraipandiyan et al., 2006). Bacterial food poisoning is the most common problem
(Soković et al., 2007). Free radicals formed in human
Citrus is one of the most essential commercial fruit crops metabolism by two ways: 1) naturally released by human
which are grown in all continents of the world (Tao et al., body to deactivate the viral and bacterial presence, or 2)
2008). In Pakistan it is cultivated on an area of due to environmental factors like pollution, smokes, and
approx.197, 000 ha with annual production of approx. others. According to Mantena et al. (2008) radical chain
1816, 000 tones (Anonymous, 2004). Pakistan is one of reactions with DNA, proteins and cell membrane cause
the largest citrus producing countries of the world. Citrus harmful effects to human body. On the other hand,
peel is used as fodder at fisheries, raw material for antioxidants, enzymes and vitamins are naturally
traditional paper, activated carbon, cosmetic products and available anti-free radical defense system that is used to
#
*Corresponding author: e-mail: drsaiqa@gmail.com Authors with equal contribution
Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239 231
Assessment of antioxidant, antibacterial and phytochemical analysis of peel of Citrus sinensis

prevent oxidative damage and also to protect the body formed in well diffusion method. The Petri dishes were
from harmful pathogens (Nabavi et al., 2013). Hence, the incubated at 37°C for 48 h. Discs, soaked with
current investigation was aimed to determine the chloroform, ethanol, methanol, and diethyl ether were
antibacterial and antioxidant activities of various extracts used as control. Microbial growth inhibition was assessed
of Citrus sinensis against pathogenic bacterial strains. by calculated the zone of inhibited diameter (mm) after
48h of incubation. Before each experiment, the optimal
MATERIALS AND METHODS density (OD) of bacterial growth 107 colony forming units
(cfu)/ml was measured through spectrophotometer at the
Plant material wavelength 600 nm (Seeley et al., 2001). Each
Citrus sinensis were purchased from the supermarket of experiment was performed in triplicate. The results of
Muzaffarabad and brought to Microbial Biotechnology sensitivity tests were denoted as (0) for no sensitivity, (1-
lab, Department of Zoology, University of Azad Jammu 3) for low sensitivity, (4-8) for moderate sensitivity and (9
and Kashmir (UAJ & K), Muzaffarabad, Pakistan. The or bigger than 9) for high sensitivity. The antibacterial
fruits were rinsed with running tap water and dried with activity of extracts with standard antibiotics was also
tissue papers. assessed by (AI) activity index (Shekhawat and
Vijayvergia, 2010).
Preparation of powder
The peel of ripened fruit was removed with sterilized Sensitivity test of antibiotics
knife and placed on white papers under shade for drying Sensitivity of antibiotics including amoxicillin (10 µg),
for 2 weeks. Dried peel was crushed to make fine powder. penicillin G (10µg), ampicillin (10µg) and trimethobrim
The powder was stored at room or at normal refrigerator (10µg) against all tested bacterial strains was assessed by
temperature before analysis. agar disc diffusion method (Baker and Pallister, 1998;
Perez et al., 1990).
Preparation of extracts
Powder (20g) was homogenized with various solvents Preliminary Phytochemical screening
viz., ethanol, methanol, chloroform, and diethyl ether The phytochemical analyses of four extracts were carried
according to high polarity for two weeks and used as out according to the methods described by Harborne,
100% concentrated extract against tested pathogens. The 1993; Sofowora, 1986; Trease and Evans, 1983.
homogenized mixtures in different solvents were Total Phenolic content
collected, filtered and evaluated for antibacterial and Phenolic contents (mg/100ml of extracts) were
antioxidant activities, and phytochemical screening. determined using the Folin-Ciocalteu reagent method
described by Zhou and Yu with slight modifications
Isolation and culturing of bacterial pathogens (Zhou and Yu, 2006). The reaction mixture was made
Bacterial pathogens such as Shigella flexneri, Serratia with extract (100µl), Folin-Ciocalteu reagent (100µl) and
odorifera, Salmonella Typhimurium and Enterobacter 20% sodium carbonate (3 ml). Reaction mixture was
amnigenus (isolated from spoiled fish), Escherichia coli, incubated at room temperature for 1h and the absorbance
Pseudomonas aeruginosa, Klebsiella pneumonia, of deep blue complex was measured at 765 nm. Gallic
Staphylococcus aureus, Streptococcus pyogenes, acid was used as a standard with varied concentration
Staphylococcus epidermidis and Serratia marcesnces from 200ppm to 1000ppm. The total phenolic content was
(P236) were isolated from human infected samples like expressed as mg gallic acid equivalents per gram extract
blood, urine and pus at Microbial Biotechnology lab, weight (mg/100gm).
Department of Zoology, UAJ&K, and their identification
was carried out at Combined Military Hospital (CMH), Total flavonoid content
Muzaffarabad, Pakistan. Estimation of total flavonoid contents of extracts was
quantified by the method illustrated by Zou et al., (2004).
Antibacterial activity of extracts The reaction mixture cocktail containing extracts (500
Agar disc and agar well diffusion methods were used to µl), distilled water (2ml), and 5% NaNO2 (0.15ml) was
determine the antibacterial activity of various extracts of incubated at room temperature for 6 min. After
C. sinensis peel as described by (Kumar et al., 2011; incubation, 10% AlCl3 (0.15ml) solution was added, the
Nwanebu et al., 2011). Nutrient agar and Nutrient Broth kept for 6 min at room temperature, followed by the
Media (NAM; Oxide CMOO3 and NBM; CM1) were addition of 2ml of 4% NaOH solution. After the addition
used for bacterial culture. The overnight culture was of water to the sample to make the final volume to 10 ml,
mixed with freshly prepared nutrient agar medium the mixture was mixed immediately and kept for 15 min
(NAM) at 45°C and was poured into the sterilized Petri further. The absorbance of the reaction mixture was
dishes. All Petri dishes were kept at room temperature in measured at 510 nm. Rutin was used as standard for the
laminar flow for solidification. The 5 mm discs were calibration curve. Total flavonoid content of extracts and
soaked with various prepared peel extracts and placed on fractions was expressed as mg rutin equivalents (RE) per
agar surface in case of disc diffusion whereas wells were gram of sample (mg/g).
232 Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239
 Saiqa Andleeb et al

