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.

Landau, Sidney I.. Webster Illustrated Contemporary Dictionary. Chicago, Illinois: J.G.

Ferguson Publishing Company,1978.

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