Antimicrobial Activity and Phytochemical

Analysis of Citrus Fruit Peels -Utilization of

Fruit Waste

1
K. Ashok kumar,
2
M. Narayani,
3
A. Subanthini and
4
M. Jayakumar
1
Department of Biotechnology,
4
Department of Chemical Engineering,
Arulmigu Meenakshi Amman College of Engineering,
Vadamavandal (Near Kanchipuram), T.V. Malai District 604 410, Tamilnadu, India.
E-Mail: ashokkumar_kh5@yahoo.com, jaimchem@gmail.com
2
Department of Chemical Engineering,
National Institute of Technology (NIT), Karnataka, India.
E-Mail: narayani@ymail.com
3
Department of Biotechnology,
Bannari Amman Institute of Technology, Sathyamangalam 638 401, Tamilnadu, India.
E-Mail: subanthini@gmail.com

Abstract:
Antibacterial activity of five different solvent extracts(ethyl acetate, acetone, ethanol, petroleum ether and
water) prepared by soxhlet extractor from two citrus fruit peel (Citrus sinensis and Citrus limon) were screened
against five pathogenic bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli , Klebsiella
pneumonia and Salmonella typhi. The highest antibacterial potentiality was exhibited by the acetone peel extract
of Citrus sinensis followed by the ethyl acetate peel extract of Citrus limon. The peel extract of Citrus sinensis
and Citrus limon can be considered to be as equally potent as the antibiotics, such as metacillin and penicillin.
MICs were tested at concentrations ranging from 50-6.25 mg/ml as wells as their MBCs. The phytochemical
analysis of the citrus peel extracts showed the presence of flavonoids, saponins, steroids, terpenoids, tannins and
alkaloids.
Keywords: Antibacterial- Soxhlet extractor-Minimum Inhibitory Concentration (MIC) Minimum
bactericidal concentration (MBC) Phytochemicals
1. Introduction
Citrus is one of the most important commercial fruit crops grown in all continents of the world [Tao et al.,
(2008)].Citrus importance is attributed to its diversified use and growing world demand with about 102.64
million tones total world production and probably stands first largest among the produced fruit [NAQVI
(2004)]. Citrus fruits are mainly used by juice processing industries while the peels are generally wasted. Since
the juice yield of citrus is less half of the fruit weight, very large amounts of byproduct wastes, such as peels are
formed every year [Manthey & Grohmann,(2001)]. Peel waste are highly perishable and seasonal, is a problem
to the processing industries and pollution monitoring agencies. There is always an increased attention in
bringing useful products from waste materials and citrus wastes are no exceptions. Suitable methods have to be
adopted to utilize them for the conversion into value-added products [Nand, (1998)]. By-product recovery from
fruit wastes can improve the overall economics of processing units. Besides this, the problem of environmental
pollution also can be reduced considerably. The citrus peels are rich in nutrients and contain many
phytochemicals, they can be efficiently used as drugs or as food supplements too. Since there is an increase in
the number of antibiotic resistance pathogens, there is always a search of an alternative drug that is regarded as
safe. Citrus peels if proved to have antibacterial activity; they can be also used in same food industry which
generates large peel wastes as a food preservative. Food processors, food safety researchers, and regulatory
agencies have been increasingly concerned with the growing number of food-borne illness outbreaks caused by
K. Ashok kumar et al. / International Journal of Engineering Science and Technology (IJEST)
ISSN : 0975-5462 Vol. 3 No. 6 June 2011 5414
some pathogens [Wilson and Droby (2000); Friedman et al. (2002); Sokovi et al (2007)]. The food industry has
tended to reduce the use of chemical preservatives in their products due to increasing pressure of consumers or
legal authorities, to either completely remove or to adopt more natural alternatives for the maintenance or
extension of product shelf life [Nychas (1995)].
The peel of Citrus fruit is a rich source of flavanones and many polymethoxylated flavones, which are very rare
in other plants [Ahmad et al. (2006)].The antimicrobial abilities of essential oils, among which citrus oils, are
