Free Radical Scavenging Activity and

In vitro Cyclooxygenase-2 AntiInflammatory Assay of Aratiles

Muntingia calabura (Linn.)
in Leaf extract

Philippines top 10 Diseases caused

by Inflammation
180
160
140
120
100
80
60
40
20
0

Rate per 100,000 Population

Rank
World life expectancy,
(2014)

Coronary Heart Disease

5%

5%

26%

6%

stroke
influenze and pneumonia
diabetes

8%

Tuberculosis
Hypertension

10%

19%

16%

Lung Disease
Kidney Disease
Violence

World life expectancy, (2014)

Inflammation
A

series of defensive biological reactions

to harmful agents that leads to pain,

redness, swelling and heat in the affected
areas of the body.
Root

of most diseases or underlying

basis of a significant number of diseases.

Inflammation

Prostaglandins

Cyclooxygenase
1

Leukotrienes

Cyclooxygenase
2

NSAIDS
(Non-Steroidal Inflammatory Drugs)
They

work by inhibiting Cyclooxygenase

enzyme.
Two forms:
COX-1:Protects stomach lining from harsh
acids and digestive chemicals. It helps in
maintaining kidney function.
COX-2: sites of Inflammation

Paired
Electron

BUT
Free radicals
Toxins
UV
rays

Diet

x
Unpaired
electron

STRESS

Drugs

Alcohol

Wrinkles
Immune
Free Radical
damage
Heart
Scavenger
problems
Alzheimers

Free Radicals

www.positivehalth.com
(2011)

Free radical scavenger

It is a vitamin, mineral, or enzyme that is able to destroy free radicals.

They work by preventing the oxidation process.

www.luckyvitamin.com

http://www.esseandco.com

Antioxidant
Enzymes
*Comes from protein and
minerals
-Superoxide dismutase
-Glutathione peroxidase
-glutathione reductase
-catalases
*requires co-factors:
Iron
Copper
Selenium
Magnesium
zinc

Antioxidant
Vitamins
*Vitamin A
*Vitamin C
*Vitamin E
*Folic acid
*Beta-carotene
*Coenzyme Q10

Antioxidant
Phytochemicals

*Carotenoids
*Flavonoids
*Allyl
sulfides
*Polyphenols

Taxonomical Hierarchy
Common name Aratiles
English name: Strawberry Tree
Kingdom: Plantae
Subkingdom: Viridiplantae
Infrakingdom: Streptophyta
Superdivision: Embryophyta
Division: Tracheophyta
Subdivision: Spermatophytina
Class: Magnoliopsida
Sub- perorder: Rosana
Order: Malvales
Family: Muntingiaceae
Genus: Muntingia
Species: calabura

Muntingia calabura Linn.

One of the most extreme fruiting trees.

It is near tropical and has the reputation of thriving with no care in poor soils and does well in
both acid and alkaline locations.

In Philippines, the Strawberry tree is called Aratiles, manzanitas or small apples which is
widely eaten by children out of hand, though it is somewhat sticky to handle.

The flowers is also used to treat headache and incipient cold or as tranquilizers, antispasmodics
and antidyspepticss and antiseptics.Often cooked in tarts and made into jam.

The leaf is considered as a tea like beverage, reduce inflammation and heat.

Muntingia calabura
(Muntingiaceae)

Used to treat
gastric ulcer;
alleviate colds

Antioxidant
property

GENERAL QUESTION:

Can the leaf extract of Muntingia calabura L. exhibit free

radical scavenging and Anti-inflammatory activity?

The Research Questions

1. What are the constituents responsible for the free radical scavenging and antiinflammatory activity of the leaf extract of Muntingia calabura?

2. At what dose is required to illicit the free radical scavenging and antiinflammatory activity of the leaf extract of Muntingia calabura?

3. Is there a correlation between the free radical scavenging activity and antiinflammatory activity?

Collection of Aratiles
(Muntingia calabura L.)

