BIBLIOGRAPHY MACARAEG– DIZA, MARIA THERESA C. November 2006. ...
BIBLIOGRAPHY
MACARAEG– DIZA, MARIA THERESA C. November 2006. Extraction and
Characterization of the Essential Oil from the Leaves of Karimbuaya (Euphorbia
neriifolia Linn). Benguet State University, La Trinidad, Benguet.
Adviser: Louisa P. Pladio, M.S.
ABSTRACT
The fresh Karimbuaya (Euphorbia neriifolia Linn.) leaf extract was determined to
be green, with leafy like odor, sweet with an after taste and contains 4.2% mass sucrose.
Phytochemical analysis showed that the fresh leaf extract contains saponin and tannin,
but it is found non-toxic. The LD50 of the fresh leaf extract using the brine shrimp
toxicity assay was 58.73 ppm.
The essential oil from the leaves of Karimbuaya (Euphorbia neriifolia Linn.) was
extracted using a combination of Soxhlet extraction and chromatographic separation
techniques. The chromatogram of the product obtained showed 4 spots with Rf values
0.20, 0.52, 0.8, and 0.95. The fatty like, odorless, and orange colored essential oil has a
melting point range of 36-40oC and is found soluble in all organic solvents used in the
solubility test. One component isolated from the essential oil is a white crystalline solid
found to have a high affinity with chloroform and melts at 142oC. The percentage yield
of the white crystal was 0.142%
The consumer panel type evaluation conducted showed that Karimbuaya mixture
as flavor enhancer for grilled chicken is favored over the use of salt or lemongrass
mixture.
TABLE OF CONTENTS
Page
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
INTRODUCTION
Background of the Study . . . . . . . . . . . . . . . . . . . . . . .
1
Importance of the Study . . . . . . . . . . . . . . . . . . . . . . .
3
REVIEW OF LITERATURE
Karimbuaya (Euphorbia neriifolia Linn.)
Botanical Description . . . . . . . . . . . . . . . . . . . . . .
4
Bioactivity
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
Flavor enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
Plant Flavor Enhancer . . . . . . . . . . . . . . . . . . . . .
6
Synthetic Flavor Enhancer . . . . . . . . . . . . . . . . . .
7
Essential Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Toxicity
Test
Brine Shrimp Assay . . . . . . . . . . . . . . . . . . . . . . . .
10
Phytochemical Screening . . . . . . . . . . . . . . . . . . . . . . . .
10
Alkaloids
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
Saponins
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
Cardenolides and Bufadienolides . . . . . . . . . . . . . .
13
Flavonoids
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
Tannins
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
ii
Anthraquinones . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
Cyanogenic glycosides . . . . . . . . . . . . . . . . . . . . . .
16
Extraction of Essential Oil . . . . . . . . . . . . . . . . . . . . . . .
17
Soxhlet Extraction . . . . . . . . . . . . . . . . . . . . . . . . . .
17
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
MATERIALS AND METHODS
Plant Material
Collection of Plant Sample . . . . . . . . . . . . . . . . . .
19
Plant Preparation . . . . . . . . . . . . . . . . . . . . . . . . . .
19
Characterization of the Fresh Karimbuaya Leaf Extract
Physico-Chemical Properties . . . . . . . . . . . . . . .
20
Phytochemical Screening . . . . . . . . . . . . . . . . . .
20
Toxicity Test
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
Hatching of Brine Shrimp Eggs . . . . . . . . . . . . . . . .
22
Preparation of Stock Solution . . . . . . . . . . . . . . . . . .
22
Dilution of the Stock Solution . . . . . . . . . . . . . . . . . .
23
Bioassay Procedure . . . . . . . . . . . . . . . . . . . . . . . .
23
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Extraction of the Essential Oil
Soxhlet Extraction . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Chromatographic Separation . . . . . . . . . . . . . . . . . . .
25
Purification of the Essential Oil Extract . . . . . . . . . .
25
iii
Characterization of the Essential Oil Extract
Physico-Chemical Test . . . . . . . . . . . . . . . . . . . . . .
29
Acceptability Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31
Preparation of Sample . . . . . . . . . . . . . . . . . . . . . . .
31
Taste Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
RESULTS AND DISCUSSION
Characterization of the Fresh Karimbuaya Leaf Extract. . . .
33
Physico-Chemical Properties . . . . . . . . . . . . . . . . . .
33
Phytochemical Screening . . . . . . . . . . . . . . . . . . . . .
33
Toxicity Test Using The Brine Shrimp Assay . . . . . . . . .
41
Extraction of Essential Oil
Crude Fat Extraction . . . . . . . . . . . . . . . . . . . . . . . . .
43
Separation of Essential Oil . . . . . . . . . . . . . . . . . . .
43
Purification of Essential Oil . . . . . . . . . . . . . . . . . . .
46
Characterization of the Essential Oil
Physico-Chemical Properties . . . . . . . . . . . . . . . . . . .
48
Acceptability of Karimbuaya Leaves as Flavor Enhancer. .
51
SUMMARY, CONCLUSION and RECOMMENDATION
55
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
58
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . . . . . . . .
67
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1
INTRODUCTION
Background of the Study
Flavor enhancers are used to intensify or improve food flavor. Essential oil
from plants and culinary herbs include a broad range of plant species that are used for
flavor enhancement in food and beverages, as well as fragrances in pharmaceutical
and industrial products. Essential oil are derived from aromatic plants of many genera
which are distributed worldwide. These oils are not only used for flavor
enhancement, but they are also used in aromatherapy, a form of herbal medicine in
which healing effects are ascribed to the aromatic compounds in essential oils and
other plant extract.
Culinary herbs refer to herbaceous aromatic plants grown and marketed fresh
or dried and include aromatic plants which are grown for their extractable essential
oils. Significant quantities of dried culinary herbs are imported into the Philippines. A
large amount of selected herbs are domestically produced for the dried spice or
condiments market.
Many herbs are commercially produced, albeit in small quantities, often in
relatively small farms. Herbs which show promise for the fresh market and as source
of essential oil are coriander (Coriandrum sativumL), oregano (Origanumspp), celery
(Apium graveolens L), lemon (citrus limon L), anise (Pimpinella anisum L.),
lemongrass (Cymbopogon citrates), and parsley (Petroselinum crispum Mill.).
Herbs and essential oils are used in a variety of meat and sausage products,
salads, stews, sauces and soups.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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Filipinos love to eat food’s mixed with flavor enhancers. They used mostly
artificial flavoring and only few use fresh herbs. Lechon, grilled whole pig, or
chicken is one among the favorites of Filipinos. This Filipino delicacy is always
present in the table during Filipino occasions. The taste of lechon is enhanced by
putting herbs inside the pig or chicken before it is grilled. Lemongrass commonly
known as tanglad is favored by Cebuanos as a flavor enhancer, Ilocanos on the other
hand use Karimbuaya.
Karimbuaya (Euphorbia neriifolia Linn) as locally known by the Ilocanos is
popular for enhancing the flavor of lechon. The leaves of this plant is used for lechon
baboy (grilled pig),and lechon manok (grilled chicken). Hearsay claims that the
plant gives a distinct aroma and sweet taste to the meat. The introduction of the use of
Karimbuaya as a food flavor enhancer would contribute to the growth of the fresh
herb market.
The extraction and characterization of the essential oil from the leaves of
Karimbuaya (Euphorbia neriifolia Linn.) using physico-chemical test, phytochemical
screening and toxicity testing was conducted at the chemistry laboratory of the
Department of Chemistry, Benguet State University, La Trinidad, Benguet. The
study was done from June to October 2006.
This study aims to:
1. Characterize the crude extract from the fresh leaves of Karimbuaya.
2. Determine the toxicity of the leaves of Karimbuaya.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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3. Extract and characterize the essential oil from the dry leaves of
Karimbuaya.
4. Determine the acceptability of Karimbuaya leaves as a flavor enhancer.
Importance of the Study.
This study finds importance for providing the scientific basis for the
utilization of Karimbuaya as a food flavor enhancer. The result of the study would
encourage local farmers to cultivate the plant and thus provide them additional source
of income for local farmers
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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REVIEW OF LITERATURE
Karimbuaya (Euphorbia neriifolia Linn.)
Botanical Description
The
plant
sample
Euphorbia neriifolia Linn. have several scientific
designation which include Euphorbia ligualaria Roxb., Euphobia pentagona Blanco,
Euphorbia trigona Merr. It is commonly known as Bait (pamp), Karimbuaya (ilk),
soro-soro (tag), and common milk hedge (eng). Karimbuaya is cultivated in gardens
and is nowhere spontaneous. As shown in Plate 1a, this plant is a shrubby, erect,
branched, fleshy, cactus like plant, 2 to 4 meters high, the trunk and older branches
being grayish and cylindric: the medium branches being slightly twisted, stout,
fleshy, and 4 or 5 angled or winged; the younger ones usually 3-winged, the wings
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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(a)
(b)
Plate 1. Karimbuaya (Euphorbia neriifolia Linn..) (a) Matured Karimbuaya tree
(b) Matured fresh leaves
lobulate, with a pair of stout, sharp, 2-4mm long spines rising from the thickened
bases at each leaf or petiole-scar. The cymes are short, solitary in the sinuses and
usually of 3 involucres. The involucres are green or pale yellow and about 6
millimeters in diameter with the lobes fimbriate (Plate 1b). This plant is cultivated in
garden as a hedge plant and it is propagated by stem cuttings (Anon., 2006).
Bioactivity
According to Nadkarni, Euphorbon, resin gum caoutchouc, malate of calcium
are its constituents (Anon, 2006). A fluid extracted from the roasted leaves is used
for earache. It is also similarly used in Malaya, as reported by Burkill and Haniff.
Nadkarni states that the milky juice of this tree is used as a drastic cathartic. The
expressed juice of the leaves is reported as very effective in relieving the paroxysm of
spasmodic asthma. The leaves are considered diuretic. The root mixed with black
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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pepper is employed in snake bites both internally and externally (Quisumbing, 1979).
The root is considered diuretic. According to folklore, the roots have been used for
snake bites. The milky juice is regarded as a purgative internally and a rubefacient
externally. Burkill quotes Dongen, who states that the latex maybe used as purgative,
diuretic and vermifuge, and for asthma. Applied to glandular swellings, it prevents
suppuration. Mixed with margosa oil, it is applied to limbs with contracted
rheumatism. Turmeric powder mixed with the juice of Euphorbia neriifolia is
recommended as an application to piles (Anon, 2006).
Euphorbia neriifolia Linn. shows wound healing activity in different
pharmacological models and patients (Rasik, 1996). The resin obtained from the plant
incorporated in the manufacture of a keratolytic ointment was proven effective in
removing warts (Banaybay, 1980).
Flavor Enhancers
These are substances used to enhance the flavor of foods or to modify the
flavor without contributing any significant flavor of their own. Controlled quantities
of these plants are safe but if ingested in large quantities, they may lead to overdose.
Generating an overall flavor experience requires more than the basic taste. Aromas
released in the mouth stimulate olfactory receptors and transmit odors to the brain.
The total flavor experience is a combination of taste, aroma and chemical feeling.
Cations produce physiological response recognized as saltiness in sodium cations. It
is believed that the cations travel through special channels in the outer membrane of
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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taste cells and enter receptor cells. This changes the voltage across the receptor cell
membrane, electrically exciting the cell and causing the release of neurotransmitters
that stimulate nerve cells to signal saltiness to the brain. In addition, anions saccharin
have been found to contribute to the sweet response in sodium saccharine (Hegenbart,
1996).
Plant Flavor Enhancer
Flavor enhancers are derived from plants and laboratory synthesis. The plant
source can be used directly in fresh, dried, or powdered form.
Lemongrass is used mainly in cheap fragrance work. Its applications include
aerosol deodorants, floor polishes, household detergents, and soaps (Robbins, 1983).
Basil is used in perfumery for its clear, sweet and mildly spicy aroma. Citronella oil,
obtained from a relative of lemongrass, is used as an insect repellant and in
perfumery. In food, several plant herbs are used to enhance the taste (Arctander,
1960).
In fish, meat and stir-fry dishes the following herbs are used as flavor
enhancers: mustard (very potent), turmeric (very potent), garlic, thyme, rosemary,
sage. Cayenne, cinnamon, cloves, ginger are not only used as flavor enhancers they
also aid in digestion. For pasta dishes flavor enhancers are cayenne, turmeric, garlic,
thyme, rosemary, ginger, mustard. Turmeric, garlic, sage, fennel, cinnamon powder,
ginger, cloves are herbs used in baking. As flavor enhancers they also aid in digestion
(Anon, 2006).
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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Synthetic Flavor Enhancer
An example of synthetic food enhancer is the monosodium glutamate (MSG).
MSG is the sodium salt of the amino acid glutamic acid. Its chemical formula is
C5H5NO4Na. It is sold as a fine white crystal substance similar in appearance to salt
or sugar. MSG is added as an ingredient to many snack foods, frozen dinners and
instant meals like instant noodles. MSG stimulates specific receptors located in taste
buds, like amino acid receptor (T1R1/T1R3), or metabotropic receptor (mGluR4 and
mGluR1) which induce the taste known as umami. Umami is a Japanese loanword,
referring to savoury or “more-ish”. Sensitivities to MSG like migraines, nausea, heart
palpitations, asthma, and anaphylactic shock are attributed to the free glutamic acid
component (Wikipedia, 2006). Chinese restaurant syndrome is often used as an
example of MSG symptom complex, a condition characterized by one or more of the
following symptoms: facial pressure, chest pain, violent dream, bronchospasm,
burning sensation in the back of the neck, forearms and chest (FDA, 1993).
