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CHAPTER I
Introduction
Diseases have grown rapidly all over the globe that is even impossible to consider
that one does not own one. Since then, different remedies were formulated by scientist.
Pharmacists and even by those we call “quack doctors.” Now, medicines and varieties of
drugs sprout out to bring instant relief to the consumer but require a costly value.
The chico or sapodilla (scientific name: Manilkara zapota L.) is believed to be
native to Yucatan and possibly other nearby parts of southern Mexico. The sapodilla is an
attractive upright, slow-growing, long-lived evergreen tree. The leaves are highly
ornamental, 3 to 4-1/2 inches long and 1 to 1-1/2 inches wide. They are medium green,
glossy, alternate and spirally clustered at the tip of forked twigs. Sapodilla flowers are
small, inconspicuous and bell-like, approximately 3/8 inch in diameter. They are borne
on slender stalks in the axil of the leaves. There are several flushes of flowers throughout
the year. The fruit is round to egg-shaped, 2-4 inches in diameter. The skin is brown and
scruffy when ripe. The flesh varies from yellow to shades of brown and sometimes
reddish-brown, and may be smooth or of a granular texture. Fruits can be seedless, but
usually have from 3 to 12 hard, black, shiny, flattened seeds about 3/4 inch long in the
center of the fruit.
Background of the Study
The study on finding the Phytochemicals on chico (Manilkara Zapota L.) rind
interest the researchers because it has much potential properties that can possibly help us
and the society about the benefits.
Chico is a prolific tree. It bears fruit most months of the year and can be grown in
many parts of the country, even during harvest peaks, chico can still command good
market price. Chico is an ugly looking fruit which has a peculiar nice smell to it. Its
sweet, brown in color, and has a juicy flesh. Some are round and some are oval with
painted ends. The slightly granular pulp is sweet when ripe.
Chico fruit may also be pulped and used for making ice cream or jam. Although a
poor source of Vitamin C, the fruit abounds in calcium phosphorous and iron. The bark
produces a milky latex, the source of chicle (a major ingredients of chewing gum), and its
wood can also be used in the manufacture of the cabinets and furniture.
The researchers decided to conduct an experiment regarding the phytochemicals
present in the chico (Manilkara Zapota L.) rind. Hopefully, we can find an alternative
chemicals appropriate enough to cure a certain disease.
Statement of the Problem
This study aims to determine the phytochemical components of chico rind.
Specifically, this aims to answer the following questions:
1. What are the phytochemical components present in chico rind?
Significance of the Study
This study is focused on determining the active components present in the chico
(Manilkara Zapota L.) rind.
The result of this study could provide information to consumers so that they
would know the chemical components found in chico rind. Consumers used to hesitate in
buying this fruit just because in their own point of view, the rind seems to be useless but
if this study will be approved; there will be no reason for us, consumers, to be doubtful
enough in buying this fruit.
Limitations of the Study
The study is limited only in studying the chico (Manilkara Zapota L.) rind
together with the nutrients present in it. It focuses on its capability with the
phytochemicals present in the chico rind and the extraction of the seven phytochemical
components namely: alkaloids, saponins, tannins, flavanoids, steroids, cyanogenic
glycoside test, and the anthraquinones; what could it do in the human body and what
would be its benefits.
Operational Definition
Phytochemicals- chemical compounds that occur naturally in plants; chemicals that may
affect, but are not yet established as essential nutrients
Chico (Manilkara Zapota L.) rind- sample of the study used in the experimentation for
the presence of the 7 phytochemicals
Alkaloids- are group of naturally occurring chemical compounds which mostly contain
basic nitrogen atoms
Flavonoids- also known as vitamin P and citrine, are a class of plant secondary
metabolites
Saponins- are amphipathic glycosides grouped phenomenologically by the soap-like
foaming they produced when shaken in aqueous solutions
Steroids- type of organic compound that contains a specific arrangement of 4 rings that
are joined to each other
Tannins- also known as a vegetable tannin, an astringent, bitter plant polyphenolic
compound that either binds and precipitates or shrinks proteins and various other organic
compounds including amino acids and alkaloids
Cyanogenic Glycosides- belong to the products of secondary metabolism, to the natural
products of plants. These compounds are composed of an alpha-hydroxynitrile type
aglycone and of a sugar moiety (mostly D-glucose).