Extraction
(Percolation)
Ethanol
Test for Solvents
Ethyl acetate

Hexane

Phytochemical
Screening

Collection
of Plant
extract

In vitro
cyclooxygenase
activity assay

Free Radical
Scavenging
method

Phytochemical
Screening

HPLC

Test for
Tannins

Test for
Saponins

Rotary
Evaporator

Test for
Flavonoids

In vitro
cyclooxygenase
inflammatory
assay

Cyclooxygenase
kit

Free Radical
Scavenging
method

Hydroxyl
radical
scavenging
activity

Nitric oxide
radical
scavenging
activity

Statistical Analysis

All results will be expressed in mean

Standard error (SEM) One-way analysis of
variance (ANOVA) will be used to compare the
groups. Tukey and LSD will also be used for
post hoc analysis.

Phytochemical
Screening
A.) Test for Tannins
NaCl

1 g of leaf extract

20 mL of distilled
water
(+) results:
Formation of brownish
green or blue black
precipitate indicate
presence of tannins.

FeCl3

B.) Test for Saponins

5 g of leaf
extract

5 mL distilled
water
(+) result:
Formation of stable foam
indicate the presence of
saponins.

Warmed in
Bunsen
burner

C.) Test for Flavonoids

Magnesium

(+) result:
Formation of purple or
red coloration indicate he
presence of flavonoids

Hydrochloric
acid

Nitric Oxide free radical

scavenging
1 mL sodium
nitroprusside

0.1 phosphate
buffer solution

Ethanolic
extract of M.
calabura

1.5 mL Greiss reagent

1% sulphanilamide
2% -0- phosphoric
acid
0.1 % (N-naphthyl)
ethylenediamine
dihydrochloride)

1.5 mL of
reaction
mixture was
removed

546 nm

Incubate at 25
C for 2 hours

HYDROXY RADICAL
SCAVENGING
FeCl3
EDTA
H2O2
2-deoxy-D-ribose

Water bath for

15mins at 95C

1mL TCA
1mL TBA

Mix

Leaf Extract

Heat

Cooled on
ice

Potassium
Phosphate
Buffer (ph 7.4)

Incubate for 1hr at 37C

Centrifuge

Absorbance (532nm)

Performing the Cayman COX Assay

Preparation of the
background values

COX
standard
wells
Background
Wells

Shake the
plate

Addition of
Colorimetric
Substrate

Inhibitor
wells

Addition of
arachidonic
acid solution

Sample Wells

Read the
absorbance
at 590 nm

References

Azmathulla Khan., et al.(2015). Antioxidant and free radical scavenging activities of root extracts
of Muntingia Calabura. Sch. J. App. Med. Sci. pp 2309-2312
Beck, C., (2014). 7 Surprising but Interesting Facts about Antioxidants. Retrieved September 11,
2016, from http://health.allwomenstalk.com/surprising-but-interesting-facts-aboutantioxidants
Bodke,Y.D., et al. (2013).Antioxidant and in vivo anti-hyperglycemic activity of Muntingia
calabura leaves extracts
Khan,A., et al.(2015). Antioxidant and free radical scavenging activities of root extracts of
Muntingia Calabura. Sch. J. App. Med. Sci. pp 2309-2312
Preethi,K., et al.(2012). Anti-inflammatory of Muntingia calabura Fruits. Pharmacognosy Journal.
Vol. 4, Issue 30, pp 51-55
Premakumari,K.B., et al.(2010). Antioxidant Activity and Estimation of Total Phenolic Content of
Muntingia Calabura by Colorimetry. Int.J. ChemTech Res. Vol.2, No.1, pp 205-208
Sani, M. H., et al.(2012). Antinociceptive Activity of Methanol Extract of Muntingia calabura
Leaves and the Mechanisms of Action Involved. Evidence-Based Complementary and
Alternative Medicine. Vol. 2012
Yadav, D. Bodke., et al.(2013).Antioxidant and in vivo anti-hyperglycemic activity of Muntingia
calabura leaves extracts