Table 1: Antibacterial activity of extracts of Citrus sinensis peel

Mean±Standard deviation of extracts

Pathogens
Diethyl ether (mm) Chloroform (mm) Ethanol (mm) Methanol (mm)
Pseudomonas aeruginosa 3.6±0.46* 4±0.81** 4.33±1.24** 5.33±2.05**
Klebsiella pneumoniae 6.3±0.47** 4.6±0.94** 11.6±1.25*** 9.3±1.24***
Serratia marcesnces 3.6±0.47* 4±0.81** 4.6±1.24** 4.6±1.7**
Staphylococcus epidermidis 2.6±0.94* 4.6±0.47** 6±2.14** 5.6±0.94**
Streptococcus Pyogenes 4.6±1.7** 4.6±0.47** 5.8±0.47** 4.3±0.47**
Escherichia coli 4±0.81** 4±1.84** 12.6±0.94*** 9±0.81***
Staphylococcus aureus 5.6±0.47** 4.6±0.47** 7.6±0.47** 6±0.81**
Salmonella Typhimurium 3.3±0.47* 5.66±1.24** 4.33±0.94** 6.0±0.81**
Shigella flexneri 2.6±0.94* 1.6±0.47* 7±2.16** 6.3±2.49**
Enterobacter amnigenus 5.3±0.47** 1.3±0.47* 4.3±0.47** 4.3±1.24**
Serratia odorifera 2.6±0.47* 3.0±0.81* 3.3±0.47* 10.0±2.16***
The results of the sensitivity tests were expressed as (0) for no sensitivity, (below 4; *) for low sensitivity, (4-8; **) for
moderate sensitivity and (9-14; ***) for high sensitivity.

Thin layer chromatography percentage scavenging activity was calculated by formula:

