SEPERATION OF CITRUS OIL FROM ORANGE PEEL AND
ANTIMICROBIAL ACTIVITY OF CITRUS OIL ON SELECTED PATHOGENS
A dissertation submitted to Calicut University in
partial fulfillment of the requirements for the award of degree of
BACHELOR OF SCIENCE IN BIOCHEMISTRY
Submitted by
MUHAMED SHAMEEM CHETTIARMALA
Reg. No: EMANSBC024
DEPARTMENT OF BIOCHEMISTRY
EMEA COLLEGE OF ARTS AND SCIENCE
KONDOTTY, MALAPPURAM
2016
EMEA
COLLEGE OF ARTS AND SCIENCE KONDOTTY
DEPARTMENT
OF BIOCHEMISTRY
CERTFICATE
This is certify that the project entitled
“Seperation of citrus oil from orange peel and antimicrobial activity of citrus
oil on selected pathogens” is a bonafide work of Mr.MUHAMED SHAMEEM CHETTIARMALA, conducted in the department of Biochemistry,
EMEA college of Arts and Science, Kondotty.
KONDOTTY Dr.T.
KRISHNAKUMAR
30-03-2016 Head
of the Department
Department
of Biochemistry
EMEA
college of Arts and Science
Kondotty,
Kerala
CERTIFICATE
This is certify that the work
entitled “SEPERATION OF CITRUS OIL FROM ORANGE PEEL AND ANTIMICROBIAL ACTIVITY
OF CITRUS OIL ON SELECTED PATHOGENS” submitted by Mr.MUHAMED SHAMEEM
CHETTIARMALA EMEA college, kondotty in partial fulfillment of the requirement
for the award of Bachelor’s Degree of University of Calicut in Biochemistry is
carried out by us under the guidance of Mr. Rajesh Assistant Professor, EMEA
College, Kondotty during the academic year 2015-2016.
Kondotty Mr.RAJESH
30-03-2016 Assistant
Professor
Department
of Biochemistry
EMEA
college of Arts and Science
DECLARATION
I
MUHAMED SHAMEEM CHETTIARMALA hereby declare that this dissertation entitled “SEPERATION OF CITRUS OIL FROM ORANGE PEEL
AND ANTIMICROBIAL ACTIVITY OF CITRUS OIL ON SELECTED PATHOGENS” is being
submitted to University of Calicut. The project is entirely the result of our
work carried out in the Department of Biochemistry, EMEA College of Arts and
Science Kondotty. This Project or any part of it has not been submitted for any
other degree, diploma or associate ship.
KONDOTTY MUHAMED SHAMEEM CHETTIARMALA
30.3.2016
ACKNOWLEDGEMENT
I
take opportunity with pride and enormous gratification to express the fitting
of thanks and gratefulness to the entire person who baked me directly or
indirectly throughout the materialization of the project work at this
magnitude.
First
I thank God Almighty for all the blessings showered upon me and for helping me
to complete my project work.
I
would like to express a deep sense of gratitude and thanks to Mr.Krishna Kumar,
Head, of Department of Biochemistry.
It
is my privilege and pleasure to express my heart full gratitude to my guide,
Mr. Shihabudheen Assistant Professor, Department of Biochemistry for his
valuable guidance.
I
would like to express my sincere thanks to Mrs. ZaheeraBanu and Mr.Rajesh T.K
for their constant support and cooperation during the entire course of work.
I
also submit my deepest sense of gratitude to the Lab Assistant Jamsheera,
Fathima, and Husna for their quite support throughout my work.