Anthraquinones- are organic compounds that have laxative effect on the body, but are
generally not recommended for regular use due to concerns about the risk of habit-
forming dependence and adverse side effects
CHAPTER II
Review of Related Literature
Chico (Manilka zapota L.)
Sapodilla or the Manilkara Zapota is an ever green tree, which is long living and
is native to the new world tropics. Though it is a native of Mexico, it was brought to the
Philippines by the Spanish Colonists. It is known by the name of chikoo or chiku, or
chickoo in India, South Asia and Pakistan. An average Sapodilla tree grows to about 30-
40m in height. The bark of the tree contains white gummy latex called the chicle. The
sapodilla trees bear fruit twice a year, though they flower all year round. The fruit, which
grows has a brown skin, resembling a potato. It grows to about 4-8 cm in diameter, and
may contain 2-10 seeds. Sapodilla has a high latex content, and does not ripen until
picked. It is extremely sweet to taste, and tastes very much like cotton candy or caramel
and has a grainy texture.
A very branched tree, growing to a height of 8 meters. Leaves are oblong to
narrowly oblong-obovate, 8 to 13 cm in length, pointed at both ends. Flowers are hairy
outside, 8 mm long and 6-parted. Fruit is brown, fleshy, ovoid to round, 3-8 cm long,
containing 5 or more shiny brown-black seeds. Fleshy is brown, soft, slightly gritty, and
sweet.
Phytochemicals
Phytochemicals are chemical compounds that occur naturally in plants, such as
beta-carotene. The term is generally used to refer to those chemicals that may affect
health, but are not yet established as essential nutrients. While there is abundant scientific
and government support for recommending diets rich in fruits and vegetables, there is
only limited evidence that health benefits are due to specific phytochemicals.
Phytochemicals as candidate therapeutics
Phytochemicals have been used as drugs for millennia. For example, Hippocrates
may have prescribed willow tree leaves to abate fever. Salicin, having anti-inflammatory
and pain-relieving properties, was originally extracted from the bark of the white willow
tree and later synthetically produced became the staple over-the-counter drug called
Aspirin. There is evidence from laboratory studies that phytochemicals in fruits and
vegetables may reduce the risk of cancer, possibly due to dietary fibers, polyphenol
antioxidants and anti-inflammatory effects. Specific phytochemicals, such as fermentable
dietary fibers, are allowed limited health claims by the US Food and Drug Administration
(FDA). An important cancer drug, Taxol (paclitaxel), is a phytochemical initially
extracted and purified from the Pacific yew tree. Among phytochemicals from edible
plants with promise for deterring disease, diindolylmethane, from Brassica vegetables
(broccoli, cauliflower, cabbage, kale, Brussels sprouts) is being tested against recurring
respiratory papillomatosis tumors (caused by the human papilloma virus), is in Phase III
clinical trials for cervical dysplasia (a precancerous condition caused by the human
papilloma virus) and is in several clinical trials for prostate cancer. Some phytochemicals
with physiological properties may be elements rather than complex organic molecules.
Abundant in many fruits and vegetables, selenium, for example, is involved with major
metabolic pathways, including thyroid hormone metabolism and immune function.
Particularly, it is an essential nutrient and cofactor for the enzymatic synthesis of
glutathione, an endogenous antioxidant.
Alkaloids
Alkaloids are a group of naturally occurring chemical compounds which mostly
contain basic nitrogen atoms. This group also includes some related compounds with
neutral and even weakly acidic properties. Also some synthetic compounds of similar
structure are attributed to alkaloids. Beside carbon, hydrogen and nitrogen, molecules of
alkaloids may contain sulfur and rarely chlorine, bromine or phosphorus. Alkaloids are
produced by a large variety of organisms, including bacteria, fungi, plants, and animals
and are part of the group of natural products (also called secondary metabolites). Many
alkaloids can be purified from crude extracts by acid-base extraction. Many alkaloids are
toxic to other organisms. They often have pharmacological effects and are used as
medications, as recreational drugs, or in entheogenic rituals. Examples are the local
anesthetic and stimulant cocaine, the stimulant caffeine, nicotine, the analgesic morphine,
or the antimalarial drug quinine. Although alkaloids act on a diversity of metabolic
systems in humans and other animals, they almost uniformly invoke a bitter taste. The
boundary between alkaloids and other nitrogen-containing natural compounds is not
clear-cut. Compounds like amino acid peptides, proteins, nucleotides, nucleic acid,
amines and antibiotics are usually not called alkaloids. Natural compounds containing
nitrogen in the exocyclic position (mescaline, serotonin, dopamine, etc.) are usually
attributed to amines rather than alkaloids. Some authors, however, consider alkaloids a
special case of amines.