The determination of major phytochemicals was further %=[(Ao-Ai)/Ao]*100; where Ao is the absorbance of
confirmed by thin layer chromatography (TLC) using pre- control and Ai is the absorbance with extracts.
coated Silica gel 60F264 plates (Wagner and Bladt,
2004). In order to get better resolution of components STATISTICAL ANALYSIS
different screening systems were used (Table 4). The
developed plates were observed under UV light (254-336 Each experiment was repeated in triplicates and mean ±
nm). Retention factor (Rf) value of each spot was standard deviation (M±SD) from absolute data were
calculated as Rf = distance travelled by the solute/ measured through on line Standard deviation calculator
distance travelled by the solvent. http://easycalculation.com/statistics/standard-deviation.
php. The percentage of bacterial growth was analyzed
ABTS.+ free radical scavenging activity statistically with One-way analysis of variance (ANOVA)
ABTS·+ or 2,2'-azino-bis(3-ethylbenzothiazoline-6- through http://www.danielsoper.com/statcalc3/calc.aspx?
sulphonic acid) free radical scavenging activity was id=43 to distinguish the treatments means (Gomez and
carried out to evaluate the antioxidant potential of C. Gomez, 2007).
sinensis extracts according to the method described by Re
et al. (1999). The ABTS stock solution was prepared by RESULTS
reacting potassium persulphate (2.5mM) and ABTS
(7mM), then the mixture was kept for at least 16 h to Since decades, herbs, trees and shrubs have used/applied
generate ABTS+ free radicals and their absorbance were by humans in many ways such as drugs, foods and
recorded at 734 nm (AoControl). For tests, 1ml of ABTS flavors. Our research is therefore aimed for the
running solution was mixed with 20-200 µg/ml of verification of the antibacterial activity of C. sinensis peel
different compounds. The absorbance of test samples extracts against some food borne organisms such as E.
(AiSample) was also observed at 734 nm. The percentage coli, P. aeruginosa, K. pneumonia, S. aureus, S.
radical scavenging activity (% RSC) was calculated using Pyogenes, S. epidermidis, S. marcesnces (P236),
the formula: %RSC=[(AoControl−AiSample)/ AoControl]×100%. S. Typhimurium (KL2), S. flexneri (KL2), E. amnigenus
(F1), and S. odorifera (F). This in vitro study was done to
DPPH Free Radical Scavenging Activity provide a direction for the awareness of C. sinensis
DPPH [(di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium] extracts to the population who utilize them to treat various
free radical scavenging activity of C. sinensis extracts was diseases caused by these bacterial pathogens. These
determined with slight modifications (You et al., 2006). pathogens were isolated and identified by the researchers
Half ml of MeOH (0.1mM) solution of DPPH was mixed of Microbial Biotechnology Laboratory, Department of
with 20-150µl of extracts, mixed with vortex vigorously Zoology, Muzaffarabad, Pakistan (Ume-Kalsoom et al.,
and left for 30 min at 37°C in an incubator The volume 2013; Andleeb et al., 2013; Rafique et al., 2012). The
was made up to 2ml by adding methanol. Methanol used results of the sensitivity tests were expressed as (0) for no
as a baseline control. The absorbance was recorded at sensitivity, (below 4*) for low sensitivity, (4-8**) for
517nm. Water (20-150 µl) was used as a control and the moderate sensitivity and (9-14***) for high sensitivity.
Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239 233
Assessment of antioxidant, antibacterial and phytochemical analysis of peel of Citrus sinensis

Table 2: Activity index analysis against human pathogenic bacteria

Effect of Citrus sinensis on human pathogens through activity index
Solvents
Pathogens Antibiotics
Diethyl ether Chloroform Ethanol Methanol
0.6 0.66 0.72 O.88 Amoxillin (6)
0.6 0.66 0.72 0.88 Penicillin G (6)
Pseudomonas aeruginosa
0.17 0.19 0.20 0.25 Trimethobrim (21)
E>A E>A E>A E>A Ampicillin (0)
1.05 0.76 1.9 1.55 Amoxillin (6)
E>A E>A E>A E>A Penicillin G (0)
Klebsiella pneumonia
0.9 0.65 1.65 1.32 Trimethobrim (7)
1.05 0.76 1.93 1.55 Ampicillin (6)
0.6 0.66 0.76 0.76 Amoxillin (6)
0.45 0.5 0.57 0.57 Penicillin G (8)
Serratia marcescens
0.45 0.5 0.57 0.57 Trimethobrim (8)
0.45 0.5 0.57 0.57 Ampicillin (8)
E>A E>A E>A E>A Amoxillin (2)
E>A E>A E>A E>A Penicillin G (0)
Staphylococcus epidermidis
0.07 0.13 0.18 0.16 Trimethobrim (33)
E>A E>A E>A E>A Ampicillin (0)
E>A E>A E>A E>A Amoxillin (0)
E>A E>A E>A E>A Penicillin G (0)
Streptococcus Pyogenes
E>A E>A E>A E>A Trimethobrim (0)
E>A E>A E>A E>A Ampicillin (0)
E>A E>A E>A E>A Amoxillin(0)
E>A E>A E>A E>A Penicillin G (0)
Escherichia coli
2.0 2.0 6.3 4.5 Trimethobrim (2)
E>A E>A E>A E>A Ampicillin (0)
E>A E>A E>A E>A Amoxillin (0)
E>A E>A E>A E>A Penicillin G (0)
Staphylococcus aureus
0.25 0.20 0.34 0.27 Trimethobrim (22)
0.8 0.65 1.08 0.85 Ampicillin (7)
E>A E>A E>A E>A Amoxillin (0)
0.36 0.62 0.48 0.66 Penicillin G (9)
Salmonella Typhimurium
0.47 0.80 0.61 O.85 Trimethobrim (7)
0.47 0.80 0.61 0.85 Ampicillin (7)
E>A E>A E>A E>A Amoxillin (0)
E>A E>A E>A E>A Penicillin G (0)
Shigella flexneri
E>A E>A E>A E>A Trimethobrim (0)
E>A E>A E>A E>A Ampicillin (0)
E>A E>A E>A E>A Amoxillin (0)
E>A E>A E>A E>A Penicillin G (0)
Enterobacter amnigenus
0.24 0.12 0.39 0.24 Trimethobrim (11)
E>A E>A E>A E>A Ampicillin (0)
E>A E>A E>A E>A Amoxillin (0)
0.65 0.75 0.82 2.5 Penicillin G (4)
Serratia odorifera
0.32 0.37 0.41 1.25 Trimethobrim (8)
E>A E>A E>A E>A Ampicillin (0)
E > A and > 1 values indicate extracts have higher effect against bacterial pathogens compared to antibiotics; less than 1 values
indicate antibiotics have higher effect against bacterial pathogens compared to extracts.

