Phytochemical studies of

Phaleria macrocarpa

Nur Shafiqah
 Introduction

 Human diet contain varied
natural mutagens and
carcinogens generated by
oxygen radicals.
 Causes DNA damage and
mutation that leads to:
a.cancer
b.heart disease
 Phaleria macrocarpa uses as
herbal medicine is exploited.
 It has anti-oxidant that
simultaneously act as anti- P.Macrocarpa tree of Thymelaceae family
cancer. (Phaleria macrocarpa,2013).
P.Macrocarpa parts: a)small flower bud, b)green tapering
leaves, c)un-ripened fruit and d)fully grown red fruit. (Altaf
R., et. al, 2013).
 Problem statement:
Phytochemical constituents and effect are still relatively unknown and
incomplete.

 Objective of research:
i. To extract and isolate pure compounds from P.macrocarpa through
column chromatography,thin layer chromatography and recrystallization.

ii. To elucidate the structure of compounds by using FTIR, 1HNMR,

13
C-NMR,GC-MS and CHN analyzer

iii. To evaluate biological activity of:

a) Toxicity
b)Anti-oxidant
c)Anti-inflammatory
d)Anti-bacteria
 Literature review

 Phaleria macrocarpa from family Thymelaceae is
utilized as medicinal plant in Indonesia and Malaysia
(Altaf R. et.al, 2013).

 Methanol extract of P.macrocaroa seeds exhibit active

anti-oxidant activity (Lay M.M. et.al, 2014).

 Mild toxic activity in HepG2 cell lines detected in

chloroform:ethyl acetate and water:acetonitrile fraction
of P.macrocarpa leaves (Yosie A. et.al, 2011).
 Sample & Methodology

 Sample preparation:
-Flowers, stems, leaves and seeds obtained are grounded
into powder.

-Acquire extracts by percolation method with each

powder part of plant weighed 180g mixed with 300ml of
MeOH solvent.

-Refraction: hexane, chloroform, ethyl acetate and

water.
 Isolation and Purification

 Thin layer chromatography (TLC):
- TLC plate dotted with sample solution and placed in
beaker containing solvent. Identify compound by calcuting
Rf.

 Column chromatography (CC):

-sample dissolved in solvent (methanol,ethyl acetate,
chloroform).
-colored bands in column exhibit separation of
compounds
 Recrystallization:
-Impure compound (2g) dissolve in beaker then placed
in ice bath for ‘seeding’process.
-Obtain crystal through vacuum filtratioin and place in
oven until crystals dry completely.
 Sampling:
Flowers, stems, leaves and seeds obtained are
grounded into powder.

Extraction and refraction:

Acquire extracts by percolation method with each powder part of plant

weighed 180g mixed with 300mlof MeOH solvent.

-Refraction: hexane, chloroform, ethyl acetate and water.

Isolation :
TLC and CC

Purification:
Recrystallization process
 Bioassay

 Brine shrimp test for
toxicity

 DppH radical scavenging

activity test for anti-
oxidant.

A.Salina hatched eggs

(Mayorga, P. et. al, 2010).
Brine shrimp test for toxicity
Obtain aerated
saltwater
containing A.salina
eggs and wait until
it hatches
-Dissolve 2mg
MeOH extract and
fractions into 0.5
ml solvent. Place
into separate vials,

10 brine shrimps and 3 ml

Monitor the test
seawater mixed with sample of
samples effect on
concentration(0.50,100,150,200
the larvae.
µg/ml). 24 hours incubation.

Calculate mortality
rate to determine
toxicity.
  DppH radical scavenging activity for anti-oxidant

1ml of MeOH extract

Incubate at room Apply
and other fraction
temperature for 30 spectrophotometer to
react with 3ml of
minutes (dark measure absorpbance
0.01mM DppH
condition). (A) at 517 nm.
solution in methanol.

Refer IC50 as Calculate free radical

concentration of test scavenging activity:
sample. [(A0-A1)X 100%])
 Characterization

 Fourier Transform Infrared Spectrophotometer (FTIR)
Dried 2mg of sample and 200mg KBr is mixed until
pellets are formed.
Data analyzed by comparing absorption frequency
bands in sample spectrum to complementary normal
vibration modes in molecules
 Carbon, Hydrogen and Nitrogen Analyzer (CHN
analyzer)
-2mg-5mg material required for duplicate run
-Mass percentage of CHN elements is determined
  Nuclear Magnetic Resonance Spectroscopy (NMR Spectroscopy)
-1H-NMR and 13C-NMR is utilised with tetramethylsilane (TMS) as
deuterated solvent
-Report chemical shifts with internal standard (CDCL3,)
 Gas-chromatography- Mass Spectrophotometry (GC-MS)
-Ions derived from vaporized sample would be recorded by mass
spectrophotometer.
-Identify components by comparison of retention time with known
standard.
 Expected Outcome

 Compound structure of Phaleria macrocarpa are
obtained and identified
 Biological activities of P.macrocarpa are evaluated
with the phytochemical constituents affecting them.
Work Schedule

 References


Altaf,R., Asmawi, M.Z.B., Dewa, A., Sadikun, A., & Umarm M.I. (2013)Phytochemistry and
medicinal properties of Phaleria Macrocarrpa (Sceff.) Boerls. Extracts. Pharmacogn Rev. 2013,
7(13):73-80. Article ID PMC3731883. Doi: 10.4103/0973-7847.112853
 Altaf,R., Asmawi, M.Z.B., Dewa, A., Sadikun, A., & Umarm M.I. (2013)Phytochemistry and
medicinal properties of Phaleria Macrocarrpa (Sceff.) Boerls. Extracts. Retrieved from:
http://www.phcogrev.com/articles/2013/7/13/images/Phcogrev_2013_7_13_73_112853_22.jpg
 Lay,M.M.,Karsani,S.,Banisalam,B.,Mohajer, S.,and Abd Malek S.N. (2014) Antioxidants,
Phytochemicals, and Cytoxicity Studies on Phaleria Maacrocarpa (Scheff.) Boerl Seeds,”
Biomed Research International, vol.2014, Article ID 410184, 2014. Doi:10.1155/2014/410184
 Phaleria macrocarpa.2013. Retrieved from: http:// garden-
frenzy.blogspot.com/2013/01/phaleria-macrocarpa.html
 Yosie, A., Effendy, M.A., Sfizul, T.M.T., & Habsah, M. (2011) Antibacterial, radical-
scavenging activities and cytotoxicity properties of Phaleria macrocarpa (Scheff.) Boerl
leaves in HepG2 cell lines. Journal of Pharmaceutical Sciences and Research,2,1700-1706.