meens ppt

Evaluation of alkaloid extracted
from carica papaya as Platelet
Stimulator
Presented by: Miss. Meena C. Thale
Under the guidance of Mrs. Tambe V. S.
Associate professor pharmaceutical chemistry
P.E.S.’s Modern college of pharmacy (for ladies)

CONTENTS
• AIM AND OBJECTIVES
• INTRODUCTION
• EXPERIMENTAL WORK AND DISCUSSION
• RESULT
• CONCLUSION
• FUTURE SCOPE

AIM :
To isolate alkaloid from carica papaya leaves
and to evaluate its effect on platelet count.
OBJECTIVES :
• To compare activity of aqueous and
methanolic extract of carica papaya leaves.
• To perform phytochemical screening of
aqueous extract of carica papaya leaves.
• To isolate alkaloids and evaluate its effect on
platelet count.

Introduction :
DENGUE FEVER:
• Spread through bite of Aedes egypti mosquito
carrying flavivirus.
• Flavivirus is divided into 4 types: DEN-1, DEN-
2, DEN-3 and DEN-4.

TRANSMISSION
•Infected person → mosquito → person
•From organ or blood donation
•From mother to child during pregnancy
RISKS
• Babies and young children than adults
• Women than men
• People with chronic disease like diabetes and asthma

MECHANISM
• In severe conditions, virus
production is increased →
affect liver and bone marrow
• Capillary permeability → fluid
from bloodstream leaks into
body cavity → low B.P.
• Dysfunction of bone marrow
→ decreased no. of platelets
( necessary for blood clotting )
→ increase risk of bleeding

SYMPTOMS: 4-10 days after the bite of infected
mosquito
• Sudden, high fever
• Severe headaches
• Pain behind the eyes
• Severe joint and muscle pain
• Nausea and Vomiting
• Skin rash
• Mild bleeding (such a nose bleed, bleeding gums, or easy
bruising)
• Severe abdominal pain
• Problems with your lungs, liver and heart

DIAGNOSIS
• Isolating the virus by collecting serum sample
• Detection of specific antibodies by ELISA from
serum
• PCR- for detecting viral genomes sequence from
serum & CSF
• Tourniquet test
• Thrombocytopenia ( i.e. Platelets < 1,00,000/mm3)

TREATMENT
• No known antiviral drugs or injections
• Antipyretics such as paracetamol & analgesics can be
used
• Papaya leaves, kiwi to increase platelet count
• Plenty of fluids can be given because dehydration occurs
• Drugs such as aspirin & ibuprofen should be avoided as
they may increase the risk of hemorrhage.

VACCINES : SANOFI PASTEUR VACCINE
• Live attenuated tetravalent vaccine
• It is in phase Ш clinical trials
Challenges to vaccine development
• Secondary heterotypic infection → increased risk of
severe disease
• Hence, vaccine development focuses on tetravalent
vaccine generation against all serotypes

PREVENTION
• Stay away from heavily populated residential areas, if
possible.
• Use mosquito repellents
• Wear long-sleeved shirts and long pants tucked into
socks.
• Use mosquito nets.
• Get rid of places where mosquitoes can breed such as old
tires, cans or flower pots that collect water.
• Bleaching powder may be used in water sources that are
not meant for drinking, as it prevents the development of
mosquito eggs
• If you have symptoms of dengue, speak to your doctor.

Use of carica papaya for treatment of
dengue fever

Carica papaya
• Classification
• Kingdom – Plantae
• Order - Brassicales
• Family - Caricaceae
• Genus - Carica
• Species - C. Papaya

NUTRITIVE VALUE OF PAPAYA
1. Proteolytic Enzymes
Eg. Papain & Chymopapain
Uses:
• In the treatment of slipped disk
• Lower inflammation and improve healing from
burns
2. Carpaine
Uses:
• Reduces blood pressure.
• It is able to kill worms and amoebas.

3. Lycopene
Uses:
• anti-cancer
• key intermediate in biosynthesis of beta-carotene
and xanthophylls.
4. Fibrin
Uses:
• Reduces the risk of blood clots
• Improves the quality of blood cells
• lmportant in preventing stroke

Pharmacological activity of each part
of papaya
LEAF:
• Treatment of dengue fever
• Cancer cell growth inhibition
• Anti-malarial activity
• Facilitates digestion
• As an acne medicine
• Increase appetite
• Meat tenderizer
• Relieves nausea

FRUITS:
• Laxative
• In indigestion
• Avoid heart attack or stroke
SEEDS:
• Nephro-protective activity
• Anti-bacterial activity
• Detoxification of liver
• Anti-helminthic and anti-amoebic property

PEELS:
• Soothing effect
• In sunscreen preparations
• Fights dandruff
• Muscle relaxant
ROOTS:
• Juice is used in urinary troubles
LATEX:
• Degumming of natural silk
• Meat tenderizer
• Lowering of blood pressure

1) Ikeyi Adachukwu (et al 2013): concluded that Carica papaya
leaves contain Alkaloid, Saponin, Tannin, Glycoside and
Flavonoids Also, from the result, it can be deduced that
phytochemicals can be extracted more when the leaves are dried
and pulverized to powered form.
2) Debarati Guha-Sapir (et al, 2005): reviewed the changing
epidemiology of the disease, focusing on host and societal
factors and drawing on national and regional journals as well as
international publications
3) Aravind. G (et al, 2013): carried phytochemical screening of
carica papaya leaf and concluded that Carica papaya is a
neutraceutical plant having a wide range of pharmacological
activities. All the prominent pharmacological activity, home
remedies and side effects of Carica papaya were studied.
Review of literature

Experimental work & discussion
• Sample collection:
leaves were collected
Washed (to remove dirt)
Dried in sunlight (1 week)
Pulverised to powder
Stored in tightly closed glass bottle

 Sterilization of Glass Wares
- Glass ware were washed with detergent, rinsed with
distilled water and air dried. They were also sterilized
on a hot air oven.