Compared with most other classes of natural compounds, alkaloids are
characterized by a great structural diversity and there is no uniform classification of
alkaloids. Historically, first classification methods combined alkaloids by the common
natural source, e.g., a certain type of plants. This classification was justified by the lack
of knowledge about the chemical structure of alkaloids and is now considered obsolete.
More recent classifications are based on similarity of the carbon skeleton (e.g.,
indole, isoquinoline and pyridine-like) or biogenetic precursor (ornithine, lysine, tyrosine,
tryptophan, etc.). However, they require compromises in borderline cases; for example,
nicotine contains a pyridine fragment from nicotinamide and pyrrolidine part from
ornithine and therefore can be assigned to both classes.
Alkaloids are often divided into the following major groups:
1. "True alkaloids", which contain nitrogen in the heterocycle and originate from
amino acids. Their characteristic examples are atropine, nicotine and morphine.
This group also includes some alkaloids which beside nitrogen heterocycle
contain terpene (e.g. evonine) or peptide fragments (e.g. ergotamine). This group
also includes piperidine alkaloids coniine and coniceine although they do not
originate from amino acids.
2. "Protoalkaloids", which contain nitrogen and also originate from amino acids.
Examples include mescaline, adrenaline and ephedrine.
3. Polyamine alkaloids – derivatives of putrescine, spermidine and spermine.
4. Peptide and cyclopeptide alkaloids.
5. Pseudalkaloids – alkaloid-like compounds which do not originate from amino
acids. This group includes, terpene-like and steroid-like alkaloids, as well as
purine-like alkaloids such as caffeine, theobromine and theophylline. Some
authors classify as pseudoalkaloids such compounds such as ephedrine and
cathinone. Those originate from the amino acid phenylalanine, but acquire their
nitrogen atom not from the amino acid but through transamination.
Some alkaloids do not have the carbon skeleton characteristic of their group. So,
galantamine and homoaporphines do not contain isoquinoline fragment, but are generally
attributed to isoquinoline alkaloids.
Saponins
Saponins are a class of chemical compounds, one of many secondary metabolites
found in natural sources, with saponins found in particular abundance in various plant
species. Specifically, they are amphipathic glycosides grouped phenomenologically by
the soap-like foaming they produce when shaken in aqueous solutions, and structurally
by their composition of one or more hydrophilic glycoside moieties combined with a
lipophilic triterpene derivative. A ready and therapeutically relevant example is the
cardio-active agent digoxin, from common foxglove.
One research use of the saponin class of natural products involves their
complexation with cholesterol to form pores in cell membrane bilayers, e.g., in red cell
(erythrocyte) membranes, where complexation leads to red cell lysis (hemolysis) on
intravenous injection. In addition, the amphipathic nature of the class gives them activity
as surfactants that can be used to enhance penetration of macromolecules such as proteins
through cell membranes. Saponins have also been used as adjuvants in vaccines.
There is tremendous, commercially driven promotion of saponins as dietary
supplements and nutriceuticals. There is evidence of the presence of saponins in
traditional medicine preparations, where oral administrations might be expected to lead to
hydrolysis of glycoside from terpenoid (and obviation of any toxicity associated with the
intact molecule). But as is often the case with wide-ranging commercial therapeutic
claims for natural products:
the claims for organismal/human benefit are often based on very preliminary
biochemical or cell biological studies; and
mention is generally omitted of the possibilities of individual chemical sensitivity,
or to the general toxicity of specific agents,) and high toxicity of selected cases.
