A Project report on,
“PHYTOCHEMICAL SCREENING AND
Anticancer Activity of MIA PAC-2 Citrus
Aurantifolia leaveS Extract “
By
Miss. Ashwini.Mudhale
Reg no:P04AW22S032002
Under the Supervision of
Dr.Soul Shekhar Pandit MSc.B.Ed.Ph.D
Department of Chemistry, Guru Nanak
CONTENTS
Introduction to Medicinal plant
Introduction to Citrus Aurantifolia
Collection and Extraction
Pharmacological activites
Phytochemical Screening
Anticancer Activity of (MIA PACA-2)
Cell line Citrus Aurantifolia
Result
Discussion
Conclusion
CHAPTER-01
INTRODUCTION TO
MEDICINAL PLANT
INTRODUCTION TO MEDICINAL PLANT:
•Medicinal plants, also called medicinal herbs, have been
discovered and used in traditional medicine practices since
prehistoric times.
• Plants synthesize hundreds of chemical compounds for
various functions, including defense and protection against
insects, fungi, diseases, and herbivorous mammals.
•Medicinal Plant Like Holy Basil,Calendula, Aloe Vera,Sage,
Fenugreek.
CHAPTER-02
INTRODUCTION OF
CITRUS AURANTIFOLIA
INTRODUCTION
Citrus aurantifolia is an important medicinal and food plant
widely cultivated in many parts Of the world. It is valued for
its nutritional qualities and numerous health benefits. The
plant is used in traditional medicine as an antiseptic,
antiviral, antifungal, anthelmintic, astringent, diuretic,
mosquito bite repellent, for the treatment of stomach
ailments, constipation, headache, arthritis, colds, coughs,
sore throats and used as appetite stimulant. These health
benefits of Citrus aurantifolia are associated with its high
amounts of photochemical and bioactive compounds such
as flavonoids, limonoids, Phenols, carotenoids minerals and
vitamins. This review examined the domestic, ethno
Scientific Classification
Kingdom Plantae
Clade Tracheophy
Clade Angiosper
m
Clade Eudicots
Order Rosids
Family Rutacease
Genus Citrus
CHAPTER-03
COLLECTION AND
EXTRACTION PROCESS
Collection and Extraction of Citrus Auratifolia
Insertion
Extraction
Working of Soxhlet Extractor
Apparatus
Collection of plant:
The leaves of Citrus Aurantifolia was collected in around our GNFG
college parking lot, in the month of May and immediately spray with
alcohol so that the leaves are free from fungus. The collected leaves
were authenticated at the department of botany GNFG college Bidar.
Extraction of plant:
The leaves of Citrus Aurantifolia were crushed into small pieces and
dried under shade which were successive extracted with the various
organic solvents vizn-hexane, chloroform and methanol from nonpolar
to polar in the Soxhlet extractor 28.325gm of leaves was inserted and
organic solvent n-hexane [1ltr] was added and allowed it to run for
48hours successively with Chloroform [1ltr], Methanol [1ltr] one after
the other.
CHAPTER-04
PHYTOCHEMICAL
ACTIVITIES and
SCREENING
• Phytochemicals (from Greek phyto, meaning
"plant") are chemicals produced by plants
through primary or secondary metabolism.
• They generally have biological activity in
the plant host and play a role in plant
growth or defense against competitors,
pathogens, or predators.
Table: Phytochemical Investigation of Crude Extract Citrus Aurantifolia
Sl.NO. TESTS n-HEXANE CHLOROFORM METHANOL
1 Alkoloids +ve -ve +ve
Mayers test
2 Steroids
a) Salkowski test -ve +ve +ve
-ve -ve -ve
b) Lieberman Burkhard
test
3 Terpenoids +ve +ve +ve
a) Lieberman Burkhrard
test
4 Glycoside
a)Killer-kill Anti test -ve -ve -ve
b)Bromine Water Test -ve -ve -ve
5 Flavonoids
a) Ferric Chloride Test -ve -ve +ve
b) Lead Acetate Test -ve -ve -ve
6 Saponins
a)Foam Test +ve -ve +ve
7 Carbohydrate
a)Moliseh’s Test -ve -ve -ve
8 Proteins
a)Xanthoproteic -ve -ve -ve
CHAPTER-05
ANTICANCER ACTIVITY
(MIA PACA-2) CELL LINE
CITRUS AURANTIFOLIA
LEAVES EXTRACT
CANCER:
Cancer is Caused By a Combination of Genetic Factors And
External Agents, Including
Genetics :
Cancer is a genetic disease that occurs when genes that control
cell growth and division are altered.
These changes can be inherited fromparents occurs as cells
divide.