• Extraction of sample
pulverised sample( 10 g) + 20 ml water and
ethanol
Stirred
Allowed to stand for 24 hrs
Filtered
Filtrate was exposed to air to obtain concentrated
extract
Stored in a clean bottle

Phytochemical screening of papaya leaves
aqueous extract
MATERIAL REAGENT COLOUR CHANGE CONFIRMATION
Carica papaya extract
( 3 ml)
Mayer’s
(0.5 ml)
Cream red ppt. Positive
( 3 ml)
Wagner’s
(0.5 ml)
Brown ppt. Positive
( 3 ml)
Dragendroff’s
( 0.5 ml )
Reddish brown Positive
(2 ml)
5% FeCl3
(0.5 ml)
Greenish Positive
Carica papaya extract Lead acetate
(0.5 ml)
White ppt. Positive
QUALITATIVE TEST FOR ALKALOID: 1 ml 1% HCl + 3ml extract → shake and filter
QUALITATIVE TEST FOR TANNINS

(2 ml)
NaOH (2 drops) +
H2SO4
Yellow ppt. Positive
( 2 ml)
Distilled water
( 3 ml)
Persistent foam after
shaking vigorously
Positive
(3 ml)
Fehling solution (A+B)
(2 ml each)
and boiled
Brick red ppt. Positive
QUALITATIVE TEST FOR FLAVONOIDS
QUALITATIVE TEST FOR SAPONINS
QUALITATIVE TEST FOR GLYCOSIDES
Carica papaya extract Distilled water, H2SO4 ,
KOH and Fehling’s
solution
Brick red ppt. Positive

Separation of alkaloid from aqueous
extract
• Preparation of mobile phase for alkaloid
Toluene : ethylacetate : diethylamine (7:2:1) was
taken in a beaker and covered with a petri plate
for 10 minutes for saturation
Separation technique
Aqueous extract spot on plates
Air dried ( 10 mins)
Kept in mobile phase for development

Air dried
dragendroff’s reagent was sprayed
Spots were obtained
Spots were scrapped out
Put into methanol
Filtered
Filtrate was air dried ( to evaporate methanol)
Alkaloid was collected

Acute oral toxicity testing
• Acute oral toxicity testing was done according to
TG 423 guidelines.
• no animal mortality was observed.

ANIMAL TESTING BY ADMINISTRATION OF ISOLATED ALKALOID
Determination of platelet count (before dosing)
6 healthy rats were weighed and blood sample was collected from
the rats. Platelet count was determined and reported.
Administration of dose to rat
The isolated alkaloid was taken and dose was prepared with
distilled water. It was administered to the rats by oral route.
Determination of platelet count (after dosing)
After 24 hours, the platelet count of the rats was measured and
reported.

Result:
Before treatment After treatment
Sr. No (Platelets /mm3) (Platelets /mm3)
1 40,000 1,26,000
2 45,000 1,32,000
3 42,000 1,29,000
4 38,000 1,20,000
5 53,000 1,33,000
6 47,000 1,28,000
mean 44,166 1,28,000

The paired student t test was used and the readings were mentioned as mean±SEM
platelets/mm3
Mean in lakhs ± SEM
Before treatment 0.4416 ±0.022
After treatment 1.28 ±0.019
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Before treatment After treatment
Plateletcount(lakhs)
***
P value is less than 0.0001 it is considered extreme significant which shows
significant increase in platelet count by the treatment with alkaloid separated from
carica papaya

CONCLUSION
• The aqueous extract of carica papaya leaves has
shown the presence of glycosides, tannins,
alkaloids, saponins, flavonoids.
• The alkaloid has shown 2.8 times increase in
platelet count (from 44,166/ mm3 to 1,28,000/mm3
• The carica papaya leaf posses platelet count
increasing activity and can be used in the treatment
of dengue fever

FUTURE SCOPE
• Further work can be performed in human
volunteers.
• HPLC
• Formulations of the aqueous extract can be
prepared.
• Potential effect of other phytoconstituents on
platelet count can be evaluated.

Reference
1. P.B. Ayoola & A. Adeyeye (2010), Phytochemical and nutrient
evaluation of carica papaya (paw paw) leaves, page no 326
2. H. Wagner, S. Bladt(1996), plant drug analysis, A thin layer
chromatography atlas, 2nd edition, page no. 5-6.
3. Khandelwal K. R. (2008), practical pharmacognosy:
techniques and experiments, 19th edition, page no. 149-156.
4. http://en.wikipedia.org/wiki/Dengue_fever
5. http://www.medindia.net/patients/patientinfo/dengue.htm
6. http://www.webmd.com/a-to-z-guides/dengue-fever-reference
7. http://www.medindia.net/patients/patientinfo/dengue.htm
8. http://en.wikipedia.org/wiki/Dengue_vaccine

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