PharChem Manuscript (Bixa Orellana)

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY
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
phytochemical tests were screenings for alkaloids, cardiac glycosides,

anthraquinones, tannins, flavonoids and cyanogenic glycosides. After
the tests were made, the researchers obtained positive results on the
tests for anthraquinones and tannins.
Keywords: Bixa orellana, phytochemicals, alkaloids, cardiac

glycosides, anthraquinones, tannins, flavonoids, cyanogenic
glycosides
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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
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5
6
7
7
7
8
9
10
11
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12
12
12
13
13
14
14
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15
16
17
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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
5. Conclusions and Recommendations

5.1 Conclusion
5.2 Recommendation
References
Curriculum Vitae
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,
cyanogenic glycosides, flavonoids, and anthraquinones based on their

structure and action. These phytochemicals have specific actions and
can be used to tell whether a plant is being used optimally for its
intrinsic effect. Examples of the pharmacological benefits of these
phytochemicals include: laxatives for anthraquinones; cardiotonic
effect for cardiac glycosides; diuretics, expectorants, and laxatives for
the saponins. Tannins have protein precipitation properties, while
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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
phytochemical tests in order to identify the different constituents

present. The method of extraction used was percolation. It utilizes a
polar solvent, methanol, to obtain the crude extract that is to be used
in the phytochemical screening. Several tests were conducted on the
crude extract to test for and identify the active constituents present in
the plant sample. Some of the tests employed were: Dragendorffs
and Mayers tests for alkaloids; Guignard test for cyanogenic
glycosides; gelatin and ferric chloride test for tannins; Keddes, KellerKillanis, and Liebermann-Burchard tests for cardiac glycosides;
Wilstatter Cyanidin and Bate-Smith and Metcalf tests for flavonoids;
and the Borntrgers tests for anthraquinones.
1.1.2 Statement of the Problem
A large percentage of medicines produced today are derived from
various phytochemicals. As pharmacy and medicine improves with
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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
advancements we have today.

Achuete, a common plant in tropical countries, and native to the
Philippines, is suspected to have significant clinical effects that may
be explored further and improved upon in order to be known if it be of
great use to mankind.
1.1.3 Objectives of the Study
to efficiently extract the active constituents found on Bixa

orellana
to effectively identify the active constituents found on Bixa

orellana
1.1.4 Significance of the Study

This study would identify the different active constituents in Bixa
orellana that could exhibit potential pharmacologic activities and
could serve as a reference for future studies.
1.1.5 Theoretical Framework
This section discusses the theoretical framework that was
developed out of the literature review to guide the researchers in the
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interpretations of the results. More so, it informs the design of the

study to address the research question: what are the phytochemical
constituents present in the leaves of Achuete or Bixa orellana?
Before testing for the presence of the bioactive compounds, a
considerate amount of the plant sample must undergo percolation to
yield an extract. This is then subjected to various standard
phytochemical tests to detect the presence of alkaloids, cyanogenic
glycosides,
tannins,
cardiac
glycosides,
flavonoids,
and
anthraquinoes. For this to be proven, the extract should appear

positive under different tests.
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1.1.6 Scope and Limitations

The study involves the extraction and identification of the
constituents of the Achuete plant (Bixa orellana) after percolation with
methanol using phytochemical screening tests. The study is restricted
to the constituents of the plant leaves only. Any other phytochemicals
found in other plant parts will not be included in the experiment and
will therefore not have a positive result. Furthermore, it is also limited
by
the
selected
phytochemical
screening
tests
used
in
the
experiment.
1.1.7 Definition of terms

Alkaloids - any of a class of nitrogenous organic compounds of plant
origin that have pronounced physiological actions on humans. They
include many drugs like morphine and poisons like atropine and
strychnine
Anthraquinone - a yellow crystalline compound obtained by
oxidation of anthracene. It is the basis of many natural and synthetic
dyes.
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Bioactive compounds - compounds that have an effect on a living