also shown to be a particularly interesting field for applications within the food and cosmetic industries
[Caccioni et al., (1998)]. It has also been used as an anti-diabetic (Hamendra and Anand (2007)]), antimicrobial
[Caccioni et al. (1998)], antifungal [Stange Jr et al. 1993], hypotensive agent [Kumamoto et al. (1986)],
antioxidant [Proteggente et al. (2003)]; [Kanaze et al. (2008)], carminative, insect repellent, antibacterial,
larvicidal, antiviral, uricosuric, anti-yeast, antihepatotoxic and antimutagenic agent [Han (1998)].
This study was aimed to focus on waste minimization in fruit juice processing industry. The combined efforts of
waste minimization during the production process and recovery of valuable product substantially reduce the
amount of waste, as well as boost the environmental profile of fruit juice processing industry. This study
investigates the antibacterial activity and the fundamental scientific basis for the use of peels of citrus fruits by
determining the chemical constituents as well as quantifying the yield percentage of crude phytochemicals.
2. Materials and Methods
2.1. Plant materials
The plants used in this study were Citrus limon (common name: Lemon) and Citrus sinensis (common
name: Sweet orange).The peels were collected from the local fruit juice shops. After collection, the peels were
shade dried at room temperature (32 - 35C) to constant weight over a period of 5 days. 15 g of each of the plant
parts were coarsely powdered using a mortar and pestle and were further reduced to powder using an electric
blender. The powder was transferred into closed containers.
2.2. Preparation of extracts
2.2.1. Soxhlet extraction:
The dried and powdered plant materials (15 g) were extracted successively with 200 ml of each solvent
separately by using soxhlet extractor for 5 h at a temperature not exceeding the boiling point of the Solvent [Lin
et al., (1999)]. The solvents used for the study were Ethyl acetate, Petroleum ether Acetone, Ethanol and Water.
The extracts were filtered and then concentrated to dryness. Yield of the extract obtained was calculated as
follows:
iclJ (%) =
wcigt o cxtroct rcco:crcJ)
wcigt o Jry powJcr
1uu
Each extract were transferred to glass vials and kept at 4 C before use. The extracts were dissolved in
25% aqueous dimethyl sulfoxide (DMSO) to produce a stock solution of 100 mg ml
-1.
2.2.2. Aqueous Extraction
The method of Dupont et al. (2005) was adopted for extraction with little modification. Briefly, 15g of
the powdered plant were soaked separately in 200 ml of distilled water at ambient temperature for 24 hour under
shaking condition at 130 rpm. The extract was then filtered using Whatman filter paper No 1 .Each extracts
were transferred to glass vials and kept at 4 C before use.
2.3. In vitro testing of extracts for antimicrobial activity:
2.3.1. Antimicrobial assay:
Overnight cultures of the Gram positive strains S. aureus, B. subtilis and the Gram negative strains E. coli, K.
pneumoniae and S. typhi were prepared on nutrient agar plates. All bacterial isolates were suspended in saline
to a turbidity equivalent to 0.5 McFarland (1.5 x 10
8
CFU/ml) and 0.1% standardized inoculum suspension was
swabbed uniformly in Mueller Hinton Agar (MHA, pH 7.3 0.1, Difco) obtained from HiMedia, Mumbai,
India. Sterile HiMedia paper disc (6mm) were soaked in 20 l of the extract diluted in 25% DMSO and dried at
37C overnight. The loaded disc was placed on the surface of medium, the compound was allowed to diffuse
and the plates were kept for incubation at 37C for 24 hrs. Antibiotic discs containing Penicillin, Methicillin and
Gentamycin (5-30g) were used as controls.
A disc loaded with 25% DMSO alone served as the negative control. The antibacterial activity was
evaluated by measuring the diameter of the inhibition zone formed around the discs. These studies were
performed in triplicate.
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2.3.2. Determination of Minimum Inhibitory Concentration (MIC) of the extracts on the test organisms
A sterile 16 well plate was labeled. 100 L of nutrient broth was added to all the wells. A volume of 100 L of
test material in 10% (v/v) DMSO or sterile water (usually a stock concentration of 100 mg/mL was pipetted into