Glutamate is found naturally in our bodies and in protein-containing foods
such as cheese, milk, meat, peas and mushrooms. Bound and unbound glutamates are
found naturally in nearly every protein-rich food. Some of the glutamate in foods is
in a “free form and this free form of glutamate enhances food flavor. Certain cheese
or fermented protein products are due to the presence of free glutamate. Studies
showed that glutamate in the body plays an important role in normal functioning of
the nervous system raising questions about whether glutamate in food could affect the
nervous system (Meadows, 2003).
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
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Glutamic acid is an amino acid commonly found in foods. It belongs to the
class of chemicals known as excitotoxins. Abnormally high levels of excitotoxins
have been shown in hundreds of animal studies to cause damage to areas of the brain
unprotected by the blood brain barrier and that a variety of chronic disease can arise
out of this neurotoxicity (FDA, 1993).
Sodium chloride, table salt, is commonly used as a flavor enhancer for food
and has been identified as one of the basic tastes (Sizer, 1997).
Essential oil
The oil derived from plants is called essential oil. Essential oils extracted
from plants are concentrated hydrophobic liquid containing volatile aromatic
compounds. It is also known as volatile oil and ethereal oil. It may also be referred
as “oil of the raw plant material” from which it was extracted, such as oil of clove or
lemongrass oil. The term essential is intended to indicate that the oil is the fragrant
essence of the plant from which it is extracted and not in the more common sense of
being indispensable (Wikipedia, 2006). Essential oils are found in plant oil cells,
glandular trichomes and oil or resin ducts (Schnaubelt, 1999).
The formation and accumulation of essential oils in plants have been
reviewed by Croteau (1986), Guenther (1972) and Runeckles and Mabry (1973).
Chemically, the essential oils are primarily composed of mono-and sesquiterpenes
and aromatic polypropanoids synthesized via the mevalonic acid pathway for
terpenes and the shikimic acid pathway for aromatic propanoids.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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The essential oils from aromatic plants are for the most part volatile and thus,
lend themselves to several methods of extraction such as hydrodistillation, steam
distillation and solvent extraction (Guenther, 1972). The specific extraction method
employed is dependent upon the plant material and the desired end-product. Essential
oil components arise via the secondary metabolism of plants.
Citrus oils, produced by the mechanical pressing of citrus peels, are also
called dry-distilled oils according to the International Standard Organization (ISO)
(Simon, 1990).
Basil oil extracted from sweet basil Ocimum basilicum from the leaves and
flowering tops are used as flavor enhancers for meat, pasta and fishes (Simon, 1990).
The European basil oils which are the highest quality contain methyl chavicol
dlinalool (Guether 1985, Simon 1990).
In aromatherapy, to receive the healing effects of essential oils, they are used
to massage into the skin, inhaled, or added to bath water (Burfield, 1990).
Toxicity Test
Brine Shrimp Assay
Toxicity means the ability of a substance to cause harmful effect. The brine
shrimp assay is a rapid, reliable, reproducible, non-tedious and inexpensive general
bioassay tool for active plant extract. The procedure allows determination of the LD50
values in microgram per milliliter of active constituents in the brine shrimp medium.
This bioassay has been used in the analysis of natural products (Guevara, 2005). This
is done by counting mortalities on artemia, adult brine shrimp.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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Artemia salina, commonly known as brine shrimp are crustaceans that live in
saline environment. Their eggs hatch quickly and the larvae are sensitive to small
doses of biologically-active chemicals (Tarpley, 1958). Brine shrimp grow faster in
slightly higher salt concentration but hatch more rapidly at lower salt concentration,
3.5% saline solution (Guevara, 2005).
Phytochemical Screening
The term “phytochemical” is derived from the Greek word phyto which
means plant. This is defined as the nonnutrient compounds in plant-derived foods
having biological activity in the body (Sizer, 1997). This include the compound that
gives hot peppers their burning taste, the compound that gives garlic its pungent
flavor, the pigments that give spinach and tomatoes their dark green and dark red
color (Criag, 1996).
Phytochemicals have health enhancing abilities and possibly curative
abilities. They number in the hundreds in most plant foods. They are found in fruits,
vegetables, legumes and grains. Phytochemicals work together with nutrients to
promote health and prevent diseases. Examples of phytochemicals include
antioxidant, enzyme stimulators, estrogen, estrogen blockers, compounds that bind
potential cancer-causing chemicals, suppressors of cancer cells (Malaspina, 1996).
Polk (1996) estimates that there may be more than 100 different
phytochemicals in just one serving of vegetables. Table 1 shows the different kinds of
food with their secondary metabolites.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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According to Bloch (1995), phytochemicals are associated with the
prevention and/or treatment of at least four of the leading causes of death in the
United States -- cancer, diabetes, cardiovascular disease, and hypertension. They are
involved in many processes including ones that help prevent cell damage, prevent
cancer cell replication, and decrease cholesterol levels.
Table 1. Most common food analyzed with their Phytochemical constituent
FOOD TYPE
OF
PHYTOCHEMICALS
Allium vegetables (garlic, onions, chives,
Allyl sulfides
leeks)
Cruciferous vegetables (broccoli, cauliflower, Indoles/glucosinolates,
cabbage, brussel sprouts, kale, turnips, bok
sulfaforaphane, Isothiocyanates/
choy, kohlrabi
Thiocyanates, Thiols
Umbelliferous vegetables (carrots, celery,
Carotenoids, Phthalides,
cilantro, parsley, parsnips)
Polyacetylenes
Citrus fruits (oranges, lemons, grapefruit)
Monoterpenes(limonene),
Glucarates
Carotenoids
Beans, grains, seeds (soybeans, oats, barley,
Flavonoids (isoflavones), Phytic
brown rice, whole wheat, flax seed) Protease
acid, Saponins
inhibitors
Herbs, spices (ginger, mint, rosemary, thyme, Gingerols, Flavonoids,
oregano, sage, basil, tumeric, caraway, fennel) Monoterpenes (limonene)
Alkaloids
Alkaloids include literally thousands of bitter, nitrogenous compounds found
throughout the plant kingdom. They often contain one or more rings of carbon atom
with a nitrogen atom in the ring. The position of the nitrogen atom in the carbon rich
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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varies with different alkaloids and with different plant families. In some alkaloids,
such as mescaline, the nitrogen atom is not in a carbon ring. The following alkaloids
include belladonna type (atropine and cocaine), LSD-type (ergine and psilocybin) and
peyote type (mescaline). Some of these have remarkable structural similarities with
neurotransmitters in the human central nervous system, including dopamine,
serotonin, and acetylcholine (Armstrong, 2001).
Saponins
Saponins are steroid/triterpenoid glycosides which are characterized by their
ability to froth when the aqueous solution is agitated. They usually exert a powerful
hemolytic action on red blood cells and when injected into the blood streams are
highly toxic. Saponins are extracted from the plant material by hot water or alcohol
and after concentration of the extract in vacuo, may undergo precipitation with ether.
A simple test for saponins is to shake an aqueous alcoholic plant extract in a test tube
and note whether persistent honeycomb froth is formed above the liquid surface
(Guevarra, 1979).
Saponins are natural surfactants or detergents extracted from plants which are
commonly used as foaming agents for beverages (Clark, 2005). They have hemolytic,
expectorative, anti-inflammatory and immune-stimulating activity. Saponin
demonstrates antimicrobial properties against fungi, bacteria and protozoa. Saponins
are found in number of herb including ginseng, paprika and red variety of onion
(Sahelian, 2006).
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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Cardenolides and Bufadienolides
Cardenolides and bufadienolides are steroids containing deoxysugars and an
unsaturated lactone ring. They are called “cardiac glycosides” as they act on the
muscle of the heart. Plant extracts are usually evaluated for cardiotonic activity either
through in-vitro or in-vivo biological tests. In phytochemical screening for cardiac
glycosides, the test may be applied either to the crude alcoholic extract or the
partially purified extract. Chemical test are designed to detect the presence of the
deoxysugars, the steroid nucleus and the unsaturated lactone (Guevarra, 2005).
Flavonoids
The flavonoids are plant pigments based on C6-C3-C5 carbon skeleton and
generally containing the γ-benzopyrone nucleus like the flavones (Figure 1a ),
isoflavones, flavonols and flavonones. Other flavonoids are the anthocyanins (Figure
1b), leucoanthocyanins, catechins, chalcones, and aurones. Flavonoid compounds
usually occur in plants as glycosides wherein one or more of the phenolic hydroxyl
groups are combined with sugar residue. Many flavonoids exhibit different biological
activities like antiviral, antifungal, anti-inflammatory and cytotoxic activities
(Guevara, 2005).
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
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(a)
(b)
Figure 1. Flavonoids (Wikipedia, 2006) (a) flavone are ketone derivatives of
flavonoid; (b) anthocyanin that occur in tissues of plants that provides
color in leaves, stem, roots, flowers and fruits
Tannins
The term “tannins” was first applied to plant constituents capable of forming
raw animal skin into leather because of their ability to cross-link with protein.
Tannins consist mainly of gallic acid residues that are linked to glucose via glycosidic
bonds.
Tannins are located mainly in the vacuoles or surface wax of the plants. In
these sites they do not interfere with plant metabolism. Only after cell breakdown and
death can they act to have metabolic effects. In leaf tissues, tannins are most common
in the upper epidermis. They serve to reduce palatability and, thus, protect against
predators (Guevara, 1979). Recent studies shows tannins have potential value as
cytotoxic or antineoplastic agents. In medicine tannins are used as an astringent and
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
16
for treatment of burns (Columbia Encyclopedia, 2006). In food, tannins are essential
to the development of flavor in red wines (Klahorst, 2006).
Anthraquinones
Anthraquinones are naturally occurring quinine pigments. They occur in
plants usually as hydroxylated, methylated, or carboxylated derivatives of
anthraquinones, anthrones or dianthrone. They are commonly used as dyes and
cathartics or purgatives (Guevara, 2005). Figure 2 shows the structural formula of
anthraquinone
Figure 2. Chemical structure of Anthraquinone
Cyanogenic Glycosides
Cyanogenic glycosides are phytotoxins which occur in at least 2000 plant
species of which a number of species are used as food in some areas of the world.
Cassava and sorghum are especially important staple foods containing cyanogenic
glycosides (Nartey, 1980). Cyanide, released from a cyanogenic glycoside in food by
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
17
ß-glucosidase either of plant or from gut microflora origin and when taken up,
follows the known cyanide metabolic pathway and toxicokinetics both for animals
and man. Cyanide is detoxified by the enzyme rhodanase, forming thiocyanate, which
is excreted by urine (Conn, 1979 a, b; Oke, 1979). Due to several factors influencing
hydrolysis of cyanogenic glycosides and the confounding influence of nutritional
status (such as riboflavin, vit. B12, sodium, methionine intake) human case studies
and epidemiological studies of the chronic toxicological effects have shown very
variable results and were not conclusive. In several studies both in animals and man
the toxicity of cyanogenic glycosides is often expressed as milligram releasable
cyanide (Conn, 1979 a, b).
Extraction of Essential Oil
Extraction refers to separating by physical or chemical means the desired
material from a plant with the aid of a solvent (Bossert, 1970). Extraction procedure
includes soxhlet extraction and column chromatography.
Soxhlet Extraction
Soxhlet extractor (Plate 2) is used to extract solutes from solids, using any
desired volatile solvents, which can be water-miscible or water-immiscible (Morrison
and Freiser, 1957). The solvent is vaporized and when it condenses, drops on the
solid substance contained in the extraction thimble and extracts the soluble
compounds. When the liquid level fills the body of the extractor, it automatically
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
18
siphons into the flask. This process is continuously repeated as the solvent in the flask
is vaporized and condensed. Soxhlet extractors are standard equipment in laboratories
that analyze fats and oils in biologic samples. Separations can be achieved at low
temperatures in inert atmospheres on a micro or macro scale by a discontinuous or
continuous process (Lo et.al., 1983).
Plate 2. Soxhlet extractor
Chromatography
Chromatography is a physical method of separation in which the components
to be separated are distributed between two phases, one of which is stationary while
the other moves in a definite direction (IUPAC, 1993). Component molecules in a
sample mixture are transported by a mobile phase over a stationary phase. Different
components will have different affinities for the stationary phase with respect to the
mobile phase and will therefore move at different rates. Thin-layer chromatography is
a separation technique in which a stationary phase consisting of an appropriate
material is spread in a uniform thin layer and fixed on a support of glass, metal or
plastic. The separation is carried out by migration through the thin layer of solutes in
a solvent or suitable mixture of solvents.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
19
Plate 3. Chromatographic separation. A mixture of compounds is separated as it
flows down a column packed with stationary phase particles. Separation is
due to different degree of interaction between sample molecules and
stationary phase. (IUPAC, 1993)
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
20
MATERIALS AND METHODS
The Plant Material
Collection of Plant Sample
Matured leaves of Euphorbia neriifolia Linn commonly known in ilocano
as Karimbuaya, were collected from Pangasinan, and Vigan, Ilocos Sur,
provinces north of Manila. The plant sample was identified at the Institute of
Biology Herbarium, at U.P. Diliman, Quezon City.
Plant Preparation
The collected mature leaves of Karimbuaya (Euphorbia neriifolia Linn.)
were washed thoroughly with tap water and air dried for 10 days. After which the
dried leaves were cut into small pieces then powdered prior to analysis. Plate 4
shows samples ready for the extraction process.
.
Plate 4. Cut and powdered Karimbuaya leaves
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
21
Characterization of the Fresh Karimbuaya Leaf Extract
Physico-Chemical Properties
The fresh leaves of Karimbuaya leaves were homogenized using a blender
and the juice was extracted by the use of several layers of cheesecloth.
Color. The color of the extract was determined based on a color chart.
Taste. About 5 drops was used to taste the extract.
Odor. This process was done by smelling after the juices have been
extracted from the leaves.
Refractive
index. The amount of sugar on the sample was measured using
Atago refractometer.
