BIBLIOGRAPHY
YVONNE B. DEGAY-BOLAYO, November 2006. PHYTONUTRIENT
ANALYSIS OF THE SHOOTS OF THE PASSION FRUIT VINE. (Passiflora edulis
Sims).Benguet State University, La Trinidad, Benguet.

Adviser: Louisa P. Pladio, M.S. Chemistry
ABSTRACT

Phytonutrient analyses of the shoots of the passion fruit revealed that this plant
contain protein, carbohydrates, fat, minerals (potassium, calcium, phosphate, sodium,
iron and zinc) and vitamins (A, C, and E). The presence of cyanogenic glycoside,
alkaloids, flavonoids, steroids, anthraquinones, sugar and essential oils were determined
in the phytochemical tests.
There is no risk associated with the consumption of the shoots of the passion fruit
as vegetable due to presence of natural toxin particularly cyanogenic glycoside because it
is easily eliminated through cooking. This is supported by the results of the brine shrimp
assay which indicated that the toxicity of the different extracts of the shoots is
insignificant.
Flavonoid, one of the compounds that impart bitter taste was partially isolated
from the sticky brown acetate extract following a thin layer chromatography directed
scheme. This fraction has a brown to yellow color and the flavonoid spot has an Rf value
of 0.467 and is indicated by the blue spot upon spraying with potassium- ferricyanide-
ferric chloride while it is a red color when sprayed with vanillin-sulfuric acid.

---

TABLE OF CONTENTS
Page
Bibliography………………………………………………………….. i
Abstract………………………………………………………………… i
Table of Contents………………………………………………………..
ii
INTRODUCTION
Background of the Study…………………………………………
1
Objectives of the Study…………………………………………..
3
Importance of the Study…………………………………………..
3
Place and Time of Study………………………………………….
3
REVIEW OF RELATED LITERATURE

Botanical Description…………………………………………….
4
Plant Nutrients

Water………………………………………………………
6
Energy-yielding nutrients…………………………………
6
Minerals……………………………………………………
6
Vitamins……………………………………………………
7
Phytochemicals

Alkaloids…………………………………………………
8
Flavonoids…………………………………………………
9
Cyanogenic glycosides……………………………………
9
Triterpenoids………………………………………………
10
Properties of Phytochemicals………………………………
11
ii

---

Natural toxins………………………………………………
11
Detoxifying Processes……………………………………..
13
Brine Shrimp Assay………………………………………………
14
Extraction Processes……………………………………………...
14
Column Chromatography…………………………………
15
Thin Layer Chromatography………………………………
16
MATERIALS AND METHODS

Materials

Plant Materials……………………………………………
17
Standard ………………...…………………………………
18
Equipment………………………………………………….
18
Phytonutrient Analyses of the shoots of Passiflora edulis Sims

Proximate Analysis…………………………………………
21
Mineral Content Analysis…………………………………..
22
Vitamin Analysis…………………………………………….
22
Sugar Analysis……………………………………………
24
Phytochemical Screening of Secondary Metabolites

Sample preparation………………………………………….
26
Phytochemical Analysis…………………………………….
26
Thin-Layer Chromatography Screening…………………...
27
Effect of Heat on Cyanogenic Glocoside…………..………
28
Toxicity test

Brine Shrimp Assay………………………………………
29
iii

---

Analysis of the Results………………………… …………
31

Extraction and Partial Isolation of Bitter Constituent of the
Shoots of the Passion Fruit
Extraction…………………………………………………..
31
Monitoring of Bitter constituent…………………………
33
Isolation (Partial)…………………………………………
33
RESULTS AND DISCUSSION

Proximate Analysis……………………………………………..
35
Mineral Analysis…………………………………………………
38
Vitamin Analysis………………………………………………….
40
Sugar Analysis…………………………………………………
43
Phytochemical Screening for Secondary Metabolites……………
48
Alkaloid……………………………………………………
48
Flavonoid…………………………………………………..
49
Cyanogenic glycoside……………………………………..
50
Effect of Heating on Cyanogenic Glycoside……………………..
56
Toxicity Test……………………………………………………...
58
Brine Shrimp Assay………………………………………..
58
Extraction of Bitter Constituents of Passion Fruit vine

Extraction of Flavonoids…………………………………
60
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary…………………………………………………………
64
Conclusions……………………………………………………….
66
iv

---

Recommendations………………………………………………..
67
LITERATURE CITED…………………………………………………
68
APPENDICES
Preparation of Test Reagents…………………………………….
73
Proximate Analysis………………………………………………
76
Thin layer chromatography………………………………………
77
Toxicity Test Using Brine Shrimp Assay……………………….
78
BIOGRAPHICAL SKETCH………………………………………….
81

v

---

1

INTRODUCTION

Background of Study
Foods of plant origin have long been regarded by folks for their
nutritional, medicinal and protective values. Vegetables are known to be a good
source of vitamins, minerals, proteins and certain hormone precursor (Shemilt,
1982). Many indigenous plants sold in the market nowadays were not used as
vegetables before.
People avoid fruits and vegetables treated with commercial pesticides but
often are unaware they are ingesting the natural toxins present in foods. There are
some reports that claim the health risks from natural chemicals are more toxic
than the risks from pesticide residues. Although this has not been conclusively
proven, they assert that some constituents of commonly consumed vegetables like
cabbage and broccoli are more toxic to human than chemical pesticides in foods
(Pimentel et al, 1996).
Man utilizes for food the different parts of the plants like the seeds, leaves,
roots and fruits. Plants are known to produce a wide array of chemicals. They
contain natural chemicals that are needed for growth and health such as
carbohydrates, sugars, proteins and vitamins. But some plants used for food
contain undesirable natural products including some amino acids, proteins,
antinutrients and glycosides (Mendoza et al, 1985). These chemicals are
considered natural toxins in plants. New Zealand Food Safety Authority (1994)
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

2

cited some commonly consumed plants to contain toxins in the leaves, fruits,
seeds and shoots.
The presence of the natural toxins in plants is to protect them from
predators, as a natural pesticide to inhibit the action of insects or to protect them
from spoilage by weather, handling, UV light and microbes (Randolph, 2005).
Ingesting foods containing toxins may produce adverse effects such as allergic
reactions or negative effects in the gastrointestinal, cardiac or nervous systems of
animals and humans.
Edible plants, full of nutrients, vitamins and fiber as recommended by
most professional health organizations are considered by people for their optimal
diet. Even plants containing toxic compounds are considered staple food because
people traditionally, developed effective processing methods to reduce or
eliminate the toxins.
In the Cordillera particularly in Benguet and in Mountain Province, shoots
of the passion fruit are utilized as vegetable. For the passion fruit, some species of
this plant like giant granadilla have been reported to contain cyanogenic glycoside
on the leaves, skin and immature seeds (Morton, 1987). The shoots of this plant
have bitter taste. The popularity of this plant for food has motivated the researcher
to study the suitability of the shoots of the passion fruit for vegetable use.


Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

3

Objectives of the Study
The main objective of the study was to determine the suitability of the
shoots of the passion fruit for vegetable use. Specifically, to:
1. Determine the phytonutrients of the shoots of the passion fruit.
2. Determine the toxicity of the shoots of the passion fruit.
3. Determine the compound responsible for the bitter taste of the shoots of
the passion fruit.

Importance of the Study

The result of this study will provide the consumers adequate information
regarding the health benefits and risks associated with the consumption of the
plant as vegetable. It will also serve as baseline information for similar studies.

Time and Place of Study
The study was conducted from May 2006 to October 2006. The
phytonutrient, phytochemical analysis, brine shrimp assay and partial isolation of
the flavonoid were conducted at the Chemistry Department, Benguet State
University and DOST Chemical Laboratories, La Trinidad, Benguet. Mineral
analyses were done by the Philippine Institute of Pure and Applied Chemistry
(PIPAC) at Ateneo de Manila, Quezon City.


Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

4

REVIEW OF RELATED LITERATURE

Passion Fruit (Passiflora edulis Sims)

Botanical Description
Passion fruit Passiflora edulis Sims (Plate 1) is also known as masaplora
or bantanas in Benguet. Morton (1987) described the plant as follows: vine of this
plant is a shallow-rooted, woody, perennial, and climbing by means of tendrils.


A. Passion Fruit Vine


B. Shoot of the Passion Fruit


C. Flower of Passion Fruit Plant
D. Passion Fruit

Plate1. The Passion Fruit Plant (Passiflora edulis Sims)
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

5

The alternate, evergreen leaves, deeply 3-lobed when mature, are finely
toothed, 3 to 8 in (7.5-20 cm) long, deep-green and glossy above, paler and dull
beneath, and like the young stems and tendrils tinged with red or purple especially
in the yellow form.
A single, fragrant flower (Plate 1c), 2 to 3 in (5-7.5 cm) wide, is borne at
each node on the new growth. The bloom, clasped by 3 large, green, leaf like
bracts, consists of 5 greenish-white sepals, 5 white petals, a fringelike corona of
straight, white tipped rays, rich purple at the base, also 5 stamens with large
anthers, the ovary, and triple-branched style forming a prominent central
structure. The nearly round or ovoid fruit ( Plate 1d) 1 1/2 to 3 inches wide has a
tough rind, smooth waxy, ranging in hue from dark-purple with faint, fine white
specks, to light -yellow. It is 1/8 inches thick, adhering to a 1/4 inches layer of
white pith. Within a cavity is filled with a mass of double-walled, membranous
sacs filled with orange-colored, pulpy juice and small, hard, dark-brown or black,
pitted seeds.
The passion fruit (Passiflora edulis Sims) is a vine known for its edible
fruit either green or ripe. This plant is also recorded for its pharmacological
action. The plant is antiscorbutic, stomachic, and refreshing when drunk as
lemonade. According to Holland and De Lanessan as mentioned by Quisumbing
(1978), the roots possess narcotic properties. Also, it is used as diuretic and
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

6

emetic. Lirio et. al. (2006) mentioned that the juice from the fruit is also used to
relieve hang-over, diarrhea, arthritis and hypertension.