the first row of the plate. Serial dilutions were performed using a micropipette to obtain dilutions: 50mg/ml,
25mg/ml, 12.5mg/ml, and finally 6.25mg/ml. Tips were discarded after use such that each well had 100 L of
the test material in serially descending concentrations. Finally, 10 L of bacterial suspension (1-2 10
8
cfu/mL)
was added to each well .Each plate was wrapped loosely with cling film to ensure that bacteria did not become
dehydrated. Negative controls were set up as follows: Nutrient broth only; Nutrient broth and sterile plant
extract; and finally positive control containing Nutrient broth, and a test organism. The plates were prepared in
duplicate, and placed in an incubator set at 37 C for 1824 h. To each well 10 L of resazurin indicator solution
was added and incubated for another 6-7 h. The color change was then assessed visually. Any color changes
from purple to pink or colorless were recorded as positive. The lowest concentration at which color change
occurred was taken as the MIC value. The average of two values was calculated and that was the MIC for the
test material and bacterial strain.
2.3.3. Determination of minimum bactericidal concentration (MBC)
To determine the MBC, for each set of well in the MIC determination(before the addition of resazurin
dye), a loopful of broth was collected from those plates well ,which did not show any visible sign of growth and
inoculated on sterile nutrient agar by streaking. Nutrient agar plates were streaked with the test organisms only
to serve as control. The plates were then incubated at 37C for 24 h. After incubation the concentration at which
no visible growth was seen was noted as the minimum bactericidal concentration.
2.4. Preliminary phytochemical analysis (Qualitative analysis):
The powered plant parts as well as the extracts were subjected to preliminary phytochemical screening
following the methodology of Sofowora (1994), Harborne (1998) and Kokate (2001).
2.4.1. Test for alkoloids: 2 ml filtrate was mixed with 1% HCl and about 6 drops of Mayors reagents. A
Creamish or pale yellow precipitate indicated the presence of respective alkaloids.
2.4.2. Test for flavonoids: 2 ml filtrate was added to conc. HCl and magnesium ribbon. Pink-tomato red color
indicated the presence of flavonoids.
2.4.3. Test for amino acids: 1 ml of the extract was treated with few drops of Ninhydrin reagent. Appearance of
purple color shows the presence of amino acids.
2.4.4. Test for tannins: 1 ml of the extract was treated with few drops of 0.1% ferric chloride and observed for
brownish green or a blue-black coloration.
2.4.5. Test for phlobatannins: Deposition of a red precipitate when an aqueous extract of each plant sample was
boiled with 1% aqueous hydrochloric acid was taken as evidence for the presence of phlobatanins.
2.4.6. Test for anthraquinones (Borntragers test): 1 ml of the extract solution was hydrolyzed with diluted
Conc. H
2
SO
4
extracted with benzene. 1 ml of dilute ammonia was added to it. Rose pink coloration suggested
the positive response for anthraquinones.
2.4.7. Test for saponins: Froth test for saponins was used. 1g of the sample was weighed into a conical flask in
which 10ml of sterile distilled water was added and boiled for 5 min. The mixture was filtered and 2.5ml of the
filtrate was added to 10ml of sterile distilled water in a test tube. The test tube was stopped and shaken
vigorously for about 30 second. It was then allowed to stand for half an hour. Honeycomb froth indicated the
presence of saponins.
2.4.8. Test for steroids: 2 ml of acetic anhydride was added to 0.5 g ethanolic extract of each sample with 2 ml
H
2
SO
4
. The color changed from violet to blue or green in some samples indicating the presence of steroids.
2.4.9. Test for phytosterol: The extract was refluxed with solution of alcoholic potassium hydroxide till
complete saponification takes place. The mixture was diluted and extracted with ether. The ether layer was
evaporated and the residue was tested for the presence of
phytosterol. The residue was dissolved in few drops of diluted acetic acid; 3 ml of acetic anhydride was added
followed by few drops of Conc. H
2
SO
4
. Appearance of bluish green color showed the presence of phytosterol.
2.4.10. Test for reducing sugars: The residue was re-dissolved in water on the water bath. To 2ml of the