Phytochemical screening
The fresh Karimbuaya leaf extract was screened for the presence of
phytochemicals using methods of Guevara (2005). The fresh extract was prepared
by liquefying the thoroughly washed and drained fresh mature leaves of
Karimbuaya (Euphorbia neriifolia Linn). Table 2 shows the different tests used
for the phytochemical screening. A confirmatory test (Table 3) for the presence of
secondary metabolites or phytochemicals using thin layer chromatography (TLC)
was also performed using the methods from Guevara (2005).
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
22
Table 2. Phytochemical Analysis
TEST COMPOUND
TESTED
Culvenor-Fitzgerald, Dragendorff’s and Mayer
Alkaloid
Froth and Liebermann Burchard
Saponins
Keller-Kiliani and Kedde
Cardenolide & bufadienolide
Bate-Smith&Metcalf and Wilstatter
Flavonoids
Gelatin and Ferric chloride
Tannins
Modified Borntrager’s
Anthraquinones
Guignard
Cyanogenic glycoside
Table 3. Confirmatory Test for Phytochemicals
VISUALIZING AGENT
COMPOUND TESTED
Antimony (III) chloride
Flavonoids/Steriods
Potassium ferricyanide-ferric chloride
Phennol/tannin/flavonoid
Dragendorff’s reagent
Alkaloid
Methanolic KOH
Phenols/Anthrone
Vanillin-sulfuric acid
Essential Oils
α-Napthol-sulfuric acid
Sugars
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
23
Toxicity Test
The toxicity of the Karimbuaya leaves was evaluated using the Brine
Shrimp Assay.
Hatching of Brine Shrimp Eggs
A hatching medium was prepared by dissolving 3.8 grams of rock salt per
100 ml distilled water. This solution has a 3.8% NaCl concentration. About 250
mg of brine shrimp egg and 80 ml hatching medium were placed in the hatching
dish. The hatching dish has two unequal compartment provided by a plastic
divider punched with several 2 mm holes. The brine shrimp eggs were sprinkled
into the larger compartment and then covered with cardboard to block the light.
The smaller compartment was illuminated for 48 hours to allow the eggs to hatch.
The nauplii were then harvested for the assay.
Preparation of Stock Solution
Fresh Karimbuaya leaves were washed thoroughly and oven dried at 60oC
for 24 hours. About 500 mg of the dried leaves were powdered using mortar and
pestle. The powdered sample was transferred to a 250 ml beaker and 50 ml of
artificial sea water (3.8% NaCl) was added. The extract was filtered using 4 layers
of cheesecloth following one hour of incubation. The filtrate collected is the stock
solution.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
24
Dilution of the Stock Solution
Five test tubes of 20 ml capacity were prepared and labeled (1to 5) for the
dilution of the stock solution. The dilution series was prepared as follows: To test
tube 1, 10 ml of the stock solution was placed while 9ml of artificial sea water
(3.8% NaCl) was placed to test tube # 2 to 5. The dilution was carried out by
transferring 1.0 ml stock solution from test tube #1 to test tube #2. From the mix
solution in test tube #2, 1.0 ml was transferred to test tube #3, 1.0ml from test
tube #3 was transferred to test tube #4 and from this, 1.0 ml was pipetted and
added to test tube #5. Table 4 presents the dilution ratio.
Table 4 Dilution of the stock solution used for the bioassay
TEST TUBE NO.
DILUTION RATIO
1 Stock
solution
2 1:
10
3 1:100
4 1:1,000
5 1:10,000
Bioassay Procedure
The diluted stock solutions in the 5 test tubes were transferred to 5
previously cleaned petri dishes and labeled similarly as the test tubes. Using a
Pasteur pipette, ten live brine shrimps (nauplii) were deposited in each Petri dish.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
25
After 24 hours, the Petri dishes were examined for brine shrimp mortality with the
aid of a magnifying lens.
Data Analysis
The toxicity of the plant extract was determined based on the value
obtained for the LD50, which refers to the amount (lethal dose) of a substance that
kills half of the test organisms. The LD50 was determined from the equation of
the line obtained by plotting the percent mortality against the log extract
concentration.
Extraction of the Essential Oil
Soxhlet Extraction
Previously cut air dried Karimbuaya leaves were powdered using a
blender. Each 15 gram batch of the powdered leaves was wrapped in filter paper
and placed in the extracting thimble of a soxhlet extractor, where 250 mL of
petroleum ether was added as the extracting solvent. The extraction process for
each batch of leaves lasted for 5 hours, until the color of the solvent in the body of
the soxhlet extractor became colorless. The petroleum ether extract was
concentrated under reduced pressure at 40oC. The concentrated plant extract was
allowed to dry to determine the percentage yield.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
26
Chromatographic Separation
A weighed amount of the concentrated petroleum ether extract was
subjected to rapid column chromatography by vacuum elution, as in Plate 5. A
column of 4.5 cm diameter was packed with 7 cm high of Silica Gel 60 Gf
(Merck). Solvent used for elution was increasing percentage of hexane-ethyl
acetate mixture starting from pure hexane to 10% ethyl acetate.
The chromatographic separation of the ether extract was monitored
through thin layer chromatography using prepared TLC plates (Merck). The
chromatograms were developed using different solvent system based on the
concentration of the solvent used for elution. The spots were first developed using
iodine vapors followed by vanillin-sulfuric acid spray and was then heated in the
oven at 110oC for color development. Components of the same Rf value were
collected and grouped as one.
Plate 6 shows the over-all extraction process.
Purification of the Essential Oil Extract
Repeated column chromatography using different column sizes and
isocratic elution yielded the oil extract. This is illustrated in Plate 7. Isocratic
elution was carried out using pure hexane (A.R. grade). The fraction identified
by TLC to contain the essential oil was further separated using a smaller column
(2.0 cm in diameter). Solubility difference using pure ethyl acetate (A.R.) and
filtration with the use of Whatman 42 separated the white crystals from the oil.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
27
1
2
3
4
5
6
7
8
9
Plate 5. Column Chromatographic Separation of the Concentrated
Petroleum ether extract from Karimbuaya leaves
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
Dried Powderized Leaves
Ether extract
Soxhlet extraction
Fresh leaves of Karimbuaya
Concentrated under vacuo
(rotary evaporator)
Essential Oil
Fractions containing
Separation by Column Concentrated Petroleum
Essential Oil
Chromatography
Ether Extract
Plate 6. Schematic diagram of the extraction of Essential oil from the leaves of Karimbuaya.
29
Plate 7. Chromatographic Separation of Essential Oil from Karimbuaya leaves
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
30
Characterization of the Essential Oil Extract
Physico-chemical Test
Solubility test. Each two drops of extracted oil was mixed with 2 ml of the
different organic solvent in a test tube. Solubility of the oil extract was
determined in different organic solvent which included chloroform, ethanol, ethyl
acetate, hexane and petroleum ether.
Melting point determination. The melting point of the oil extract was
analyzed as follows: About 0.1ml of the viscous oil was placed in a 50 ml beaker
that was suspended in a water bath. This set-up is shown in Plate 8. The
temperature of the water bath starting from 10oC was gradually increased until the
oil melted.
Plate 8. Melting point set-up for the essential oil
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
31
The melting point of the white crystals separated from the extracted oil
was analyzed using the Thiele-Dennis tube. About 1 mm height of the white
crystals was placed inside the sealed capillary tube. A thermometer was attached
to the capillary tube containing the sample and was then placed inside the Thiele-
Dennis tube filled with glycerol. The tube was heated until the white crystals
melted. The set-up is presented in Plate 9.
Plate 9 A Thiele-Dennis tube for melting point determination of the
crystal (Visconde A.D. et al, 2003)
Acid Number. About 100 mg of oil was mixed with mixture containing
12.5 ml of ethanol (95%) and 12.5 ml ether. The mixture was titrated with 0.1M
KOH until a red color remains after swirling. Phenolphthalein indicator was used
to indicate the end point of the titration. Plate 10 presents the color of the sample
solution before and after titration.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
32
A. Before KOH Titration
B. After KOH titration
Plate 10. Acid number determination of the Essential Oil
Acceptability Test
A consumer type panel consisting of 48 members ranging in age from 20
to 60 years was asked to evaluate the acceptability of the aroma and taste of
lechon manok, grilled with Karimbuaya leaves. Common spices for flavoring
lechon sold in the market were the standard ingredients in the acceptability test
conducted.
Preparation of Sample.
Dressed chickens previously washed and drained were used for the three
treatments. The chickens were then stuffed with the respective treatment
ingredients as shown in Table 5. The stuffed chickens were grilled at the same
time.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
33
Table 5. Treatments used in the chicken for the acceptability test
Treatment Ingredients
T1 Salt
T2
Karimbuaya leaves, 5 pieces chopped
onion bulbs, salt and pepper
T3
Lemongrass, 5 pieces chopped onion
bulbs, salt and pepper
Taste Test
Before the taste test, a sensory evaluation form (Appendix B) was given to
each consumer to mark after tasting. The grilled chickens were cut into bite sizes
and given to the members of the panel. The consumers were asked to rinse their
mouth with water after tasting each of the treated samples.
Data Analysis
One way ANOVA test was used to determine if there was a significant
difference between the three treatments. Computer software SPSS was used in the
data analysis. This software computes using the P-value approach.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
34
RESULTS AND DISCUSSION
Characterization of the Fresh Karimbuaya Leaf Extract
Physico-chemical Properties
The liquid squeezed from the homogenized fresh leaves of Karimbuaya
was green in color, with a leafy like odor, and sweet with an after taste. The
percentage (%) mass sucrose content of the fresh extract was 4.2%. This was
obtained using a refractometer.
Phytochemical Screening
To identify the secondary metabolites present in the extract,
phytochemical analysis was done. The results summarized in Table 6, showed
that the fresh extract contains only the secondary metabolites, saponin and tannin.
Although a slight turbidity was observed in the Culvenor-Fiztgerald test for
alkaloid, this result is considered negative according to Guevara (2005). Presence
of alkaloid in the sample is indicated by a heavy precipitation evaluated as (+++).
The result of on the Laboratory test tube method, no formation of precipitates,
confirms that alkaloid is not present in the sample.
Plate 11 represents the above results.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
35
Table 6. Phytochemical Analyses of Fresh Karimbuaya Leaf Extract
TESTS CLASS
OF RESULTS
COMPOUNDS
Culvenor-Fitzgerald
Alkaloid
Dragendorff
(+) slight turbidity yellow
precipitate
Mayer
(+) slight turbidity yellow
precipitate
Laboratory test tube
Dragendorffs
(-) yellow liquid
Mayer’s
(-) colorless liquid
Froth
Saponins
(+) frothing
Liebermann-
(+) green liquid
Burchard
Guignard
Cyanogenic glycosides (-) yellow color
Keller-Kiliani
Cardenolides and
(-) orange ring at the
Bufadienolides
interface
Kedde
(-) orange brown liquid
Bate-Smith&Metcalf
Flavonoids
(-) orange brown liquid
Wilstatter
(-) light orange liquid
“Cyanidine”
Gelatin
Tannin
(+)jelly precipitate
Ferric chloride
(+) brownish green liquid
Modified Borntragers
Anthraquinones
(-) colorless in the
ammoniacal layer
To confirm the above results, phytochemical analyses using thin-layer
chromatography (TLC) was also performed. The tests confirmed the presence of
saponin and tannin. The absence of alkaloids and other secondary metabolites
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
36
A. Alkaloid Test (negative result)
Plate 11. Phytochemical Screening Result
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
37
Plate 11. Continued…
B. Saponin (positive result)
C. Cyanogenic glycosides
Plate 11. Continued…
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
38
D. Cardenolides and Bufadienolides (negative result)
Plate 11. Continued…
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
39
E. Flavonoids
Plate 11. Continued …
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
40
F. Tannins (positive result)
Plate 11. Continued….
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
41
G. Anthraquinone (negative result)
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
42
were also confirmed. The results are presented in Table 7. The same table also
shows that essential oil and sugar are present in the fresh extract. The violet spots
observed upon spraying the developed TLC plates with the vanillin-sulfuric acid
visualizing agent, indicate that the extract contains essential oil. Using the α-
Napthol-sulfuric spray reagent, a blue spot was observed which confirmed the
presence of sugar in the Karimbuaya leaves. Results collaborates the finding
using refractometer for the presence of sugar.
Table 7. Phytochemical Analyses using Thin-Layer Chromatography (TLC)
SPRAY REAGENTS
CONSTITUENT TESTED
RESULT
Antimony(III) chloride
Flavonoids/steroids
(-) no spot
Potassium ferricyanide-
Phenol/tannin/flavonoid
(-) yellow spot
ferric chloride
Dragendorff’s reagent
Alkaloid
(-) yellow spot
KOHmethanolic
Phenols/anthrone
(-) no spot
Vanillin-sulfuric acid
Essential oils
(+) wide range of color
α-Napthol-sulfuric acid
Sugars
(+) blue spot
Toxicity Test Using The Brine Shrimp Assay
Table 8 showed that the concentrated stock solution of Karimbuaya has an
average of 13.3 % mortality after 24 hours. The diluted concentration of the stock
solution showed 0% mortality on the nauplii.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
43
Table 8. Average % mortality after 24 hours
CONC. OF EXTRACT
AVERAGE % MORTALITY
(ppm)
(24 hours)
11363.63 13.3
1136.36 0
113.63 0
11.36 x 10-3
0
11.36 x 10-7
0
From the graph (Figure 3) the LD50 of the plant solution was computed to be
58.73 ppm. Activities are considered significant if the LD50 value is less than 30
ppm (Saupe, 2006). The result indicates that the Karimbuaya extract is non-toxic.