While such statements require constant review (and despite the myriad web
claims to the contrary), it appears that there are very limited US, EU, etc. agency-
approved roles for saponins in human therapy. In their use as adjuvants in the production
of vaccines, toxicity associated with sterol complexation remains a major issue for
attention. Even in the case of digoxin, therapeutic benefit from the cardiotoxin is a result
of careful administration of an appropriate dose. Very great care needs to be exercised in
evaluating or acting on specific claims of therapeutic benefit from ingesting saponin-type
and other natural products.
Tannin
A tannin (a.k.a a vegetable tannin, i.e. a type of biomolecule, as opposed to
modern synthetic tannin) is an astringent, bitter plant polyphenolic compound that either
binds and precipitates or shrinks proteins and various other organic compounds including
amino acids and alkaloids. The astringency from the tannins is what causes the dry and
puckery feeling in the mouth following the consumption of unripened fruit or red wine.
Likewise, the destruction or modification of tannins with time plays an important role in
the ripening of fruit and the aging of wine. The term tannin (from tanna, an Old High
German word for oak or fir tree, as in Tannenbaum) refers to the use of wood tannins
from oak in tanning animal hides into leather; hence the words "tan" and "tanning" for the
treatment of leather. However, the term "tannin" by extension is widely applied to any
large polyphenolic compound containing sufficient hydroxyls and other suitable groups
(such as carboxyls) to form strong complexes with proteins and other macromolecules.
The compounds are widely distributed in many species of plants, where they play a role
in protection from predation, and perhaps also in growth regulation. Tannins have
molecular weights ranging from 500 to over 3,000 (gallic acid esters) and up to 20,000
(proanthocyanidins). Tannins are incompatible with alkalis, gelatin, heavy metals, iron,
lime water, metallic salts, strong oxidizing agents and zinc sulfate, since they form
complexes and precipitate in aqueous solution.
Tannins have been shown to precipitate proteins, which inhibits in some ruminant
animals the absorption of nutrients from high-tannin grains such as sorghum.
In sensitive individuals, a large intake of tannins may cause bowel irritation,
kidney irritation, liver damage, irritation of the stomach and gastrointestinal pain. With
the exception of tea, long-term and/or excessive use of herbs containing high
concentrations of tannins is not recommended. A correlation has been made between
esophogeal or nasal cancer in humans and regular consumption of certain herbs with high
tannin concentrations.
Many plants employ tannins to deter animals. It has not been determined whether
tannin was produced for another purpose, e.g. as pesticide, or whether it evolved
specifically for the purpose of inhibiting predation. Animals that consume excessive
amounts of these plants fall ill or die. Acorns are a well known problem in cattle
breeding. The lethal dose is said to be around 6% of the animal's body weight. This is
only an approximate figure since acorns from Red Oak were shown to contain on average
two to four times the tannins than those from White Oak. Some deer and moose were
found to have perished due to ingesting acorns. Symptoms include ataxia and shortness of
breath. Some animals, like squirrels and mule deer have developed the ability to consume
high concentrations of tannins without ill effects. Humans would usually find the bitter
taste of foods containing high amounts of tannins unpalatable. (Some humans were found
to be unable to taste bitter foods.) Tannins are leached from acorns before they are used
for human consumption.
Flavonoids
Flavonoids (or bioflavonoids), also collectively known as Vitamin P and citrin,
are a class of plant secondary metabolites. According to the IUPAC nomenclature, they
can be classified into:
flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone)
structure (examples: quercetin, rutin).
isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone)
structure
neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone)
structure.
The three flavonoid classes above are all ketone-containing compounds, and as
such, are flavonoids and flavonols. This class was the first to be termed "bioflavonoids."
The terms flavonoid and bioflavonoid have also been more loosely used to describe non-
ketone polyhydroxy polyphenol compounds which are more specifically termed
flavanoids, flavan-3-ols, or catechins (although catechins are actually a subgroup of
flavanoids).
Flavonoids (specifically flavanoids such as the catechins) are "the most common
group of polyphenolic compounds in the human diet and are found ubiquitously in
plants". Flavonols, the original bioflavonoids such as quercetin, are also found
ubiquitously, but in lesser quantities. Both sets of compounds have evidence of health-
modulating effects in animals which eat them.