solution, in the test tube was added, 1ml each of Fehling's solutions A and B. The mixture was shaken and
heated in a water bath for 10min. The color obtained was recorded. A brick-red precipitate indicates reducing
sugar
K. Ashok kumar et al. / International Journal of Engineering Science and Technology (IJEST)
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2.4.11. Test for terpenoids (Salkowski test):5 ml of each extract was mixed in 2 ml of chloroform, and
concentrated H
2
SO
4
(3 ml) was carefully added to form a layer. A reddish brown coloration of the inter face was
formed to show positive results for the presence of terpenoids.
2.4.12. Test for cardiac glycosides (Keller-Killani test): 5 ml of each extracts was treated with 2 ml of glacial
acetic acid containing one drop of ferric chloride solution. This was underlayed with 1 ml of concentrated
sulphuric acid. A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. A violet ring
may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually
throughout thin layer.
2.4.13. Test for Chalcones: 2 ml of Ammonium hydroxide was added to 0.5 g ethanolic extract of each sample.
Appearance of reddish color showed the presence of chalcones.
3. Results and discussion:
The soxhlet extract of the citrus peel using different solvents yielded different results in each of the experiment
conducted in the this study. There existed, a difference in the percentage yield of the extract obtained between
various solvents. Figure 1, shows the comparison of the percentage yield of extracts obtained from different
solvent with respect to various sources. For Citrus limon extract, the ethyl acetate extract showed the highest
yield of about 18% followed by the ethanol extract and the acetone extract showing the least percentage yield.
Citrus sinensis peel extract showed highest yield when acetone was used as a solvent with a yield percentage of
about 17% followed by ethyl acetate (12%). The aqueous extract of both the citrus peel showed a moderate
percentage yield. This variation in yield between ranges of solvents explains that solubility of different plant
compound in different solvent.
Citrus peel extracts showed a significant antibacterial activity against all the test organisms. Citrus sinensis peel
extracts showed a very good antimicrobial activity when compared to Citrus limon .Acetone extract of Citrus
sinensis showed a maximum zone of inhibition against E.coli (16mm) followed by S. typhi (15mm), B.subtilis
and K.pnemonia (14mm) and S.aureus (13mm) whereas the ethanol and aqueous extract of Citrus sinensis did
not show such high antibacterial activity. This antibacterial activity may be indicative of the presence of
metabolic toxins or broad spectrum antibiotic compounds. In case of Citrus limon, ethyl acetate and acetone
showed more or less the same antibacterial activity. Aqueous extracts showed very less antibacterial activity
when compared to other solvents. Petroleum ether did not show any significant effect against all the tested
strains except E.coli. This shows that petroleum ether has the capability of extracting one such antibacterial
agent which may be very toxic to E.coli. Thus it indicates that different extracts may have diverse antibacterial
agent that has different modes of action or the bacteria may have a special metabolism to overcome or adapt its
activity. Ethyl acetate proves to be a good solvent for the extraction of antibacterial agents from both the sources
as it has shown better yield as well as antibacterial activity relating that higher yield means high concentration
of single or variety of phytochemicals and therefore high antibacterial activity. This statement can be validated
as acetone has shown highest yield as well antibacterial activity in Citrus sinensis.
Ekwenye and Edeha (2010) reported antibacterial activity of the ethanol and aqueous extract of Citrus sinensis
leaf against the test organisms taken in their study (E.coli: aqueous extract -7mm, ethanol extract -3mm;
K.pneumoniae: 3mm for both the extracts; S.aureus: aqueous extract -1mm, ethanol extract-2mm). Comparing
their results with our study with the same test organism and solvent shows that Citrus sinensis peel