Plant Nutrients

Water
Most fruits and vegetables contain up to 90 percent water. Strawberries,
watermelon, lettuce, cabbage, celery, spinach and broccoli contain 90-99% water.
Functions of the water in the body are: carries nutrients and waste products;
maintains large molecules such as proteins and glycogen; participates in
metabolic properties; aids in the regulation of normal body temperature and
maintains blood volume (Whitney and Rolfes, 2005).

Energy Yielding Nutrients
The body uses carbohydrates, fats, and protein for energy to fuel all its
activities. When these nutrients are used by the body, the bonds between each
atom break, releasing energy. Some of the energy is released as heat, but some is
used to send electrical impulses through the brain and nerves, to synthesize body
compounds and to move muscles (Whitney and Rolfes, 2005).

Minerals
Minerals are inorganic elements (Whitney and Rolfes, 2005) required in
physiological processes: they regulate fluid balance, muscle contractions and
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

7

nerve impulses (Encyclopedia, 2003). The major minerals which are needed in
large amounts are calcium, phosphorus, potassium, sodium, chloride, magnesium
and sulfur. Trace minerals, such as chromium, copper, fluoride, iodine, iron,
selenium and zinc are needed in smaller quantities (Encyclopedia, 2003).

Vitamins
Whitney and Rolfes (2005) defined vitamins as organic, essential nutrients
required in tiny amounts to perform specific functions that promote growth,
reproduction, or the maintenance of health and life. Vitamins differ from
carbohydrates, fats and proteins in the following ways: a) their structure- they are
individual units and are not linked together as molecules of glucose or amino
acids; b) function- they do not yield energy when broken down; they assist the
enzymes that release energy from carbohydrates, fats and proteins; c) food
contents- the amount of vitamins ingested from food are measured in terms of
micrograms. Vitamins maybe water-soluble (like B vitamins and Vit. C) or fat-
soluble (such as Vit. A, D, E, and K).

Phytochemicals

Scholbe (2001) defined phytochemicals as non-nutritive plant chemicals
that contain protective, disease-preventing compounds. Guevara (2005) cited that
these phytochemicals are some of the secondary plant metabolites like alkaloids,
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

8

saponins, steroids, flavonoids, tannins and polyphenols, anthraquinones,
cyanogenic glycosides and terpenoids.

Alkaloids
These are small organic molecules, comprising several carbon rings with
side chains in which one or more of the carbon atoms have been replaced by
nitrogen. Examples of these bitter compounds are nicotine, quinine, srychnine,
ergotamine atropine and caffeine. The action of alkaloids on the nervous system is
to disrupt electrochemical transmission at nerve junctions, either preventing
transmission or enhancing it inappropriately (Randolph, 2005).
Glycoalkaloids are found in all potatoes. Higher level of this toxin is
found in the sprouts and the peel of the potatoes that taste bitter. These toxins are
produced by the plant due to stress like presence of microorganisms, UV light and
damage such as bruising (Delicious Organics Inc, 2004). Solanine is a
cholinesterase inhibitor found in the surface of the potatoes exposed to light
which turned green from chlorophyll production. Highest concentration is found
beneath the peel. Peeling deeply to remove the green splotches and cooking in a
steam or water reduces the toxin. Solanine can cause gastric upset and respiratory
problems including the fatal depression of the activity of the central nervous
system (Pimentel et. al., 1996). Spontaneous abortions in laboratory animals have
been reported so pregnant women should avoid green potatoes even the cooked
ones with bitter taste (Guilbert, 2001).
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

9

Flavonoids
These are phenolic plant pigments generally containing the γ-benzopyrone
nucleus. Flavonoids include the anthocyanins, leucoanthocyanins, catechins,
aurones and chalcones. Anthocyanins make up the most important coloring matter
in plants (Guevarra, 2005). Drewnoski and Carneros (2005) stated that flavonoids
that impart the bitter taste found in citrus fruits include flavanones (naringin),
flavones (nobelitin) and flavonols (quercitin). In the report of Chang (1990) as
mentioned by the same authors, genistin, a bitter and astringent isoflavone
glucoside is thought to be responsible for the objectionable taste of soy protein.
They also reported that catechin and epicatechin occur in tea. Epicatechin is more
bitter than catechin. The bitter taste of chocolate may also be due to catechins,
present in higher amounts than in milk chocolates.

Cyanogenic Glycosides

According to Food Standards Australia New Zealand (2004) glycosides
are widely distributed in plants. Cassava and bamboo shoots contain cyanogenic
glycosides, linamarin and taxiphillin, respectively. Cyanogenic glycosides are the
glycosides of the alpha- hydroxynitriles. The toxicity of cyanogenic plant depends
on the hydrogen cyanide that may be released upon consumption. If the
cyanogenic plant is inadequately detoxified during processing or preparation of
food, the potential hydrogen cyanide concentration, which may be released, is
high. This compound is contained in kernels of almonds, lemons, limes, apples,
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

10

pears, cherries, apricots, prones and palms. Kidney, black-eyed peas and lima
beans also contain bitter cyanogenic glycoside.
The other class of glycoside is the mustard oil glycoside or glucosinates,
which are found exclusively in plants belonging to the family of Crucifereae.
There is abundant evidence relating goiter as a result of excessive consumption of
cruciferous plants. Other foods with antithyroid activity include plants in the
genus Allium (onion group) and other vegetables. However, it has not been
proven that these foods are goitrogenic unless they comprise an excessive high
proportion of the diet (Pimentel, et. al. 1996).
Saponin is a glycoside, which occurs in many legumes. According to
Mendoza et. al. (1985) these glycosides of hydroxylated steroids or tripenoid tend
to depress the growth of experimental animals. The same author reports that
cycasin, another toxic glycoside, when fed to rats, is a potent carcinogen
producing tumors in the liver, kidney, intestine and lung.

Triterpenoids
According to Drewnoski and Carneros (2005) some dietary
phytochemicals are bitter and toxic. They mentioned that some plants in the
cucurbitaceae family like cucumbers and zucchini are bitter and inedible due to
triterpenes, cucurbitacins or oxygenated tetracyclic triterpenes present in them.


Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

11

Properties of Phytochemicals
Whitney and Rolfes (2005) mentioned that phytochemicals impart tastes,
aromas and colors and other characteristics to foods. The burning sensation of hot
peppers, the pungent flavor of garlics and onions, the bitter tang of chocolates and
the dark red color of tomatoes are due to the phytochemicals present on them.
The same authors further enumerated some phytochemicals found in fruits and
vegetables: sulforaphane, cancer-fighting found in broccoli; flavonoid, protection
against lung cancer found in apples; ellagic acid of strawberries may inhibit
certain type of cancer; carotenoids, lutein and zeaxanthin found in spinach and
other colorful vegetables which help to protect the eyes against mascular
degeneration. The distinctive taste and smell of celery and its property of
lowering high blood pressure and cholesterol levels are given by butyl phthalide
(New Zealand Food Authority, 1994).

Natural Toxins
Plants utilized by man as food contain undesirable natural products called
toxin. Harborne (1982) as cited by Mendoza et. al. (1985) lists some of the
common natural products involved in the interaction of plants, animals and man.
These includes proteins (protease inhibitors, amylase inhibitors,
hemagglutinins/lectins, allergens); glycosides (cyanogenic glycosides,
glucosinolates, saponins, vicine); amino acids (lathyrogens, hypoglycemic agents,
mimosine) and others which include cycasin, pyrroligtine alkaloids, gossypol and
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

12

antivitamin. Different parts of the plant (stem, roots, leaves, and seeds) may
contain varying concentrations of a toxin. The age of the plant also contributes to
the difference in concentration. Young plants may contain lesser or more
constituent toxins than mature ones (Klaassen, 2001). According to Trefry and
Jameson-Jones (2004) plant diseases and environmental stress like drought, heat,
cold and mineral deficiencies can affect toxin concentration in plants. Different
varieties of the plant have also different concentrations of toxins. Liener (1983) as
cited by Mendoza et. al. (1985) points out that evidence for a particular food
constituent being toxic to man is only presumptive since most of the research in
the toxicity of plant stuff is performed on animals. Such studies revealed the
effects as inhibition of growth, decrease in food efficiency, goitrogenic response,
pancreatic hypertrophy, hypoglycemia, and liver damage. Food poisoning due to
tomatine from green tomatoes and dioscorine from yams have been observed in
humans and domestic animals. The toxin in tomatoes causes their bitter taste. The
leaves and stems of tomato also contain glycoalkaloids, making them inedible
(Elpel, 2005). The toxicity of oxalic acid, which is present in rhubarb, depends on
the age of the plant, the season, the climate and the type of the soil. The leaves of
the plant contain the highest concentrations of oxalic acid. Poisoning of this type
may cause muscle twitching, cramps, decreased breathing and heart action, pain
headache, vomiting, convulsions and coma (New Zealand Food Safety Authority,
1994).
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