The widespread distribution of flavonoids, their variety and their relatively low
toxicity compared to other active plant compounds (for instance alkaloids) mean that
many animals, including humans, ingest significant quantities in their diet. Results from
experimental evidence suggest that flavonoids may modify allergens, viruses, and
carcinogens indicating flavonoids have potential to be biological "response modifiers", in
vitro studies of flavonoids have displayed anti-allergic, anti-inflammatory, anti-microbial
and anti-cancer activities.
Flavonoids (both flavonols and flavanols) are most commonly known for their
antioxidant activity in vitro.
Consumers and food manufacturers have become interested in flavonoids for their
possible medicinal properties, especially their putative role in prevention of cancers and
cardiovascular diseases. Although physiological evidence is not yet established, the
beneficial effects of fruits, vegetables, tea, and red wine have sometimes been attributed
to flavonoid compounds rather than to known micronutrients, such as vitamins and
dietary minerals.
Alternatively, research conducted at the Linus Pauling Institute and evaluated by
the European Food Safety Authority indicates that, following dietary intake, flavonoids
themselves are of little or no direct antioxidant value. As body conditions are unlike
controlled test tube conditions, flavonoids and other polyphenols are poorly absorbed
(less than 5%), with most of what is absorbed being quickly metabolized and excreted.
The increase in antioxidant capacity of blood seen after the consumption of flavonoid-
rich foods is not caused directly by flavonoids themselves, but most likely is due to
increased uric acid levels that result from metabolism of flavonoids. According to Frei,
"we can now follow the activity of flavonoids in the body, and one thing that is clear is
that the body sees them as foreign compounds and is trying to get rid of them."
Steroids
A steroid is a type of organic compound that contains a specific arrangement of
four rings that are joined to each other. Examples of steroids include cholesterol, the sex
hormones estradiol and testosterone, and the anti-inflammatory drug dexamethasone.
The sterane core of steroids is composed of seventeen carbon atoms bonded together to
form four fused rings: three cyclohexane rings (designated as rings A, B, and C in the
figure to the right) and one cyclopentane ring (the D ring). The steroids vary by the
functional groups attached to these rings and by the oxidation state of the rings. Sterols
are special forms of steroids, with a hydroxyl group at position-3 and a skeleton derived
from cholestane. Hundreds of distinct steroids are found in plants, animals, and fungi.
All steroids are made in cells either from the sterols lanosterol (animals and fungi) or
from cycloartenol (plants). Both lanosterol and cycloartenol are derived from the
cyclization of the triterpene squalene. Taxonomical/Functional
Some of the common categories of steroids:
Animal steroids
o Insect steroids
Ecdysteroids such as ecdysterone
o Vertebrate steroids
Steroid hormones
Sex steroids are a subset of sex hormones that produce sex
differences or support reproduction. They include
androgens, estrogens, and progestagens.
Corticosteroids include glucocorticoids and
mineralocorticoids. Glucocorticoids regulate many aspects
of metabolism and immune function, whereas
mineralocorticoids help maintain blood volume and control
renal excretion of electrolytes. Most medical 'steroid' drugs
are corticosteroids.
Anabolic steroids are a class of steroids that interact with
androgen receptors to increase muscle and bone synthesis.
There are natural and synthetic anabolic steroids. In
popular language, the word "steroids" usually refers to
anabolic steroids.
Cholesterol, which modulates the fluidity of cell membranes and is
the principal constituent of the plaques implicated in
atherosclerosis.
Plant steroids
o Phytosterols
o Brassinosteroids
Fungus steroids
o Ergosterols
Cyanogenic Glycosides
In this case, the aglycone contains a cyanide group. In many plants, these
glycosides are stored in the vacuole but if the plant is attacked they are released and
become activated by enzymes in the cytoplasm. These remove the sugar part of the
molecule and release toxic hydrogen cyanide. Storing them in inactive forms in the
cytoplasm prevents them from damaging the plant under normal conditions. An example
of these is amygdalin from almonds. They can also be found in the fruits (and wilting
leaves) of the rose family (including cherries, apples, plums, almonds, peaches, apricots,
raspberries, and crabapples). Cassava, an important food plant in Africa and South
America, contains cyanogenic glycosides and therefore has to be washed and ground
under running water prior to consumption. Sorghum (Sorghum bicolor) expresses
cyanogenic glycosides in its roots and thus is resistant to pests such as rootworms
(Diabrotica spp.) that plague its cousin maize (Zea mays L.). It was once thought that
cyanogenic glycosides might have anti-cancer properties, but this idea was disproven, see
Amygdalin. A recent study may also show that increasing CO2 levels, caused by
anthropogenic emissions, may result in much higher levels of cyanogenic glycoside
production in Sorghum and Cassava plants, making them highly toxic and inconsumable.