Antibacterial activity of extracts solvents of different polarity were employed in research

The conventional solvent extraction of the citrus peel work as polar: ethanol and methanol; and non-polar:
using different solvents had yielded different results in chloroform and diethyl ether. The antibacterial activity of
each of the experiment conducted in this study. Hence a C. sinensis extracts was assayed in vitro by agar disc
234 Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239
 Saiqa Andleeb et al

Table 3: Phytochemical screening of extracts of Citrus sinensis peel

Solvents
Phytochemicals
Ethanol Methanol Diethyl ether Chloroform
Saponins + + - -
Tannins + + - -
Terpenoids + + + +
Alkaloids + + + +
Steroids + + + +
Cardiac glycoisdes + + + +
Flavonoids + + + +
+ indicates presence, - indicates absence

Table 4: Thin Layer Chromatography of C. sinensis peel extracts

Used Solvent systems
Extracts used CM (5:5) BAW (4:1:5) BEW (4:1:2. 2) BAEW (9:6:5:1)
Rf value Spot color Rf value Spot color Rf value Spot color Rf value Spot color
S1= 0.3 Yellow S1= 0.4 Yellow
Diethyl Ether S= 0.8 Yellow No result
S2=0.7 Purple S2= 0.7 Purple
S1= 0.2 Blue S1= 0.7 Purple
Chloroform S= 0.3 Blue No result
S2= 0.5 Yellow S2= 0.8 Blue
S1= 0.2 Yellow
Ethanol S= 0.3 Yellow S= 0.3 Pink No result
S2= 0.4 Purple
S1= 0.5 Yellow
S1= 0.7 Yellow
Methanol S2= 0.5 Purple S= 0.4 Pink 0.6 Purple
S2= 0.4 Pink
S3= 0.9 Purple
Solvents: CM= Chloroform/ Methanol (5:5), BAW = Butanol / Acetic acid / Water (4:1: 5),
BEW = Butanol /Ethanol / Water (4:1:2. 2), BAEW = Butanol / Acetic acid / Ether / Water (9:6:5:1).

Fig. 1: Anti-bacterial activity of extracts of C. sinensis peel against food borne pathogens through agar disc diffusion
method. Salmonella typhimurium (KL1), Shigella flexneri (KL2), Enterobacter amnigenus (F1), Serratia odorifera (F),
Pseudomonas aeruginosa (Psuedo), Staphylococcus aureus (U272), Streptococcus Pyogenes (Strep), Escherichia coli,
Staphylococcus epidermidis (Staphlo) and Seratia marcesnces (P236)
and well diffusion against food-borne bacterial pathogens mm) and E. coli (12.6±0.94 mm, 9±0.81 mm; table 1).
(fig. 1). Table 1 summarizes the microbial growth Methanolic extract of C. sinensis also showed significant
inhibition of all extracts of citrus peel. There was no activity against S. odorifera (10.0±2.16 mm) whereas
inhibition zone formed in control viz., ethanol, methanol, ethanol extract had low effect (3.3±0.47 mm) (table 1).
chloroform, and diethyl ether. Among the solvent Chloroform extracts exhibited reasonable bioactivity
extracts, ethanol and methanol extracts exhibited the between 4-8 mm against all tested bacterial pathogens
highest antibacterial activity. These extracts were except S. flexneri, E. amnigenus and S. odorifera (table
effective against K. pneumoniae (11.6±1.25 mm, 9.3±1.24 1).
Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239 235
Assessment of antioxidant, antibacterial and phytochemical analysis of peel of Citrus sinensis

Fig. 2: Scavenging activities of peel extracts of Citrus sinensis towards ABTS.+ and DPPH free radicals.

Fig. 3: Gallic acid equivalent phenolic contents in mg/100 ml of Citrus sinensis extracts absorbance recorded at 765
nm. A) Gallic acid equivalent phenolic contents of C. sinensis extracts. B) Calibration Curve using gallic acid as a
standard. O. Ether (extract of C. sinensis in diethyl ether); O. Chl. (extract of C. sinensis in chloroform); O.E. (extract
of C. sinensis in ethanol); O.M. (extract of C. sinensis in methanol).