13

Detoxifying Processes
Shemilt (1982) reports cyanogenic glycosides can be detoxified by
adequate processing techniques or traditional methods of preparation. A common
processing method is heating or cooking of food. Mendoza, et. al. (1985)
disclosed that proper cooking and processing should be done in plants containing
protease inhibitors to make it safe for consumption.
Lectins can be readily denatured by sufficient heat treatment thus, beans
classified as Phaseolus Vulgaris should be heated sufficiently at least 15 minutes
in boiling water to make it safe for consumption (Shemilt, 1982).
Salda (1999) reported that many wild yam species possess sapogenins and
alkaloids making them inedible. She also mentioned that the simplest method to
detoxify tubers is by chipping the peeled tubers into uniformly thin slices, soaking
these chips in brine solution and rasping them several times in running water.
Elpel (2005) mentioned that young milkweed shoots contain a toxic, bitter
alkaloid that has to be properly cooked out to render the plants safe to eat. He
also stated that the proper way of cooking is to plunge the herbs into boiling water
for two minutes, drain the water, then repeat the process two more times until all
bitterness is gone which makes it safe.
Processes employed to make plants containing cycasin safe for human
consumption are fermentation, heating, water extraction or semi- drying (New
Zealand Food Safety Authority, 1994).
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

14

Brine Shrimp Assay

The brine shrimp assay has been standardized for natural screening of
bioactive substances in plant extracts. This uses the larvae (nauplii) of brine
shrimp, Artemia Salina Leach. The lethal concentration for 50% mortality after 6
hours of exposure or after 24 hours of exposure is determined as the measure of
the toxicity of the extract or compound. The solubility of the extract is of
convenience to make the test simple and rapid. The use of the polar extracts
would be of shorter exposure, whereas, lower concentrations is achieved in
nonpolar extracts require longer time (Colegate & Molyneux, 1993).

Extraction Processes

Constituents of phytochemical and biological interests are soluble in 80%
to 95% ethyl alcohol. Organic constituents from the plant can be extracted by
alcohol and serial extraction with solvents of increasing polarity (Guevara, 2005).
Nonpolar compounds can be extracted by nonpolar solvents like hexane and polar
compounds can be extracted by ethyl acetate, a polar solvent (Pladio, 1999).
Extraction is a transfer of one solvent into another by means of
distribution process. When a solution is shaken with a second solvent with which
it is immiscible, the solute will distribute itself between the two liquid phases.
When the two phases have separated again into distinct solvent layers, an
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

15

equilibrium situation will be achieved such that the ratio of the concentration of
the solute in each layer will define a constant called distribution coefficient. The
distribution coefficient has a constant value for each solute and is dependent on
the nature of the solvents used in each case. The solute distributes itself between
the two solvents so that its chemical activity (effective concentration) is the same
in each phase.

Column Chromatography
Column chromatography is used to separate the mixture of two or more
different compounds by distribution between two phases (one is stationary and the
other is moving). In a solid-liquid phase partitioning technique, the solid are those
materials that will not dissolve in the associated liquid phase such as silica gel and
alumina. The more polar the functional group, the more strongly it will bind to
alumina or silica gel. Elutants or the materials to be separated, move down the
column at differing rates on their relative affinity for their adsorbent and the
solvents or eluents .The versatility results of the chromatography can be varied
due to the following: the adsorbent chosen, the polarity of the solvents chosen, the
size of the column (both length and diameter) relative to the amount of the
material to be chromatographed and the rate of elution or flow (Pavia et. al.,
1976).


Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

16

Thin Layer Chromatography
According to Guevarra (2005), thin layer chromatography can analyze
most of the different classes of characteristic compounds present in the crude dry
extract. This also provides semi-quantitative information on the active
constituents of an extract enabling an assessment of plant quality. Thin layer
chromatography (TLC) is also suitable for comparing different batches of sample
plant and can indicate possible adulterations of material. Pavia et. al. (1976) stated
that thin layer chromatography is related to column chromatography, however,
rather than allowing the moving liquid phase to percolate down the adsorbent, it is
allowed to ascend on a thin layer of adsorbent.
Rf value (ratio of front) is a constant value for any compound,
corresponding to its physical property.









Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

17

MATERIALS AND METHODS

Materials

Plant Materials
The plant material used in the study were the young shoots ( edible part,
about 3 to 4 leaves from the top) and mature leaves (inedible portion) of the
passion fruit (Passiflora edulis Sims.) as shown in Plate 2.


Plate 2. Shoots of the Passiflora edulis Sims



Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

18

Standard
Table 1 presents the materials used as standards or positive control in the
phytonutrient analysis.

Table 1. Standard substances used in the phytonutrient analyses

STANDARD
ANALYSIS
(Positive Control)
A. Vitamin

A
Aflaxin (2500 IU)
E
Myra E 400
B. Sugar
(1 % aqueous solutions)
Molisch Test
Starch
Benedict’s Test
Fructose
Barfoed’s Test
Lactose

Sucrose

Ribose
C. Cyanogenic Glycoside
Cassava tuber peeling

Equipment
The following laboratory equipment were used in the analyses: analytical
balance, blender (Plate 3A), separatory funnel (Plate 3B), rotary evaporator (Plate
3C), water bath, refractometer, oven and column chromatography (Plate 3D).
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

19


A. Blender (sample preparation)

B. Separatory Funnel (partitioning
of crude extract)


Plate 3. Equipment used in the extraction of bitter
constituent of the passion fruit
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

20

Plate 3 continued…

C. Rotary Evaporator (concentration of solutions
under vacuo )







D. Column Chromatography (separation
and isolation)
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

21

Phytonutrient Analyses of the Shoots
of Passiflora edulis Sims.



Proximate Analysis
Sample Preparation. Fresh shoots of the passion fruit plant were washed
and finely chopped prior to analysis.
Classical methods of analyses (Table 2) were used to determine the
moisture, ash, crude fat, and crude protein contents of the shoots of the passion
fruit. All analyses were replicated three times.

Table 2. Proximate analysis of the shoots of the passion fruit vine


PROXIMATE ANALYSIS
METHOD OF ANALYSIS
(Wet Basis)



% Moisture
Oven- drying


% Ash
Gravimetric


% Crude Protein
Kjeldahl


% Crude Fats
Soxhlet


% Nitrogen free-extract
By difference

*Source: Official Methods of Analysis of AOAC International, Lees, (1975)




Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

22

Mineral Content Analysis

About 1 kg of the fresh shoots of the passion fruit was sent to the
Philippine Institute of Pure and Applied Chemistry (PIPAC), Ateneo de Manila
for mineral content analysis. The analytical methods used were Ashing-Acid
Digestion/Atomic Absorption Spectrophotometry for Potassium, Calcium,
Sodium, Iron and Zinc and Ashing-Acid Digestion/ Spectrophotometry for
Phosphate.

Vitamin Analysis
The presence of Vitamin A and E in the sample was determined
qualitatively while Vitamin C was quantitatively analyzed. Table 3 presents the
different methods used.

Table 3. Vitamin analysis
VITAMINS METHOD
Carr-Price Reaction
Vitamin A
Modified Furter- Meyer’s Test
Vitamin E
Titrimetric Method/
Vitamin C
Oxidation-Reduction

*Source: Biochemistry Laboratory Manual in Benguet State University (2006)


Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

23

Carr-Price Reaction. The content of Vitamin A capsule, Aflaxin 2500 IU,
was transferred in a test tube. This served as first positive control. Another
positive control was prepared by mixing two ml of fresh extract with another
capsule of Aflaxin. One ml of chloroform was added to each control, afterwards
were allowed to cool in an ice bath. Two ml of cold saturated chloroform solution
of antimony (III) chloride was then added. Formation of a blue violet precipitate
indicates a positive result. The same procedure was applied to two ml of the fresh
extract of the shoots.
Modified Furter-Meyer Test. Two positive controls were prepared in
separate test tubes, a capsule of Vitamin E (Myra E) and two mL of fresh extract
mixed with Myra E. To each test tube, one ml of each chloroform, n-butyl alcohol
and concentrated HNO3 were added, and then was heated in a water bath for ten
minutes. A positive result was indicated by the formation of a yellow solution.
The same procedure was followed using only the fresh extract sample.
Oxidation-Reduction Titration. Five grams of fresh shoots of the passion
fruit vine was homogenized in a blender with 10 mL 1% oxalic acid. The
resulting solution was filtered and the filtrate was collected in a 50 ml volumetric
flask. A volume of 25 ml filtrate was transferred to a burette. Two ml of 2,6-
dichlorophenolindophenol dye was titrated with the solution until the end point,
from red to light pink color was attained. The volume of the titrant (filtrate) was
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

24

noted. This procedure was repeated for the second and third trial. The vitamin C
content was computed using the formula;
mg Vitamin C/ g sample = 0 .3 mg of Vit. C 50 mL _________



Average vol. of sol’n needed to 5 g sample
Consume
the
dye

Sugar Analysis
The tests used for the determination of sugar presence in the sample are
presented in Table 4.