CONTENT
SL NO
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CONTENT
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PAGE NO
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1
|
INTRODUCTION
|
7
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2
|
REVIEW OF LITERATURE
|
8
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2.1
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PHYTOCONSTITUENTS
|
10
|
2.2
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PHARMACOLOGICAL PROFILE
Antioxidant property
Protection against cardio
vascular disease
Anti carcinogenic property
Reduced risk of kidney
stones
Antiulcer property
Anti-anxiety effect
Anti-typhoid
activity
Antibacterial activity
Larvicidal activity
Anti-diabetic activity
|
11
11
11
12
12
12
12
13
13
13
13
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3
|
AIM & OBJECTIVE
|
14
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4
4.1
4.2
4.3
4.4
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MATERIALS &
METHODS
MATERIALS
REQUIEREMENTS
REAGENTS
METHODS
|
15
15
15
15
16
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5
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RESULT&DISCUSSION
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18
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6
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CONCLUSION
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20
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7
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BIBILIOGRAPHY
|
21
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INTRODUCTION
An orange ,specifically ,the
sweet orange (citrus sinensis)is the
most commonly grown tree fruit in the
world the present work addresses to the development of the part of the
processes needed for the extraction of value added products like oleoresin from
orange peel, which is the waste of orange processing industry. The outcome of
the present work highlighted that the orange peels are good source of oleoresin
which contain essential oil does have the potential to become important raw
material for food processing industries. The simplest method of oleoresin
extraction using orange peel is the solvent extraction method.it is found
through solvent extraction gives higher yield than Other methods. It is the process in which, this method also help
us to check the antimicrobial activity of essential oil(citrus oil )present in
oleoresin. These results demonstrated that the successful extraction of
oleoresin and providing information that
citrus oil have an antimicrobial activity on both gram positive and gram
negative bacteria of selected pathogens and these also providing potential
benefits for industrial extraction of oleoresin from an economic and
environmental point of view .this also give us to the information that the
replacement of oilment, waxes contain chemicals have an allergic effect by the
citrus oil.
REVIEW OF LITERATURE
Orange
tree is mostly cultivated and rarely found in the forests, it was first
cultivated in southern china and northeastern India. The Persian orange grown
widely in southern Europe after it’s introduction to Italy in the eleventh
century was bitter.it was primarily grown for it’s medicinal purposes Italian
traders might have introduced it to the
Mediterranean area after 1450 AD Christopher Columbus took the seeds of oranges, lemons, and citrons to Haiti and
the Caribbean on his second journey of sea in 1493 AD ,.Portuguese navigators
have also been credited with bringing orange trees to the Mediterranean region
around 1500AD .after identification of the orange fruits ,it was quickly
adopted has an edible fruit.
FRUIT
The orange fruit is hesperidium.it is a
type of berry that ranges widely in size, colour, shape, juice quality .fruits
are globose to ovoid in shape. Wild orange fruit has a smooth skin and the
petiole wings are entire, the petioles of sour orange leaves are much larger
than that of sweet orange. The word “orange” is derived from sanskrith term
narang, mainly 11 individual pieces are present in a typical fruit. Oranges are
round citrus fruits with finely-textured skins that are of course orange in
colour just like their pulpy flesh. Oranges usually range from
approximately two to three inches in
diameter.
PHYTOCONSTITUENTS
Orange fruit contain 1.5% essential
oil, the main phytoconstituents present in orange fruit are
D-limonene(90%),citrall, citronellal, nootkaton, sinesal
,n-nonanal,n-decanal,n-dodecanal,linalylacetate,geranylacetate,citronelyl
acetate and anthranil acid methyl ester. lipophilic flavonoids and
furanocumarines are reported in pressed oils. There is some evidence that
active ingredients of orange stimulates the secretion of gastric juice, orange also
contains several bitter flavor glycosides like neohespiridine and naringin, whose
sugar component is rutinose. both sugars are disaccharide of
glucoseandrhamnose(6-desoxymannose).
sr
|
Phytoconstituents
|
|
1.
|
Flavone, glycosides, neohespiridine,
naringin,
Hesperidine, narirutin,
triterpene, limonene,
Citrol,pigment:anthocyanine,beta-cryptoxanthine,cryptoxanthine,zeaxanthine,
Rutin,eriocitrin,homocysteine,polymethoxylatedflavones,tangertin,nobiletin,
Flavonoids: citracridone,
citbrasin and noradrenalin
|
Fruit
Peel
|
2.
|
Terpanoids: linalool,
betaelemene
|
Leaves
|
3.
|
Triterpenes, limonene
|
Flowers
|
4.
