PharChem Manuscript (Bixa Orellana)
PharChem Manuscript (Bixa Orellana)
PharChem Manuscript (Bixa Orellana)
PHYTOCHEMICAL ANALYSIS OF
Bixa orellana
A Research Paper
Presented to the
Faculty of Pharmacy
University of Santo Tomas
In Partial Fulfillment
Of the Requirements of the Degree
Bachelor in Pharmacy
by
ALAVA, PAUL JAMES AMBIDA
ALCAUSIN, DENISE ANNE REYES
ANDAL, MARY IRIS MENDOZA
BAGON, NICOLE EILEEN MONTALES
BARRETTO, DANIELLE PARAS
BAUTISTA, CALVIN EJ ROBLEDO
November 2014
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ABSTRACT
This study centers on the different phytochemicals present in
Bixa Orellana, commonly known as Achuete. It is a native of tropical
America and is also cultivated and naturalized in other tropical and
subtropical countries. Achuete is considered a pantropic plant. Its
leaves are known to be an entire-ovate, with a length of 8 to 20 cm,
and a width of 5 to 12 cm. The researchers used the leaves, which
were collected from Laguna, Philippines, in the belief that this part of
the plant contains more constituents than the others. The leaves
were dried in open air, were grinded to fine particles, and were
subjected to percolation. The extract obtained underwent different
phytochemical tests in order to obtain knowledge about the different
phytochemical
constituents
present
in
Bixa
orellana.
These
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TABLE OF CONTENTS
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Abstract
1. The Problem Rationale
1.1 Introduction
1.1.1 Background of the Study
1.1.2 Statement of the Problem
1.1.3 Objectives
1.1.4 Significance of the Study
1.1.5 Theoretical Framework
1.1.6 Scope and Limitations
1.1.7 Definition of Terms
1.2 Research Impediments
2. The Research Questions
2.1 Literature Review
2.1.1 About Plant
2.1.1.1
Plant Name
(Synonyms/Vernacular Names)
2.1.2 Botanical Descriptions
2.1.2.1
Taxonomical Classification
2.1.2.2
Botanical Description
2.1.2.3
Chemical Composition
2.1.3 Ethnopharmacologic survey
2.1.4 Pharmacologic activities
2.1.4.1
Anti-convulsant activity
2.1.4.2
Analgesic activity
2.1.4.3
Antidiarrheal activity
2.2 Research Question
3. The Research Methods
3.1 Preparation of Stock Plant Extract
3.2 Percolation Setup
3.3 Methodology and Schematic Diagrams
4. Results and Discussion
4.1 Alkaloids
2
5
6
7
7
7
8
9
10
11
11
12
12
12
13
13
14
14
14
15
16
17
18
19
34
4.2
4.3
4.4
4.5
4.6
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Cardiac Glycosides
Anthraquinones
Flavonoids
Tannins
Cyanogenic glycosides
37
39
41
42
44
46
46
48
49
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CHAPTER 1
THE PROBLEM RATIONALE
1.1 Introduction
1.1.1 Background of the study
Plants, specifically medicinal plants, are of great importance in the
broad field of medicine, especially in the pharmaceutical industry.
These medicinal plants are considered as rich resources of ingredients
which can be used in drug development and synthesis. Besides that
these plants play a critical role in the development of human culture
around the whole world, they also contain active constituents or
phytochemicals, which cause various physiologic and pharmacologic
actions on the human body. These bioactive compounds can be
classified
into
alkaloids,
saponins,
tannins,
cardiac
glycosides,
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some alkaloids in their salt forms are used as stimulants, but most
alkaloids are powerful poisons. Flavonoids are known for their
antioxidant properties.
The study focuses on Achuete or Bixa orellana, generally known in
developing countries as folk medicine for the treatment of common
infections in the form of decoctions, teas, juices, etc. The leaves of
the
plant
selected
for
the
study
was
subjected
to
different
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time, there is always the need and desire to find better, safer and
more clinically effective sources of drugs.
One of the ever-present concerns of the pharmaceutical industry is
how we can improve the quality, efficacy, and safety of these
products,
despite
the
different
medical
and
pharmaceutical
PAGE
tannins,
cardiac
glycosides,
flavonoids,
and
PAGE
the
selected
phytochemical
screening
tests
used
in
the
experiment.