Brine Shrimp Assay
15
y = 0.8061x + 2.66
10
R2 = 0.303
% Mortality
5
0
-8
-6
-4
-2
0
2
4
6
-5
Log concentration
Figure 3. Determination of LD50
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
44
Extraction of Essential Oil
Crude Fat Extraction
The petroleum ether extract obtained from the soxhlet extraction using 100
grams of powdered dried Karimbuaya leaves was dried under vacuo at 40oC to
yield 5.1664 gram of crude fat or concentrated petroleum ether extract. The
recovered crude fat is solid and colored dark green. Plate 12 shows the sample of
the crude fat
Plate 12. Extracted crude Fat from
Karimbuaya
Separation of Essential Oil
The essential oil was obtained from the crude fat or petroleum ether
extract after repeated chromatographic processes guided by thin layer
chromatographic techniques. Isocratic elution using pure hexane was performed
to elute the essential oil. Table 9 shows the chromatographic characteristics of the
nine fractions obtained from the crude fat. These fractions are shown in Plate 13.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
45
Essential oils, because they are non-polar are the first to be eluted from the
column. Based on the TLC chromatogram, fractions I to III which contain the oil
were pooled and concentrated under vacuo at 40oC.
Table 9. Thin-Layer Chromatography of the Petroleum Ether Extract
FRACTION DEVELOPING
CHROMATOGRAPHIC
NUMBER
SOLVENT
CHARACTERISTIC
Rf values
Color of Spots
(Vanillin-Sulfuric Acid Spray)
I
100% Hexane
0.73
Violet
0.88
Violet
0.93
Violet
II
0.2% E-H
0.7
Violet
III
0.2% E-H
0.43
Violet
0.68
Violet
IV
0.4%E-H
0.13
Brown-violet
V
0.4%E-H
0.08
Brown-violet
VI
0.4%E-H
0.05
Brown-violet
VII
10%E-H
0.22
Brown
0.3
Brown violet
0.4
Violet
0.48
Violet
0.6
Violet
0.82
Violet
VIII
10%E-H
0.13
Green-violet
0.23
Violet
0.30
Violet
IX
15%E-H
0.07
Brown-violet
0.12
Brown-violet
* E-H Ethyl acetate in hexane
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
46
Plate 13. Eluates from the Column Chromatography from the
concentrated petroleum extract of the Karimbuaya leaves
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
47
The pooled Fractions (I-III) was again subjected to rapid column
chromatography to yield 9 fractions as shown in Figure 4. After several
extraction using chromatographic techniques, an orange-colored fat-like substance
was obtained. This is the essential oil.
The chromatogram (Plate 14) of the essential oil showed four spots,
indicating the presence of four components. The over-all extraction process for
the separation of the essential oil from the leaves of Karimbuaya is summarized in
Figure 4.
Purification of Essential Oil
The essential oil extracted showed presence of white crystals. The large
spot (Plate 14) located at the lower part of the chromatogram with Rf value of
0.20 corresponds to the white crystals.
Plate 14. Chromatogram of the essential oil using
Vanillin-sulfuric acid as visualizing agent
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
48
100 grams Dried Karimbuaya leaves
Petroleum ether
Petroleum Ether extract
(5.1664%)
Concentrate under vacuo
Column chromatography
I II III IV V IV VII VIII IX
Column chromatography
( hexane)
1 2 3 4 5 6 7 8 9
Column chromatography
(hexane)
A B C D
Column
chromatography
Essential Oil (0.30%)
white crystal (0.142%)
Figure 4. Schematic diagram of the Over- all Extraction of Essential oil
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
49
The presence of the crystals probably explains why the essential oil is solid at
room temperature. Applying selective solubility difference followed by filtration,
the white crystals were separated from the oil. This is shown in Plate 15. Pure
ethyl acetate solution was used to recover the oil from the mixture. Percent
recovery for the white crystals was 0.142%.
Plate 15. White Crystal Isolate from the leaves of Karimbuaya
Characterization of the Essential Oil
Physico-chemical Properties
Table 10 gave a summary of the physico-chemical properties of the
essential oil and the white crystals isolated from the leaves of Karimbuaya.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
50
Table 10. Physico-chemical Properties of the Essential Oil and Crystal
PARAMETERS
PHYSICO-CHEMICAL PROPERTIES
Essential Oil
Crystals
Color
Orange
White
Odor
Odorless
Odorless
Melting point
36oC – 40oC
142 oC
Solubility
Hexane
Chloroform
Ethyl acetate
Ethanol
Petroleum ether
Chloroform
Physical state
solid
solid
Acid value
2.9
Essential Oil. The essential oil extracted was odorless and orange in color.
This is shown in Plate 16. The melting point determined ranges from 36-40oC.
The wide range of melting point denotes that the oil has other components, which
is confirmed by the presence 4 spots observed in the chromatogram revealed in
Plate 14. The essential oil is soluble in all of the organic solvents used in the
solubility test. The oil has an acid value of 2.9. The acid value measures the
amount of the fatty acids hydrolyzed. The higher acid number indicates the
degree of degradation of the oil (Anon, 2006).
Crystals. The solid separated from the essential oil is white in color and is
odorless. It has a melting point of 142oC. The solubility of the crystal was tested
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
51
A
B
C
Plate 16. A. Essential oil extracted from Karimbuaya leaves
B. top view of the oil in the beaker
C. The essential oil in a Florence flask after concentrating in vacuo
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
52
in several organic reagents and is found soluble in chloroform. as shown in Table
11.
Table 11. – Solubility of the White Crystal Isolate
REAGENTS
RESULT REAGENTS RESULT
70% ethanol
Insoluble
Ethyl acetate
Insoluble
80% ethanol
Insoluble
Hexane
Insoluble
85% ethanol
Insoluble
Diethyl ether
Insoluble
90% ethanol
Insoluble
Chloroform
Soluble
Petroleum ether
Insoluble
2%EtOAc-Hexame
Insoluble
Acceptability of Karimbuaya Leaves
as a Flavor Enhancer
The sensory attributes of foods are of paramount importance as people
take pleasure from a combination of their visual, olfactory, taste and tactile
perception. Overall flavor requires not just the basic taste: sweet, salty, bitter, and
sour. According to Hegenbart(1996) the total flavor experience is a combination
of taste, aroma and the chemical feeling. Modifications on the flavors of food are
achieved by addition of flavor enhancers.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
53
The acceptability of fresh Karimbuaya leaves as a flavor enhancer in
lechon preparation was compared to the common commercial ingredients. The
aroma, taste and overall acceptability were evaluated.
Table 12. Multiple Comparison on Aroma
SAMPLE MEAN
SIG.
DIFFERENCE
Karimbuaya mixture vs. salt only
0.38
0.202
Karimbuaya mixture vs. Lemongrass mixture
0.52*
0.046
Salt only vs. Lemongrass mixture
0.15
0.784
* significant at 0.05
Aroma
Table 12 shows that the mean difference of 0.38 between Karimbuaya
mixture and salt only is not significant at the 0.05 level. This indicates that there
is no difference on the aroma of the grilled chicken stuffed with Karimbuaya
mixture and salt only, respectively. On the other hand, between Karimbuaya
mixture and lemongrass mixture, the mean difference (0.52) is significant. This
implies that the aroma of grilled chicken as enhanced by Karimbuaya leaves is
preferred by the consumer panel. Between the two standard ingredients, salt only
and lemongrass mixture, the mean difference is not significant. This shows that
the aroma of grilled chicken stuffed with lemongrass is the same as that of the
salt.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
54
Table 13. Multiple Comparison on Taste
SAMPLE MEAN
SIG.
DIFFERENCE
Karimbuaya mixture vs. salt only
0.15
0..820
Karimbuaya mixture vs. Lemongrass mixture
0.96*
0.000
Salt only vs. Lemongrass mixture
0.81*
0.002
* significant at 0.50
Taste
Comparing the taste of the grilled chicken treated with Karimbuaya
mixture to the one treated with salt only, the mean difference (0.15) obtained is
not significant. The result implies that the taste is the same. Karimbuaya mixture
when compared with lemongrass mixture, the mean difference is significant. This
means the consumers prefer the taste of chicken flavored with Karimbuaya
mixture than the lemongrass mixture. The standard ingredients used in the
analysis, salt only and lemongrass mixture, revealed a significant difference on
the taste of the grilled chicken.
Overall Acceptability
The overall acceptability shown in Table 14 reveals a significant
difference only between Karimbuaya mixture and lemongrass mixture. This
implies that the Karimbuaya mixture as flavor enhancer on grilled chicken is
accepted by consumers.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
55
Table 14. Multiple Comparisons on Overall acceptability
SAMPLE MEAN
SIG.
DIFFERENCE
Karimbuaya mixture vs. salt only
0.15
0..820
Karimbuaya mixture vs. Lemongrass mixture
0.96*
0.000
Salt only vs. Lemongrass mixture
0.81*
0.002
* significant at 0.05
Table 15. Acceptability test of Karimbuaya mixture
PARAMETERS Fcomputed
SIG.
Aroma
2.996*
0.05
Taste
9.020*
0.00
Overall acceptability
3.486*
0.03
* significant at 0.05
In table 15, statistical analysis using the F test (one-way ANOVA) showed
that the computed F values for the aroma, taste and overall acceptability were
higher than the F value (2.66) at the 0.05 level of significance. The results imply
that addition of chopped Karimbuaya leaves to the usual spices for lechon
preparation, is favored by the consumer panel. This denotes that the grilled meat
with Karimbuaya leaves produces an aroma and taste that is much preferred than
lemongrass and the salt.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
56
SUMMARY, CONCLUSION and RECOMMENDATION
Summary
This study aims to characterize the extracts (fresh leaf and the essential
oil) from the leaves of Karimbuaya (Euphorbia neriifolia Linn). The toxicity and
acceptability as a flavor enhancer of the fresh leaves of Karimbuaya was also
determined.
The characterization of the fresh Karimbuaya leaf extract was found to be
green with leafy like odor and sweet with an after taste. The sweetness was
confirmed with the presence of 4.2% mass sucrose using a refractometer. Results
of the phytochemical analysis of the leaf extract showed the presence of saponin
and tannin. The same metabolites were confirmed present in the sample from the
results of the thin-layer chromatography (TLC). The presence of the essential oil
in the sample was also revealed in the chromatogram which was developed using
a vanillin-sulfuric spray reagent.
The LD50 equivalent to 58.73 ppm obtained from the brine shrimp assay
indicates that the Karimbuaya leaves are non-toxic. This indicates that the
concentration of the saponin and tannin present is below lethal dose (30 ppm).
The percentage yield of the recovered essential oil of the Karimbuaya
leaves obtained from repeated rapid column chromatography was 0.30%. The
chromatogram of the orange colored essential oil indicates the presence of four
(4) components with Rf values 0.20, 0.52, 0.8, and 0.95. The essential oil
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
57
extracted is solid at room temperature with a melting point range of 36o – 40oC
and is soluble to all the organic solvents (ethanol, ethyl acetate, hexane, petroleum
ether, chloroform) used in the test.
The percentage yield of the white crystal isolate was 0.142%. The
chromatogram shows the crystals to have an Rf value of 0.20. It has high
chloroform affinity and a melting point of 142oC.
As a flavor enhancer, the fresh Karimbuaya leaves were compared with
the standard ingredients, salt and lemongrass. Results indicated that the
Karimbuaya leaves were preferred by the consumer panel as a flavor enhancer on
grilled chicken.
Conclusion
Based on the results, the following conclusions were drawn:
1.
The Karimbuaya leaf extract is a green solution with leafy like odor,
sweet with an after taste and is non-toxic as indicated by the
LD50 value of 58.73 ppm;
2.
The orange colored essential oil extracted from the leaves of
Karimbuaya was fatty like with a wide melting point range of
36-40oC. The chromatogram of the oil shows 4 spots indicating
that the oil is a mixture. One component is a white crystalline
mixture with a melting point of 142oC;
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
58
3.
The Karimbuaya leaves as a flavor enhancer for grilled chicken was
preferred over the standard ingredients, salt and lemongrass.
Recommendation
The use of fresh Karimbuaya (Euphorbia neriifolia Linn.) leaves as a
flavor enhancer on grilled chicken is recommended.
Further study should be done to determine the following:
1.
effect of the fresh Karimbuaya leaves as a flavor enhancer on
other meat and fish products;
2.
the constituents responsible for the other properties of the plant
not included in the study;
3.
isolate and identify other constituents of the essential oil;
4.
and to analyze the bioactivity of the plant.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
59
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Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
63
Appendix A-1
Analysis of Variance (ANOVA) for AROMA
Sum
of
df Mean F Sig
Squares
Square
Between Groups
6.931
2
3.465
2.996 .05*
Within Groups
163.063
141
1.156
Total
169.993
143
Multiple Comparison
Dependent Variable : AROMA
Tukey HSD
Mean
Difference
Std. Error
(I) TREAT
(J) TREAT
(I-J)
Sig.
1
2
.38
.22
.202
3
.15
.22
.784
.
2
1
38
.22
.202
3
.52*
.22
.046
3
1
.15
.22
.784
2
.52*
.22
.046
* mean difference is significant at 0.5
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
MACARAEG– DIZA, MARIA THERESA C. November 2006. Extraction and
Characterization of the Essential Oil from the Leaves of Karimbuaya (Euphorbia
neriifolia Linn). Benguet State University, La Trinidad, Benguet.
Adviser: Louisa P. Pladio, M.S.
ABSTRACT
The fresh Karimbuaya (Euphorbia neriifolia Linn.) leaf extract was determined to
be green, with leafy like odor, sweet with an after taste and contains 4.2% mass sucrose.
Phytochemical analysis showed that the fresh leaf extract contains saponin and tannin,
but it is found non-toxic. The LD50 of the fresh leaf extract using the brine shrimp
toxicity assay was 58.73 ppm.