Activity index (AI) of extracts BAEW system was not favorable for this separation as
Four antibiotics were used against all tested bacterial only methanolic extract of orange had showed separation
pathogens. The maximum inhibition was recorded by in this system (table 4).
trimethobrim against P. aeruginosa, S. epididermis, S.
aureus, and E. amnigenus. On the other hand, ampicillin, Antioxidant activity of extracts
amoxillin, and penicillin G showed both moderate and The antioxidant activity of methanol and ethanol extracts
low effects against all tested pathogens as mentioned in of C. sinensis peel showed a significant free radical
table 2. The sensitivity of antibiotics was compared with scavenging activity generated by ABTS (55.8% and
different C. sinensis extracts through activity index (table 60.7%) as compared to other solvent extracts i.e
2). Results confirmed the potential use of all extracts as chloroform and diethyl ether (fig. 2). Similar results were
compared to antibiotics except trimethobrim. also found by DPPH assay. The methanolic and ethanolic
extracts of C. sinensis peel showed 70 to 80% DPPH
Phytochemical screening of extracts scavenging activity based on its capability as hydrogen
Phytochemical screening indicated the presence of donator (fig. 2).
terpenoids, alkaloids, steroids, glycosides and flavonoids
in all solvents (table 3). On the other hand, saponins and Total phenolic contents
tannins were present in polar solvents as compared to Phenolic antioxidants are included in the category of free
non-polar. The results of thin layer chromatography of radical terminators. Natural antioxidants are primarily
various C. sinensis peel extracts showed a common plant phenolics and polyphenolic compounds that may
occurrence of different phytochemicals when separated present in different parts of plants. Current research
using four solvent systems such as Chloroform/ Methanol determined the concentration of phenolic contents in C.
(CM: 5:5), Butanol / Acetic acid / Water (4:1: 5), Butanol sinesis extracts as these contents are higher in methanol
/Ethanol / Water (BEW: 4:1:2.2), and Butanol / Acetic extracts, followed by ethanol extracts, moderate in ether
acid / Ether / Water (BAEW: 9:6:5:1). CM and BEW extracts and very low in chloroform extracts (fig. 3A).
systems showed significantly high separation followed by Calibration Curve using gallic acid as a standard is also
BAW system which revealed moderate separation. shown in fig. 3B.
236 Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239
 Saiqa Andleeb et al

Fig. 4: Rutin equivalent flavonoid contents in mg/100 ml of Citrus sinensis extracts absorbance recorded at 510 nm. A)
Rutin equivalent flavonoid contents in mg/100 ml of C. sinensis extracts. B) Calibration Curve using rutin as a
standard. O. Ether (extract of C. sinensis in diethyl ether); O. Chl. (extract of C. sinensis in chloroform); O.E. (extract
of C. sinensis in ethanol); O.M. (extract of C. sinensis in methanol)

Total flavonoid content Alterable antibacterial activity of different extracts of