Table 4. Sugar analysis
SUGAR
METHOD
General
Molisch test
Reducing/non reducing
Benedict’s test
Monosaccharide
Barfoed’s test
% Sugar
Refractometer

Sample preparation. Fresh shoots were homogenized using a blender and
the juice was squeezed using cheese cloth. The extract was filtered using an
ordinary filter paper.
Molisch test. This was used to determine the presence of all types of
carbohydrates. To each of separate ten test tubes ten drops of 1 % of a standard
sugar sample (fructose, lactose, ribose, sucrose, and starch) was added. A drop of
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

25

Molisch reagent was mixed followed by gradual addition of one ml of
concentrated sulfuric acid. The color at the junction of the two liquids was noted.
A positive result was indicated by the formation of a violet purple color. The
same procedure was followed for the fresh extract and the water soluble fraction
of the alcohol extract from the plant sample.

Benedict’s Test. This was used to determine the presence of reducing
sugars in the sample before and after hydrolysis. Five ml of the fresh shoot
extract, water soluble fraction, and starch were first subjected to hydrolysis by
adding 5 drops of concentrated hydrochloric acid to each sample and then boiled
for thirty minutes. To each ten test tubes containing sugar samples (fructose,
lactose, ribose, sucrose, and hydrolysate from starch), two mL of Benedict’s
reagent was added and mixed. The mixture was boiled in a water bath for three
minutes. Formation of precipitate after three minutes indicated the positive result.
The same procedure was also followed for the hydrolyzed and unhydrolyzed
filtrates from the fresh shoots extract and water soluble extracts.
Barfoed’s
Test. This was used to distinguish between monosaccharide
and reducing disaccharide sugars. To one ml of each standard sugar sample,
hydrolysate of the starch, fresh extract and water soluble fraction from the shoots,
2 ml of Barfoed’s reagent was added and boiled for three minutes. A positive
result is indicated by the formation of the precipitate after three minutes. The
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

26

same procedure was employed for the unhydrolyzed fresh extract and water-
soluble fraction of the shoots.

Phytochemical Screening for Secondary Metabolites

Sample Preparation
The fresh shoots of the passion fruit were air- dried at room temperature
and then ground using a blender. A volume of 450 mL of 80% ethyl alcohol was
used to completely submerge about 100 g of the ground plant material. The plant
materials were soaked for 48 hours and then filtered using a Buchner funnel. The
filtrates collected were concentrated using the rotary evaporator. The filtrates
were used in the phytochemical analysis.

Phytochemical Analysis
Phytochemical tests (Table 5) were done to determine the possible class of
compounds present in the extracts of the shoots of the Passiflora edulis Sims.
Procedure used was based from the methods of Guevara (2005).






Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

27

Table 5. Phytochemical tests using test tuber reactions
TEST COMPOUND


Mayer's, Dragendorff's
and Wagner’s*
Alkaloids

Keller-Kiliani
Cardenolides and Bufadienolides
Kedde
Cardenolides and Bufadienolides
Bate-Smith and Metcalf
Flavonoids
Willstatter Flavonoids
Froth Saponins
Gelatin
Tannins and Polyphenolic compounds
Ferric Chloride
Tannins and Polyphenolic compounds
Guignard
Cyanogenic glycoside


*Mojab et al, (2003)

Thin- layer Chromatography (TLC) Screening
To confirm the presence of the phytochemicals, thin-layer
chromatography was performed on the crude extract using procedures as shown in
Table 6 (Guevarra, 2005).





Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

28

Table 6. Phytochemical tests using Thin- Layer Chromatography
(confirmatory test)

COMPOUND
OBSERVABLE RESULT
SPRAY REAGENT
TESTED
FOR A POSITIVE TEST



Flavonoids
Antimony(III)
Intense yellow to orange visible
Steroids
Chloride
zones appear on spraying for
glycosidic flavonoids

Phenols, Tannins
Potassium

Flavonoids
ferrricyanide-ferric
Blue Spots
chloride

Alkaloids Dragendorff’s
reagent
Brown-orange visible spots
immediately on spraying

Coumarins
Methanolic potassium
Anthraquinones- orange color
Anthraquinones,
hydroxide
Anthrones- yellow
Anthrones, Phenols
(Borntrager’s reagent)
Coumarins- blue

Higher alcohols

Triterpenes and sterols appear
Phenols

as blue- violet spots
Steroids
Vanillin- sulfuric Acid Essential oils- zones with wide
Essential Oils
range of colors

Sugars
α-naphthol-sulfuric
Blue spots
acid


Effect of Heat on Cyanogenic Glycoside
To determine the effect of heat on the cyanogenic glycoside content of the
sample, the shoots were blanched for 2-3 minutes. The cyanogenic glycoside
content of the blanched and the uncooked shoots were tested using the Guignard
test. The same test was also performed on samples of uncooked and blanched
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

29

mature leaves. The colors of the picrate papers used in the samples and standards
were observed and compared. The standard used for positive test was cassava
tuber peelings.

Toxicity Test

Brine Shrimp Assay
The larvae (nauplii) of Brine Shrimps were used in the bioassay. The eggs
of the Brine shrimps were obtained in a pet shop.
Hatching of Brine Shrimp. The plastic dish and the artificial sea water
(3.8 g of rock salt per 100 ml distilled water) were prepared for hatching the
shrimp’s eggs. Incubation lasted for 36 hours after sowing the cysts in the dish
containing artificial sea water.
Dilution Series. Different concentrations (parts per million) of the plant
extract were prepared by dilution. Three replications were done for every
concentration. Table 7 shows the dilution series.






Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

30

Table 7. Dilution series

TEST TUBE NUMBER
DILUTION SERIES
(ppm)

1 10,000
2 1:10
3 1:100
4 1:1000
5 1:10,000


Assay. The assay began 36 hours after sowing the cysts. Twenty nauplii
were collected using a pipette from the hatching dish and transferred using the
minimum amount of artificial seawater to a petri dish containing the sample
extract. A control containing artificial sea water was used.
The numbers of dead nauplii after 24 hours were counted in every dish.
The nauplii were considered dead if they were immobile at the bottom of the dish.
A hand lens was used to check the inactivity. To confirm the number of shrimps,
nauplii that were alive were also counted.
Series of tests were conducted on the different concentrations of fresh,
ethanol, hexane, ethyl acetate and the water-soluble extracts.



Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

31

Analysis of Results
Extract concentrations were converted to log concentrations to make the
wide range of values easier to deal with. The numbers of dead nauplii were
counted and the percent mortality (death) was computed. The % mortality versus
log extract concentration was plotted and from the equation of the line obtained,
the LD50 for each sample extract were determined. The LD50 is the dose that is
lethal to half of the animals tested. Activities or plant toxicity were considered
significant if the LD50 is less than 30 µg/ ml. or 0.03 mg/ ml (Saupe, 2006).

Extraction and Partial Isolation of Bitter Constituent
of the Shoots of the Passion Fruit


The overall process for the extraction and partial isolation of bitter
constituent of the shoots of the passion fruit is shown in Figure 1.

Extraction
The plant materials were prepared prior to extraction. Batch of passion
fruit shoots were gently washed and air-dried. About 1.8 kg of the air-dried
materials was homogenized with 2.845 L of distilled technical grade of ethyl
alcohol. This was allowed to soak overnight. The liquid was filtered using
buchner funnel and ordinary filter paper.

Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

32


1.8 kg Air-dried Shoots of the Passion Fruit

* Homogenize with 2.845 L EtOH
* Stand overnight, filter
* Repeat 5x


Residue
Filtrate

* Rotary evap

Ethanol Extract
Ethanol

(90 g, 8.10%)
(recovered)

* Add 100 ml Water
* Extract with 600 ml Hexane
* Brine shrimp assay

Hexane Layer
Aqueous Layer

* Extract with 400 ml Ethyl Acetate


Hexane Extract
Water-Soluble
EtOAc Layer
(22.2 g, 1.998%)
Fraction

* Rotary evap

* Brine shrimp assay
* Brine shrimp assay
Ethyl Acetate Extract
* TLC
* TLC
(10 g, 0.55%)


*Brine shrimp assay


*TLC (Phytochemical

Analysis)
Column chromatography

Repeat 3x

Partial Isolate


* TLC
Figure 1. Schematic Diagram of Extraction Process
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

33

The filtrate was concentrated using rotary evaporator with the water bath
maintained at 40°C. The crude ethanol extract obtained was partitioned using
solvents of increasing polarity, hexane (Absolute Reagent) followed by distilled
technical grade ethyl acetate. An ordinary separatory funnel was used in
partitioning the crude ethanol extract. The hexane and ethyl acetate extracts were
concentrated under rota-evaporator. To maximize the yield, the soaking of the
plant residue was repeated five times using ethanol recovered from the rota-
evaporator.

Monitoring of Bitter Constituent.
To determine the presence of the bitter constituents of the plant, the
hexane and ethyl acetate extract were subjected to phytochemical analysis using
thin layer chromatography (TLC). The extract that gave the distinct positive result
to the flavonoid test was subjected to chromatographic separation.

Isolation (Partial)
To separate the bitter constituent, the extract was subjected to repeated
column chromatography using vacuum elution. Two different column sizes, 4.5
and 2.0 cm diameters were used. The column was packed with silica gel 60 G
(Merck) to a height of 8.0-cm. Starting with pure hexane and increasing at 5-10%
gradient ethyl acetate- hexane mixture up to 100% ethyl acetate, eluates were
collected in about 20 ml fraction using test tubes. The collected eluates were
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

34

spotted on a TLC and were developed using different concentrations of the ethyl
acetate –hexane mixture based on the polarity of the fraction. The spots were first
visualized with potassium ferricyanide-ferric chloride and then with vanillin-
sulfuric acid. Fractions that showed positive result with the flavonoid test were
pooled together and were again subjected to column chromatography using
isocratic elution (40% ethyl- acetate hexane mixture).
The Rf value of the spot positive for the flavonoid test was determined by
dividing the distance traveled by the component by the distance traveled by the
solvent.
Distance traveled by component
Rf =
Distance traveled by solvent











Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

35

RESULTS AND DISCUSSIONS


Phytonutrient Analyses


Proximate Analysis

The proximate compositions of the shoots of the Passiflora edulis Sims as
shown in Table 8 were determined on a wet basis.