The essential oil from the leaves of Karimbuaya (Euphorbia neriifolia Linn.) was
extracted using a combination of Soxhlet extraction and chromatographic separation
techniques. The chromatogram of the product obtained showed 4 spots with Rf values
0.20, 0.52, 0.8, and 0.95. The fatty like, odorless, and orange colored essential oil has a
melting point range of 36-40oC and is found soluble in all organic solvents used in the
solubility test. One component isolated from the essential oil is a white crystalline solid
found to have a high affinity with chloroform and melts at 142oC. The percentage yield
of the white crystal was 0.142%
The consumer panel type evaluation conducted showed that Karimbuaya mixture
as flavor enhancer for grilled chicken is favored over the use of salt or lemongrass
mixture.
TABLE OF CONTENTS
Page
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
INTRODUCTION
Background of the Study . . . . . . . . . . . . . . . . . . . . . . .
1
Importance of the Study . . . . . . . . . . . . . . . . . . . . . . .
3
REVIEW OF LITERATURE
Karimbuaya (Euphorbia neriifolia Linn.)
Botanical Description . . . . . . . . . . . . . . . . . . . . . .
4
Bioactivity
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
Flavor enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
Plant Flavor Enhancer . . . . . . . . . . . . . . . . . . . . .
6
Synthetic Flavor Enhancer . . . . . . . . . . . . . . . . . .
7
Essential Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Toxicity
Test
Brine Shrimp Assay . . . . . . . . . . . . . . . . . . . . . . . .
10
Phytochemical Screening . . . . . . . . . . . . . . . . . . . . . . . .
10
Alkaloids
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
Saponins
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
Cardenolides and Bufadienolides . . . . . . . . . . . . . .
13
Flavonoids
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
Tannins
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
ii
Anthraquinones . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
Cyanogenic glycosides . . . . . . . . . . . . . . . . . . . . . .
16
Extraction of Essential Oil . . . . . . . . . . . . . . . . . . . . . . .
17
Soxhlet Extraction . . . . . . . . . . . . . . . . . . . . . . . . . .
17
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
MATERIALS AND METHODS
Plant Material
Collection of Plant Sample . . . . . . . . . . . . . . . . . .
19
Plant Preparation . . . . . . . . . . . . . . . . . . . . . . . . . .
19
Characterization of the Fresh Karimbuaya Leaf Extract
Physico-Chemical Properties . . . . . . . . . . . . . . .
20
Phytochemical Screening . . . . . . . . . . . . . . . . . .
20
Toxicity Test
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
Hatching of Brine Shrimp Eggs . . . . . . . . . . . . . . . .
22
Preparation of Stock Solution . . . . . . . . . . . . . . . . . .
22
Dilution of the Stock Solution . . . . . . . . . . . . . . . . . .
23
Bioassay Procedure . . . . . . . . . . . . . . . . . . . . . . . .
23
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Extraction of the Essential Oil
Soxhlet Extraction . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Chromatographic Separation . . . . . . . . . . . . . . . . . . .
25
Purification of the Essential Oil Extract . . . . . . . . . .
25
iii
Characterization of the Essential Oil Extract
Physico-Chemical Test . . . . . . . . . . . . . . . . . . . . . .
29
Acceptability Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31
Preparation of Sample . . . . . . . . . . . . . . . . . . . . . . .
31
Taste Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
RESULTS AND DISCUSSION
Characterization of the Fresh Karimbuaya Leaf Extract. . . .
33
Physico-Chemical Properties . . . . . . . . . . . . . . . . . .
33
Phytochemical Screening . . . . . . . . . . . . . . . . . . . . .
33
Toxicity Test Using The Brine Shrimp Assay . . . . . . . . .
41
Extraction of Essential Oil
Crude Fat Extraction . . . . . . . . . . . . . . . . . . . . . . . . .
43
Separation of Essential Oil . . . . . . . . . . . . . . . . . . .
43
Purification of Essential Oil . . . . . . . . . . . . . . . . . . .
46
Characterization of the Essential Oil
Physico-Chemical Properties . . . . . . . . . . . . . . . . . . .
48
Acceptability of Karimbuaya Leaves as Flavor Enhancer. .
51
SUMMARY, CONCLUSION and RECOMMENDATION
55
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
58
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . . . . . . . .
67
iv
1
INTRODUCTION
Background of the Study
Flavor enhancers are used to intensify or improve food flavor. Essential oil
from plants and culinary herbs include a broad range of plant species that are used for
flavor enhancement in food and beverages, as well as fragrances in pharmaceutical
and industrial products. Essential oil are derived from aromatic plants of many genera
which are distributed worldwide. These oils are not only used for flavor
enhancement, but they are also used in aromatherapy, a form of herbal medicine in
which healing effects are ascribed to the aromatic compounds in essential oils and
other plant extract.
Culinary herbs refer to herbaceous aromatic plants grown and marketed fresh
or dried and include aromatic plants which are grown for their extractable essential
oils. Significant quantities of dried culinary herbs are imported into the Philippines. A
large amount of selected herbs are domestically produced for the dried spice or
condiments market.
Many herbs are commercially produced, albeit in small quantities, often in
relatively small farms. Herbs which show promise for the fresh market and as source
of essential oil are coriander (Coriandrum sativumL), oregano (Origanumspp), celery
(Apium graveolens L), lemon (citrus limon L), anise (Pimpinella anisum L.),
lemongrass (Cymbopogon citrates), and parsley (Petroselinum crispum Mill.).
Herbs and essential oils are used in a variety of meat and sausage products,
salads, stews, sauces and soups.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
2
Filipinos love to eat food’s mixed with flavor enhancers. They used mostly
artificial flavoring and only few use fresh herbs. Lechon, grilled whole pig, or
chicken is one among the favorites of Filipinos. This Filipino delicacy is always
present in the table during Filipino occasions. The taste of lechon is enhanced by
putting herbs inside the pig or chicken before it is grilled. Lemongrass commonly
known as tanglad is favored by Cebuanos as a flavor enhancer, Ilocanos on the other
hand use Karimbuaya.
Karimbuaya (Euphorbia neriifolia Linn) as locally known by the Ilocanos is
popular for enhancing the flavor of lechon. The leaves of this plant is used for lechon
baboy (grilled pig),and lechon manok (grilled chicken). Hearsay claims that the
plant gives a distinct aroma and sweet taste to the meat. The introduction of the use of
Karimbuaya as a food flavor enhancer would contribute to the growth of the fresh
herb market.
The extraction and characterization of the essential oil from the leaves of
Karimbuaya (Euphorbia neriifolia Linn.) using physico-chemical test, phytochemical
screening and toxicity testing was conducted at the chemistry laboratory of the
Department of Chemistry, Benguet State University, La Trinidad, Benguet. The
study was done from June to October 2006.
This study aims to:
1. Characterize the crude extract from the fresh leaves of Karimbuaya.
2. Determine the toxicity of the leaves of Karimbuaya.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
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3. Extract and characterize the essential oil from the dry leaves of
Karimbuaya.
4. Determine the acceptability of Karimbuaya leaves as a flavor enhancer.
Importance of the Study.
This study finds importance for providing the scientific basis for the
utilization of Karimbuaya as a food flavor enhancer. The result of the study would
encourage local farmers to cultivate the plant and thus provide them additional source
of income for local farmers
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
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REVIEW OF LITERATURE
Karimbuaya (Euphorbia neriifolia Linn.)
Botanical Description
The
plant
sample
Euphorbia neriifolia Linn. have several scientific
designation which include Euphorbia ligualaria Roxb., Euphobia pentagona Blanco,
Euphorbia trigona Merr. It is commonly known as Bait (pamp), Karimbuaya (ilk),
soro-soro (tag), and common milk hedge (eng). Karimbuaya is cultivated in gardens
and is nowhere spontaneous. As shown in Plate 1a, this plant is a shrubby, erect,
branched, fleshy, cactus like plant, 2 to 4 meters high, the trunk and older branches
being grayish and cylindric: the medium branches being slightly twisted, stout,
fleshy, and 4 or 5 angled or winged; the younger ones usually 3-winged, the wings
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
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(a)
(b)
Plate 1. Karimbuaya (Euphorbia neriifolia Linn..) (a) Matured Karimbuaya tree
(b) Matured fresh leaves
lobulate, with a pair of stout, sharp, 2-4mm long spines rising from the thickened
bases at each leaf or petiole-scar. The cymes are short, solitary in the sinuses and
usually of 3 involucres. The involucres are green or pale yellow and about 6
millimeters in diameter with the lobes fimbriate (Plate 1b). This plant is cultivated in
garden as a hedge plant and it is propagated by stem cuttings (Anon., 2006).
Bioactivity
According to Nadkarni, Euphorbon, resin gum caoutchouc, malate of calcium
are its constituents (Anon, 2006). A fluid extracted from the roasted leaves is used
for earache. It is also similarly used in Malaya, as reported by Burkill and Haniff.
Nadkarni states that the milky juice of this tree is used as a drastic cathartic. The
expressed juice of the leaves is reported as very effective in relieving the paroxysm of
spasmodic asthma. The leaves are considered diuretic. The root mixed with black
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pepper is employed in snake bites both internally and externally (Quisumbing, 1979).
The root is considered diuretic. According to folklore, the roots have been used for
snake bites. The milky juice is regarded as a purgative internally and a rubefacient
externally. Burkill quotes Dongen, who states that the latex maybe used as purgative,
diuretic and vermifuge, and for asthma. Applied to glandular swellings, it prevents
suppuration. Mixed with margosa oil, it is applied to limbs with contracted
rheumatism. Turmeric powder mixed with the juice of Euphorbia neriifolia is
recommended as an application to piles (Anon, 2006).
Euphorbia neriifolia Linn. shows wound healing activity in different
pharmacological models and patients (Rasik, 1996). The resin obtained from the plant
incorporated in the manufacture of a keratolytic ointment was proven effective in
removing warts (Banaybay, 1980).
Flavor Enhancers
These are substances used to enhance the flavor of foods or to modify the
flavor without contributing any significant flavor of their own. Controlled quantities
of these plants are safe but if ingested in large quantities, they may lead to overdose.
Generating an overall flavor experience requires more than the basic taste. Aromas
released in the mouth stimulate olfactory receptors and transmit odors to the brain.
The total flavor experience is a combination of taste, aroma and chemical feeling.
Cations produce physiological response recognized as saltiness in sodium cations. It
is believed that the cations travel through special channels in the outer membrane of
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
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taste cells and enter receptor cells. This changes the voltage across the receptor cell
membrane, electrically exciting the cell and causing the release of neurotransmitters
that stimulate nerve cells to signal saltiness to the brain. In addition, anions saccharin
have been found to contribute to the sweet response in sodium saccharine (Hegenbart,
1996).
Plant Flavor Enhancer
Flavor enhancers are derived from plants and laboratory synthesis. The plant
source can be used directly in fresh, dried, or powdered form.
Lemongrass is used mainly in cheap fragrance work. Its applications include
aerosol deodorants, floor polishes, household detergents, and soaps (Robbins, 1983).
Basil is used in perfumery for its clear, sweet and mildly spicy aroma. Citronella oil,
obtained from a relative of lemongrass, is used as an insect repellant and in
perfumery. In food, several plant herbs are used to enhance the taste (Arctander,
1960).
In fish, meat and stir-fry dishes the following herbs are used as flavor
enhancers: mustard (very potent), turmeric (very potent), garlic, thyme, rosemary,
sage. Cayenne, cinnamon, cloves, ginger are not only used as flavor enhancers they
also aid in digestion. For pasta dishes flavor enhancers are cayenne, turmeric, garlic,
thyme, rosemary, ginger, mustard. Turmeric, garlic, sage, fennel, cinnamon powder,
ginger, cloves are herbs used in baking. As flavor enhancers they also aid in digestion
(Anon, 2006).
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Synthetic Flavor Enhancer
An example of synthetic food enhancer is the monosodium glutamate (MSG).
MSG is the sodium salt of the amino acid glutamic acid. Its chemical formula is
C5H5NO4Na. It is sold as a fine white crystal substance similar in appearance to salt
or sugar. MSG is added as an ingredient to many snack foods, frozen dinners and
instant meals like instant noodles. MSG stimulates specific receptors located in taste
buds, like amino acid receptor (T1R1/T1R3), or metabotropic receptor (mGluR4 and
mGluR1) which induce the taste known as umami. Umami is a Japanese loanword,
referring to savoury or “more-ish”. Sensitivities to MSG like migraines, nausea, heart
palpitations, asthma, and anaphylactic shock are attributed to the free glutamic acid
component (Wikipedia, 2006). Chinese restaurant syndrome is often used as an
example of MSG symptom complex, a condition characterized by one or more of the
following symptoms: facial pressure, chest pain, violent dream, bronchospasm,
burning sensation in the back of the neck, forearms and chest (FDA, 1993).
Glutamate is found naturally in our bodies and in protein-containing foods
such as cheese, milk, meat, peas and mushrooms. Bound and unbound glutamates are
found naturally in nearly every protein-rich food. Some of the glutamate in foods is
in a “free form and this free form of glutamate enhances food flavor. Certain cheese
or fermented protein products are due to the presence of free glutamate. Studies
showed that glutamate in the body plays an important role in normal functioning of
the nervous system raising questions about whether glutamate in food could affect the
nervous system (Meadows, 2003).
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Glutamic acid is an amino acid commonly found in foods. It belongs to the
class of chemicals known as excitotoxins. Abnormally high levels of excitotoxins
have been shown in hundreds of animal studies to cause damage to areas of the brain
unprotected by the blood brain barrier and that a variety of chronic disease can arise
out of this neurotoxicity (FDA, 1993).
Sodium chloride, table salt, is commonly used as a flavor enhancer for food
and has been identified as one of the basic tastes (Sizer, 1997).