A reasonable quantity of flavonoids was found in all Citrus was in concurrence with previous studies
tested C. sinensis extracts (fig. 4A). The presence of (Palakawong et al., 2010; Pandey et al., 2010;
flavonoids was gradually increased as moving towards Nannapanenl et al., 2008; Soković et al., 2007). The
more polar solvent extracts as compared to non-polar. difference in anti-bacterial activity could be due to
Methanolic extracts expressed higher flavonoid contents variation in chemical compositions and their penetration
than rest of all tested extracts (fig. 4A). The calibration through the cell wall and cell membrane of bacterial
Curve using rutin as a standard was also shown in fig. 4B. pathogens (Palakawong et al., 2010; Soković et al.,
2007). In previous literatures, it has been demonstrated
DISCUSSION that citrus plants have been used as an antidiabetic
(Hamendra and Anand (2007), antioxidant (Kanaze et al.,
Antibacterial activity 2008; Proteggente et al., 2003), insect repellent,
In current research the extracts of C. sinensis were antibacterial, antiviral, antiyeast, and anti-mutagenic
prepared through conventional solvent extraction method agent (Han, 1998).
using both polar: ethanol and methanol; and non-polar:
chloroform and diethyl ether solvents which revealed the Phytochemical screening
significant results. These extracts showed considerable The most important bioactive compounds of plants are
antibacterial activity against tested pathogens. Similar alkaloids, flavonoids, tannins and phenolic compounds
results were obtained by Ekwenye and Edeha, (2010) (Duraipandiyan et al., 2006; Edeoga et al., 2005). Our
against K. pneumoniae, E. coli, and S. aureus. On the results were consistent with Ahmad et al. (2006) that the
other hand, moderate bacterial growth inhibition was peel of Citrus fruit was a rich source of phytochemicals,
recorded with diethyl ether extract against K. which were very rare in other plants and were active
pneumoniae, S. Pyogenes, S. aureus, E. coli and E. against various pathogens. The difference in the
amnigenus (table 1). It was found that diethyl ether antibacterial activity with the same source when extracted
extract showed low sensitivity of S. epidermidis, S. with different solvent proves that not all phytochemicals
marcesnces, and P. aeruginosa. Kabra et al. (2012) also that are responsible for antibacterial activity are soluble in
found that the ethanolic extract of citrus peel was a single solvent.
effective against S. aureus, K. pneumonia, B. subtilis, P. Antioxidant activity
vulgaris, E. coli and P. aeruginosa. Our results are not Similar results were obtained by both ABTS.+ and DPPH
agreed with other studies such as non-polar solvents did free radical scavenging methods. Our results were
not show any significant effect against bacterial consistent with the findings of Kanaze et al. (2008).
pathogens except E. coli. This may indicate that these Foods rich in antioxidant phytochemicals were important
solvents have the capacity of extracting the antibacterial for the prevention of diseases related to oxidant stress i.e.
agent, which may be extremely toxic to K. pneumoniae, S. heart and cancer (Nabavi et al., 2013; Seerama et al.,
Pyogenes, S. aureus, E. coli, and E. amnigenus. Thus, it 2005).
points out that diverse extracts may have different
antibacterial agent, which may have different modes of CONCLUSIONS AND FUTURE WORK
action. C. sinensis peel oil showed inhibition of E. coli
and B. subtilis (Amandeep et al., 2009). In conclusions, C. sinensis peel extracts have a wide
range of antibacterial and antioxidant activity. These
Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239 237
Assessment of antioxidant, antibacterial and phytochemical analysis of peel of Citrus sinensis