Table 8. Proximate Composition of the Shoots of the Passion Fruit



COMPOSITION (%)
PARAMETER
(wet basis)

Moisture
81.03


Ash
1.5


Crude Protein
7.73


Crude Fats
0.9 3


Nitrogen free-extract
8.81

Moisture content. The moisture or water content of the shoots is very high
(81.03%), which suggests that it is very susceptible to spoilage. Bennion and
Scheule (2000) stated that the perishability of the food is related to its water
content. The food with higher water content is more perishable. This relationship
occurs because microorganisms require water for their growth. Fresh vegetables
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

36

have usual water activity values of 0.95 to 0.99 causing them to be more
perishable.
Ash content. The ash content of the shoots is 1.5 %. According to the
Official Methods of Analysis of (AOAC) International, the ash content shows the
inorganic constituent of the shoots. James (1995) states that the ash content is the
residue that remains after the moisture has been removed and the organic
materials (fats, protein, carbohydrates, vitamins, organic acids) have been
oxidized to volatile materials through ignition at very high temperature. This
represents the mineral content of the plant.
Crude protein. The percent crude protein content of the shoots was
7.73%. This value was computed by multiplying the total nitrogen by the protein
factor suitable to the food (Portugal et. al., 1997). Total nitrogen was determined
by the Kjeldahl method and the protein factor used is 6.25. Aurand and Woods,
(1973) mentioned that proteins are essential components of every living cell and
are utilized in the formation and regeneration of tissue. They also pointed out that
certain proteins serve as enzymes, antibodies and provide functions in metabolic
regulation and contractile processes. The percent crude protein value of the
passion fruit shoots is low compared to plant foods with relatively high proteins
(20% to 30%) such as legumes (Bennion and Scheule, 2000). As reported by
Wills .et al, (1989) protein is mostly functional, for example as enzymes rather
than storage pool as in grains and nuts. The low value for percent crude protein
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

37

implies that the shoot of passion fruit is not an important contributor of protein in
the diet.
Crude fat. The amount of the crude fat extracted from the shoots was
found to be 0.93%. This value does not represent the true fat content, but rather
the lipid fraction of the food which are the constituents soluble in non-polar
organic solvents. The crude fat includes fats, phospholipids, sphingolipids, waxes,
steroids, terpenes and fat soluble vitamins (James, 1995). As reported by Brody
(1994), dietary fats and oils are excellent sources of energy and they contribute to
the palatability of the diet. In the study, the value obtained showed that the shoots
contain low amount of fats compared to 46% in groundnut as reported by Ezeagu
et al, (2006). This means that the shoots are not a good source of energy. Bennion
and Scheule (2000) explained that the leaf is actively working or metabolizing
part of a plant and does not generally store energy.
Nitrogen free extract or carbohydrates. The nitrogen free extract or
carbohydrates according to James,(1995), was calculated “by difference” using
the formula suggested by Lees, R. (1975):
% Nitrogen free extract = 100-(%water + %protein + % ash +% fat)
This calculation included the indigestible carbohydrate component as well as
other components of the food item that are not measured as water, protein, fat and
ash (Portugal et. al, 1997). The computed value of the nitrogen free extract of the
shoots was 8.81%. This value is higher than what was reported by Portugal et. al,
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

38

(1997) on the carbohydrate content of some green leafy vegetables such as
chayote leaves (2.3%), pechay leaves (3.2%) and saluyot leaves (7.5%).
Compared to cereals with high carbohydrate contents, the shoots of the passion
fruit is not a good source of supplemental carbohydrates.

Mineral Content
The mineral contents of the shoots of Passiflora edulis Sims is presented in
Table 9. These were determined from the ash content. Differences in composition
may occur as a result of the loss of some volatile inorganic constituents such as
chlorides during the ashing process and the presence in the ash residue of
constituents, such as sulfur in protein (James, 1995). He also classified the
minerals as major elements if there values are greater than 100 ppm or 0.01% and
trace elements if the values are lesser than 100 ppm or 0.01%.

Table 9. Mineral content of the shoots of the passion fruit
COMPOSITION
MINERAL
(ppm)
Potassium 5100

Calcium 980

Sodium 15

Phosphate as P
1070

Iron 22

Zinc 10
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

39

Major elements. Based from the results obtained (Table 9), potassium
(5100 ppm), calcium (980 ppm) and phosphate (1070 ppm) are considered major
elements since their values were higher than 100 ppm. Wills et. al. (1989) stated
that potassium is the major mineral found in fruits and vegetables, especially the
green leafy vegetables with parsley containing the highest amount. They further
reported that health authorities in many countries are urging increased
consumption of potassium from fruits and vegetables, to counter the effects of
sodium in the diet.
Encyclopedia (2003) gives the importance of the different dietary
minerals. Potassium is important for the heart and other muscles. It helps muscle
contraction and maintains fluid and electrolyte balances in the body. It also helps
transmit nerve impulses and release energy from fat, protein and carbohydrates
during metabolism. Aurand and Woods (1973) stated that calcium is most needed
during growth periods, pregnancy and lactation. Calcium is also essential for
building strong bones and teeth, as well as maintaining bone strength and density.
It also plays a role in muscle contraction, blood clotting and maintenance of cell
membranes (Encyclopedia, 2003). The same source mentioned that another key
element for building strong bones and teeth is phosphorus. It also helps the body
release energy from fat, protein, and carbohydrate during metabolism. It also
plays a role in the formation of genetic material, cell membranes and certain
enzymes.
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

40

Trace elements. Sodium (15 ppm), Iron (22 ppm), and zinc (10 ppm) are
considered trace elements since they have values lower than 100 ppm. Whitney
and Rolfes (2005) stated that sodium is the main cation outside the cells. Sodium
is used by the body to produce muscle contractions, maintain fluid balance,
conduct nerve impulses, and carry nutrients to cells (Encyclopedia, 2003).
Aurand and Woods (1973) pointed out that iron is an indispensable element for
the normal function of hemoglobin of red blood cells, myoglobin of heart muscle
and the cytochromes of respiration. Finally, the role of zinc, an elemental form of
protein is to promote wound healing (Encyclopedia, 2003).

Vitamin Analysis
Shown in Table 10, Vitamin A, E and C were found present in the extracts
of fresh shoots of the passion fruit vine.

Table 10. Vitamin content of fresh shoot extract
TREATMENT
VITAMIN
Standard Standard Fresh
extract
VITAMIN A
(Aflaxin)
(Aflaxin +fresh extracts)

+

+
+
VITAMIN E
(Myra E)
(Myra E + fresh extract)


+
+
+
VITAMIN C
1.43mg/g
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

41

Vitamin A. In the Carr Price reaction (Plate 4), it was observed that the
shoots gave yellow green and violet precipitate, the same as that of the two
standards solutions. The results indicate the presence of
f Vitamin A in the fresh
extract shoot. Aurand and Woods (1973) reported the importance of Vitamin A;
plays an important part in vision, essential for the integrity of the epithelial tissues
of the body and necessary for normal growth and development. Wills et. al,
(1989) reported that prolonged deficiency to Vitamin A can lead to blindness. He
also mentioned that Retinol, a vitamin A compound, is not present in vegetables
but some carotenoids found in dark green leafy vegetables can be converted to
retinol by man.



Plate 4. Vitamin. A analysis
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

42

Vitamin E. Result of the Modified Furter –Meyer test for Vitamin E
(Plate 5) showed that the fresh shoot extract gave a yellow green to dark yellow
solution, which is similar to the results obtained in the two standards used. The
yellow colors of the solutions indicate a positive result. Thus, the shoots of the
passion fruit contains Vitamin E. This Vitamin as cited by Whitney and Rolfes
(2005) is a fat soluble oxidant and one of the body’s primary defenses against the
adverse effects of free radicals. He also mentioned that it could reduce the risk of
heart disease by protecting low density lipoproteins against oxidation. Brody
(1994) stated that good sources of
f Vitamin E are vegetable oils. Dark green leaves
and nuts are also rich in this vitamin (Aurand and Woods, 1973).


Plate 5. Vitamin E analysis
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

43

Vitamin C. The amount of Vitamin C in the passion shoots is 1.43 mg/g.
This value is very low compared to Amti leaves (58mg/g), kangkote leaves (35
mg/g) and chayote leaves (20mg/g) as reported by Portugal (1997). Although
Vitamin C is only a minor constituent of vegetables, it has a major importance to
man by preventing diseases (Wills et.al, 1989). Vitamin C is important for the
gums, arteries and other soft tissues, bone, for nerve and brain function, for
nutrient metabolism and for antioxidant defense against free radicals (Kohlmeier,
2003).
Morton (1987) cited that the pulp and seeds of passion fruit are rich in
Vitamins A and C. This means edible portions of the passion fruit vine are rich in
these vitamins.