|
Vitamins:B1,B2,B3,B4,B5,B6
And vitamin C
Minerals: calcium, iron,
magnesium, zinc, phosphorous, potassium
|
fruits
|
PHARMACOLOGICAL PROFILE
Antioxidant
property
Oranges
form a rich source of vitamin C ,flavonoids phenolic compounds and pectins. The
main flavonoids found in citrus species are hesperidin, narirutin, naringin,
and eriocitrin. Just one orange provides 116% of the daily requirement for
vitamin C. vitamine C is the primary water soluble antioxidant, which prevent
free radicle generation in the body and damage to the tissues in the aqueous
environment both inside and outside cells. Drinking of orange juice with out
salt and sugar is associated with reduced severity of inflammatory conditions, likeasthma,
Osteo arthritis and rheumatoid arthritis. Vitamin C is also necessary for the
proper functioning of immune system. Vitamin C is good for preventing cold, cough and recurrent ear
infection.
Protection
against cardio vascular disease
According
toward health organizations recent
report, citrus fruits often protection against cardiovascular disease by
reducing levels of homocysteine. Orange
fruit contains vitamin C ,carotenoids and flavonoids which are cardio
protective. Cholesterol lowering effect of orange is produced by limonene. Poly methoxylated
flavones(PMFs) are present in citrus fruit peel, which can lower cholesterol
more effectively than some prescription drugs, without showing any side effect.
Although a variety of citrus fruit contain PMFs, The most common PMFs are
tangeretin and nobiletin, which are found in the peels of orange .PMFs work
like statin drugs that inhibit the synthesis of cholesterol and triglyserides
inside the liver. However grating a tablespoon or so of the peel of orange each
day and using it to flavor tea, salad, yogurt, soups, snacks or rice may be
practical way of achieving some cholesterol lowering benefits.
Anti
carcinogenic property
Limonene,
one of the main constituents of orange, reduces the risk of mouth ,skin, lung, breast,
stomach and colon cancer. Another constituents of orange is hesperidine and
it’s flavone analogue,diosmin has also exhibited anticarcinogenic activities in
various in vivo studies. Anticarcinogenic activity mainly depend on anti
oxidant properties of the molecules, as well as their ability to modulate the
detoxifying hepatic enzymes. The poly methoxylated flavones have shown strong
antiproliferative action against cancer cells and antigen activated t
lymphocytes. beta-cryptoxanthine(an orange red carotenoid)is present in highest
amount in oranges. It may significantly lower one’s risk of developing lung
cancer.
Reduced
risk of kidney stones
A
study published in the British journal of nutrition found that when women drank
½ liter of orange juice daily, their urinary pH value and citric acid axcretion
increased thereby diminishing the risk
of forming calcium oxalate stones significantly.
Antiulcer
property
Intake
of orange juice on regular basis reduced the infection incidence with
helicobacter pylori thus preventing development of ulcers.
Anti-anxiety
effect
Aroma-therapists use orange oil as a
tranquilizer, researchers have found evidence that sweet orange oil is an
anxiolytic agent.
Anti-typhoid activity
Typhoid
fever (TF) caused by salmonella typhis a major public health problem, particularly
in developing countries. Constituents of orange fruit responsible for
anti-typhoid activity include flavonoids like citacridone, citbrasin and saponins.
Antibacterial
activity
Orange
are eaten to allay fever the roasted pulp is prepared as a poultice for skin
diseases. The fresh peel is rubbed on acne. A decoction of the dried leaves and
flowers is taken in Italy and France as an antispasmodic, cardio protective and
anti-emetic agent, a decoction of husked orange seeds is prescribed for urinary
aliments in china. Orange peel oil produces lethal effect on fleas, fire
ants and house flies due to it’s
90-95% limonene .orange peel is
medicinally used against fungi.