Essential oil
The oil derived from plants is called essential oil. Essential oils extracted
from plants are concentrated hydrophobic liquid containing volatile aromatic
compounds. It is also known as volatile oil and ethereal oil. It may also be referred
as “oil of the raw plant material” from which it was extracted, such as oil of clove or
lemongrass oil. The term essential is intended to indicate that the oil is the fragrant
essence of the plant from which it is extracted and not in the more common sense of
being indispensable (Wikipedia, 2006). Essential oils are found in plant oil cells,
glandular trichomes and oil or resin ducts (Schnaubelt, 1999).
The formation and accumulation of essential oils in plants have been
reviewed by Croteau (1986), Guenther (1972) and Runeckles and Mabry (1973).
Chemically, the essential oils are primarily composed of mono-and sesquiterpenes
and aromatic polypropanoids synthesized via the mevalonic acid pathway for
terpenes and the shikimic acid pathway for aromatic propanoids.
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The essential oils from aromatic plants are for the most part volatile and thus,
lend themselves to several methods of extraction such as hydrodistillation, steam
distillation and solvent extraction (Guenther, 1972). The specific extraction method
employed is dependent upon the plant material and the desired end-product. Essential
oil components arise via the secondary metabolism of plants.
Citrus oils, produced by the mechanical pressing of citrus peels, are also
called dry-distilled oils according to the International Standard Organization (ISO)
(Simon, 1990).
Basil oil extracted from sweet basil Ocimum basilicum from the leaves and
flowering tops are used as flavor enhancers for meat, pasta and fishes (Simon, 1990).
The European basil oils which are the highest quality contain methyl chavicol
dlinalool (Guether 1985, Simon 1990).
In aromatherapy, to receive the healing effects of essential oils, they are used
to massage into the skin, inhaled, or added to bath water (Burfield, 1990).
Toxicity Test
Brine Shrimp Assay
Toxicity means the ability of a substance to cause harmful effect. The brine
shrimp assay is a rapid, reliable, reproducible, non-tedious and inexpensive general
bioassay tool for active plant extract. The procedure allows determination of the LD50
values in microgram per milliliter of active constituents in the brine shrimp medium.
This bioassay has been used in the analysis of natural products (Guevara, 2005). This
is done by counting mortalities on artemia, adult brine shrimp.
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Artemia salina, commonly known as brine shrimp are crustaceans that live in
saline environment. Their eggs hatch quickly and the larvae are sensitive to small
doses of biologically-active chemicals (Tarpley, 1958). Brine shrimp grow faster in
slightly higher salt concentration but hatch more rapidly at lower salt concentration,
3.5% saline solution (Guevara, 2005).
Phytochemical Screening
The term “phytochemical” is derived from the Greek word phyto which
means plant. This is defined as the nonnutrient compounds in plant-derived foods
having biological activity in the body (Sizer, 1997). This include the compound that
gives hot peppers their burning taste, the compound that gives garlic its pungent
flavor, the pigments that give spinach and tomatoes their dark green and dark red
color (Criag, 1996).
Phytochemicals have health enhancing abilities and possibly curative
abilities. They number in the hundreds in most plant foods. They are found in fruits,
vegetables, legumes and grains. Phytochemicals work together with nutrients to
promote health and prevent diseases. Examples of phytochemicals include
antioxidant, enzyme stimulators, estrogen, estrogen blockers, compounds that bind
potential cancer-causing chemicals, suppressors of cancer cells (Malaspina, 1996).
Polk (1996) estimates that there may be more than 100 different
phytochemicals in just one serving of vegetables. Table 1 shows the different kinds of
food with their secondary metabolites.
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According to Bloch (1995), phytochemicals are associated with the
prevention and/or treatment of at least four of the leading causes of death in the
United States -- cancer, diabetes, cardiovascular disease, and hypertension. They are
involved in many processes including ones that help prevent cell damage, prevent
cancer cell replication, and decrease cholesterol levels.
Table 1. Most common food analyzed with their Phytochemical constituent
FOOD TYPE
OF
PHYTOCHEMICALS
Allium vegetables (garlic, onions, chives,
Allyl sulfides
leeks)
Cruciferous vegetables (broccoli, cauliflower, Indoles/glucosinolates,
cabbage, brussel sprouts, kale, turnips, bok
sulfaforaphane, Isothiocyanates/
choy, kohlrabi
Thiocyanates, Thiols
Umbelliferous vegetables (carrots, celery,
Carotenoids, Phthalides,
cilantro, parsley, parsnips)
Polyacetylenes
Citrus fruits (oranges, lemons, grapefruit)
Monoterpenes(limonene),
Glucarates
Carotenoids
Beans, grains, seeds (soybeans, oats, barley,
Flavonoids (isoflavones), Phytic
brown rice, whole wheat, flax seed) Protease
acid, Saponins
inhibitors
Herbs, spices (ginger, mint, rosemary, thyme, Gingerols, Flavonoids,
oregano, sage, basil, tumeric, caraway, fennel) Monoterpenes (limonene)
Alkaloids
Alkaloids include literally thousands of bitter, nitrogenous compounds found
throughout the plant kingdom. They often contain one or more rings of carbon atom
with a nitrogen atom in the ring. The position of the nitrogen atom in the carbon rich
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varies with different alkaloids and with different plant families. In some alkaloids,
such as mescaline, the nitrogen atom is not in a carbon ring. The following alkaloids
include belladonna type (atropine and cocaine), LSD-type (ergine and psilocybin) and
peyote type (mescaline). Some of these have remarkable structural similarities with
neurotransmitters in the human central nervous system, including dopamine,
serotonin, and acetylcholine (Armstrong, 2001).
Saponins
Saponins are steroid/triterpenoid glycosides which are characterized by their
ability to froth when the aqueous solution is agitated. They usually exert a powerful
hemolytic action on red blood cells and when injected into the blood streams are
highly toxic. Saponins are extracted from the plant material by hot water or alcohol
and after concentration of the extract in vacuo, may undergo precipitation with ether.
A simple test for saponins is to shake an aqueous alcoholic plant extract in a test tube
and note whether persistent honeycomb froth is formed above the liquid surface
(Guevarra, 1979).
Saponins are natural surfactants or detergents extracted from plants which are
commonly used as foaming agents for beverages (Clark, 2005). They have hemolytic,
expectorative, anti-inflammatory and immune-stimulating activity. Saponin
demonstrates antimicrobial properties against fungi, bacteria and protozoa. Saponins
are found in number of herb including ginseng, paprika and red variety of onion
(Sahelian, 2006).
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Cardenolides and Bufadienolides
Cardenolides and bufadienolides are steroids containing deoxysugars and an
unsaturated lactone ring. They are called “cardiac glycosides” as they act on the
muscle of the heart. Plant extracts are usually evaluated for cardiotonic activity either
through in-vitro or in-vivo biological tests. In phytochemical screening for cardiac
glycosides, the test may be applied either to the crude alcoholic extract or the
partially purified extract. Chemical test are designed to detect the presence of the
deoxysugars, the steroid nucleus and the unsaturated lactone (Guevarra, 2005).
Flavonoids
The flavonoids are plant pigments based on C6-C3-C5 carbon skeleton and
generally containing the γ-benzopyrone nucleus like the flavones (Figure 1a ),
isoflavones, flavonols and flavonones. Other flavonoids are the anthocyanins (Figure
1b), leucoanthocyanins, catechins, chalcones, and aurones. Flavonoid compounds
usually occur in plants as glycosides wherein one or more of the phenolic hydroxyl
groups are combined with sugar residue. Many flavonoids exhibit different biological
activities like antiviral, antifungal, anti-inflammatory and cytotoxic activities
(Guevara, 2005).
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(a)
(b)
Figure 1. Flavonoids (Wikipedia, 2006) (a) flavone are ketone derivatives of
flavonoid; (b) anthocyanin that occur in tissues of plants that provides
color in leaves, stem, roots, flowers and fruits
Tannins
The term “tannins” was first applied to plant constituents capable of forming
raw animal skin into leather because of their ability to cross-link with protein.
Tannins consist mainly of gallic acid residues that are linked to glucose via glycosidic
bonds.
Tannins are located mainly in the vacuoles or surface wax of the plants. In
these sites they do not interfere with plant metabolism. Only after cell breakdown and
death can they act to have metabolic effects. In leaf tissues, tannins are most common
in the upper epidermis. They serve to reduce palatability and, thus, protect against
predators (Guevara, 1979). Recent studies shows tannins have potential value as
cytotoxic or antineoplastic agents. In medicine tannins are used as an astringent and
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for treatment of burns (Columbia Encyclopedia, 2006). In food, tannins are essential
to the development of flavor in red wines (Klahorst, 2006).
Anthraquinones
Anthraquinones are naturally occurring quinine pigments. They occur in
plants usually as hydroxylated, methylated, or carboxylated derivatives of
anthraquinones, anthrones or dianthrone. They are commonly used as dyes and
cathartics or purgatives (Guevara, 2005). Figure 2 shows the structural formula of
anthraquinone
Figure 2. Chemical structure of Anthraquinone
Cyanogenic Glycosides
Cyanogenic glycosides are phytotoxins which occur in at least 2000 plant
species of which a number of species are used as food in some areas of the world.
Cassava and sorghum are especially important staple foods containing cyanogenic
glycosides (Nartey, 1980). Cyanide, released from a cyanogenic glycoside in food by
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ß-glucosidase either of plant or from gut microflora origin and when taken up,
follows the known cyanide metabolic pathway and toxicokinetics both for animals
and man. Cyanide is detoxified by the enzyme rhodanase, forming thiocyanate, which
is excreted by urine (Conn, 1979 a, b; Oke, 1979). Due to several factors influencing
hydrolysis of cyanogenic glycosides and the confounding influence of nutritional
status (such as riboflavin, vit. B12, sodium, methionine intake) human case studies
and epidemiological studies of the chronic toxicological effects have shown very
variable results and were not conclusive. In several studies both in animals and man
the toxicity of cyanogenic glycosides is often expressed as milligram releasable
cyanide (Conn, 1979 a, b).
Extraction of Essential Oil
Extraction refers to separating by physical or chemical means the desired
material from a plant with the aid of a solvent (Bossert, 1970). Extraction procedure
includes soxhlet extraction and column chromatography.
Soxhlet Extraction
Soxhlet extractor (Plate 2) is used to extract solutes from solids, using any
desired volatile solvents, which can be water-miscible or water-immiscible (Morrison
and Freiser, 1957). The solvent is vaporized and when it condenses, drops on the
solid substance contained in the extraction thimble and extracts the soluble
compounds. When the liquid level fills the body of the extractor, it automatically
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siphons into the flask. This process is continuously repeated as the solvent in the flask
is vaporized and condensed. Soxhlet extractors are standard equipment in laboratories
that analyze fats and oils in biologic samples. Separations can be achieved at low
temperatures in inert atmospheres on a micro or macro scale by a discontinuous or
continuous process (Lo et.al., 1983).
Plate 2. Soxhlet extractor
Chromatography
Chromatography is a physical method of separation in which the components
to be separated are distributed between two phases, one of which is stationary while
the other moves in a definite direction (IUPAC, 1993). Component molecules in a
sample mixture are transported by a mobile phase over a stationary phase. Different
components will have different affinities for the stationary phase with respect to the
mobile phase and will therefore move at different rates. Thin-layer chromatography is
a separation technique in which a stationary phase consisting of an appropriate
material is spread in a uniform thin layer and fixed on a support of glass, metal or
plastic. The separation is carried out by migration through the thin layer of solutes in
a solvent or suitable mixture of solvents.
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Plate 3. Chromatographic separation. A mixture of compounds is separated as it
flows down a column packed with stationary phase particles. Separation is
due to different degree of interaction between sample molecules and
stationary phase. (IUPAC, 1993)
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MATERIALS AND METHODS
The Plant Material
Collection of Plant Sample
Matured leaves of Euphorbia neriifolia Linn commonly known in ilocano
as Karimbuaya, were collected from Pangasinan, and Vigan, Ilocos Sur,
provinces north of Manila. The plant sample was identified at the Institute of
Biology Herbarium, at U.P. Diliman, Quezon City.
Plant Preparation
The collected mature leaves of Karimbuaya (Euphorbia neriifolia Linn.)
were washed thoroughly with tap water and air dried for 10 days. After which the
dried leaves were cut into small pieces then powdered prior to analysis. Plate 4
shows samples ready for the extraction process.
.
Plate 4. Cut and powdered Karimbuaya leaves
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Characterization of the Fresh Karimbuaya Leaf Extract
Physico-Chemical Properties
The fresh leaves of Karimbuaya leaves were homogenized using a blender
and the juice was extracted by the use of several layers of cheesecloth.
Color. The color of the extract was determined based on a color chart.
Taste. About 5 drops was used to taste the extract.
Odor. This process was done by smelling after the juices have been
extracted from the leaves.
Refractive
index. The amount of sugar on the sample was measured using
Atago refractometer.
Phytochemical screening
The fresh Karimbuaya leaf extract was screened for the presence of
phytochemicals using methods of Guevara (2005). The fresh extract was prepared
by liquefying the thoroughly washed and drained fresh mature leaves of
Karimbuaya (Euphorbia neriifolia Linn). Table 2 shows the different tests used
for the phytochemical screening. A confirmatory test (Table 3) for the presence of
secondary metabolites or phytochemicals using thin layer chromatography (TLC)
was also performed using the methods from Guevara (2005).