Sugar Analysis
Table 11 shows the results of the qualitative sugar analyses performed on
the shoots of the passion fruit (Passiflora edulis Sims).
General test for all types of carbohydrates. In the Molisch test (Plate 9)
for determining the presence of carbohydrates, glycosidic bonds are hydrolyzed
by concentrated sulfuric acid to monosaccharides which are dehydrated to
furfural, hydroxymethylfurfural and other decomposition products. These
products react with alpha-naphthol forming violet color. Although this is not a
specific test for carbohydrate because furfural forming substances give positive
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

44

result, a negative result is an indication of the absence of carbohydrates
(Biochemistry Manual for use at Benguet State University, 2006).

Table 11. Sugar Analysis of the shoots of the Passiflora edulis Sims
QUALITATIVE TEST
QUANTITATIVE
TEST

General
Test for
Test for
% Sugar
SAMPLE
Test
Reducing
Monosaccharide


Sugar

(Molisch’s) (Benedict’s)
(Barfoed’s)
Standard

Starch
+
No ppt (-)
Blue, no ppt

(-)
Fructose
+
Red (+)
Red ppt

(+)
Lactose
+
Red (+)
Blue, no ppt

(-)
Sucrose
+
No ppt (-)
Blue, no ppt

(-)
Ribose
+
Red (+)
Red ppt

(+)
Fresh Extract






Yellow-
Yellow-green



green ppt
ppt
Hydrolyzed
+
(+)
(+)


Yellow-
Yellow-green

Unhydrolyzed

green ppt
ppt

+
(+)
(+)
10 ˚Brix
Water-soluble




extract


Yellow-
Yellow-green

Hydrolyzed
+
green ppt
ppt

(+)
(+)


Yellow-
Yellow-green

Unhydrolyzed
+
green ppt
ppt
15 ˚Brix

(+)
(+)

Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

45

Khadem (1988) states that the carbohydrates were first thought to be hydrated
carbons but today, it applies to a large number of organic compounds,
monomeric, oligomeric and polymeric in nature which do not necessarily have
their hydrogen and oxygen atoms in the molecular ratio of 2:1 but which can be
either synthesized from or hydrolyzed to monosaccharides. The formation of the
violet ring at the junction of the solutions in the Molisch test reveals the presence
of carbohydrates in the shoot extracts as shown in Plate 6.


Plate 6. General test for carbohydrate (Molisch Test)

Benedict’s test. Reducing sugars are carbohydrates that react with mild
oxidizing agents under basic conditions to give an aldonic acid (Bettelheim et.al.
2004). In this test for reducing sugars, the cupric ion was reduced to cuprous ion.
The cuprous ion was less soluble thus it precipitated out of the alkaline
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

46

(Biochemistry Manual for use at Benguet State University, 2006)). Khadem
(1988) revealed that all monosaccharides are reducing but in disaccharides there
are reducing and nonreducing. The results of the test reveal the presence of
reducing sugar in the shoots. In the test, yellow -green and brown precipitate was
formed in the sample since the extracts were colored. The basis for the positive
result was the formation of the precipitate after heating in the water bath. These
results are shown in Plate 7.




Plate 7. Test for Reducing Sugar (Benedict’s Test)

Barfoed’s test. Barfoed’s test was done to distinguish between the
monosaccharides and reducing disaccharides. Results of the test showed that the
extracts formed precipitates after three minutes of heating which implied that the
sample contain monosaccharide carbohydrates. In contrast, the disaccharide
standards gave a blue solution indicating a negative result (Plate 8). In acidic
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

47

medium, monosaccharides are more easily oxidized than disaccharides
(Biochemistry Manual for use at Benguet State University, 2006).


Plate 8. Test for Reducing Sugars (Barfoed’s Test)

Refractometry. The total soluble solid or % sugar content of the fresh
crude extract determined was 10° Brix at 20°C and 15.2° Brix at 20°C for the
water soluble extract. The Brix value refers to % used as the soluble solid content
in a solution such as sugar, salt, protein acid, etc. which dissolve in water. James,
(1995) explains that as the light passes from one medium to the aqueous solution,
the light rays are refracted. The refractive index of the solution is dependent on
the concentration of materials in a solution. Hence, refractive index is used as a
measure of the % sugar in a sample or solution.

Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

48

Phytochemical Screening for Secondary Metabolites.

Literature search reveals that the bitter taste and toxins of many food
sources are due to the presence of phytochemicals like alkaloids, flavonoids,
cyanogenic glycosides and triterpenes. Phytochemicals which are considered
nonnutrient compounds found in plants impart their tastes (Cataldo et. al, 2003).
Drewnoski and Carneros (2005) reported some dietary phytochemicals that are
bitter, toxic and lethal; flavonoids, cyanogenic glycosides and alkaloids are bitter
compounds.
The phytochemical analysis of air-dried shoots extract using test tube
reactions are presented in Table 12. The results revealed that only alkaloid,
flavonoid, steroids and cyanogenic glycoside are found in the crude extract of the
shoots.
Alkaloid

Observation of heavy precipitation (+++) in the Mayer’s and Wagner’s
tests, indicates presence of alkaloids in the sample. The negative precipitation on
the Dragendorff’s test may be due to interfering factors like the sensitivity of the
reagent.




Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

49

Table 12. Phytochemical analysis (air- dried shoots extract)
COMPOUND TEST
OBSERVATION RESULT
Alkaloids
Mayer's
yellow precipitate
+++
Wagner’s test
brown precipitate
+++
Draggendorrf’s
no orange ppt
-
Steroids
Keller-Kiliani
reddish brown color
+
at the interface

Flavonoids
Bate-Smith and
strong red-violet
+
Metcalf
color

Willstatter
dark brown color
+
Saponins
Froth
froth is less than 1
-
cm

Tannins and
Gelatin
no precipitate
-
Polyphenolic
compounds
Ferric Chloride
no black solution
-
Cyanogenic
Guignard
orange color of
+
glycoside
picrate paper



Flavonoid

The ethanolic exctract of the shoots of the passion fruit gave a positive
result for the Bate-Smith and the Metcalf test, which indicates that the one type of
flavonoid present in the shoots is leucoanthocyanins. Also, positive result in the
Wilstatter test signifies that another flavonoid with an γ- benzopyrone nucleus is
found in the shoots.
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

50

Cyanogenic Glycoside.
Result of the Guignard test shows that cyanogenic glycoside is found in
the shoots and mature leaves of the passion fruit. Color comparison on the change
of color in the picrate paper was done to determine the presence of cyanogenic
glycosides in the sample. A change in color from yellow to orange of the picrate
paper implies a positive result.
The mature leaves have higher amount than the shoots as indicated by the
dark orange color of the picrate paper compared to the lighter orange shade
obtained from the shoots (Plate 9). This result is confirmed by Klaasen (2001)
who reported that the age of the plant contributes to the variability of chemical
constituents. He further mentioned that in spring pokeweed, sprouts and young
leaves are safer to eat than mature leaves because of lesser toxins.



Plate 9. Guignard test for cyanogenic glycoside
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

51

Using thin-layer chromatography (TLC), the presence of the same
metabolites was confirmed. These results are shown in Table 13. The color of the
chromatograms shown in Plate 10 indicates the presence of alkaloids and
flavonoids. The intense blue color is for the flavonoids (Plate 10A and B) while
the brown orange color (Plate 10C) refers to the alkaloid. Other compounds which
tested positive are steroids (Plate 10A), phenols, anthraquinones (Plate 10D),
anthrones (Plate 10D), triterpenes, essential oils (Plate 10E) and sugars (Plate
10F).















Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

52

Table 13 Phytochemical analysis using Thin Layer Chromatography method

COMPOUND
SPRAY REAGENT
OBSERVATION RESULT
TESTED
Flavonoids
Antimony(III) Chloride Intense
yellow
(+)
Steroids
upon spraying



Phenols,
Potassium
Blue Spots
Flavonoids
ferrricyanide-ferric
(+)
Flavonoids
chloride

Alkaloids Dragendorff’s
reagent

Brown-orange
visible spots
immediately on
(+)
spraying

Coumarins,
Methanolic potassium Anthraquinones-
(+)
Anthraquinones,
hydroxide
orange color

Anthrones
(Borntrager’s reagent)
Anthrones-

Phenols

yellow
(+)
Coumarins- no

blue color
(-)

Higher alcohols,
Vanillin- sulfuric Acid Triterpenes and
Phenols
sterols appear as
(+)
Steroids, Essential
blue- violet spots

Oils
Essential oils-

zones with wide
(+)
range of colors


Sugars
alpha-naphthol-sulfuric Blue spots
(+)
acid








Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

53

A. Test for flavonoids and steroids


B. Test for phenols and flavonoids

Plate 10. Chromatograms for Phytochemical Analysis
using Thin Layer Chromatography


Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

54


Plate 10. Continued…

C. Test for alkaloids



D. Test for anthraquinones, anthrones
and phenols



Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

55

Plate 10. Continued…


E. Test for phenols, steroids,



essential oils





F. Test for sugars


Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

56

Effect of Heat on Cyanogenic Glycoside
Table 14 presents the results of the Guignard test performed to determine
the effect of the heat on the cyanogenic glycoside content of the shoots of the
passion fruit vine. Plate 11 shows the color change in the picrate paper used. In
the uncooked shoots and cassava tuber peeling, the picrate paper changed to
orange (+) while there was no change in the cooked sample (-). This shows that
the cyanogenic glycoside can be lost during heating or cooking. This finding
corroborates the report of Shemilt (1982) that cooking process either by boiling,
roasting or sun-drying inactivates the enzyme linamarase found in cassava which
liberates hydrogen cyanide.