organism, tissue or cell. In the field of nutrition, they are distinguished
from essential nutrients.
Cardiac glycoside - a chemical compound that has effects on the
heart, stomach, intestines, and nervous system. It is the active
ingredient in many different heart medicines. It can be poisonous if
taken in large amounts.
Chlorophyll - a green pigment, present in all green plants and in
cyanobacteria, responsible for the absorption of light to provide
energy for photosynthesis. Its molecule contains a magnesium atom
held in a porphyrin ring.
Cyanogenic glycoside - glycoside in which the aglycone moiety
contains a cyanide group. A cyanogenic glycoside can release
poisonous hydrogen cyanide if acted upon by some enzyme.
Diuretics - any substance that promotes the production of urine.
Expectorant - a medicine that promotes the secretion of sputum by
the air passages, used especially to treat coughs.
Glycoside - a compound formed from a simple sugar and another
compound by replacement of a hydroxyl group in the sugar molecule.
Many drugs and poisons derived from plants are glycosides.
Laxative - (chiefly of a drug or medicine) tending to stimulate or
facilitate evacuation of the bowels.
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Saponins amphipathic glycosides grouped phenomenologically by

the soap-like foaming they produce when shaken in aqueous
solutions, and structurally by having one or more hydrophilic
glycoside moieties combined with alipophilic triterpene derivative.
Tannin - yellowish or brownish bitter-tasting organic substance
present in some galls, barks, and other plant tissues, consisting of
derivatives of gallic acid, used in leather production and ink
manufacture.
Phytochemicals - any of various biologically active compounds
found in plants. Flavonoid - organic compound, any member of a
class of biological pigments containing no nitrogen that are found in
many plants. Flavonoids are the most important plant pigments for
flower coloration, producing yellow or red/blue pigmentation in petals
designed to attract pollinator animals.
1.2 Research Impediments
It is possible that the desired results in this experiment may not be
achieved due to phytochemical loss during air-drying up to performing
the different screening tests. The extracts may not be pure since the
methanol may have not fully evaporated, which results to the
retainment of the pigment, chlorophyll. Chlorophyll needs to be
separated to avoid interfering with the color of the test results. Skill,
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dexterity and strict compliance to the procedures are also needed, to

avoid error and loss in getting the results of the tests.
CHAPTER 2
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THE RESEARCH QUESTIONS
2.1 Review of Related Literature

2.1.1 About Bixa orellana
2.1.1.1 Plant name (Synonymous/Vernacular Names
Bixa orellana has the following vernacular names: Achiti (Ilk),
Achote (Tag), Asuti (Tag), Sotis (Bis) and Asuite (Ilk). In English, it is
most commonly known as Annatto or Lipstick plant.
Figure 1 Bixa orellana
2.1.2 Botanical description

2.1.2.1 Scientific names
Other scientific names of Bixa orellana:
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Bixa acuminata Linn

Bixa americana Linn
Bixa arborea Linn
Bixa upatensis Linn
Bixa urucurana Linn
2.1.2.2 Taxonomical Classification
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Malvales
Family: Bixaceae
Genus: Bixa
Species: Bixa orellana
2.1.2.3 Botanical description
Bixa orellana can be found in regions spanning the globe. Grown
from either seed or cutlings, B. orellana requires full sunlight and
protection from the wind (Morton 2004). The plant grows equally well
in lowlands and mountainous regions or areas of higher elevation
(Bruggeman 2007). Native to the tropical American area, B. orellana
is found in largest quantities from Mexico to Ecuador and Brazil. This
plant is cultivated in warm regions of the world, such as Philippines,
India and Sri Lanka mainly for the dye which the seeds yield.
Bixa orellana L. is a shrub or bushy tree which ranges from 3 to 10
meters in height. Its glossy, ovate leaves are evergreen with reddish
veins; they have a round, heart-shaped base and a pointed tip. With a
thin, long stem, the leaves are between 8 and 20 cm long and 5 and
14 cm wide. The twigs are covered with rust colored scales when
young and bare when older. Bixas flowers are pink, white, or some
combination, and are 4 to 6 cm in diameter.
From the flower
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15
protrudes a striking two-valved fruit, covered either with dense soft