extracts provide the possibilities to discover new effective Ghasemi K, Ghasemi Y and Ebrahimzadeh MA (2009).
antibacterial agents. Further studies are required to purify Anti-oxidant activity, phenol and flavonoid contents of
and characterize the bioactive compounds of both polar 13 citrus species peels and tissues. Pak. J. Pharma.
and non-polar extracts of the C. sinensis peel. Sci., 22: 277-281.
Gomez KA and Gomez AA (2007). Statistical Procedures
ACKNOWLEDGEMENT for Agricultural Research. 2nd ed. Translation:
Sjamsudin E. & Baharsjah J. S. UI Press, Jakarta, pp.
The authors are grateful to Directorate of Advance Study 134-176.
and Research of Azad Jammu and Kashmir University for Hamendra SP and Anand K (2007). Anti-diabetic
providing funds for the study. potential of Citrus sinensis and Punica granatum peel
extracts in alloxan treated male mice. Bio. Factors, 31:
REFERENCES 17-24.
Han ST (1998). Medicinal plants in the south pacific,
Ahmad MM, Salim-ur-Rehman, Iqbal Z, Anjum FM and world health organization, (WHO), Regional
Sultan JI (2006). Genetic variability to essential oil Publications West Pacific Series, No. 19, Manila, pp.
composition in four citrus fruit species. Pak. J. Bot., 1-254.
38(2): 319-324. Harborne JB (1993). Phytochemical method, 3rd Edition,
Amandeep S, Ahmed R, Bilal and Anil D (2009). In vitro Chapman and Hall, London. pp. 135-203.
antibiotic activity of isolated volatile oil of Citrus Kabra AO, Bairagi GB, Mahamuni AS and Wanare RS
sinensis. Int. J. Pharma. Res. Develop., 1(7): 1-4. (2012). In vitro Anti-microbial activity and
Andleeb S, Ghous T, Riaz N, Shahzad N, Ghous S and phytochemical analysis of the peels of Citrus medica.
Awan UA (2013). Assessment of antibacterial activity Inter. J. Res. Pharma. Biomed. Sci., 3(1): 34.
of Momordica charantia extracts and antibiotics Kanaze FI, Termentzi A, Gabrieli C, Niopas I,
against fecal contaminated water associated Georgarakis M and Kokkalou E (2008). The
Enterococcus spp. Pak. J. Zool., 45(2): 555-558. phytochemical analysis and antioxidant activity
Anonymous (2004). Government of Pakistan Economic assessment of orange peel (Citrus sinensis) cultivated
Survey. Islamabad: Finance Division. in Greece-Crete indicates a new commercial source of
Ayoola GA, Johnson OO, Adelowotan T, Aibinu IE, hesperidin. Biomedi. Chromato., 23: 239-249.
Adenipekun E, Adepoju AA, Bello-Coker HAB and Kim HG, Lim HA, Kim SY, Kang SS, Lee HY and Yun
Odugbemi TO (2008). Evaluation of the chemical PY (2007). Development of functional Hanji added
constituents and the anti-microbial activity of the citrus peel. J. Korea Tappi., 9: 38-47.
volatile oil of Citrus reticulata fruit (Tangerine fruit Kim JY, Oh TH, Kim BJ, Kim SS, Lee NH and Hyun CG
peel) from South West Nigeria. Afr. J. Biotechnol., (2008). Chemical composition and anti-inflammatory
7(13): 2227-2231. effects of essential oil from Faefugium japonicum
Baker FJ and Pallister CJ (1998). Baker and the flower. J. Oleo Sci., 57: 623-628.
Siverton’s Introduction to Medical Labotatory Kumamoto H, Matsubara Y, Iizuka Y, Okamoto K and
Technology. Seventh Edition. Butterworth Heinous Yokoi K (1986). Structure and hypotensive effect of
Main, Oxford, pp. 241-440. flavonoid glycosides in orange (Citrus sinensis
Caccioni DR, Guizzardi M, Biondi DM, Renda A and OSBECK) Peelings. Agri. Biol. Chem., 50: 781-783.
Ruberto G (1998). Relationship between volatile Kumar KA, Narayani M, Subanthini A and Jayakumar M
components of citrus fruit essential oils and (2011). Anti-microbial activity and phytochemical
antimicrobial action on Penicillium digitatum and analysis of citrus fruit peels utilization of fruit waste.
Penicillium italicum. Int. J. Food Microbiol., 43:73-79. Int. J. Engin. Sci. Tech., 3: 5414-5421.
Das K, Tiwari RKS and Shrivastava DK (2010). Mantena SK, King AL, Andringa KK, Eccleston HB and
Techniques for evaluation of medicinal plant products Bailey SM (2008). Mitochondrial dysfunction and
as antimicrobial agent: Current methods and future oxidative stress in the pathogenesis of alcohol- and
trends. J. Med. Plants Res., 4(2): 104-111. obesity-induced fatty liver diseases. Free Radical Bio.
Duraipandiyan V, Ayyanar M and Ignacimuthu S (2006). Med., 44: 1259-1272.
Anti-microbial activity of some ethno medicinal plants Nabavi SF, Nabavi SM, Setzer WN, Nabavi SA, Nabavi
used by Paliyar tribe from Tamil Nadu, India. BMC SA and Ebrahimzadeh MA (2013). Anti-oxidant and
Complement. Alt. Med., 6: 35-41. anti-hemolytic activity of lipid-soluble bioactive
Edeoga HO, Okwu DE and Mbabie BO (2005). substances in avocado fruits. Fruits, 68(3): 185-193.
Phytochemical constituents of some Nigerian Nannapanenl R, Muthaiyan A, Crandall P and Johnson
Medicinal Plant. Afr. J. Biotechnol., 4: 685-688. MG (2008). Anti-microbial activity of commercial
Ekwenye UN and Edeha OV (2010). The anti-bacterial citrus-based natural extracts against Escherichia coli
activity of crude leaf extract of Citrus sinensis (sweet 0157: H7 isolates and mutant strains. Food Borne
orange). Int. J. Pharm. Bio. Sci., 1(4): 742-750. Pathogen dis., 5: 695-699.
238 Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239
 Saiqa Andleeb et al