External Agents :
These include:
Physical carcinogens : These includes ultraviolet and ionizing
radiation, such as x-rays and radon
Chemical carcinogens : These include asbestos,components of
tobacco smoke, alcohol,aflatoxin,and arsenic.
Anticancer Activity:
In past decades, anticancer research has led to remarkable results despite many of the approved
drugs still being characterized by high systemic toxicity mainly due to the lack of tumor selectivity
and present pharmacokinetic drawbacks, including low water solubility, that negatively affect the
drug circulation time and bioavailability .
MIA PACA-2:
It is a Human Pancreatic Cancer cell Line used extensively in pancreatic cancer research and
therapy development. In 1977, MIA PACA-2 cell were derived from the carcinoma of a 65-year old
male. The cells exhibit Ck5.
MTT Assay:
Experimental procedure:
Cells were incubated at a concentration of 1 X 10*cells/ml in culture medium for 24 h at 37C and
5% CO2.
Cells were seeded at a concentration (7Oul)_10*cells/well in 100 ul culture medium and 100ul sample of
LM series in (1000ug/ml) into micro plates respectively (tissue culture grade, and 96 wells).
Control wells were incubated with DMSO (0.2% in PBS) and cell line. All samples were incubated in
triplicate. Controls were maintained to determine the control cell survival and the percentage of live cells
after culture.
Cell cultures were incubated for 24 h at 37°C and 5% CO2 in CO2 incubator (Thermo scientific
BB150)
After incubation, the medium was completely removed and Added 20 ul of MTT reagent (5mg/min PBS).
After addition of MTT, cells incubated for 4 hrs at 37°C in CO, incubator.
Observed the wells for formazan crystal formation under microscope. The yellowish MTT was reduced to
dark colored formazan by viable cells only.
After removing the medium completely. Added 200ul of DMSO (kept for 10 min) and incubate
at 37 'C (wrapped with aluminium foil).
Triplicate samples were analyzed by measuring the absorbance of each sample by a microplate reader
(Benesphera E21)at a wavelength of 550 nm.
CHAPTER-06
RESULT AND DISCUSSION
Result:
Sl. Name of the Average IC50 Standard Concentration
No. Compound Deviation
1 CAH 110.5 ± 0.84 400-3.12µM
2 CAC 89.92 ±0.05 400-3.12µM
3 CAM 40 ±05.47 400-3.12µM
4 Doxorubicin 28.00 ± 05.27 400 – 3.12uM
Anticancer activity of various extract of Citrus aurantifolia Leaves against Mia Paca-2 Cancer cell
line. CAH: Citrus aurantifolia n- hexane extract, CAC: Citrus aurantifolia chloroform extract,
CAM: Citrus aurantifolia methanol extract.
The results in above Table shows that activity of Citrus aurantifolia leaves acts as
anticancer agent which is as illustrated in the Table, the methodology used to carry
out the anticancer activity is by MTT assay. The most efficient cytotoxic activity of
Citrus aurantifolia leaves extract was against Mia Paca-2 cell line with IC50 =
40.00 is methanol extract followed by chloroform extract with IC50 = 89.92 and n
hexane extract with IC50 = 110.5. The methanol extracts is relatively more potent
than other solvent extracts when compared with the standard doxorubicin with
IC50 = 28.0 which is found to be nearly equal effective against the cancer cell line
Mia Paca-2with minimum deviation of ± 5.27, all the cell viabilities are carried
with 400 – 3.12uM concentration.
Discussion:
Cancer is regarded as one of the most significant public health- issue and
medicinal plant are now considered as natural source of anticancer medicine
The plant material has active phytoconstituents which are known as
medicinally valuable give promising results for various disease. Our study
focused on evaluating the cytotoxic effect of Citrus aurantifolia leaves on
pancreatic cancer cell lines Mia Paca-2 using various solvent extracts viz. n-
hexane, chloroform and methanol. As per the result is concerned, the
methanol extracts is relatively more potent with IC50 = 40.00 than other
solvent extracts when compared with the standard doxorubicin with
IC50 = 28.0 which is found to be nearly equal effective against the
cancer cell line Mia Paca-2 with minimum deviation of ± 05.47 for n-
CHAPTER-07
CONCLUSION
Conclusion:
The present experimental study revealed that MTT assay is
one of the most widely used method for evacuating
preliminary cancer activity of natural produce extracts. Based
on the result and discussion we conclude that the Citrus
aurantifolia leaves are considered as best remedy for
inhibiting the cancer cell. The presence of various
phytoconstituents in citrus aurantifolia methanol extract may
act as important source of drug against Mia Paca-2 cell lines
and further study is also required for isolating the particular
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