A doubling of CO2 concentration was found to double the concentration of cyanogenic
glycosides in the leaves. Dhurrin, linamarin, lotaustralin, and prunasin are also classified
as cyanogenic glycosides.
Anthraquinone
Anthraquinone, also called anthracenedione or dioxoanthracene is an aromatic
organic compound with formula C14H8O2, that can be viewed as a diketone derivative of
anthracene (with loss of one of the central pi-bonds in the anthracene). The term usually
refers to one specific isomer, 9,10-anthraquinone or 9,10-dioxoanthracene, whose ketone
groups are on the central ring. This compound is an important member of the quinone
family. It is a building block of many dyes and is industrially used in bleaching pulp for
papermaking. It is a yellow highly crystalline solid, poorly soluble in water but soluble in
hot organic solvents. For instance, it is almost completely insoluble in ethanol near room
temperature but 2.25 g will dissolve in 100 g of boiling ethanol. Several other
anthraquinone isomers are possible, such as 1,2-, 1,4-, and 2,6-anthraquinone, but they
are of comparatively minor importance. The term is also used in the more general sense
of any compound that can be viewed as an anthraquinone with some hydrogen atoms
replaced by other atoms or functional groups. These derivatives include many substances
that are technically useful or play important roles in living beings.
A large industrial application of anthraquinones is for the production of hydrogen
peroxide. 2-Ethyl-9,10-anthraquinone or a related alkyl derivatives is used, rather
anthraquinone itself.
Catalytic hydrogen peroxide production with the anthraquinone process
Derivatives of 9,10-anthraquinone include many important drugs (collectively called
anthracenediones). They include
laxatives like dantron, emodin, and aloe emodin, and some of the senna
glycosides
antimalarials like rufigallol
antineoplastics used in the treatment of cancer, like mitoxantrone, pixantrone, and
the anthracyclines.
CHAPTER III
Methodology
This chapter deals on the procedure involved in the determination of the
Phytochemicals present in the chico (Manilkara Zapota L.).
Materials, Equipments and Chemicals
Beaker
Tongs
Test tubes
Test tube rack
Filter paper
Picrate paper
Litmus paper
Spatula
Cork
Buncher funnel
Dropper/pipette
Stoppered vials
Evaporating dish
Analytical balance
Rotary evaporator
Stirring rod
Separatory funnel
Electric stove
200 mL distilled water
Magnesium turnings
Drgendorff’s reagent
Mayer’s reagent
Gelatin-salt solution
1 L Ethanol/ Ethyl alcohol
1 mL sulfuric acid
5 mL ammonia solution
Hexane
0.5 g NaCl solution
Ferric chloride solution
Concentrated sulfuric acid
1% Emulsion solution
5 mL Benzene
10 mL chloroform
3 mL FeCl3
General Procedure
I. Preparation of the Plant Extracts
A g of chico rind was weighed and placed in the Erlenmeyer flask. It was soaked
for 2428 hours using 00 ml or sufficient 80% ethyl alcohol. It was filtered through a
Buncher funnel preferably with a gentle suction.
II. Phytochemical Analysis
(A.)Alkaloids
A.1 Preliminary test
An aliquot portion of the extract was taken and evaporated to syrupy form over
a steam bat. A 5 ml of 2M hydrochloric acid was added with stirring for about 5 minutes
and cooled. The syrupy form was divided into two. A portion was taken and tested with
2-3 drops of Dragendorff’s reagent and another portion with 2-3 drops of Mayer’s
reagent. A positive result is indicated by an orange precipitate with Dragendorff’s reagent
and a white precipitate with Mayer’s test.
