PharChem Manuscript (Bixa Orellana)

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

10

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


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


23


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.

27


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


29


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.


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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


3.3.5.3 Wilstater “Cyanidin” Test

31


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.


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.

32


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.

34


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

35


Dragendorff’s Test Orange precipitate

Red brown solution

(-)

Mayer’s Test White precipitate

Light brown solution

(-)

B. Primary Assay


Red brown solution

(-)


Light brown solution

(-)

C. Confirmatory Test


Orange yellow solution

(-)

36



Light yellow solution

(-)

D. Quaternary Bases


Brick red solution

(-)


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,

37


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.

38


4.2.2 Results


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,

39


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


Borntrager's Test Red coloration (++)

40


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.

41


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


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

(-)

42


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


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

44


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


45


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.

46


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

47


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.

48


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


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


Name: Denise Anne Reyes Alcausin

Date of Birth: August 23, 1995

Place of Birth: Cotabato

Age: 19

Religion: Roman Catholic

Civil Status: Single


Address: Santiago St., Town & Country West, Molino III, Bacoor City, Cavite



Secondary: Divine Light Academy (2008 - 2012)

Tertiary:University of Santo Tomas (2012 - Present)

Curriculum Vitae

51


Name: Mary Iris Mendoza Andal

Date of Birth: February 14, 1996

Place of Birth: Batangas City

Age: 18




Address: Balagtasin I, San Jose, Batangas



Secondary: St. Bridget College ( 2008 - 2012 )

Tertiary: University of Santo Tomas ( 2012 - Present )

Curriculum Vitae

52


Name: Nicole Eileen M. Bagon

Date of Birth: August 16, 1995

Place of Birth: Sta. Cruz, Laguna

Age: 19




Address: Lotus de Cataluna Dormitory, Tolentino St., Sampaloc, Manila



Secondary: De La Salle - Lipa Integrated School (2008 - 2012)

Tertiary: University of Santo Tomas (2012 - Present)

Curriculum Vitae

53


Name: Danielle Paras Barretto

Date of Birth: November 25 1995

Place of Birth: Manila

Age: 18




Address: Aston Martin st., St. Dominic Villa, City of San Fernando, Pampanga



Secondary: Pampanga High School (2008-2012)

Tertiary: University of Santo Tomas (2012-present)

Curriculum Vitae

54


Name: Calvin EJ Robledo Bautista

Date of Birth: January 26, 1996

Place of Birth: Lipa city, Batangas

Age: 18

Religion: Iglesia Ni Cristo



Address: Tower 1 Robinsons Place Manila, Padre Faura St., Ermita, Manila



Secondary: De La Salle - Lipa Integrated School (2008 - 2012)

Tertiary: University of Santo Tomas (2012 - Present)

55

PharChem Manuscript (Bixa Orellana)

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