Table 14. Effect of Heat on the Cyanogenic Glycoside Content

TREATMENT OBSERVATION
(color of picrate paper)

Control (+)
Brick red (+)
cassava tuber peeling
Shoots

a. Uncooked
Orange (+)
b. Cooked
No change (-)
Mature Leaves

a. Uncooked
dark orange (+)
b. Cooked
No change (-)

Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

57



Plate 11. Guignard Test for Cooked and Uncooked Shoots

According to Food Standards Australia New Zealand, (2004) all parts of
the cassava are cyanogenic and the starch –rich tuber must be properly processed
before it can be eaten. Sweet varieties of cassava with low cyanide content can be
processed adequately by peeling and cooking.
In cassava when its cellular integrity is disrupted as what occurs in
grinding and maceration, a highly specified β- glucosidase catalyzes the
hydrolysis of the glycosidic bond; to produce acetone cyanohydrin and glucose
are formed. Hydroxynitrilyase or oxynitrilase are the second enzyme catalyzing in
the dissociation of cyanohydrin to form HCN and acetone. The acetone and HCN
are resembled and volatiles can be leached away with water from the starchy
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

58

residue. Drying the residue after sufficient washings or boiling can be used to
remove the volatile (Food Standards Australia New Zealand, 2004).

Toxicity Test

Brine Shrimp Assay
LD50 (Lethal Dose). Using the brine shrimp assay, the toxicity of the
different extracts (fresh, ethanol, hexane, and water- soluble) were determined at
different concentrations. Toxicity was measured in terms of LD50. Calculation of
LD50 is presented in appendix C.
Table 15 presents the percent mortality and LD50 obtained from the brine
shrimp assay. All the extracts have LD50 values higher than 30 ppm, which
according to Saupe means that the toxicity of the plant extracts were not
significant. This means that the shoots of the passion fruit vine is not toxic. From
the table, the water soluble extract has the lowest LD50 or the most toxic fraction,
but still the value is insignificant. This finding supports the result of the Guignard
test which revealed the presence of cyanogenic glycoside, a toxic substance. This
substance is maybe responsible for the toxicity revealed in the assay, but the
amount present is low, which explains the low toxicity observed.

Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

Table 15. Brine shrimp assay (after 24 hours)
% MORTALITY (after 24 hours)
LD50 (ppm)
EXTRACTS
Concentration (ppm)

1 10 100 1000
10,000
Fresh
10
23.35
43.35
81.65
93.35
1.985
Ethanol
16.65
33.35
45
71.65
96.65
73.45
Hexane
8.35
16.65
38.35
66.65
95
173.38
Ethyl
15 23.35 48.35 85 98.35
1.824
Acetate
Water Soluble
16.5 30 55 78.35 98.35 54.203
Extract

---

60

Extraction of Bitter Constituents
of Passion Fruit Vine


Extraction of the Flavonoids

The yield obtained in hexane extract (22.2g or 1.998%) and ethyl acetate
extract (10 g or 0.55%) from 1.8 kg of air-dried shoots of the passion fruit vine
are very low. This caused the difficulty in isolation of the bitter constituents. The
presence of the plant constituents; alkaloids, flavonoids, cyanogenic glycosides
and triterpenes in the shoots of the Passiflora edulis Sims was revealed by
phytochemical analysis and was confirmed using thin layer chromatography
except for the cyanogenic glycoside. Phytochemical tests showed that the
alkaloid, triterpenes and flavonoids are present in the ethyl acetate extract. From
the observable results in the thin layer chromatography, the flavonoid showed a
very distinct color spot on the different spray reagents used. This may imply
higher amount of flavonoid is present in the extract.
Table 16 shows that the ethyl acetate extract is positive for alkaloid and
flavonoid while the hexane extract is negative to both tests.
The sticky brown acetate extract was subjected to chromatographic
separation monitored by thin layer chromatography. Fractions that were positive
to the flavonoid test were pooled and further subjected to repeated column
chromatography to yield the Bb2 fraction. This fraction has a brown to yellow
color. Table 17 shows there are several components of the Bb2 fraction. The
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

61

flavonoid is indicated by the blue-colored spot upon spraying with potassium-
ferricyanide-ferric chloride and red color when sprayed with vanillin -sulfuric
acid.




Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

Table 16. Phytochemical Analysis Using Thin Layer Chromatography Method

RESULT
COMPOUND TESTED
Hexane extract
Ethyl Acetate Extract
Flavonoids, Steroids
No Intense yellow appear (-) Intense yellow appear on sprayin
Phenols, Flavonoids
No blue Spots (-) Blue Spots
Alkaloids
No brown-orange spots upon spraying (-) Brown-orange spots upon spra
Coumarins
Anthraquinones- orange color (+) Anthraquinones- orange color
Anthraquinones,
Anthrones- yellow (+) Anthrones- yellow
Anthrones,Phenols
Coumarins- no blue color (-) Coumarins- no blue color

Higher alcohols,Phenols Triterpenes and sterols appear as blue- violet Triterpenes and sterols appear
Steroids, Essential Oils
spots (+) violet spots
Essential oils- wide range of colors (+) Essential oils- wide range of col

Sugars
Blue spots (+) Blue spots

---

63


Table 17. Characteristics of the Bb2 Fraction
SOLVENT
COLOR
SYSTEM
FRACTION
(Ethyl
RF
Vanillin-
Potassium
Acetate –
Iodine
Sulfuric
Ferricyanide –
Hexane)
Vapor
Acid
Ferric Chloride


0.883 Violet Yellow

Bb2
65%
0.567 yellow Yellow brown
-green

0.467 Red
blue
0.367 Green Yellow Yellow
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

64

SUMMARY, CONCLUSION AND RECOMMENDATION

Summary
This research aimed to determine the suitability of the shoots of the
passion fruit for vegetable use. The specific objectives were to determine the
phytonutrients present in the shoots, determine the toxicity of the shoots and to
determine the compound responsible for the bitter taste of the shoots. This study
was conducted from April to October 2006 at the Benguet State University,
D.O.S.T .and P.I.P.A.C. laboratories.
Varied sample preparations were followed for every analysis performed.
Proximate analysis showed that for every 100 g fresh sample, there were 81.03 %,
moisture, 1.5 % ash, 7.73 % crude protein, 0.93 % crude fat and 8.81 % nitrogen
free- extract.
The mineral content of the shoots were: potassium (5,100) ppm, calcium
(980 ppm), sodium (15 ppm), phosphate as phosphorus (1070 ppm), iron
(22 ppm) and zin (10 ppm). Ashing-acid digestion and Atomic Absorption
Spectrophotometry were used in the analysis.
Qualitative vitamin analysis for A and E were employed to reveal their
presence. Vitamin C in the shoots was quantitatively determined and was found to
contain 1.43 mg/g sample.
The presence of sugar was also determined using general and specific test
for reducing sugars and monosaccharides. The tests showed the presence of
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

65

reducing sugar and a monosaccharide. Refractive index was also measured. The
presence of sugar was also confirmed in the thin-layer chromatography using the
alpha-naphthol as the spray reagent.
Phytochemical analysis revealed that flavonoids, steroids, phenols,
alkaloids, anthraquinones, anthrones, triterpenes, essential oils, sugar and
cyanogenic glycoside are present in the shoot samples. The bitter taste of the
shoots of passiflora maybe attributed to the presence of alkaloids, flavonoids and
cyanogenic glycosides. Through cooking, cyanogenic glycoside were volatilized
or volatilized.
The toxicity of the shoots was determined by the brine shrimp assay. The
assay revealed that the LD 50 obtained from the different extracts (fresh, ethanol,
hexane, ethyl acetate and water soluble extracts) were not significant, since they
are higher than 30 ppm. Activity or toxicity is significant if obtained values of
LD 50 is lower than 30 ppm.
The flavonoid, one of the bitter constituent of the shoots was extracted and
partially isolated by repeated vacuum column chromatography, monitored by
thin-layer chromatography using potassium ferricyanide-ferric chloride as spray
reagent.




Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

66

Conclusion
Based on the results obtained, the following conclusions were drawn:
1. The shoots of the passion fruit is a rich source of phytonutrients such as
proteins, carbohydrates, vitamins (A, E and C), minerals and the
phytochemicals cyanogenic glycoside, alkaloids, flavonoids, steroids,
anthraquinones, sugar and essential oils.
2. There is no risk due to the presence of the natural toxin particularly
cyanogenic glycoside because it is easily eliminated through heating or
cooking.
3. Brine shrimp assay showed that toxicity of the shoot extracts are
insignificant.
4. Phytochemical analysis determined the presence of flavonoid, one of
several compounds that impart bitter taste to food. Repeated column
chromatography yielded the yellow brown fraction Bb2, whose TLC
showed the flavonoid spot to have an Rf value of 0.467. The
chromatogram was developed using 65% ethyl acetate-hexane solvent
system and spray reagent vanillin sulfuric acid.





Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

67

Recommendations
Based on the findings and conclusions, the following are recommended
1. The shoots of the passion fruit is highly recommended for vegetable
consumption.
2. Studies on the bioactivity of the plant should be done.
3. Further study should be done on the isolation and characterization of the
bitter constituents of the passion fruit.
4. Other similar studies should be done on the shoots of other indigeneous
plants used as vegetables.

















Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

68

APPENDICES

Appendix A. Preparation Of Test Reagents

Reagents for Proximate Analysis:
Bromocresol green solution: Dissolve 100 mg bromocresol green in 100
mL ethanol
Methyl red indicator: Dissolve 0.1 g methyl red in 100 mL methanol
Receiver solution: Dissolve 40 g of boric acid in about 500-600 ml very
hot de-ionized water. Mix and add more de-ionized water to volume of
900 mL. Cool the solution and add 10 mL of bromocresol green solution
and 7 mL of methyl red solution. Dilute to 1 L with de-ionized water and
mix carefully.
Sodium hydroxide, 40%: Dissolve 400 g NaOH in water and make up to 1
liter.
Reagents for Phytochemical Analysis
Ammoniacal Chloroform: To 1 L chloroform add 3.6 mL of 28%
ammonia. Add sufficient anhydrous sodium sulphate to take up the water.
Filter or decant.
1M sulfuric acid: Add 2.8 mL of the concentrated sulfuric acid to enough
distilled water to make up 100 mL solution.
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

69

Mayer’s reagent: Dissolve 1.4 g mercuric iodide in 60 mL water. Pour the
resulting mixture into a solution of 5.0 g potassium iodide dissolved in 10
mL water. Add enough water to make 100 mL .
Dragendorff’s reagent:
Solution A- dissolve 0.85 g bismuth(III) nitrate in a mixture of 10
ml acetic acid and 40 mL water
Solution B- dissolve 8 g potassium iodide in 20 mL water
Stock solution- mix equal parts of Solution A and Solution B. The
resulting mixture can be stored in dark bottle for long time at room
temperature.
To prepare the reagent mix 1 mL stock solution with 2 mL of acetic acid
and 10 mL water.
2M Hydrochloric acid: Add 17 mL of concentrated HCl to enough
distilled water to make 100 mL solution
0.3 M Hydrochloric Acid: Add 2.5 mL of concentrated HCl to enough
distilled water to make 100 mL solution.
Iron(III) chloride reagent: Dissolve 3 mL of 1% Ferric chloride in 50 mL
glacial acetic acid
Iron(III) chloride solution, 1%: Dissolve 1.0 g Ferric chloride in 100 mL
distilled water.
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

70

Sodium Chloride, 10%: Dissolve 10 g NaCl in enough distilled water to
make 100 mL solution
Gelatin-salt reagent: Mix an equal amount of 1% gelatin solution with
10% sodium chloride
Sodium picrate solution: Dissolve 5 g sodium carbonate and 0.5 g picric
acid to enough water to make 100 mL solution
Potassium-ferricyanide-ferric chloride- Mix equal volume of 1%
potassium ferricyanide and 1% ferric chloride solution
Antimony(III) chloride: Prepare solution of 10% antimony chloride in
chloroform( dissolve 10 g of antimony(III) chloride in enough chloroform
to make 100 mL solution)
Methanolic Potassium Hydroxide,: Dissolve 5 g potassium hydroxide in
enough methanol to make 100 mL solution
Vanillin-sulfuric acid: Add 50 mL of concentrated sulfuric acid to 10 mL
of 2.5% vanillin in ethyl alcohol. Mix with cooling
alpha-naphthol-sulfuric acid: Mix 10.5 mL of 15% ethanolic Α-naphthol
with 6.5 mL concentrated sulfuric acid. Dilute with 40.5 mL ethanol and 4
mL water.



Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

71

Appendix B. Proximate Analysis

Oven- drying for moisture content. Moisture content was determined on
the basis of weight loss of water from the samples after oven-drying at specified
temperature. Ten gram of fresh sample was accurately weighed using an
analytical balance. The sample was placed in an oven maintained at 105°C and
was dried for four hours. The sample was then removed from the oven and cooled
in a desiccator and weighed to constant weight.
Gravimetric method for ash determination. Five gram of the fresh sample
was weighed in a crucible and then placed in a temperature controlled furnace
preheated to 600°C. Heating was maintained for 8 hours. The crucible was
removed and was directly transferred to cool in a desiccator. The crucible was
weighed until constant weight was obtained.
Kjeldahl method for crude protein. The weighed sample was placed in a
digestion tube. Two Kjeltabs Cu 3.5 was added. Twelve ml of sulfuric acid was
gently added. A blank was also prepared in the same manner. The sample and the
blank were digested until a clear solution was obtained. After digestion, the tubes
were removed from the digester, cooled and diluted with water. The tubes were
then placed in a Kjeltec (distillation unit). A conical flask containing 25 ml of
boric acid (containing bromocresol green and methyl red indicator) was placed
under the condenser outlet. Twenty five ml of 40% NaOH was dispensed to the
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

72

tubes and distilled for 4 minutes. The ammonium borate solutions formed were
titrated with 0.1 M HCl to purplish-gray end-point (James, 1995).
Soxhlet method for crude fat. About 2 grams of dry sample from the
moisture analysis was extracted with anhydrous ether in a Soxhlet apparatus for
four hours. After extraction, the ether was recovered and the flask with fat was
dried in an oven (110°C) for 30 minutes. The weight of the crude fat was
obtained.
Thin-Layer Chromatography: Phytochemical Analysis


Preparation of plant extract. Two grams of the dried powdered shoots was
defatted with 10 mL hexane and was heated over a water bath for 5 minutes. The
solvent was decanted. The defatted plant residue was treated with 10 mL mixture
of chloroform and acetic acid (99:1 v/v) and was heated for 5 to 10 minutes over a
water bath. This was filtered and labeled as solution A. The resulting residue from
solution A was treated with 10 mL mixture of chloroform, methanol and acetic
acid (49.5:49.5:1) and was heated for 5 to 10 min. over a water bath. This was
filtered and labeled as solution B. The residue from solution B was extracted with
10 ml of a mixture of methanol and water (1:1) and was heated for 5 to 10
minutes. This was filtered and labeled finally as solution C. Solutions A, B, and C
were spotted on a TLC plate and visualized with the spray reagents.



Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

73

Appendix C Toxicity test using Brine
ShrimpAssay



FRESH EXTRACT
120
100
H
80
AT
y = 22.5x + 5.34
60
R2 = 0.9671
DE
%
40
20
0
0
1
2
3
4
5
LOG CONCENTRATION


Figure 2. Fresh Extract



ETHANOL EXTRACT
120
100
80
H
y = 19.83x + 13
AT
60
R2 = 0.9772
DE
%
40
20
0
0
1
2
3
4
5
LOG CONCENTRATION


Figure 3. Ethanol Extract
Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

74

HEXANE EXTRACT
100
80
H
y = 22.33x + 0.34
60
AT
R2 = 0.9664
DE
40
%
20
0
0
1
2
3
4
5
LOG CONCENTRATION


Figure 4. Hexane Extract




ETHYL ACETATE
120
100
H
80
AT
y = 22.835x + 8.34
60
DE
R2 = 0.962
%
40
20
0
0
1
2
3
4
5
LOG CONCENTRATION


Figure 5. Ethyl acetate Extract

Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

75

Water Soluble Extract
120
100
H
80
y = 21.205x + 13.23
AT
60
R2 = 0.9927
% DE
40
20
0
0
1
2
3
4
5
LOG CONCENTRATION


Figure 6. Water Soluble Extract















Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

76

BIOGRAPHICAL SKETCH

The author was born on November 3, 1977 at Sagada, Mountain Province
She is the second child among the four children of Mr. Manuel C. Degay and Mrs.
Stella B. Degay .
She finished her elementary education at Bangaan Elementary School. Her
secondary education was obtained at MPGCHS- Bangaan Annex (now Bangaan
National High School), Sagada, Mountain Province. She was consistently an
honor student. In March 1999, she received her degree of Bachelor of Science in
Chemistry at Saint Louis University, Baguio City.
On June 2004, she was given the opportunity to teach chemistry subject at
Benguet State University for two months as a substitute. One year later, she was
again hired for the same position of the University for one semester. She is
currently an instructor in chemistry at the College of Arts and Sciences .
She is married to Crispolo N. Bolayo of Tublay, Benguet. They are
blessed with three daughters: Craishel, Zai and Chelsea.



Phytonutrient Analysis Of The Shoots Of The Passion
Fruit Vine / Yvonne B. Degay-Bolayo. 2006

---

Document Outline

• PHYTONUTRIENTANALYSIS OF THE SHOOTS OF THE PASSION FRUIT VINE
  • BIBLIOGRAPHY
  • ABSTRACT
  • TABLE OF CONTENTS
  • INTRODUCTION
    • Background of Study
    • Objectives of the Study
    • Importance of the Study
    • Time and Place of Study
  • REVIEW OF RELATED LITERATURE
    • Passion Fruit (Passiflora edulis Sims)
    • Plant Nutrients
    • Phytochemicals
    • Brine Shrimp Assay
    • Extraction Processes
  • MATERIALS AND METHODS
    • Materials
    • Phytonutrient Analyses of the Shootsof Passiflora edulis Sims.
      • Toxicity Test
      • Extraction and Partial Isolation of Bitter Constituentof the Shoots of the Passion Fruit
  • RESULTS AND DISCUSSIONS
    • Phytonutrient Analyses
    • Phytochemical Screening for Secondary Metabolites
    • Toxicity Test
    • Extraction of Bitter Constituentsof Passion Fruit Vine
  • SUMMARY, CONCLUSION AND RECOMMENDATION
    • Summary
    • Conclusion
    • Recommendations
  • APPENDICES
    • Appendix A
    • Appendix B
    • Appendix C
  • BIOGRAPHICAL SKETCH

---