bristles or a smooth surface. These round fruits, approximately 4 cm
wide, appear in a variety of colors: scarlet, yellow, brownish-green,
maroon, and most commonly bright red. When ripe, they split open
and reveal numerous amount of small, fleshy seeds, about 5 mm in
diameter and covered with red-orange pulp, the embryo of which is
poisonous (Chopra 2009).
2.1.2.4 Chemical composition
Bixa orellana seeds are one of the only natural source of bixin, a
carotenoid widely used in food industry as colorant. Its seeds contain
a fatty oil with palmitin, stearin, and phytosterol. A study of
carotenoid pigments in the seeds identified bixin, norbixin, carotene,
cryptoxanthin,
lutein,
zeaxanthin
and
methyl
bixin.
Phytochemical screening yielded carbohydrates, steroids, alkaloids,

proteins, flavonoids, terpenoids, phenolics, tannins and glycosides
(Stuart, 2013).
2.1.3 Ethnopharmacologic survey
Bixa orellana is commonly used as antipyretic, laxative and
expectorant in traditional medicine in Brazil (Mariath, 2008).
In
addition to that, it is said to have an anti-inflammatory activity used

for bruises and wounds. It can also be used for the treatment of
Bronchitis to partially reduce the swelling of the Bronchi. Usually, the
infusion of the leaves of the plant has been shown to be effective
against sore throat, and eye inflammation (Barbosa, 2009). In South
and Central America, most of the natives use it as an aphrodisiac and
insect repellant, while the pulp, which includes the seed, is used to
color beverages and other delicacies all over the world.
The seeds of Bixa orellana are slightly astringent and when
decocted are very good remedy for Gonorrhea. Its seeds also posses
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16
antigonorrheal and antipyretic properties but to a lesser extent

(Newman, 2006). The pulp surrounding the seeds is also astringent
and slightly purgative which is given for patients with dysentery. Its
pulp, if applied immediately to burns, is believed to prevent the
formulation of blisters and even scars (Zegarra, et.al., 2005). The pulp
is also prescribed for stomach ache in Netherlands and Mexico, in
which the seeds and leaves of the plant is official in the Pharmacopeia
of each country (Wolf, 2007).
2.1.4 Pharmacologic activities
2.1.4.1 Anticonvulsant activity
Shilpi, et al. (2006) determined the anticonvulsant activity of the
Bixa orellana leaves. The methanol extract of Bixa orellana leaves
was prepared to investigate whether it had any effect on the central
nervous system and any role in controlling seizures in mice. A number
of tests were employed to evaluate neuropharmacological and
anticonvulsant activity.
Neuropharmacological
activity
was
monitored
using
the
pentobarbitone-induced hypnosis in an open-field and hole-cross

tests. A test substance with CNS-depressant activity can reduce time
for the onset of sleep and/or prolong the duration of sleep. In the
reduction in time for the onset of sleep and increase in the duration of
total sleeping time caused by Bixa orellana leaves extract was almost
comparable to the standard drug diazepam. This result suggests
that Bixa orellana leaves extract has a depressing effect on the CNS.
The anticonvulsant activity was further monitored using the
strychnine-induced anticonvulsant test. The extract significantly
increased the survival time after strychnine administration at the
doses of 250 and 500 mg/kg compared to the control but failed to
prevent the mortality of the test animals. In both the open-field and
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17
hole-cross tests, which evaluate the behavioral effects of a test

substance on the CNS, Bixa orellana leaves extract exhibited a
decrease in locomotor activity in test animals.
2.1.4.2 Analgesic activity
Uddin, et al. (2006) determined the analgesic effect of the Bixa
orellana leaves. Extracts of Bixa orellana leaves have been reported
to be useful in headaches. The extract was also investigated for
analgesic activity using the acetic acid-induced model. When
administrated intraperitoneally to mice, acetic acid causes algesia by
liberating
noxious
endogenous
substances,
including
serotonin,
histamine, prostaglandin, bradykinin and substance P that sensitize

pain nerve endings. Among the prostanoids, mainly prostacyclin (PGI 2)
has been held responsible for the causation of pain following acetic
acid administration. It has been suggested that acetic acid stimulates
the vanilloid (VR1) and bradykinin (B2) receptors in the pathway
comprising sensory afferent C-fibers. Therefore, the observed activity
of Bixa orellana leaves extract might stem from its ability to interfere
with the synthesis or release of those endogenous substances of the
nerve fibers involved in the pain transmission pathway.
2.1.4.3 Antidiarrheal activity
Sadhu, et al. (2006) observed the antidiarrheal properties of Bixa
orellana. Numerous reports of traditional use of Bixa orellana leaves in
treating diarrhea were confirmed when the extract was screened for
antidiarrheal
performed
activity.
using
Evaluation
castor
of
antidiarrheal
oil-induced
diarrhea
activity
was
model
and
gastrointestinal motility test in mice.