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10
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11
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CHAPTER 2
12
PAGE
13
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14
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15
cryptoxanthin,
lutein,
zeaxanthin
and
methyl
bixin.
In
PAGE
16
activity
was
monitored
using
the
PAGE
17
noxious
endogenous
substances,
including
serotonin,
activity.
using
Evaluation
castor
of
antidiarrheal
oil-induced
diarrhea
activity
was
model
and
PAGE
Methanolic
extract
of
B.
Strychnine
induced mices
nt
2006
Acetic
Analgesic
orellana
Methanolic
extract
of
B.
acid
induced mices
2006
orellana
Methanolic
extract
of
Castor
B.
oil
induced mices
orellana
2.2 Research Question/s (or Hypotheses)
al. 2006
18
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CHAPTER 3
THE RESEARCH METHODS
19
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extracts
were
evaporated
spontaneously
in
large
evaporating dishes until a thick, syrupy liquid remained. This was the
20
PAGE
Receiver
21
PAGE
22
PAGE
23
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24
Test
tube
served
as
the
control.
Two
drops
of
PAGE
Mayers reagent was added to test tube C. The color reaction was
observed and recorded.
25
PAGE
26
PAGE
27
PAGE
reaction at the interface of the acid and aqueous layer was observed
and recorded.
28
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29
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3.3.3 Anthraquinones
The materials used were a 250-mL beaker, a filter paper, a funnel,
a separatory funnel, a test tube, a hot plate, litmus paper, a dropping
pipette, and a 20-mL graduated cylinder.
3.3.3.1 Borntragers Test
Ten milliliters of distilled water and 6 mL of crude extract was
placed in a beaker, mixed then filtered. Aqueous filtrate was collected
and the residue discarded. The filtrate was extracted thrice with 5 mL
portions of benzene in a separatory funnel. Benzene extracts were
combined and divided into 2 portions. Test tube A served as the
control while 5 mL ammonia solution was added to test tube B. The
color reaction was observed and recorded.
30
3.3.3.2
PAGE
31
PAGE
served as control while 5mL ammonia solution was added to test tube
B. The color reaction was observed and recorded.
32
PAGE
35-40 oC in a water bath. Any color change in the picrate paper was
observed.
3.3.5. Flavonoids
The materials used were test tube, dropping pipette, filter paper,
funnel, and 10-mL graduated cylinder.
3.3.5.1 Preparation of Sample
Six milliliters of hexane and 3 mL of water were added to 6 mL of
plant extract placed in a test tube. The upper hexane layer was
pipetted and discarded while 5 mL of 80% ethyl alcohol was added to
the defatted aqueous layer. The solution was filtered and divided into
3 portions. Test tube A served as control.
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34
PAGE
the solution was diluted with equal volume of water and 1 mL octyl
alcohol. The color change was observed.
3.3.6. Tannins
The materials used were filter paper, test tube, dropping pipette,
evaporating dish, 250-mL beaker, hot plate, and 20-mL graduated
cylinder.
3.3.6.1 Preparation of Sample
Twenty milliliters of hot distilled water was added to 6 mL of
extract. Five drops of 10% sodium chloride solution was also added
then filtered. The filtrate was divided into 3 portions. Test tube A
served as control.
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36
PAGE
37
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CHAPTER 4
RESULTS AND DISCUSSION
38
Name of Test
Positive Result
A. General Tests
Dragendorffs
Experiment
Inferenc
Result
Orange
Test
precipitate
Mayers Test
White
PAGE
(-)
precipitate
Light brown
solution
B. Primary Assay
Dragendorffs
Orange
Test
precipitate
Mayers Test
White
(-)
precipitate
Light brown
solution
C.
Confirmatory Test
39
Dragendorffs
Orange
Test
precipitate
PAGE
(-)
Orange yellow
solution
Mayers Test
White
(-)
precipitate
Light yellow
solution
D.
Quaternary Bases
Dragendorffs
Orange
Test
(-)
precipitate
White
(-)
precipitate
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PAGE
Alkaloid-precipitating
reagents
Dragendorff's,
which
alkaloids.
Negative
results
were
obtained
from
the
41
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4.2.1 Description
Cardiac glycosides are glycosides, which consist of a lactone
ring, a steroid nucleus and a sugar moiety. They are classified
according to their sugar moiety: cardenolides (5-membered ring)
and bufadienolides (6-membered ring). Cardiac glycosides are also
called cardiotonic glycosides because of their pharmacologic action
on the heart and are used for the treatment of congestive heart
failure and cardiac arrhythmia.