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Table 2. Phytochemical Analysis
TEST COMPOUND
TESTED
Culvenor-Fitzgerald, Dragendorff’s and Mayer
Alkaloid
Froth and Liebermann Burchard
Saponins
Keller-Kiliani and Kedde
Cardenolide & bufadienolide
Bate-Smith&Metcalf and Wilstatter
Flavonoids
Gelatin and Ferric chloride
Tannins
Modified Borntrager’s
Anthraquinones
Guignard
Cyanogenic glycoside
Table 3. Confirmatory Test for Phytochemicals
VISUALIZING AGENT
COMPOUND TESTED
Antimony (III) chloride
Flavonoids/Steriods
Potassium ferricyanide-ferric chloride
Phennol/tannin/flavonoid
Dragendorff’s reagent
Alkaloid
Methanolic KOH
Phenols/Anthrone
Vanillin-sulfuric acid
Essential Oils
α-Napthol-sulfuric acid
Sugars
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Toxicity Test
The toxicity of the Karimbuaya leaves was evaluated using the Brine
Shrimp Assay.
Hatching of Brine Shrimp Eggs
A hatching medium was prepared by dissolving 3.8 grams of rock salt per
100 ml distilled water. This solution has a 3.8% NaCl concentration. About 250
mg of brine shrimp egg and 80 ml hatching medium were placed in the hatching
dish. The hatching dish has two unequal compartment provided by a plastic
divider punched with several 2 mm holes. The brine shrimp eggs were sprinkled
into the larger compartment and then covered with cardboard to block the light.
The smaller compartment was illuminated for 48 hours to allow the eggs to hatch.
The nauplii were then harvested for the assay.
Preparation of Stock Solution
Fresh Karimbuaya leaves were washed thoroughly and oven dried at 60oC
for 24 hours. About 500 mg of the dried leaves were powdered using mortar and
pestle. The powdered sample was transferred to a 250 ml beaker and 50 ml of
artificial sea water (3.8% NaCl) was added. The extract was filtered using 4 layers
of cheesecloth following one hour of incubation. The filtrate collected is the stock
solution.
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Dilution of the Stock Solution
Five test tubes of 20 ml capacity were prepared and labeled (1to 5) for the
dilution of the stock solution. The dilution series was prepared as follows: To test
tube 1, 10 ml of the stock solution was placed while 9ml of artificial sea water
(3.8% NaCl) was placed to test tube # 2 to 5. The dilution was carried out by
transferring 1.0 ml stock solution from test tube #1 to test tube #2. From the mix
solution in test tube #2, 1.0 ml was transferred to test tube #3, 1.0ml from test
tube #3 was transferred to test tube #4 and from this, 1.0 ml was pipetted and
added to test tube #5. Table 4 presents the dilution ratio.
Table 4 Dilution of the stock solution used for the bioassay
TEST TUBE NO.
DILUTION RATIO
1 Stock
solution
2 1:
10
3 1:100
4 1:1,000
5 1:10,000
Bioassay Procedure
The diluted stock solutions in the 5 test tubes were transferred to 5
previously cleaned petri dishes and labeled similarly as the test tubes. Using a
Pasteur pipette, ten live brine shrimps (nauplii) were deposited in each Petri dish.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
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After 24 hours, the Petri dishes were examined for brine shrimp mortality with the
aid of a magnifying lens.
Data Analysis
The toxicity of the plant extract was determined based on the value
obtained for the LD50, which refers to the amount (lethal dose) of a substance that
kills half of the test organisms. The LD50 was determined from the equation of
the line obtained by plotting the percent mortality against the log extract
concentration.
Extraction of the Essential Oil
Soxhlet Extraction
Previously cut air dried Karimbuaya leaves were powdered using a
blender. Each 15 gram batch of the powdered leaves was wrapped in filter paper
and placed in the extracting thimble of a soxhlet extractor, where 250 mL of
petroleum ether was added as the extracting solvent. The extraction process for
each batch of leaves lasted for 5 hours, until the color of the solvent in the body of
the soxhlet extractor became colorless. The petroleum ether extract was
concentrated under reduced pressure at 40oC. The concentrated plant extract was
allowed to dry to determine the percentage yield.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
26
Chromatographic Separation
A weighed amount of the concentrated petroleum ether extract was
subjected to rapid column chromatography by vacuum elution, as in Plate 5. A
column of 4.5 cm diameter was packed with 7 cm high of Silica Gel 60 Gf
(Merck). Solvent used for elution was increasing percentage of hexane-ethyl
acetate mixture starting from pure hexane to 10% ethyl acetate.
The chromatographic separation of the ether extract was monitored
through thin layer chromatography using prepared TLC plates (Merck). The
chromatograms were developed using different solvent system based on the
concentration of the solvent used for elution. The spots were first developed using
iodine vapors followed by vanillin-sulfuric acid spray and was then heated in the
oven at 110oC for color development. Components of the same Rf value were
collected and grouped as one.
Plate 6 shows the over-all extraction process.
Purification of the Essential Oil Extract
Repeated column chromatography using different column sizes and
isocratic elution yielded the oil extract. This is illustrated in Plate 7. Isocratic
elution was carried out using pure hexane (A.R. grade). The fraction identified
by TLC to contain the essential oil was further separated using a smaller column
(2.0 cm in diameter). Solubility difference using pure ethyl acetate (A.R.) and
filtration with the use of Whatman 42 separated the white crystals from the oil.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
27
1
2
3
4
5
6
7
8
9
Plate 5. Column Chromatographic Separation of the Concentrated
Petroleum ether extract from Karimbuaya leaves
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
Dried Powderized Leaves
Ether extract
Soxhlet extraction
Fresh leaves of Karimbuaya
Concentrated under vacuo
(rotary evaporator)
Essential Oil
Fractions containing
Separation by Column Concentrated Petroleum
Essential Oil
Chromatography
Ether Extract
Plate 6. Schematic diagram of the extraction of Essential oil from the leaves of Karimbuaya.
29
Plate 7. Chromatographic Separation of Essential Oil from Karimbuaya leaves
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
30
Characterization of the Essential Oil Extract
Physico-chemical Test
Solubility test. Each two drops of extracted oil was mixed with 2 ml of the
different organic solvent in a test tube. Solubility of the oil extract was
determined in different organic solvent which included chloroform, ethanol, ethyl
acetate, hexane and petroleum ether.
Melting point determination. The melting point of the oil extract was
analyzed as follows: About 0.1ml of the viscous oil was placed in a 50 ml beaker
that was suspended in a water bath. This set-up is shown in Plate 8. The
temperature of the water bath starting from 10oC was gradually increased until the
oil melted.
Plate 8. Melting point set-up for the essential oil
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
31
The melting point of the white crystals separated from the extracted oil
was analyzed using the Thiele-Dennis tube. About 1 mm height of the white
crystals was placed inside the sealed capillary tube. A thermometer was attached
to the capillary tube containing the sample and was then placed inside the Thiele-
Dennis tube filled with glycerol. The tube was heated until the white crystals
melted. The set-up is presented in Plate 9.
Plate 9 A Thiele-Dennis tube for melting point determination of the
crystal (Visconde A.D. et al, 2003)
Acid Number. About 100 mg of oil was mixed with mixture containing
12.5 ml of ethanol (95%) and 12.5 ml ether. The mixture was titrated with 0.1M
KOH until a red color remains after swirling. Phenolphthalein indicator was used
to indicate the end point of the titration. Plate 10 presents the color of the sample
solution before and after titration.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
32
A. Before KOH Titration
B. After KOH titration
Plate 10. Acid number determination of the Essential Oil
Acceptability Test
A consumer type panel consisting of 48 members ranging in age from 20
to 60 years was asked to evaluate the acceptability of the aroma and taste of
lechon manok, grilled with Karimbuaya leaves. Common spices for flavoring
lechon sold in the market were the standard ingredients in the acceptability test
conducted.
Preparation of Sample.
Dressed chickens previously washed and drained were used for the three
treatments. The chickens were then stuffed with the respective treatment
ingredients as shown in Table 5. The stuffed chickens were grilled at the same
time.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
33
Table 5. Treatments used in the chicken for the acceptability test
Treatment Ingredients
T1 Salt
T2
Karimbuaya leaves, 5 pieces chopped
onion bulbs, salt and pepper
T3
Lemongrass, 5 pieces chopped onion
bulbs, salt and pepper
Taste Test
Before the taste test, a sensory evaluation form (Appendix B) was given to
each consumer to mark after tasting. The grilled chickens were cut into bite sizes
and given to the members of the panel. The consumers were asked to rinse their
mouth with water after tasting each of the treated samples.
Data Analysis
One way ANOVA test was used to determine if there was a significant
difference between the three treatments. Computer software SPSS was used in the
data analysis. This software computes using the P-value approach.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
34
RESULTS AND DISCUSSION
Characterization of the Fresh Karimbuaya Leaf Extract
Physico-chemical Properties
The liquid squeezed from the homogenized fresh leaves of Karimbuaya
was green in color, with a leafy like odor, and sweet with an after taste. The
percentage (%) mass sucrose content of the fresh extract was 4.2%. This was
obtained using a refractometer.
Phytochemical Screening
To identify the secondary metabolites present in the extract,
phytochemical analysis was done. The results summarized in Table 6, showed
that the fresh extract contains only the secondary metabolites, saponin and tannin.
Although a slight turbidity was observed in the Culvenor-Fiztgerald test for
alkaloid, this result is considered negative according to Guevara (2005). Presence
of alkaloid in the sample is indicated by a heavy precipitation evaluated as (+++).
The result of on the Laboratory test tube method, no formation of precipitates,
confirms that alkaloid is not present in the sample.
Plate 11 represents the above results.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
35
Table 6. Phytochemical Analyses of Fresh Karimbuaya Leaf Extract
TESTS CLASS
OF RESULTS
COMPOUNDS
Culvenor-Fitzgerald
Alkaloid
Dragendorff
(+) slight turbidity yellow
precipitate
Mayer
(+) slight turbidity yellow
precipitate
Laboratory test tube
Dragendorffs
(-) yellow liquid
Mayer’s
(-) colorless liquid
Froth
Saponins
(+) frothing
Liebermann-
(+) green liquid
Burchard
Guignard
Cyanogenic glycosides (-) yellow color
Keller-Kiliani
Cardenolides and
(-) orange ring at the
Bufadienolides
interface
Kedde
(-) orange brown liquid
Bate-Smith&Metcalf
Flavonoids
(-) orange brown liquid
Wilstatter
(-) light orange liquid
“Cyanidine”
Gelatin
Tannin
(+)jelly precipitate
Ferric chloride
(+) brownish green liquid
Modified Borntragers
Anthraquinones
(-) colorless in the
ammoniacal layer
To confirm the above results, phytochemical analyses using thin-layer
chromatography (TLC) was also performed. The tests confirmed the presence of
saponin and tannin. The absence of alkaloids and other secondary metabolites
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
36
A. Alkaloid Test (negative result)
Plate 11. Phytochemical Screening Result
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
37
Plate 11. Continued…
B. Saponin (positive result)
C. Cyanogenic glycosides
Plate 11. Continued…
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
38
D. Cardenolides and Bufadienolides (negative result)
Plate 11. Continued…
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
39
E. Flavonoids
Plate 11. Continued …
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
40
F. Tannins (positive result)
Plate 11. Continued….
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
41
G. Anthraquinone (negative result)
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
42
were also confirmed. The results are presented in Table 7. The same table also
shows that essential oil and sugar are present in the fresh extract. The violet spots
observed upon spraying the developed TLC plates with the vanillin-sulfuric acid
visualizing agent, indicate that the extract contains essential oil. Using the α-
Napthol-sulfuric spray reagent, a blue spot was observed which confirmed the
presence of sugar in the Karimbuaya leaves. Results collaborates the finding
using refractometer for the presence of sugar.
Table 7. Phytochemical Analyses using Thin-Layer Chromatography (TLC)
SPRAY REAGENTS
CONSTITUENT TESTED
RESULT
Antimony(III) chloride
Flavonoids/steroids
(-) no spot
Potassium ferricyanide-
Phenol/tannin/flavonoid
(-) yellow spot
ferric chloride
Dragendorff’s reagent
Alkaloid
(-) yellow spot
KOHmethanolic
Phenols/anthrone
(-) no spot
Vanillin-sulfuric acid
Essential oils
(+) wide range of color
α-Napthol-sulfuric acid
Sugars
(+) blue spot
Toxicity Test Using The Brine Shrimp Assay
Table 8 showed that the concentrated stock solution of Karimbuaya has an
average of 13.3 % mortality after 24 hours. The diluted concentration of the stock
solution showed 0% mortality on the nauplii.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
43
Table 8. Average % mortality after 24 hours
CONC. OF EXTRACT
AVERAGE % MORTALITY
(ppm)
(24 hours)
11363.63 13.3
1136.36 0
113.63 0
11.36 x 10-3
0
11.36 x 10-7
0
From the graph (Figure 3) the LD50 of the plant solution was computed to be
58.73 ppm. Activities are considered significant if the LD50 value is less than 30
ppm (Saupe, 2006). The result indicates that the Karimbuaya extract is non-toxic.
Brine Shrimp Assay
15
y = 0.8061x + 2.66
10
R2 = 0.303
% Mortality
5
0
-8
-6
-4
-2
0
2
4
6
-5
Log concentration
Figure 3. Determination of LD50
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
44
Extraction of Essential Oil
Crude Fat Extraction
The petroleum ether extract obtained from the soxhlet extraction using 100
grams of powdered dried Karimbuaya leaves was dried under vacuo at 40oC to
yield 5.1664 gram of crude fat or concentrated petroleum ether extract. The
recovered crude fat is solid and colored dark green. Plate 12 shows the sample of
the crude fat
Plate 12. Extracted crude Fat from
Karimbuaya
Separation of Essential Oil
The essential oil was obtained from the crude fat or petroleum ether
extract after repeated chromatographic processes guided by thin layer
chromatographic techniques. Isocratic elution using pure hexane was performed
to elute the essential oil. Table 9 shows the chromatographic characteristics of the
nine fractions obtained from the crude fat. These fractions are shown in Plate 13.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
45
Essential oils, because they are non-polar are the first to be eluted from the
column. Based on the TLC chromatogram, fractions I to III which contain the oil
were pooled and concentrated under vacuo at 40oC.