Castor oil causes diarrhea through its active metabolite ricinoleic
acid, which stimulates the peristaltic activity of small intestine leading
to changes in electrolyte permeability of intestinal mucosa. Its action
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is also associated with stimulation of release of endogenous

prostaglandins. Bixa orellana leaves extract significantly and dosedependently decreased the severity of castor oil-induced diarrheal
episodes in the test animals. The extract also reduced the total
number of feces as well as the total number of wet feces both
significantly and dose-dependently. In the gastrointestinal motility
test, the extract was found to reduce the movement of charcoal meal
in mice to a statistically significant level (P < 0.01) only at the highest
dose tested (500 mg/kg). Therefore, it could be interpreted that the
observed antidiarrheal activity of Bixa orellana leaves extract may be
attributed to a possible inhibition of prostaglandin biosynthesis and to
a lesser extent to its retardation of gastrointestinal transit.
Table 1 Pharmacologic studies involving Bixa orellana
Extracts used
Test subjects Pharmacologi References

c
Activity
Methanolic
extract
of
B.
Strychnine
Anticonvulsa Shilpi, J., et al.
induced mices
nt
2006
Acetic
Analgesic
Uddin, S., et al.
orellana
Methanolic
extract
of
B.
acid
induced mices
2006
orellana
Methanolic
extract
of
Castor
B.
oil
Antidiarrhea Sadhu, S., et
induced mices
orellana
2.2 Research Question/s (or Hypotheses)
al. 2006
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The researchers expect to obtain positive results on the different

phytochemical tests such as Borntrgers tests for Anthraquinones;
Ferric Chloride and Gelatin Tests for Tannins; and the General Test,
Primary Assay, Confirmatory Test and Test for Quaternary Base for
Alkaloids.
CHAPTER 3
THE RESEARCH METHODS
This chapter presents the preparation of the plant extract, the

percolation setup, and the methods and materials in phytochemical
tests of different plant constituents such as alkaloids, cardiac
glycosides, cyanogenic glycosides, flavonoids, and tannins.
3.1 Preparation of Stock Plant Extract
An approximately 300 grams of Bixa orellana was collected and
air-dried for two weeks. The principle behind air-drying was to
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preserve the plant constituents without subjecting it to heat which

might destroy the heat-labile components of the plant. The dry
sample was then cut into small pieces and was ground to increase
the surface area of the sample and to expose the tissues and cells
containing the phytochemicals using the Wiley Mill.
Prior to grinding, a percolation set up was prepared using a 1 liter
amber bottle, cork, glass tubing, and rubber tubing. A cotton plug
was first placed into the inverted amber bottle, followed by the
powdered leaves of Bixa orellana, filling up to 2/3 of the bottle. A
filter paper was then placed on top of the powdered leaves, followed
by marbles to hold the filter paper in place. Methanol was used
instead of ethanol as the solvent for extraction because it is more
polar and has a lower boiling point, therefore would produce a higher
percentage yield and ease the evaporation phase. The set-up was
then covered using a clean sheet of paper to prevent the methanol
from evaporating.
The ground plant material was allowed to macerate in the
methanol. The extracts were collected every day during the span of
the percolation procedure. After each collection, the percolator was
filled once again with the solvent.
These
extracts
were
evaporated
spontaneously
in
large
evaporating dishes until a thick, syrupy liquid remained. This was the
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stock plant extract containing concentrated plant constituents, which

was utilized in the different phytochemical screening tests.
3.2 Percolation Setup
Owing to its efficiency as well as its simplicity, percolation was
used to extract the active constituents of Bixa orellana. The figure
below illustrates the percolation set-up:
Rubber tubing
Filter paper with marble on top
Iron stand with iron ring
Ground material with methanol as solvent