extract(E.coli :aqueous extract -9mm, ethanol extract -8mm; K.pneumoniae : aqueous extract -7mm, ethanol
extract - 8mm; S.aureus: aqueous extract -9mm, ethanol extract-7mm) has high degree of antibacterial activity
as compared to the leaf extract. However, this difference may be because of the difference in the phytochemical
composition in various part of the plant or may be also due to the extraction method used and/or environmental
factors or difference in the genotypes of the citrus plant used. The citrus peel extract exhibited similar or higher
antibacterial activity as that of the standard antibiotics used in the study. However, gentamycin showed higher
activity relatively.
The zone of inhibition for Citrus limon and Citrus sinensis peel oil against E. coli was 14mm and 13mm
respectively; S. aureus was 14mm and 12mm respectively; K. pneumonia was 13mm and 11mm respectively [F.
Glay kirbalar et al (2009)]. Amandeep et al (2009) reported that Citrus sinensis peel oil showed zone of
inhibition of 13mm and 17mm against E.coli and B. subtilis respectively.
MIC and MBC of different solvent extracts of Citrus sinensis peel and Citrus limon are shown in table 2 and
table 3 respectively. The extracts showed significant activity. MIC by broth dilution showed good results
compared to disc diffusion method as there may be a problem with the diffusion of the biological component
into the agar. The hydrocarbon components either remain on the surface of the medium or evaporate [Griffin et
al. (2000)]. That could be the reason for the better results obtained by the microdilution method. Broth method,
carried out in microtitre trays, has the advantage of lower workloads for a larger number of replicates and the
K. Ashok kumar et al. / International Journal of Engineering Science and Technology (IJEST)
ISSN : 0975-5462 Vol. 3 No. 6 June 2011 5417
use of small volumes of the test substance and growth medium [Sokovi et al. (2007)].Lack of activity can
thus only be proven by using large doses (Farnsworth, 1993).Alternatively, if the active principle is present in
large quantities, there could be other constituents exerting antagonistic effects or negating the positive effects of
the bioactive agents [Jager et al.,(1996)].
The difference in the antibacterial activity with the same source when extracted with different solvent has
proven that not all phytochemicals that are responsible for antibacterial activity are soluble in a single solvent.
Hence solvents of different polarity should be employed as discussed in this study (polar: water, acetone,
ethanol; non-polar: ethyl acetate, petroleum ether). Sequential or successive solvent extraction is as good option
for better solubility of many of the phytochemcials but it is always necessary to know the phytochemicals
extracted by each individual solvent so as to avoid the inclusion of unnecessary solvents for extraction process
as well as to understand the role of each solvent in the extraction of a individual or class of phytochemicals.
With no antibacterial activity, extracts may be active against other bacterial species which were not tested [Shale
et al., (1999)].
The medicinal value of these plants lies in bioactive phytochemical constituents that produce definite
physiological action on the human body [Akinmoladun et al., (2007)]. The preliminary phytochemcial
investigation revealed the presence of various constituents of citrus peels. The results are shown in the table.
Different solvent showed different class of phytochemicals .they showed the presence of flavanods, saponins
etc. antraquiones were completely absent in both the citrus peels. These constituents could account for the
antibacterial activity but it is difficult to correlate their action to a specific phyochemical.
The presence of phenol further indicated that Citrus limon and Citrus sinensis peels could act as anti-
inflammatory, anti clotting, antioxidant, immune enhancers and hormone modulators. Citrus lemon and Citrus
sinensis peels have high quantity of saponin which has hemolytic activity and cholesterol binding properties
.Therefore, in addition to their use as drugs, citrus peels can be used as a food preservative or even as food
supplement as many literature says that they are highly nutritive.


