Njoroge SM, Koaze H, Karanja PN and Sawamura M against plant and human pathogens. J. Pharma. Res.,
(2005). Volatile constituents of red blush grapefruit 3(4): 700-702.
(Citrus paradisi) and pummel (Citrus grandis) peel Sofowora A (1993). Screening plants for bioactive agents.
essential oils from Kenya. J. Agric. Food Chem., 53: In: medicinal plants and traditional Medicinals in
9790-9794. Africa, 2nd Ed. Spectrum books Ltd, Sunshine house,
Nwanebu FC, Obi RK, Ogbulie C, Nwachukwu IN and Ibadan, Nigeria. pp. 134-156.
Chukwu F (2011). Anti-microbial activity of leaf and Sokmen A, Gulluce M, Akpulat HA, Deferera D, Tepe B,
root extracts of Vernonia amygdalina (Bitter leaf) on Polissiou M, Sokmen M and Sahin F (2004). In vitro
some bacterial isolates. Int. J. Environ. Health Human anti-microbial and anti-oxidant activities of the
Develop., 12(1): 51-62. essential oils and methanol extracts of endemic Thymus
Palakawong C, Sophanodora P, Pisuchpen S and spathulifolius. Food control, 15: 627-634.
Phongpaichit S (2010). Anti-oxidant and anti-microbial Soković M, Marin PD, Brkić D and van Griensven LJLD
activities of crude extracts from mangosteen (Garcinia (2007). Chemical composition and anti-bacterial
mangostana L) parts and some essential oils. Int. Food activity of essential oils of ten aromatic plants against
Res. J., 17: 583-589. human pathogenic bacteria. Food, 1: x-y.
Pandey RR, Dubey RC and Saini S (2010). Stange RR Jr, Midland SL, Eckert JW and Sims JJ
Phytochemical and anti-microbial studies on essential (1993). An anti-fungal compound produced by
oils of some aromatic plants. Afr. J. Biotechnol., 9: grapefruit and valencia orange after wounding of the
4364-4368. wounding of the peel. J. Nat. Prod., 56: 1627-1629.
Perez RM, Avila JG, Perez S, Martinez A and Martinez G Tao N-guo, Liu Y-jin, Zhang J-hong, Zeng H-yan, Tang
(1990). Anti-microbial activity of some American Y-fang and Zhang M-ling (2008). Chemical
algae. J. Ethnopharmacol, 29: 111-116. composition of essential oil from the peel of satsuma
Proteggente AR, Saija A, De PA and Rice-evans CA mandarin. Afr. J. Biotechnol., 7: 1261-1264.
(2003). The compositional characterization and Thenmozhi M and Rajeshwari S (2010). Phytochemical
antioxidant activity of fresh juices from sicilian sweet analysis and anti-microbial activity of Polyalthia
orange (Citrus sinensis l. osbeck) varieties, Free longifolia. Int. J. Pharm. Biol. Sci., 1(3): 1-7.
Radical Res., 37: 681-687. Trease GE and Evans WC (2002). Pharmacognosy, 15th
Rafique A, Andleeb S, Ghous T, Shahzad N and Shafique Ed, Vol 42-44, Saunders Publishers, London. pp. 221-
I (2012). Antibacterial activity of traditional herbs and 229, 246-249, 304-306, 331-332, 391-393.
standard antibiotics against poultry associated Ume-Kalsoom, Siddique S, Shahzad N, Ghous T and
Psuedomonas aureginosa. J. Pharma. Sci. Inno., 1: 12- Andleeb S (2013). In vitro screening of herbal extracts
16. and antibiotics against bacteria isolated from Fish
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M products at retail outlets. Brit. Microbiol. Res. J., 3(1):
and Rice-Evans CA (1999). Anti-oxidant activity 19-31.
applying an improved ABTS radical cation Upadhyay RK, Dwivedi P and Ahmad S (2010).
decolorization assay. Free Radical Bio. Med., 26(9- screening of anti-bacterial activity of six plant essential
10): 1231-1237. oils against pathogenic bacterial strains. Asian J. Med.
Seeley HW, Vandemark PJ and Lee JJ (2001). Microbes Sci., 2: 152-158.
in action: A Laboratory Manual of Microbiology. 4th Wagner H and Bladt S (2004). Plant drug analysis-A thin
Edition. W.H. Freeman and Co., New York, pp. 57- layer chromatography atlas 2nd edition. Thompson
130. Press Ltd., New Dehli, India, pp. 14-247.
Seerama NP, Adamsa LS, Henninga SM, Niua Y, Zhang You WC, Brown LM, Zhang L, Li JY, Jin ML, Chang YS
Y, Nair MG and Hebera D (2005). In vitro anti- Ma JL, Pan KF, Liu WD, Hu Y, Crystal-Mansour S,
proliferative, apoptotic and antioxidant activities of Pee D, Blot WJ, Fraumeni JF, Xu GW and Gail MH
punicalagin, ellagic acid and a total pomegranate (2006). Randomized double-blind factorial trial of
tannin extract are enhanced in combination with other three treatments to reduce the prevalence of
polyphenols as found in pomegranate juice. J. precancerous gastric lesions. J. Nat. Cancer Inst., 98:
Nutritional Biochem., 16(6): 360-367. 974-983.
Sharma N, Kalra KL, Oberoi HS and Bansal S (2007). Zhou K and Yu L (2006). Total phenolic contents and
Optimization of fermentation parameters for anti-oxidant properties of commonly consumed
production of ethanol from kin now waste and banana vegetables grown in Colorado. LWT - Food Sci. Tech.,
peels by simultaneous saccharifi cation and 39:1155-1162.
fermentation. Indian J. Microbiol., 47: 310-316. Zou Y, Lu Y and Wei D (2004). Anti-oxidant activity of a
Shekhawat N and Vijayvergia R (2010). Evaluation of flavonoid-rich extract of Hypericum perforatum L. in
anti-microbial potential of some medicinal plants vitro. J. Agric. Food Chem., 52: 5032-5039.

Pak. J. Pharm. Sci., Vol.28, No.1, January 2015, pp.231-239 239

View publication stats