Table 9. Thin-Layer Chromatography of the Petroleum Ether Extract
FRACTION DEVELOPING
CHROMATOGRAPHIC
NUMBER
SOLVENT
CHARACTERISTIC
Rf values
Color of Spots
(Vanillin-Sulfuric Acid Spray)
I
100% Hexane
0.73
Violet
0.88
Violet
0.93
Violet
II
0.2% E-H
0.7
Violet
III
0.2% E-H
0.43
Violet
0.68
Violet
IV
0.4%E-H
0.13
Brown-violet
V
0.4%E-H
0.08
Brown-violet
VI
0.4%E-H
0.05
Brown-violet
VII
10%E-H
0.22
Brown
0.3
Brown violet
0.4
Violet
0.48
Violet
0.6
Violet
0.82
Violet
VIII
10%E-H
0.13
Green-violet
0.23
Violet
0.30
Violet
IX
15%E-H
0.07
Brown-violet
0.12
Brown-violet
* E-H Ethyl acetate in hexane
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
46
Plate 13. Eluates from the Column Chromatography from the
concentrated petroleum extract of the Karimbuaya leaves
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
47
The pooled Fractions (I-III) was again subjected to rapid column
chromatography to yield 9 fractions as shown in Figure 4. After several
extraction using chromatographic techniques, an orange-colored fat-like substance
was obtained. This is the essential oil.
The chromatogram (Plate 14) of the essential oil showed four spots,
indicating the presence of four components. The over-all extraction process for
the separation of the essential oil from the leaves of Karimbuaya is summarized in
Figure 4.
Purification of Essential Oil
The essential oil extracted showed presence of white crystals. The large
spot (Plate 14) located at the lower part of the chromatogram with Rf value of
0.20 corresponds to the white crystals.
Plate 14. Chromatogram of the essential oil using
Vanillin-sulfuric acid as visualizing agent
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
48
100 grams Dried Karimbuaya leaves
Petroleum ether
Petroleum Ether extract
(5.1664%)
Concentrate under vacuo
Column chromatography
I II III IV V IV VII VIII IX
Column chromatography
( hexane)
1 2 3 4 5 6 7 8 9
Column chromatography
(hexane)
A B C D
Column
chromatography
Essential Oil (0.30%)
white crystal (0.142%)
Figure 4. Schematic diagram of the Over- all Extraction of Essential oil
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
49
The presence of the crystals probably explains why the essential oil is solid at
room temperature. Applying selective solubility difference followed by filtration,
the white crystals were separated from the oil. This is shown in Plate 15. Pure
ethyl acetate solution was used to recover the oil from the mixture. Percent
recovery for the white crystals was 0.142%.
Plate 15. White Crystal Isolate from the leaves of Karimbuaya
Characterization of the Essential Oil
Physico-chemical Properties
Table 10 gave a summary of the physico-chemical properties of the
essential oil and the white crystals isolated from the leaves of Karimbuaya.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
50
Table 10. Physico-chemical Properties of the Essential Oil and Crystal
PARAMETERS
PHYSICO-CHEMICAL PROPERTIES
Essential Oil
Crystals
Color
Orange
White
Odor
Odorless
Odorless
Melting point
36oC – 40oC
142 oC
Solubility
Hexane
Chloroform
Ethyl acetate
Ethanol
Petroleum ether
Chloroform
Physical state
solid
solid
Acid value
2.9
Essential Oil. The essential oil extracted was odorless and orange in color.
This is shown in Plate 16. The melting point determined ranges from 36-40oC.
The wide range of melting point denotes that the oil has other components, which
is confirmed by the presence 4 spots observed in the chromatogram revealed in
Plate 14. The essential oil is soluble in all of the organic solvents used in the
solubility test. The oil has an acid value of 2.9. The acid value measures the
amount of the fatty acids hydrolyzed. The higher acid number indicates the
degree of degradation of the oil (Anon, 2006).
Crystals. The solid separated from the essential oil is white in color and is
odorless. It has a melting point of 142oC. The solubility of the crystal was tested
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
51
A
B
C
Plate 16. A. Essential oil extracted from Karimbuaya leaves
B. top view of the oil in the beaker
C. The essential oil in a Florence flask after concentrating in vacuo
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
52
in several organic reagents and is found soluble in chloroform. as shown in Table
11.
Table 11. – Solubility of the White Crystal Isolate
REAGENTS
RESULT REAGENTS RESULT
70% ethanol
Insoluble
Ethyl acetate
Insoluble
80% ethanol
Insoluble
Hexane
Insoluble
85% ethanol
Insoluble
Diethyl ether
Insoluble
90% ethanol
Insoluble
Chloroform
Soluble
Petroleum ether
Insoluble
2%EtOAc-Hexame
Insoluble
Acceptability of Karimbuaya Leaves
as a Flavor Enhancer
The sensory attributes of foods are of paramount importance as people
take pleasure from a combination of their visual, olfactory, taste and tactile
perception. Overall flavor requires not just the basic taste: sweet, salty, bitter, and
sour. According to Hegenbart(1996) the total flavor experience is a combination
of taste, aroma and the chemical feeling. Modifications on the flavors of food are
achieved by addition of flavor enhancers.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
53
The acceptability of fresh Karimbuaya leaves as a flavor enhancer in
lechon preparation was compared to the common commercial ingredients. The
aroma, taste and overall acceptability were evaluated.
Table 12. Multiple Comparison on Aroma
SAMPLE MEAN
SIG.
DIFFERENCE
Karimbuaya mixture vs. salt only
0.38
0.202
Karimbuaya mixture vs. Lemongrass mixture
0.52*
0.046
Salt only vs. Lemongrass mixture
0.15
0.784
* significant at 0.05
Aroma
Table 12 shows that the mean difference of 0.38 between Karimbuaya
mixture and salt only is not significant at the 0.05 level. This indicates that there
is no difference on the aroma of the grilled chicken stuffed with Karimbuaya
mixture and salt only, respectively. On the other hand, between Karimbuaya
mixture and lemongrass mixture, the mean difference (0.52) is significant. This
implies that the aroma of grilled chicken as enhanced by Karimbuaya leaves is
preferred by the consumer panel. Between the two standard ingredients, salt only
and lemongrass mixture, the mean difference is not significant. This shows that
the aroma of grilled chicken stuffed with lemongrass is the same as that of the
salt.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
54
Table 13. Multiple Comparison on Taste
SAMPLE MEAN
SIG.
DIFFERENCE
Karimbuaya mixture vs. salt only
0.15
0..820
Karimbuaya mixture vs. Lemongrass mixture
0.96*
0.000
Salt only vs. Lemongrass mixture
0.81*
0.002
* significant at 0.50
Taste
Comparing the taste of the grilled chicken treated with Karimbuaya
mixture to the one treated with salt only, the mean difference (0.15) obtained is
not significant. The result implies that the taste is the same. Karimbuaya mixture
when compared with lemongrass mixture, the mean difference is significant. This
means the consumers prefer the taste of chicken flavored with Karimbuaya
mixture than the lemongrass mixture. The standard ingredients used in the
analysis, salt only and lemongrass mixture, revealed a significant difference on
the taste of the grilled chicken.
Overall Acceptability
The overall acceptability shown in Table 14 reveals a significant
difference only between Karimbuaya mixture and lemongrass mixture. This
implies that the Karimbuaya mixture as flavor enhancer on grilled chicken is
accepted by consumers.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
55
Table 14. Multiple Comparisons on Overall acceptability
SAMPLE MEAN
SIG.
DIFFERENCE
Karimbuaya mixture vs. salt only
0.15
0..820
Karimbuaya mixture vs. Lemongrass mixture
0.96*
0.000
Salt only vs. Lemongrass mixture
0.81*
0.002
* significant at 0.05
Table 15. Acceptability test of Karimbuaya mixture
PARAMETERS Fcomputed
SIG.
Aroma
2.996*
0.05
Taste
9.020*
0.00
Overall acceptability
3.486*
0.03
* significant at 0.05
In table 15, statistical analysis using the F test (one-way ANOVA) showed
that the computed F values for the aroma, taste and overall acceptability were
higher than the F value (2.66) at the 0.05 level of significance. The results imply
that addition of chopped Karimbuaya leaves to the usual spices for lechon
preparation, is favored by the consumer panel. This denotes that the grilled meat
with Karimbuaya leaves produces an aroma and taste that is much preferred than
lemongrass and the salt.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
56
SUMMARY, CONCLUSION and RECOMMENDATION
Summary
This study aims to characterize the extracts (fresh leaf and the essential
oil) from the leaves of Karimbuaya (Euphorbia neriifolia Linn). The toxicity and
acceptability as a flavor enhancer of the fresh leaves of Karimbuaya was also
determined.
The characterization of the fresh Karimbuaya leaf extract was found to be
green with leafy like odor and sweet with an after taste. The sweetness was
confirmed with the presence of 4.2% mass sucrose using a refractometer. Results
of the phytochemical analysis of the leaf extract showed the presence of saponin
and tannin. The same metabolites were confirmed present in the sample from the
results of the thin-layer chromatography (TLC). The presence of the essential oil
in the sample was also revealed in the chromatogram which was developed using
a vanillin-sulfuric spray reagent.
The LD50 equivalent to 58.73 ppm obtained from the brine shrimp assay
indicates that the Karimbuaya leaves are non-toxic. This indicates that the
concentration of the saponin and tannin present is below lethal dose (30 ppm).
The percentage yield of the recovered essential oil of the Karimbuaya
leaves obtained from repeated rapid column chromatography was 0.30%. The
chromatogram of the orange colored essential oil indicates the presence of four
(4) components with Rf values 0.20, 0.52, 0.8, and 0.95. The essential oil
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
57
extracted is solid at room temperature with a melting point range of 36o – 40oC
and is soluble to all the organic solvents (ethanol, ethyl acetate, hexane, petroleum
ether, chloroform) used in the test.
The percentage yield of the white crystal isolate was 0.142%. The
chromatogram shows the crystals to have an Rf value of 0.20. It has high
chloroform affinity and a melting point of 142oC.
As a flavor enhancer, the fresh Karimbuaya leaves were compared with
the standard ingredients, salt and lemongrass. Results indicated that the
Karimbuaya leaves were preferred by the consumer panel as a flavor enhancer on
grilled chicken.
Conclusion
Based on the results, the following conclusions were drawn:
1.
The Karimbuaya leaf extract is a green solution with leafy like odor,
sweet with an after taste and is non-toxic as indicated by the
LD50 value of 58.73 ppm;
2.
The orange colored essential oil extracted from the leaves of
Karimbuaya was fatty like with a wide melting point range of
36-40oC. The chromatogram of the oil shows 4 spots indicating
that the oil is a mixture. One component is a white crystalline
mixture with a melting point of 142oC;
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
58
3.
The Karimbuaya leaves as a flavor enhancer for grilled chicken was
preferred over the standard ingredients, salt and lemongrass.
Recommendation
The use of fresh Karimbuaya (Euphorbia neriifolia Linn.) leaves as a
flavor enhancer on grilled chicken is recommended.
Further study should be done to determine the following:
1.
effect of the fresh Karimbuaya leaves as a flavor enhancer on
other meat and fish products;
2.
the constituents responsible for the other properties of the plant
not included in the study;
3.
isolate and identify other constituents of the essential oil;
4.
and to analyze the bioactivity of the plant.
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
59
LITERATURE CITED
ANON. 2006. Chemistry and Extraction of essential oils, 2006 accessed on June
19, 2006 at WWW.hort.purdue.edu/newcrop/proceedings1990/VI-472
ANON. 2006. Monosodium glutamate accessed on June 2, 2006 at
http://en.wikipedia.org
ANON. 2006. Soro-Soro(Euphorbia neriifolia Linn.) accessed on June 3, 2006 at
http://stuartxchange.com
ANON. 2006. Soro-soro accessed on June 3, 2006 at
www.bpi.da.gov.ph/Publications/mp/pdf/soro-soro.pdf
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Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
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63
Appendix A-1
Analysis of Variance (ANOVA) for AROMA
Sum
of
df Mean F Sig
Squares
Square
Between Groups
6.931
2
3.465
2.996 .05*
Within Groups
163.063
141
1.156
Total
169.993
143
Multiple Comparison
Dependent Variable : AROMA
Tukey HSD
Mean
Difference
Std. Error
(I) TREAT
(J) TREAT
(I-J)
Sig.
1
2
.38
.22
.202
3
.15
.22
.784
.
2
1
38
.22
.202
3
.52*
.22
.046
3
1
.15
.22
.784
2
.52*
.22
.046
* mean difference is significant at 0.5
Extraction and Characterization of the Essential Oil from the Leaves of Karimbuaya
(Euphorbia neriifolia Linn) / Maria Theresa C. Macaraeg – Diza. 2006
Document Outline
- Extraction andCharacterization of the Essential Oil from the Leaves of Karimbuaya (Euphorbianeriifolia Linn)
- BIBLIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- Background of the Study
- Importance of the Study.
- REVIEW OF LITERATURE
- Karimbuaya (Euphorbia neriifolia Linn.)
- Flavor Enhancers
- Toxicity Test
- Phytochemical Screening
- Extraction of Essential Oil
- MATERIALS AND METHODS
- RESULTS AND DISCUSSION
- Characterization of the Fresh Karimbuaya Leaf Extract
- Toxicity Test Using The Brine Shrimp Assay
- Extraction of Essential Oil
- Charactererization of the Essential Oil
- Acceptability of Karimbuaya Leavesas a Flavor Enhancer
- SUMMARY, CONCLUSION and RECOMMENDATION
- Summary
- Conclusion
- Recommendation
- LITERATURE CITED
- Appendix