Glass tubing
Receiver
Figure 2 Percolation Setup

3.3 Methodology and Schematic Diagrams
This study was conducted to isolate and determine the presence
of the various phytochemical constituents in the leaves of Bixa
orellana. The plant sample was gathered from a single location,
Batangas City, to ensure that the sample grew under the same
environmental conditions. It was then air dried in order to preserve
the constituents present before it was ground in the Wiley Mill to be
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used for constituent extraction. A percolator was used to extract the

constituents using a polar solvent, methanol, to maximize the
percentage yield. After collecting the crude extract, the sample was
subjected to different phytochemical screening procedures as shown
below.
3.3.1 Alkaloids
The materials used were 250-mL beaker, test tube, funnel,
dropping pipette, hot plate, stirring rod, litmus paper, and 20-mL
graduated cylinder.
3.3.1.1 General Test for Alkaloids
Six milliliters of crude extract added with 10 mL ammoniacal
chloroform was placed on a beaker. The solution was mixed and
filtered. To the filtrate, 1mL of 1M sulfuric acid was added. It was
shaken and left to stand for 2 minutes. The upper layer was pipetted
and divided into 3 portions. Test tube A served as the control. Two
drops of Dragendorffs reagent was added to test tube B while 2
drops of Mayers reagent was added to test tube C. The color reaction
was observed and recorded.
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3.3.1.2 Primary Assay

In a test tube, 5 mL of HCl was added to 6 mL of crude extract.
The test tube was then placed in a water bath for 5 minutes with
constant stirring. After the water bath, 0.5 g sodium chloride was
dissolved to the solution and then filtered. The residue was washed
with enough volume of 2M HCl to bring the filtrate volume to 6 mL.
Filtrate was divided to 4 portions. One milliliter for test tube A served
as the control. To test tube B, 2 drops of Dragendorffs reagent was
added to 1mL of filtrate while 2 drops of Mayers reagent was added
to 1mL of filtrate in test tube C. Three milliliters of filtrate for test
tube D was used for the confirmatory test. The color reaction was
observed and recorded.
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24
3.3.1.3 Confirmatory Test

For the confirmatory test, 28% ammonia was added to test tube D
until the solution became alkaline. The alkalinized solution was
extracted thrice with small amount of chloroform. Upper layer was
pipetted and set aside for the test for Quaternary Base while the
lower layer was evaporated to dryness over steam bath placed under
the hood. After drying, 5 mL of 2M HCl was added to the residue. The
solution was stirred for 2 minutes, cooled, and then divided into 3
portions.
Test
tube
served
as
the
control.
Two
drops
of
Dragendorffs reagent was added to test tube B while 2 drops of
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Mayers reagent was added to test tube C. The color reaction was
3.3.1.4 Test for Quaternary Base

The upper aqueous layer from the confirmatory test was used in
this test. It was acidified using 2M HCl. The solution was filtered and
divided into 3 portions. Test tube A served as the control. Two drops
of Dragendorffs reagent was added to test tube B while 2 drops of
Mayers reagent was added to test tube C. The color reaction was
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PAGE
3.3.2. Cardiac Glycoside

The materials used were separatory funnel, test tube, dropping
pipette, 250-mL beaker, hot plate, funnel, filter paper, and 20-mL
graduated cylinder.
3.3.2.1 Preparation of Sample
Six milliliters of plant extract was placed in a separatory funnel
and 6 mL hexane with 2 mL of water was added. The solution was
gently shaken and allowed to separate. The upper hexane layer was
removed from the defatted aqueous layer. The latter was extracted
with hexane and water (2:1) until most of the pigment was removed.
Hexane layer was discarded. Defatted aqueous layer was heated over
a water bath for about 5 minutes then cooled at room temperature.
Solution was divided into 4 portions. Test tube A served as control.
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PAGE
3.3.2.2 Keller Killanis Test

To test tube B, 3mL of ferric chlored was added. One milliliter of
concentrated sulfuric acid was cautiously added by tilting the test
tube and allowing it to trickle along the side of the tube. The color
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PAGE
reaction at the interface of the acid and aqueous layer was observed
and recorded.
3.3.2.3 Keddes Test

To test tube C, 2 mL of dichloromethane was added, mixed, and
allowed to stand to separate. Upper layer was removed while 4 drops
of Keddes reagent was added to the lower DCM layer. The color
reaction was observed and recorded.
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PAGE
3.3.2.4 Libermann Burchards Test