4.2.2 Results
Name of Test
Positive
Experiment
Result
Result
Reddish
Inference
(+)
brown color
which may
turn blue or
purple
Dark brown
solution with
purple precipitate
Liebermann
Blue to
Burchard Test
green, red,
(-)
pink, purple
or violet
Light yellow
solution
42
Kedde's Test
Blue violet
PAGE
(-)
coloration
2 layers: Red
brown solution and
Light yellow
solution with oil
droplets in the
middle
4.2.3 Discussion
Cardiac glycosides are insoluble in non-polar solvents, thus
hexane was used to defat the sample from its non-polar portion like
chlorophyll. The Keller-Killiani test, which was conducted to test for
the presence of deoxy sugar, yielded a positive result with the
formation of a purple precipitate. However, both Liebermann
Burchard test, which was performed to determine the presence of
unsaturated sterol group, and Kedde's test, which was carried out to
test for the presence of unsaturated lactone, yielded negative
results with the absence blue/green coloration and blue-violet
coloration, respectively. Nevertheless, the Liebermann Burchard test
determined the presence of a saturated sterol group, with the light
yellow coloration of the solution.
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PAGE
Even though the sample did show a positive result to one test, it
cannot be concluded that it contains cardiac glycosides.
4.3 Anthraquinones
4.3.1 Description
Anthraquinones are glycosides, which are soluble in dilute
alcohol and boiling water that gives a characteristic red, violet, and
green color with a base. They are orange-red compounds that are
used as dyeing agents. Anthraquinones are important in the
pharmaceutical industry for their cathartic/laxative effect. There are
5 types of anthraquinones: anthraquinone, anthranol, dianthrone,
oxanthrone and aloin type.
4.3.2 Results
Name of Test
Positive
Experiment
Result
Borntrager's Test
Result
Red
coloration in
(No photo
the lower
available)
(++)
ammonical
layer
Inference
44
Modified
Pink color
PAGE
(+)
Borntrager's Test
2 layers: Light
yellow and Red
orange with oil
droplets
4.3.3 Discussion
The sample was defatted using benzene, a non-polar solvent.
The Borntrager's test, which is a test for the presence of an Oglycoside or a free anthraquinone, yielded a double positive result
with a red ring layer on the lower ammoniacal layer. The Modified
Borntrager's test, which is a test for the presence of O-glycosides or
very stable types of antraquinones, also yielded a positive result
with a red-orange coloration. The two tests confirmed the presence
of anthraquinones in the Bixa orellana.
4.4 Flavonoids
4.4.1 Description
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PAGE
Positive
Experiment
Result
Bate-Smith &
Result
Strong red or
Metcalf Test
violet color
Inference
(-)
Greenish brown
solution
Wilstater
Color ranging
"Cyanidin" Test
from orange
(-)
to crimson
and magenta
and
occasionally
to green or
blue
2 layers of light
brown and dark
brown solution
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47
4.4.3 Discussion
The sample was defatted with the use of a non-polar solvent,
hexane, like in cardiac glycosides. The Bate-Smith and Metcalf's test
was conducted to test for the presence of leucoanthocyanins. The
acidification of the extract did not yield a strong red or violet color,
thus denying the presence of leucoanthocyanins. The Wilstatter or
Cyanidin test, which identifies the presence of -benzopyrone
through the acidification and reduction of flavonoids, also yielded
negative results, without the orange to crimson and magenta
decoloration of the solution.
4.5 Tannins
4.5.1 Description
Tannins are polyphenolic compounds, which are able to combine
with protein of animal hides that prevents them from putrefaction
and convert them into leather. They are pale-yellow to light brown in
color and are amorphous substances, which are slightly acidic due to
the presence of the phenolic portion. They are classified according
to
their
phenolic
nuclei:
hydrolyzable,
non-hydrolyzable
or
PAGE
4.5.2 Results
Name of Test
Positive
Experiment
Result
Gelatin Test
Result
Formation of
Inference
(+++)
a jelly
precipitate
Yellow solution
with jelly
precipitate
Ferric chloride
Blue-black
Test
(hydrolysabl
(+++)
e tannin)
Brownish
green
(condensed
tannins)
Matchstick Test
Wood will
(-)
turn red or
(No photo
pink in color
available)
Dark green in color
4.5.3 Discussion
48
PAGE
treated
with
acids
and
enzymes,
are
converted
or
49
PAGE
anti-neoplastics,
sedatives,
and
expectorants
to
the
Positive
Experiment
Result
Guignard's Test
Result
Appearance
Inference
(-)
of various
shade of red
within 15
minutes
Yellow brown
coloration of the
strip
4.6.3 Discussion
Chloroform was added to the crude extract to free the
cyanogenic glycosides from the sample. The test conducted to test
for the presence of cyanogenic glycosides was Guignard's test.