Larvicidal
activity
The
saponins present in the peel possess larvicidal activity
Anti-diabetic
activity
Antidiabetic
activity of orange is due to bioflavonoids such as hesperidine and naringin
present in citrus fruit peel thus peels play an antidiabetic role in
C57BL/KsJ-db/db mice via regulation of glucose regulatory enzymes. They
decrease the activity of glucose -6-phosphatase and phophoenolpyruvate.the
anti-diabetic potential of orange peel and juice appear to be mediated via anti
peroxidation, inhibition of alpha amylase
enzyme activity that is responsible for the conversion of complex
carbohydrates to glucose ,increased hepatic glycogen content ,stimulation of
insulin secretion and repair of secretory defects of pancreatic beta cells.
AIM
& OBJECTIVE
The
aim of the work includes “the extraction of oleoresin contain citrus oil and to
determine it’s antimicrobial activity”
1. Extraction
of oleoresin from fine powder of dried orange peel.
2. To
determine the antimicrobial activity of oleoresin extract from orange peel.
MATERIALS
& METHODS
MATERIALS:
REQUIEREMENTS
Glass beaker, dried and fined ground
orange peel powder, burette, petriplate,
distilled water, micropipette, waterbath, weighingbalance, incubator, reagents,
swab,
REAGENTS
EXTRCTION:
Acetone-250 ml
Methanol 10 ml
ANTIMICROBIAL ACTIVITY:
Peptone
Agar
Beef
extract
NaCl
Distilled
water
Cultures
of staphylococcus aureus and E.coli
METHODS
Extraction:
COLLECTION OF RAW
MATERIAL
The
raw material taken under examination for the extraction of oleoresin from
orange peel. Orange peels are removed from fresh oranges which are procured
from local market.
METHOD
Oleoresin content of the sample was extracted
by the simplest method.the dry fine powdered orange peel sample was taken in
burette, acetone was added and kept over night. The extract was drained in to a
pre weighed beaker and the solvent was evaporated to dryness. The difference in
weight will give the amount of oleoresin present in the sample.
ANTIMICROBIAL ACTIVITY:
The
antimicrobial activity of oleoresin containing citrus oil extracted from peels
of orange fruits(citrus sinensis) was screened against some medically important
microorganisms. gram-positive bacteria (staphylococcus aureus),gram negative
bacteria(Escherichia choli) were used.
Source of microbial
culture: cultures
were from clinical sources.they were obtained
from the medical microbiology and parasitology laboratory. They are two
strains of gram positive bacteria(staphylococcus aureus) gram negative bacteria
(Escherichia coli) .these are used and tested along with the organisms .the
bacterial cultures were maintained in their respective agar slants at four
degree Celsius through out the course of study and used as the stock culture.
Inoculum standardization:
Medium preparation:
The materials required for the
nutrient agar preparation are peptone, beafextract,NaCl ,agar ,distilled water
1.Weigh
all the ingredient and dissolve in distilled water except agar.
2.
Check the pH and adjust to 7.2 using NaoH or Hcl
3.Dissolve
the agar
4.Prepare
the cotton plug and wrapped it with paper.
5.It
was then sterilized using an autoclave at 15 lbs pressure for 15-20 minutes.
6.The
medium was prepared
SCREENING
OF OLEORESIN FOR ANTIMICROBIAL ACTIVITY:
Screening of oleoresin for antimicrobial
activity was done by the agar well diffusion method .each labeled medium plate
was uniformly seeded with a test
organism by means of sterile swab rolled in suspension and spread over the
plate surface, wells of 5mm in diameter and 2cm apart were punched on the
culture medium. The extract was used to stock and the methanol was used for
diluting the oil and as control. The small amount of oleoresin is taken by
micropipette and were dropped in to well to fullness, each plate was kept in
incubator for 24 hour .zones of inhibition around the wells, measured in(mm)
were used as a positive bio activity.
RESULT&DISCUSSION
DISCUSSION:
On
the basis of simple extraction of oleoresin and to determine the antimicrobial
activity of citrus oil in the oleoresin and following can be inferred.
Extraction
of oleoresin:
The
yield of pectin obtained is highest in turbid extract, but this might be due to
some suspended impurities present in the extract, therefore in the process of
extraction of oleoresin(citrus oil) from the orange peel, It is recommended on
the basis of results obtained.