To test tube D, 10 mL of dichloromethane was added then stirred
for a few minutes. The upper DCM layer was removed. Lower DCM
layer was dried by passing it through an anhydrous sodium sulfate
placed over a dry filer paper in a funnel. The filtrate was divided into
2 portions. Test tube A served as the control. Three drops of acetic
anhydride and 1 drop of concentrated sulfuric acid (trickled along the
side of the tube) was added to test tube B. The immediate color
reaction was observed and recorded.
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PAGE
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
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Modified Borntragers Test
Six milliliters of plant extract was placed in a test tube with 10 mL

of 0.5M potassium hydroxide and 1 mL of 5% hydrogen peroxide. The
solution was stirred and heated under water bath for 10 minutes. It
was cooled then filtered. The filtrate was acidified with glacial acetic
acid then extracted twice with 5 mL portion of benzene. Benzene
extracts were combined and divided into 2 portions. Test tube A
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served as control while 5mL ammonia solution was added to test tube
B. The color reaction was observed and recorded.
3.3.4 Cyanogenic Glycosides

The materials used were test tube, filter paper, cork, hot plate,
250-mL beaker, and 10-mL graduated cylinder
3.3.4.1 Guignard Test
Few drops of chloroform were added to 6 mL plant extract placed
in a test tube. A cork with a yellow picrate pater suspended on it was
used as the stopper of the test tube. The test tube was warmed at
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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.
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.
33
PAGE
3.3.5.2 Bate-Smith and Metcalfs Test

To test tube B, 0.5 mL concentrated HCl was added. The color
change was observed and recorded.
3.3.5.3 Wilstater Cyanidin Test

To test tube C, 0.5 mL concentrated HCl was added. Three to four
pieces of Magnesium turnings was placed and color change was
observed then recorded. When no definite color change was visible,
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.
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.
35
PAGE
3.3.6.2 Ferric Chloride Test

To test tube B, 3 drops of ferric chloride reagent was added. Three
drops of ferric chloride reagent was also added to a prepared
aqueous tannic acid solution. The color reaction was observed and
recorded.
36
PAGE
3.3.6.3 Gelatin Test

To test tube C, 3 drops of gelatin salt reagent was added. Three
drops of gelatin salt reagent was also added to prepared aqueous
tannic acid solution. Formation of a jelly precipitate was observed.
3.3.6.4 Matchstick Test

The matchstick was dipped in the plant extract the dried. It was
moistened with HCl acid and warmed near the flame. The color of the
matchstick wood was observed.
37
PAGE
CHAPTER 4
RESULTS AND DISCUSSION
This chapter presents the results and discussions regarding the

different phytochemical tests conducted on the crude extract of the
plant Bixa orellana.
4.1. Alkaloids
4.1.1 Description
Alkaloids are naturally occurring organic compounds, which
contain one or more nitrogen in a heterocyclic ring and are
synthesized by plants from amino acids. They are mostly white basic
solids, and usually exist as crystals, which unite with acids to form
salts. Their free forms are insoluble in water, but soluble in organic
solvents like alcohol, benzene, ether and chloroform. Their salts
behave otherwise. Although bitter tasting, alkaloids have a wide
range of marked pharmacologic action on man and on animals. They
can be classified according to their source and according to their
ring structure.
4.1.2 Results
38
Name of Test
Positive Result
A. General Tests
Dragendorffs
Experiment
Inferenc
Result
Orange
Test
precipitate
Mayers Test
White
PAGE
(-)
Red brown solution

(-)
precipitate
Light brown
solution
B. Primary Assay
Dragendorffs
Orange
Test
precipitate
Mayers Test
White
(-)
Red brown solution

(-)
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
Brick red solution

Mayers Test
White
(-)
precipitate
Brick red solution

4.1.3 Discussion
The general tests and primary assays conducted to determine the
presence of alkaloids were Dragendorff's test and Mayer's test. In
the general tests, the free forms of alkaloids, because of their
40
PAGE
lipophilic nature, were extracted from the sample using chloroform,

an organic solvent, with the help of ammonia, a base, which
increases the ability of the solvent to penetrate the cell. In the
primary assays, the salt forms of alkaloids were extracted through
the addition of hydrochloric acid, a mineral acid, and sodium
chloride.
Alkaloid-precipitating
reagents
Dragendorff's,
which
contain potassium bismuth iodide, and Mayer's, which contain