Guignard's test is a test for cyanophores. However, this test is nonspecific for there are other substances, which can liberate H 2S, SO2
50
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CHAPTER 5
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
Research showed that Bixa orellana contains alkaloids, flavonoids,
tannins and glycosides. Following the phytochemical screenings
conducted for each plant constituent, the plant extracts obtained
from
the
leaves
of
Achuete
(Bixa
orellana)
contained
only
51
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of the plant utilized in the study from the plant in the reference. The
phytochemicals in the plant may also have been damaged or lost
during air-drying or during the experiments, and only negligible
amounts of alkaloids, flavonoids, cardiac glycosides, and cyanogenic
glycosides were present in the extract, requiring more sensitive tests.
5.2 Recommendations
The researchers have recommendations that could improve
further the study. In removing the methanol from the plant extract,
one should completely evaporate the methanol in a water bath to
dryness. This must be done to gather concentrated crude extract
needed for the phytochemical screenings and to avoid errors that
could be caused by the presence of methanol. Once through with the
percolation, the crude extracts must be stored in a cool place, as to
prevent the acquisition of molds. Other phytochemical screenings
should also be conducted, like the hemolysis test for saponins, so as
to gain more knowldge regarding the phytochemical compounds
found in Bixa orellana.
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REFERENCES
Delhi, India.
Clements, J. (2005). Antimicrobial Agents and
de Janeiro, Brazil.
Deshmukh, S. (2013). Pharmacognostical and Phytochemical
Investigation of leaves of Bixa orellana Linn. International
Journal of Pharmaceutical Sciences Review and
Research,Volume 22(Issue 1), 247-252. Retrieved November 3,
53
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2014, from
http://www.globalresearchonline.net/journalcontents/v22
1/45.pdf
Elias, M. (2006). Mineral Nutrition, Growth and Yields of Tropical
Medicinal Plants.
Shilpi, J., et al. (2006). Preliminary pharmacological screening of
Bixa orellana L. leaves. Journal of Ethnopharmacology, Volume
108(Issue 2), 264-271. Retrieved November 3, 2014, from
http://www.sciencedirect.com/science/article/pii/S03788741060
02571
Stuart, G. (2013, October 1). Achuete. Retrieved November 3,
2014, from http://www.stuartxchange.org/Asuete.html
Curriculum Vitae
54
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Educational Background
Secondary: Batangas State University (2008-2010)
Sovereign Shepherd School of Values and Learning (20102012)
Tertiary: University of Santo Tomas (2012-present)
Curriculum Vitae
55
PAGE
Address: Santiago St., Town & Country West, Molino III, Bacoor City,
Cavite
E-mail: [email protected]
Educational Background
Secondary: Divine Light Academy (2008 - 2012)
Tertiary:University of Santo Tomas (2012 - Present)
Curriculum Vitae
56
Educational Background
Secondary: St. Bridget College ( 2008 - 2012 )
Tertiary: University of Santo Tomas ( 2012 - Present )
Curriculum Vitae
PAGE
57
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Educational Background
Secondary: De La Salle - Lipa Integrated School (2008 - 2012)
Tertiary: University of Santo Tomas (2012 - Present)
Curriculum Vitae
58
PAGE
Address: Aston Martin st., St. Dominic Villa, City of San Fernando,
Pampanga
E-mail: [email protected]
Educational Background
Secondary: Pampanga High School (2008-2012)
Tertiary: University of Santo Tomas (2012-present)
Curriculum Vitae
59
PAGE
Educational Background
Secondary: De La Salle - Lipa Integrated School (2008 - 2012)
Tertiary: University of Santo Tomas (2012 - Present)
60