Antimicrobial
activity:
It
was depicted that oleoresin(citrus oil) had a more consistent antimicrobial
activity when used along with organic solvent(methanol) that the neat
extract(citrus oil). This is probably because methanol had the ability to aid the diffusion of the
extract in the media for the assay. This also corroborates the fact that water
is almost universally the solvent used to prepare herbs.
In addition staphylococcus aureus have the positive activity with citrus oil. This
also shows the importance of methanol in the dilution of the extracts. which
states that some traditional medicines are more
effective when used alone jafari
et al.(2011) showed the antimicrobial activity of essential oil of Citrus
aurantifolia against specific pathogens. According to this study ,essential oil
of c.aurantifolia can inhibit spoilage bacteria and consequently, reduce the
risk of diseases associated with consumption of contaminated products.
previous
studies have shown the various constituents of citrus oil. Veriotti and sacks
2002 carried out a high speed characterization and analysis of peels of five
species of orange oil.orange oil was fount to be more effective antimicrobial
agent than lemon oil.all other conditions being identical,2000 ppm of orange
oil had effect on all the gram positive cultures tested,including the spores of
staphylococcus aureas. gram negative cultures, that is E. coli. The anti
bacterial activity of natural spices on multi drug resistant Escherichia coli
carried out by Rahman et al(2010)also revealed that all bacteria tested were
susceptible to undiluted lime juice. This confirms the fact that natural
species might have antibacterial activity against enteric pathogens and could
be used for prevention of diarrheal diseases.
CONCLUSION
Nagpur
region is well known in central Asia as largest orange producing region .it is
aklso known as the California ,producing excellent quality oranges in large
number though it has great production of oranges ,the down stream processing
and value added product manufacturing
technology is not yet developed .the present work is dedicated for the
development of the part of the process technology needed for the extraction of
value added products that is orange oil(oleoresin) from the orange peel, which
is the waste of orange juice processing industry and orange peel is not use
even biogas plants ,because of the citrus content. The present work revealed
that the sweet orange peels are good source of citrus oil and does have the
potential to become important raw
material for food processing industries.it is found from the experimentation
that the peel source ,for extraction of oleoresin .so it can be concluded that
the process in which orange oil is extracted using the technique of simple
extraction. These results demonstrates the successful extraction of citrus oil,
providing potential benefits for industrial extraction of citrus oil from an economic and environmental
point of view.
This study also demonstrates that
the antimicrobial activity against the oleoresin from the orange peel limonene
has been implicated to be an effective antimicrobial agent .the antimicrobial
activity of orange oil may therefore, be harnessed against medically important
pathogens that are implied in various infections.
BIBILIOGRAPHY
*Martin M.A,siles J.A.I,EI Bari H,Chica
.university of Ibntofail .faculte dos sciences ,kenitra .orange peel:organic
waste or energetic resource.
* Tobias I NdubuisiEzejiofor,N V Eke (2011)
waste to wealth:industrial raw materials
potential of peels of Nigerian
sweet orange.
* Subba,M.S,T.C.Somithri and R.A.O,suryanarayana
,2006.Antimicrobial action of citrus oil
* Trevor .G.2001.Analysis of citrus oils.
*Lamikanra
,A.1999 .Essential microbiology .Second edition AMKRA,Lagos,Nigeria
*Jigna,P,J
Darshana and A Sumithra ,2005.Efficacy of aqueous and methanol extracts of some
Medicinal plants for potential antibacterial
activity.turk
*Text
book of Medicinal and pharmaceutical chemistry.Harkishan singh,V.K.Kapoor
*
Bioactive extracts of orange researchgate.P Benelli,CAS Riehl
*Analysis
of antioxidants from orange juice obtained by countercurrent supercritical
fluid extraction.
*Extraction
separation and identification of volatile organic compounds from paprika
oleoresin JM Guadayol,J Caixach ,J Ribe
- journal of agriculture
1977 –ACS Publication
* Text book of Exraction of natural products
using near-critical solvents- Edited by
M.B.King and T.R.Bott
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