mercuric potassium iodide, were added to the different test tubes
containing the extracts. Both reagents would induce precipitation in
the presence of a heavy metal, yielding double salts. Negative
results were obtained, as the formation of an orange precipitate and
a white precipitate respectively were not observed.
The confirmatory tests were performed to determine the
presence of 1, 2, and 3 alkaloids. Negative results were obtained
from the Dragendorff's and Mayer's test as the formation of an
orange precipitate and a white precipitate respectively were not
observed.
The quaternary tests were carried out to determine the presence
of
alkaloids.
Negative
results
were
obtained
from
the
Dragendorff's and Mayer's test as the formation of an orange

precipitate and a white precipitate respectively were not observed.
4.2 Cardiac Glycosides
41
PAGE
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
Keller Killani's Test
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.
43
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
Red ring layer

Light golden
yellow solution
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
45
PAGE
Flavonoids are glycosides, which contain one or more phenolic

hydroxyl group combined with sugar residues. In most plants, benzopyrone is found in their flavonoid structure. Flavonoids are
used in the medicine industry as anti-oxidants, anti-cancer, antimicrobial, liver protectant, and a free radical scavenger. They
include anthocyanins, leucoanthocyanins, catechins, aurones and
chalcones.
4.4.2 Results
Name of Test
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
46
PAGE
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
condensed, complex and pseudotannins. Tannins are used in the

medicine industry as astringents because of their ability to
precipitate proteins as a defense mechanism against pathogens.
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
Blue black solution
Wood will
(-)
turn red or
(No photo
pink in color
available)
Dark green in color
4.5.3 Discussion
48
PAGE
The tannins were extracted from the crude extract by the

addition of sodium chloride, which turns the tannins into their watersoluble salts. Tannins are known to precipitate proteins. This was the
principle behind the gelatin test, which was carried out to test for
the presence of tannins. Gelatins are a mixture of proteins and
peptides, therefore are precipitated by tannins. The test yielded a
positive result, with the presence of a jelly precipitate. This
confirmed the presence of tannins in Bixa orellana.
The ferric chloride test was conducted to determine the
presence of hydrolyzable and condensed tannins. This yielded a
positive result with a blue-black precipitate, indicating the presence
of hydrolyzable tannins. However, no brownish green precipitate
formed. This indicates the absence of condensed tannins. This was
further confirmed with the matchstick test, which was carried out to
test for the presence of condensed tannins. Condensed tannins,
when
treated
with
acids
and
enzymes,
are
converted
or
polymerized into a red insoluble compound called phlobaphene.

This red coloration on the matchstick was not obtained, thus
indicating the absence of condensed tannins in Bixa orellana.
4.6 Cyanogenic glycosides
4.6.1 Description
49
PAGE
Cyanogenic glycosides are compounds, which undergo hydrolysis

when chewed or digested, resulting to the release of hydrogen
cyanide. They are used in the pharmaceutical industry as flavoring
agents,
anti-neoplastics,
sedatives,
and
expectorants
to
the
respiratory tract. They have a lethal dose on humans of 1 mg/kg.

4.6.2 Results
Name of Test
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
PAGE
or aldehydes. The test yielded a negative result, with the absence

of yellow to brick red decoloration of the sodium picrate paper.
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
anthraquinones and tannins. Based on the researchers hypothesis,

all the phytochemical screening tests should have yielded positive
results. This error may have been due to the difference in the source
51
PAGE
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.
52
PAGE
REFERENCES
Bruggerman, L. (2008). Tropical Plants and Their Cultivation.

Chopra, R. (2009). Poisonous Plants of India (pp. 203-210).
Delhi, India.
Clements, J. (2005). Antimicrobial Agents and
Chemotherapy (pp. 1793-1799).