(B.)Saponins
B. 1 The Froth Test
A portion of the extract was taken for Froth Test, adding a volume of 80% ethyl
alcohol and shake vigorously for 30 seconds. “Honeycomb” froths greater than 3 cm.
from the surface of the liquid persists after 30 minutes the sample is considered positive
for saponins. But if the froth is les than 3 cm. it is considered as negative.
(C.)Tannin
C. 1 Gelatin Test
An extract was prepared with the same procedure as in for Alkaloid Test and
tested with three drops of gelatin-salt solution. Formation of precipitate indicates the
presence of tannins.
C. 2 Chloride Test
Another portion of the extract was treated and poured with 3 drops of ferric
chloride solution. A blue-black color indicates the presence of condensed tannins.
(D.)Flavonoids
D. 1 Bate-smith and Metacalf Test for Leucoanthocyanins
An aliquot portion was treated with 0.5 mL concentrated HCl and for any color
changes for 15 minutes in water bath. After an hour, a change in color was observed. A
strong red or violet color indicates the presence of Leucoanthocyanins.
(E.)Steroids
E. 1 Keller-kiliani Test
An aliquot portion of the extract was taken and was evaporated to incipient
dryness over warm bath. It was defatted by triturating the residue with hexane. The
hexane extract was decanted and the treatment was repeated until most of the colored
pigments have been removed. The hexane extract was discarded. The defatted residue
was placed over a warm bath to remove the hexane. A 3 mL FeCl3 reagent was added.
The mixture was stirred and transferred to a test tube. A 1 mL of concentrated sulfuric
acid was added cautiously and allows the mixture to stand for a few minutes. A reddish-
brown, which may turn blue or purple, will indicate the presence of 2 deoxysugars.
(F.)Cyanogenic Glycoside Test
F. 1 The Guignard Test
An aliquot portion of the extract was placed in a test tube. It was moisten with
enough water, and was added a few drops of chloroform to enhance the enzyme activity.
A 1 mL of 1% emulsion solution must be added to insure the hydrolysis of the glycoside.
The test tube must be stopped with a cork, from which a piece of picrate paper is
suspended. The paper strip must not touch the inner side of the test tube. The test tubes
must be warmed at 35-40 degree Celsius or must be kept at room temperature for 3 hours
and was observed for any color change in the paper. The relative concentrations of the
cyanogenic glycosides were measured and various shades of red appeared within 15
minutes.
(G.)Anthraquinones
G. 1 Borntrager’s Test
An aliquot portion of the extract was taken and evaporated to inapient dryness
over a warmth bath. A 10 mL distilled water was added to the residue and fitered. The
filtrate was extracted twice with 5 mL portion of benzene. The benzene extracts were
divided into two portions. One portion was reserved as the control. The other portion was
treated with 5 mL ammonia solution and was shaked. A red coloration in the lower
alkaline layer indicates the presence of anthraquinones.
Bibliography
Online Sources
http://en.wikipedia.org/wiki/Phytochemical
http://www.stuartxchange.org/Chico.html
http://en.wikipedia.org/wiki/Alkaloid
http://www.herbs2000.com/h_menu/alkaloids.htm
http://www.phytochemicals.info/phytochemicals/saponins.php
http://en.wikipedia.org/wiki/Tannin
http://en.wikipedia.org/wiki/Flavonoid
http://en.wikipedia.org/wiki/Steroid
http://en.wikipedia.org/wiki/Glycoside
http://en.wikipedia.org/wiki/Anthraquinone
http://www.agriculturalproductsindia.com/fruits/fruits-sapodilla.html http://
www.ncbi.nlm.nih.gov/pubmed/10669009
Books
Bahr, Lauren S., Johnston, Bernard, and Bloomfield, Louise A.. Collier’s Encyclopedia.
New York: P.F. Collier, 1996.
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Phytochemical Analysis of
Chico (Manilkara zapota L.) Rind
In Partial Fulfillment
For Research III
Researchers:
Arong, Lyndy C.
Clitar, Sheila A.
Flores, Nerissa T.
Lagrada, Nina Jane J.
Mariquit, Ernelyn B.
Zacal, Dova Salome V.
November, 2010