Figure 1 Percentage yield of citrus peel extracts








0
2
4
6
8
10
12
14
16
18
20
C. limon C.sineses
Y
i
e
l
d
(
%
)
(
w
/
w
)
Various extracts
Ac
EtOAc
EtOH
Pet ether
Aq
K. Ashok kumar et al. / International Journal of Engineering Science and Technology (IJEST)
ISSN : 0975-5462 Vol. 3 No. 6 June 2011 5418
Table 1. Zone of inhibition (mm) of Citrus limon peel and citrus sinensis peel extract against test bacteria on Mueller-Hinton agar medium
using disc diffusion method


Bacteria
Zone of inhibition(mm)
Citrus peel extracts* Standard
antibiotics
Acetone

Petroleum
ether
Ethanol Ethyl
acetate
Aqueous Pen Met Gen

C.l

C.s

C.l


C.s

C.l


C.s

C.l


C.s

C.l


C.s
E. coli 12 16 13 10 11 8 11 9 10 9 7 7 29
S.aureus 10 13 7 - 9 7 10 11 - 9 6 6 18
S. typhi 10 15 - 7 9 8 10 9 7 - - - 24
B. subtilis 9 14 7 - 9 7 10 10 9 9 8 9 20
K.Pneumonia 12 14 8 9 10 8 12 11 - 7 - - 25
*disc concentration 100 mg/ml
C.l- Citrus limon; C.s- Citrus sinensis
- indicates < 5mm

Table 2. MIC values of Citrus limon and Citrus sinensis peel extract for different bacterial strains


Bacteria
Citrus lemon peel extract
(mg/ml)
Citrus sinensis peel extract
(mg/ml)
Acetone

Petroleum
ether
Ethanol
Eth
yl
acet
ate
Aqueou
s
Acetone

Petroleum
ether
Ethano
l
Ethyl
acetate
Aqueo
us
E.coli 12.5 6.25 25 50 50 6.25 25 25 50 50
S.aureus 50 50 50 50 - 25 - 50 12.5 50
S. typhi 50 - 50 25 50 6.25 50 50 50 50
B. subtlis 50 50 25 50 50 6.25 - 50 50 50
K.pneumonia 25 50 25 25 - 6.25 50 50 25 50


Table 3. MBC values of Citrus limon and Citrus sinensis peel extract for different bacterial strains


Bacteria
Citrus lemon peel extract
(mg/ml)
Citrus sinensis peel extract
(mg/ml)
Aceto
ne

Petroleum
ether
Ethano
l
Ethyl
acetate
Aqueo
us
Aceton
e

Petroleum
ether
Ethano
l
Ethyl
acetate
Aqueous
E.coli 50 12.5 50 - - 12.5 50 50 - -
S.aureus 50 - - - - 50 - - 25 -
S. typhi - - - 50 - 25 - - - -
B. subtlis - - 50 - - 25 - - - -
K.pneumonia 50 - - 50 - 25 - - 50 -



K. Ashok kumar et al. / International Journal of Engineering Science and Technology (IJEST)
ISSN : 0975-5462 Vol. 3 No. 6 June 2011 5419
Table 4. Phytochemical analysis of Citrus limon and Citrus sinensis peel extracts

+ indicates presence, - indicates absence
C. lim- Citrus limon , C.sin- Citrus sinensis
5. Conclusions
Recycling of fruit waste is one of the most important means of utilizing it in a number of innovative ways
yielding new products and meeting the requirements of essential products required in human, animal and plant
nutrition as well as in the pharmaceutical industry. This work has identified the antibacterial activity against the
test organisms and phytochemical constituents in Citrus limon and Citrus sinensis peels extracts obtained from
different solvents. However, further evaluation performed with the pure compounds is required for the definite
conclusion of the bioactive compounds contributing to the antimicrobial activity, although the nature and
number of active components involved in each extract are not clear, however they are promising. This finding
can form the basis for further studies to prepare an optimize preparation of the herbal extract.

Acknowledgement

The authors sincerely thank the authorities of Arulmigu Meenakshi Amman College of Engineering for the
successful completion of the work.

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Phytochemicals

Solvents
Acetone Petroleum
ether
Ethyl acetate Ethanol Aqueous
C.lim

C.sin C.lim

C.sin C.lim

C.sin C.lim

C.sin C.lim

C.sin
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Chalcones - - - - + - - - - -
Alkaloids + - + + - + + + + -
Steriods + - + + - + + + + -
Terpenoids - + - + - + + + + -
Phytosterol - - - - - - + - + -
Cardiac glycosides - + - + - + + + + -
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K. Ashok kumar et al. / International Journal of Engineering Science and Technology (IJEST)
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