Correa, M. (2007). Traditional Herbs in Brasil (pp. 157-159). Rio
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
PAGE
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
Name: Paul James Ambida Alava

Date of Birth: July 16 1995
Place of Birth: Lipa
Age: 19
Religion: Christian
Nationality: Filipino
Address: Villa Neneng Subd., Kumintang Ibaba, Batangas City
E-mail: pauljamesalava@yahoo.com
54
PAGE
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
Name: Denise Anne Reyes Alcausin

Date of Birth: August 23, 1995
Place of Birth: Cotabato
Age: 19
Religion: Roman Catholic
Civil Status: Single
55
PAGE
Address: Santiago St., Town & Country West, Molino III, Bacoor City,
Cavite
E-mail: izalcausin@yahoo.com
Secondary: Divine Light Academy (2008 - 2012)
Tertiary:University of Santo Tomas (2012 - Present)
Curriculum Vitae
Name: Mary Iris Mendoza Andal

Date of Birth: February 14, 1996
Place of Birth: Batangas City
Age: 18
56
Address: Balagtasin I, San Jose, Batangas

E-mail: iris_pink14@yahoo.com
Secondary: St. Bridget College ( 2008 - 2012 )
Tertiary: University of Santo Tomas ( 2012 - Present )
Curriculum Vitae
Name: Nicole Eileen M. Bagon

Date of Birth: August 16, 1995
Place of Birth: Sta. Cruz, Laguna
Age: 19
PAGE
57
PAGE
Address: Lotus de Cataluna Dormitory, Tolentino St., Sampaloc,

Manila
E-mail: calvin_bautista@yahoo.com
Secondary: De La Salle - Lipa Integrated School (2008 - 2012)
Tertiary: University of Santo Tomas (2012 - Present)
Curriculum Vitae
Name: Danielle Paras Barretto

Date of Birth: November 25 1995
Place of Birth: Manila
Age: 18
58
PAGE
Address: Aston Martin st., St. Dominic Villa, City of San Fernando,
Pampanga
E-mail: dnielle04@gmail.com
Secondary: Pampanga High School (2008-2012)
Tertiary: University of Santo Tomas (2012-present)
Curriculum Vitae
Name: Calvin EJ Robledo Bautista

Date of Birth: January 26, 1996
Place of Birth: Lipa city, Batangas
Age: 18
Religion: Iglesia Ni Cristo
59
PAGE
Address: Tower 1 Robinsons Place Manila, Padre Faura St., Ermita,

Manila
E-mail: calvin_bautista@yahoo.com
Secondary: De La Salle - Lipa Integrated School (2008 - 2012)
Tertiary: University of Santo Tomas (2012 - Present)
60

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

phytochemical tests were screenings for alkaloids, cardiac glycosides,

Keywords: Bixa orellana, phytochemicals, alkaloids, cardiac

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

5. Conclusions and Recommendations

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

cyanogenic glycosides, flavonoids, and anthraquinones based on their

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

phytochemical tests in order to identify the different constituents

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

advancements we have today.

to efficiently extract the active constituents found on Bixa

to effectively identify the active constituents found on Bixa

1.1.4 Significance of the Study

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

interpretations of the results. More so, it informs the design of the

anthraquinoes. For this to be proven, the extract should appear

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

1.1.6 Scope and Limitations

1.1.7 Definition of terms

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

Bioactive compounds - compounds that have an effect on a living

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

Saponins amphipathic glycosides grouped phenomenologically by

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

dexterity and strict compliance to the procedures are also needed, to

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

THE RESEARCH QUESTIONS

2.1 Review of Related Literature

Figure 1 Bixa orellana

2.1.2 Botanical description

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

Bixa acuminata Linn

From the flower

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

protrudes a striking two-valved fruit, covered either with dense soft

Phytochemical screening yielded carbohydrates, steroids, alkaloids,

addition to that, it is said to have an anti-inflammatory activity used

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

antigonorrheal and antipyretic properties but to a lesser extent

pentobarbitone-induced hypnosis in an open-field and hole-cross

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

hole-cross tests, which evaluate the behavioral effects of a test

histamine, prostaglandin, bradykinin and substance P that sensitize

gastrointestinal motility test in mice.

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

is also associated with stimulation of release of endogenous

Test subjects Pharmacologi References

Anticonvulsa Shilpi, J., et al.

Uddin, S., et al.

Antidiarrhea Sadhu, S., et

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

The researchers expect to obtain positive results on the different

This chapter presents the preparation of the plant extract, the

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

preserve the plant constituents without subjecting it to heat which

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY

stock plant extract containing concentrated plant constituents, which

Ground material with methanol as solvent

Figure 2 Percolation Setup

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY