pharchem manuscript (bixa orellana)
TRANSCRIPT
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY PAGE
PHYTOCHEMICAL ANALYSIS OF Bixa orellana
A Research PaperPresented to the
Faculty of PharmacyUniversity of Santo Tomas
In Partial FulfillmentOf 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 Page
Abstract 21. The Problem Rationale
1.1 Introduction1.1.1 Background of the Study1.1.2 Statement of the Problem1.1.3 Objectives1.1.4 Significance of the Study1.1.5 Theoretical Framework1.1.6 Scope and Limitations1.1.7 Definition of Terms
1.2 Research Impediments
5677789
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2. The Research Questions2.1 Literature Review
2.1.1 About Plant2.1.1.1 Plant Name (Synonyms/Vernacular Names)
2.1.2 Botanical Descriptions2.1.2.1 Taxonomical Classification2.1.2.2 Botanical Description2.1.2.3 Chemical Composition
2.1.3 Ethnopharmacologic survey2.1.4 Pharmacologic activities
2.1.4.1 Anti-convulsant activity2.1.4.2 Analgesic activity2.1.4.3 Antidiarrheal activity
2.2 Research Question
111112121213131414141516
3. The Research Methods3.1 Preparation of Stock Plant Extract3.2 Percolation Setup3.3 Methodology and Schematic Diagrams
171819
4. Results and Discussion4.1 Alkaloids4.2 Cardiac Glycosides4.3 Anthraquinones
343739
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4.4 Flavonoids4.5 Tannins4.6 Cyanogenic glycosides
414244
5. Conclusions and Recommendations5.1 Conclusion5.2 Recommendation
4646
References 48Curriculum Vitae 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
some alkaloids in their salt forms are used as stimulants, but most alkaloids are
powerful poisons. Flavonoids are known for their antioxidant properties.
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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:
Dragendorff’s and Mayer’s tests for alkaloids; Guignard test for cyanogenic glycosides;
gelatin and ferric chloride test for tannins; Kedde’s, Keller-Killani’s, and Liebermann-
Burchard tests for cardiac glycosides; Wilstatter “Cyanidin” and Bate-Smith and
Metcalf tests for flavonoids; and the Bornträger’s 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 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.
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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 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.
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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.
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.
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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.
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,
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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, dexterity and
strict compliance to the procedures are also needed, to avoid error and loss in getting
the results of the tests.
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CHAPTER 2
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
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2.1.2 Botanical description
2.1.2.1 Scientific names
Other scientific names of Bixa orellana:
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,
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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. Bixa’s flowers are pink, white, or some
combination, and are 4 to 6 cm in diameter. From the flower 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,
ß-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 antigonorrheal and antipyretic properties
but to a lesser extent (Newman, 2006). The pulp surrounding the seeds is also
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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 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
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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 (PGI2) 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 activity. Evaluation of
antidiarrheal activity was performed using castor oil-induced diarrhea 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 is also associated with stimulation of
release of endogenous prostaglandins. Bixa orellana leaves extract significantly and
dose-dependently 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.
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Table 1 Pharmacologic studies involving Bixa orellana
Extracts used Test subjects Pharmacologic
Activity
References
Methanolic extract
of B. orellana
Strychnine
induced mices
Anticonvulsant Shilpi, J., et al.
2006
Methanolic extract
of B. orellana
Acetic acid
induced mices
Analgesic Uddin, S., et al.
2006
Methanolic extract
of B. orellana
Castor oil induced
mices
Antidiarrhea Sadhu, S., et al.
2006
2.2 Research Question/s (or Hypotheses)
The researchers expect to obtain positive results on the different phytochemical
tests such as Bornträger’s 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.
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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 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
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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 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:
Figure 2 Percolation Setup
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Iron stand with iron ring
Filter paper with marble on top
Ground material with methanol as solvent
Receiver
Rubber tubing
Glass tubing
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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 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 Dragendorff’s reagent was added to test tube B while 2 drops of Mayer’s 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 Dragendorff’s reagent was added to 1mL of filtrate while 2 drops of
Mayer’s 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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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
A served as the control. Two drops of Dragendorff’s reagent was added to test tube B
while 2 drops of Mayer’s reagent was added to test tube C. The color reaction was
observed and recorded.
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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 Dragendorff’s reagent was added to test tube B
while 2 drops of Mayer’s reagent was added to test tube C. The color reaction was
observed and recorded.
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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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3.3.2.2 Keller Killani’s 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 reaction at the interface of the acid and aqueous
layer was observed and recorded.
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3.3.2.3 Kedde’s 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 Kedde’s reagent was added to
the lower DCM layer. The color reaction was observed and recorded.
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3.3.2.4 Libermann Burchard’s 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.
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.
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3.3.3.1 Borntrager’s 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.
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3.3.3.2 Modified Borntrager’s 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 served as
control while 5mL ammonia solution was added to test tube B. The color reaction was
observed and recorded.
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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 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
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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.
3.3.5.2 Bate-Smith and Metcalf’s 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
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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, 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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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.
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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.
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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
Name of Test Positive Result Experiment Result Inference
A. General Tests
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Dragendorff’s Test Orange precipitate
Red brown solution
(-)
Mayer’s Test White precipitate
Light brown solution
(-)
B. Primary Assay
Dragendorff’s Test Orange precipitate
Red brown solution
(-)
Mayer’s Test White precipitate
Light brown solution
(-)
C. Confirmatory Test
Dragendorff’s Test Orange precipitate
Orange yellow solution
(-)
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Mayer’s Test White precipitate
Light yellow solution
(-)
D. Quaternary Bases
Dragendorff’s Test Orange precipitate
Brick red solution
(-)
Mayer’s 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 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,
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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 4° 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
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.
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4.2.2 Results
Name of Test Positive Result Experiment Result Inference
Keller Killani's Test Reddish brown
color which may
turn blue or
purple
Dark brown solution
with purple precipitate
(+)
Liebermann Burchard
Test
Blue to green,
red, pink, purple
or violet
Light yellow solution
(-)
Kedde's Test Blue violet
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,
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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.
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 Result Experiment Result Inference
Borntrager's Test Red coloration (++)
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in the lower
ammonical layer
(No photo available)
Red ring layer
Light golden yellow
solution
Modified Borntrager's
Test
Pink color
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 O-glycoside 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.
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4.4 Flavonoids
4.4.1 Description
Flavonoids are glycosides, which contain one or more phenolic hydroxyl group
combined with sugar residues. In most plants, γ-benzopyrone is found in their
flavonoid structure. Flavonoids are used in the medicine industry as anti-oxidants,
anti-cancer, anti-microbial, liver protectant, and a free radical scavenger. They
include anthocyanins, leucoanthocyanins, catechins, aurones and chalcones.
4.4.2 Results
Name of Test Positive Result Experiment Result Inference
Bate-Smith & Metcalf
Test
Strong red or
violet color
Greenish brown
solution
(-)
Wilstater "Cyanidin"
Test
Color ranging
from orange to
crimson and
magenta and
occasionally to
green or blue2 layers of light brown
and dark brown
solution
(-)
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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 γ-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.
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4.5.2 Results
Name of Test Positive Result Experiment Result Inference
Gelatin Test Formation of a
jelly precipitate
Yellow solution with
jelly precipitate
(+++)
Ferric chloride Test Blue-black
(hydrolysable
tannin)
Brownish green
(condensed
tannins)
Blue black solution
(+++)
Matchstick Test Wood will turn
red or pink in
color
(No photo available)
Dark green in color
(-)
4.5.3 Discussion
The tannins were extracted from the crude extract by the addition of sodium
chloride, which turns the tannins into their water-soluble salts. Tannins are known to
precipitate proteins. This was the principle behind the gelatin test, which was carried
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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
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 Result Experiment Result Inference
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Guignard's Test Appearance 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 non-specific for there are other substances, which can liberate H2S, SO2
or aldehydes. The test yielded a negative result, with the absence of yellow to brick
red decoloration of the sodium picrate paper.
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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 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 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
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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
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,
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/S0378874106002571
Stuart, G. (2013, October 1). Achuete. Retrieved November 3, 2014, from
http://www.stuartxchange.org/Asuete.html
Curriculum Vitae
49
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY PAGE
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: [email protected]
Educational Background
Secondary: Batangas State University (2008-2010)
Sovereign Shepherd School of Values and Learning (2010-2012)
Tertiary: University of Santo Tomas (2012-present)
Curriculum Vitae
50
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Name: Denise Anne Reyes Alcausin
Date of Birth: August 23, 1995
Place of Birth: Cotabato
Age: 19
Religion: Roman Catholic
Civil Status: Single
Nationality: Filipino
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
51
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Name: Mary Iris Mendoza Andal
Date of Birth: February 14, 1996
Place of Birth: Batangas City
Age: 18
Religion: Roman Catholic
Civil Status: Single
Nationality: Filipino
Address: Balagtasin I, San Jose, Batangas
E-mail: [email protected]
Educational Background
Secondary: St. Bridget College ( 2008 - 2012 )
Tertiary: University of Santo Tomas ( 2012 - Present )
Curriculum Vitae
52
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Name: Nicole Eileen M. Bagon
Date of Birth: August 16, 1995
Place of Birth: Sta. Cruz, Laguna
Age: 19
Religion: Roman Catholic
Civil Status: Single
Nationality: Filipino
Address: Lotus de Cataluna Dormitory, Tolentino St., Sampaloc, Manila
E-mail: [email protected]
Educational Background
Secondary: De La Salle - Lipa Integrated School (2008 - 2012)
Tertiary: University of Santo Tomas (2012 - Present)
Curriculum Vitae
53
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Name: Danielle Paras Barretto
Date of Birth: November 25 1995
Place of Birth: Manila
Age: 18
Religion: Roman Catholic
Civil Status: Single
Nationality: Filipino
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
54
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Name: Calvin EJ Robledo Bautista
Date of Birth: January 26, 1996
Place of Birth: Lipa city, Batangas
Age: 18
Religion: Iglesia Ni Cristo
Civil Status: Single
Nationality: Filipino
Address: Tower 1 Robinsons Place Manila, Padre Faura St., Ermita, Manila
E-mail: [email protected]
Educational Background
Secondary: De La Salle - Lipa Integrated School (2008 - 2012)
Tertiary: University of Santo Tomas (2012 - Present)
55