Far Eastern University
Institute of Arts and sciences
Department of Medical Technology
A.Y. 2013-2014
An Investigation on the Possible Utilization of Red Onion Extract as Blood Detector
MT1041
Astillo, Rio Roane
Keh, Kent Edbert
Ladera, Graciella
Perez, Patria Niňa
Paz, Gerald John
Sioson, Berona Faith
Chapter I
The Problem and Its Background
Introduction
Crime is still very rampant despite the laws governing such cases. It is true not
only in the Philippines but in the whole world as well. Different crimes happen every day
which include but not limited to rape, murder, robbery, massacre, hostage and
homicide. Such crimes were not new which in fact, has been done a lot of written
literature from Sir Arthur Conan Doyle (22 May 1859 – 7 July 1930) up to the present
time and other authors in crimes. The works of those writers involved solving mystery
crimes through careful observation of the crime scene, reenactment of possible
happening and analysis of fragments and evidences found in the crime scene. The
analysis part has become evidently useful in most cases which are now termed as
forensics. Forensic investigations refer to the use of science and technology in the
investigation and establishment of facts or evidence to be used in criminal justice or
other proceedings (utb.edu). In case of rape, forensic experts analyze the presence of
sperm in a vaginal wash specimen through a panel of chemical laboratory tests. Aside
from that, crimes involving killings use various rapid tests for blood detection including
Kastle-Meyer test and Guaiac test. It takes just a swab on the surface suspected with
presence of blood and the swab will instantly change color if blood is positive
(chemistry.about.com).
Tests for presumptive blood used by the forensic scientists give clue and an
initial, on-site detection or confirmation of blood component. If it is confirmed blood,
there are also some tests that serologists do to further evaluate; for instance the origin
of the blood (Saferstein, 2011). These tests for blood has been modified and improved
so that it can also be utilized for various clinical laboratory tests. Laboratory analysis of
urine and feces includes detection for the presence of blood. Using reagents for blood
detection is a great help to easily know if blood is present in the specimen. Fecal blood
contamination often use guaiac test to detect blood and may pathologically indicate
bleeding in the gastrointestinal tract (Strasinger and Di Lorenzo, 2008). The basic
principle is that the hemoglobin part of the red blood cell has pseudoperoxidase reaction
to Guaiac test reagent that produces blue color. Urine tests use hydrogen peroxide
1
(H2O2) and an addition of chromogen to produce a distinct color (Strasinger and Di
Lorenzo, 2008). In forensics, Kastle-Meyer test is presumptive of blood which uses
phenolphthalein as an indicator (chemistry.about.com). However, Kastle-Meyer test is
not readily available because phenolphthalein which is its indicator is hard to find.
Nevertheless, the substance anthocyanin which is almost structurally similar in
phenolphthalein is also seen in many other plants.
The Philippines is blessed with the availability of such plants that can provide
alternatives to products that are not readily available at use. This can be attributed to it
being a tropical country. Its nature is enriched with many different natural resources that
can be made as food, shelter, clothes, medicines and even fuels. Food is an essential
factor that humans need in order to survive the everyday life. Vegetables like cabbage,
carrot, squash, potato, cucumber and radish are just the common products sold in the
market since these are included in the primary needs of human. Spices like ginger,
pepper, chili, garlic and onion are also commonly sold in which primary ingredient to
make meals. With the abundant production of these ingredients for the people’s food
annually, huge amount of their waste products are thrown away as well.
Analysis of the different components of plant parts were done in order to utilize
as an alternative for many medicines, fuels, reagents use in the laboratory or anything
that is useful to man. Chemicals that occur naturally in plants like phenolic compounds
include but not limited to lycopene, saponins, tannins, anthocyanin, flavonoids and
others are the usually component used in researches. These components are
commonly seen in the of leaves, fruits, trunks, peels, roots of plants like guava, tomato,
garlic, onions, atis and cabbage (Harborne, 2000). However utilization of peels or even
the outer layers of the flesh of the fruit (and vegetables) is rare. Red Onion is one of the
major ingredients in every meal for most Filipinos. In the study conducted by Lanzotti,
2006, onion is characterized by polar compounds of phenolic and steroidal origin, often
glycosylated, showing interesting pharmacological properties. The flavonoids in onion
tend to be more concentrated in the outer layers of the flesh. The quercetin and
anthocyanin content of onions will be lost if it were "over-peeled" (whfoods.com).
Anthocyanin, which is comparable to phenolphthalein when used as pH indicator, is
2
responsible for the various colors of plant leaves, fruits and flowers. Red onion (Allium
cepa) has that substance in its outer layers of the flesh (National Onion Association).
Despite the inexpensiveness of the Guaiac and Kastle- Meyer solution, the
researchers have tried to devise a substance derived from plants to be used as an
indicator of blood. The outer layer of the red onion will be of used in the experiment to
test for the presence of blood in different surfaces. The central core of this study is to
determine whether red onion (bulb), Allium cepa can be utilized as an indicator for
blood.
Background of the Study
Allium cepa, commonly termed as onion; in Filipino terms—Sibuyas; in Bisayan
—Cebuyas Bombay and; in Ilocano—Lasona. It belongs to the family Mililiaceae and
has been widely use for culinary and medicinal purposes. Onion bulbs exhibit a lot of
medicinal uses as stated in CRC Handbook of Medicinal Spices (Duke, 2003). Onion
bulbs itself contains anthocyanins, organosulfur compounds and quercetin. Onion bulbs
are also said to be aphrodisiac, diuretic, expectorant, emmenagogue, hypoglycemic,
and stimulant. The scales outside the onion bulb are one of the richer sources of
quercetin. This flavonoid is said to be an antioxidant, deactivating molecules that are
injurious to cells in the body (National Onion Association, n.d). In addition to the
flavonoid quercetin onions also have anthocyanin that is mainly responsible for the color
of the bulb (Duke, 2003). Furthermore, the dried outer skin of the bulb reduces a
bacteriocide and an excellent yellow dye (Asis, 2001). The qualitative anthocyanin
content of red onion includes a wide structural assortment including several unique
flavonoid structures. Agric, 2007 first mentioned red onion as a rich source of
anthocyanin.
There are several sections of medical laboratory science apart from hematology
wherein blood detection is done. It is widely done in the urine and fecal analysis.
Forensics also utilized blood detection schemes somewhat parallel to what is done in a
medical laboratory.
In the routine urinalysis section of the laboratory, reagent strip is utilized for the
chemical screening of the patient urine. The reagent strip has chemically impregnated
3
absorbent pads attached to a plastic strip. These pads are color producing when the
reaction takes place upon contact in the urine (Strasinger, 2008).
Blood in the urine is detected via reagent strip wherein heme compound reacts
on the reagent strip. This method is based on the liberation of oxygen from peroxide in
the reagent strip by the peroxidase-like activity of heme in free hemoglobin, lysed
erythrocytes, or myoglobin. Intact erythrocytes are lysed on the strip, causing the
hemoglobin to react. Further, Heme catalyzes the oxidation of tetramethylbenzidine to
produce a green color. The strip is read at 60 seconds following sample application
(McPherson and Pincus, 2011).
Most tests for occult blood in feces use gum guaiac, a phenolic compound that
produces a blue color when oxidized. The peroxidase-like activity of hemoglobin
molecule, as in the reagent strip principle, results in the liberation of oxygen from
hydrogen peroxide and the gum guaiac is oxidized by the oxygen released from the
reaction producing blue oxidation product. Various interfering substances may give
false positive results. These are mostly enzyme peroxidase from hemoglobin and
myoglobin found in red meat, vegetables like horseradish, turnips and brocoli, and fruits
like bananas, black grapes, pears, plums and melons. In addition, WBCs and bateria
also have peroxidase activity (Turgeon, 2008).
However, blood presumptive test can rule out the possibility if the fluid studied is
blood. These tests relies on the use of chemicals that will change color when in the
presence of blood like phenolphthalein which turn from colorless to pink when added to
surfaces suspected of blood. This is the Kastle-Meyer test and this color test is more
commonly used in forensics (Lerner and Lerner, 2006). A guaiac test kit uses hydrogen
peroxide and guaiac cards. The guaiac cards contain phenolic compounds. Despite the
guaiac test and Kastle-Meyer test which are the affordable and can be a self-service
test for detection of blood, the researchers come up with an idea to make a blood
detector from natural resources which can be as efficient as the first two mentioned
blood detector agents. An article published in American Journal of Pathology published
an article regarding the efficacy of phenolphthalein as blood detector compared to other
three substances—Benzidine, guaiac and o-toluidine. The phenolphthalein (Kastle-
Meyer) test, first described in 1903, if properly carried out is much more reliable and
4
specific for hemoglobin than the three tests in general use. In the chemical laboratories
of Bellevue Hospital have made a comparative study of all four tests for several years
and are convinced that the phenolphthalin test is the most specific for hemoglobin
(Gettler and Kaye, nd). Phenolphthalein compound which is hard to find must have an
alternative in order that the presumptive blood test would not be delayed or prolonged,
so the researchers come up with an idea to substitute anthocyanin to phenolphthalein
wherein they have almost similar molecular structure and pH indicator property.
According to the Electronic Journal of Environmental, Agriculture and Food Chemistry,
2010, anthocyanin changes color when the ph is 2-9 and the color will be from dark pink
to mehdi green. Phenolphthalein is an organic compound (C20H14O4) used as an acid-
base indicator. The compound is colorless in acidic solution and pinkish in basic
solution (with the transition occurring around pH 9) (http://digipac.ca, retrieved 2013). It
is widely used as pH indicator.
Although guaiac test is interfered by many factors still this is widely used today in
the laboratory for its low cost and sensitivity. So for many Filipinos relying on this test
they could, upon establishing, perform this at home through the use of anthocyanins of
the red onion extract from the outer layers of its flesh. This is more helpful for people
with low income for this will lessen their expenses. Conversely, the principle and the
significant basis for the use of red onion extract of anthocyanins on red onion’s outer
layers of the flesh should be established. However, the principle that the researchers
found out about this study is that, in an alkaline solution, H+ ions from the anthocyanin
are removed by excess hydroxide ions. This allows electrons in the anthocyanin to
spread out in oxygen’s p-orbitals, causing a hypsochromic shift, but also leaving a
bonding site open. Metal ions such as Mg2+ , Fe2+ , Fe3+, Ca2+,and Al3+ are known
to bond with anthocyanin compounds, and the addition of these metal ions could cause
a change in color as well. Some of these metals will chelate with multiple anthocyanins,
which can produce a very different color than is typically exhibited by the metal ion, or
the anthocyanin itself. This also emphasizes the idea that chelation requires a pH above
the pKa of the phenolic group, because the H+ ions need to be removed for the metal
ion(s) to have an open bonding site. Because the acidified anthocyanins are generally
5
accepted as red in color, deprotonated anthocyanins must be present either alone, or
chelated with certain metal ions to change the color (math.ufl.edu, retrieved 2010).
This research aims to investigate on the possible utilization of red onion extract
as blood detector.
Statement of the Problem
This research aims to investigate on the possible utilization of red onion extract as
blood detector. It aims to answer the following questions:
1. What is the anthocyanin extraction procedure of red onions that is suitable for
presumptive blood detection?
2. Will there be a color produced by the red onion extract against blood?
3. What is the color produced by the red onion extract against the following :
a. Stained fresh blood on:
a.1 wood
a.1.1 wiped (dried) blood
a.1.2 blood droplets
a.2 white fabric
a.2.1 wiped (dried) blood
a.2.1 blood droplets
a.3 metal (knife)
a.3.1 wiped (dried) blood
a.3.2 blood droplets
a.4 concrete (floor)
a.4.1 wiped (dried) blood
a.4.2 blood droplets
a.5 filter paper
a.5.1 wiped (dried) blood
a.5.2 blood droplets
b. Stained diluted blood on:
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b.1 wood
b.1.1 wiped (dried) blood
b.1.2 blood droplets
b.2 white fabric
b.2.1 wiped (dried) blood
b.2.1 blood droplets
b.3 metal (knife)
b.3.1 wiped (dried) blood
b.3.2 blood droplets
b.4 concrete (floor)
b.4.1 wiped (dried) blood
b.4.2 blood droplets
b.5 filter paper
b.5.1 wiped (dried) blood
b.5.2 blood droplets
c. Red Liquid on:
c.1 wood
c.2 white fabric
c.3 metal (knife)
c.4 concrete (floor)
c.5 filter paper
Hypothesis
There is a color reaction produced by the red onion extract when stained on
blood samples.
Objectives
This research aims to
1. Determine the anthocyanin extraction procedure of red onions that is suitable for
presumptive blood detection.
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2. Determine if there will be a color produced by the red onion extract against blood
3. Determine the color produced by the red onion extract against the following :
a. Stained fresh blood on:
a.1 wood
a.1.1 wiped (dried) blood
a.1.2 blood droplets
a.2 white fabric
a.2.1 wiped (dried) blood
a.2.1 blood droplets
a.3 metal (knife)
a.3.1 wiped (dried) blood
a.3.2 blood droplets
a.4 concrete (floor)
a.4.1 wiped (dried) blood
a.4.2 blood droplets
a.5 filter paper
a.5.1 wiped (dried) blood
a.5.2 blood droplets
b. Stained diluted blood on:
b.1 wood
b.1.1 wiped (dried) blood
b.1.2 blood droplets
b.2 white fabric
b.2.1 wiped (dried) blood
b.2.1 blood droplets
b.3 metal (knife)
b.3.1 wiped (dried) blood
b.3.2 blood droplets
b.4 concrete (floor)
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b.4.1 wiped (dried) blood
b.4.2 blood droplets
b.5 filter paper
b.5.1 wiped (dried) blood
b.5.2 blood droplets
c. Red Liquid on:
c.1 wood
c.2 white fabric
c.3 metal (knife)
c.4 concrete (floor)
c.5 filter paper
Significance of the Study
The results of this research will benefit many sectors including educational, health
and government institutions. The research will provide information on the results of the
investigation on the possible utilization of red onion extract as blood detector. This will
thereby guide them in utilizing red onion outer flesh. Among the people who will benefit
are the following:
The people in red onion farming. The result of this study will boost their market
income as their product is given another means to be used—particularly in making it a
reagent that will detect blood.
The clinical technicians. To widen their perceptive on the other benefits one can get
from red onions in the clinical settings. Likewise, the results of this study may give them
insights about the lower cost of this reagent to probably use in presumptive blood
detection. In addition, it may encourage them to further explore the other application of
the red onion outer layer extract in the medicinal field.
Crime laboratory scientists. This study can contribute to the methods of presumptive
blood detection in crime scenes. The effectiveness of the red onion extract as blood
detector can provide substitute to the traditional reagent that is phenolphthalein and
guaiac’s reagent in a crime scene investigation.
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The community. Onion peelings and outer flesh turns to be a waste product when
used in cooking. Upon proving the efficacy of the red onion extract as blood detector,
people can apply presumptive blood detection on their households and minimize the
waste products from red onions.
And finally to the future researchers. The results of this study will provide them ideas
to continue and further explore different aspects of the research and modify the
methods used to create more convenient and low cost processes. Furthermore this can
serve as their reference in finding other possible blood detector agents.
Delimitations of the Study
This study is focused on the investigation on the possible utilization of red onion
extract as blood detector. Furthermore, the molecular components of the onion bulb are
not covered throughout the study.
Red onion extract will be utilized as only presumptive blood detector in possible
crime scene surfaces just like the Kastle-Meyer and Guaiac’s reagent. It is not intended
to be used in more specific circumstances like animal blood detection, DNA typing, etc.
Red onion outer layers and no other parts of the onion bulb will be used in this
study. It will be collected at Central Market, Sampaloc, Manila. Only the fresh, matured
red onion bulb outer layer will be subjected to extraction. The methods will include only
ethanolic, methanolic and aqueous extraction and will utilize various apparatuses for the
separation of the solvents. These apparatuses are the soxhlet, magnetic stirrer or
hotplate and rotary evaporator.
Blood samples for detection in surfaces will be obtained from selected students
of Medical Technology of Far Eastern University, Manila. Other materials to be used in
this study as enumerated in the statement of the problem number 3 will be acquired
from places in convenience to the researchers. .
The efficacy of the blood detection of the red onion peel and outer layer extract
will be compared only against Guaiac test and not in the Kastle-Meyer test because
both tests utilizes the same principle and it might cause insignificant redundancy.
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Chapter II
Theoretical Background
Review of Related Literature
This chapter provides the related literatures about the problem presented which
serves as its basis for its underlying principles on the study.
Red Onions
Books
According to Duke, 2003, Allium cepa, commonly termed as onion; in Filipino
terms—Sibuyas; in Bisayan—Cebuyas Bombay and; in Ilocano—Lasona. It belongs to
the family Mililiaceae and has been widely use for culinary and medicinal purposes. The
dried outer skin of the bulb reduces a bactericide and an excellent yellow dye. The
scales outside the onion bulb are one of the richer sources of quercetin, a very useful
phytochemical also shared with evening primrose. Scales contain a heart stimulant that
increases pulse volume, affects the uterus, promotes bile production, and reduces blood
sugar. Onion bulbs are said to be aphrodisiac, diuretic, expectorant, emmenagogue,
hypoglycemic, and stimulant. Onion juice demonstrated anti-aggregator and
hypocholesterolemic activities in humans’ subjects. Onions are alleged to stimulate bile
production, to speed healing of gunshot wounds, and to cure scorpion bites, freckles,
and the common cold.
Harborne, 2000 added that chemicals that occur naturally in plants like phenolic
compounds include but not limited to lycopene, saponins, tannins, anthocyanin,
flavonoids and others are the usually component used in researches. These
components are commonly seen in the of leaves, fruits, trunks, peels, roots of plants
like guava, tomato, garlic, onions, atis and cabbage.
Libster, 2000, stated the various uses of onion in different parts of the world. In
Asian, Indians eat raw onions, spiced up with lemon, pepper, and salt, for bronchitis,
colic, edema, fever, and scurvy. Some people with parasites macerate an onion in white
wine and drink it on an empty stomach in the morning. Or pediatric patients drink water
in which onion has stepped overnight to kill parasites. Cooked onions are consumed by
11
Japanese macrobioticists to calm the nervous system and alleviate irritability and sore
muscles after heavy labor. A cut raw onion is placed under the pillow to aid insomnia.
Reputed to be hypotensive, onions have recently been shown to contain the
antihypertensive agent prostaglandin A1, but only at ca. 1 ppm. Juice of the bulb is used
for coughs and earache. Macerated in gin the bulbs are used for dropsy and gravel. In
India, onions are believed to be aphrodisiac, especially if retained in a cow dung year in
a well-stoppered pot for four months. Even wilder, in an Indian formula for acute
dysentery, one buries a grain of opium in an onion bulb and then roasts the onion. Most
of the real and folk medicinal attributes of onion are shared with garlic and other lesser
known members of the genus Allium. Garlic is popular with organic gardeners and
naturopaths for its biological activities. For millennia, onions have been famous for food,
condiments, and medicine. Green onions are eaten raw with meats, fish, cheese, or as
a vegetable, or chopped and added to cottage cheese, or cooked. Onions are eaten
raw, boiled, baked, creamed, broiled, fried, French-fried, roasted, or pickled, and in
soups, stews, dressings, or salads, but perhaps more importantly, added to other
ingredients for innumerable dishes. Dry onions may be served as a vegetable dish or to
flavor meat, fish, and poultry dishes and are also used in salt substitutes such as Spike,
Mrs. Dash and Vegit. A thick layer of cooked onion is used on the French dish
pissaladiere, sometimes called “Provencal pizza”. Onions are used in the Catalan
sauces sofregit and samfaina. In Tunisia, a fermented onion paste called “hrous” is
used to flavor couscous, soups, and stews. The papery outer skins, called “shuski” in
slavic Macedonia, are used as a dye for coloring Easter eggs, and in Egypt they are
used to color and flavor eggs called “hamine”. The leaves of some cultivars are widely
used as scallions. In Catalonia, the large shoots called “calcots” or “sprunzale”,
sprouted from bulbs planted in trenches, are blanched and eaten raw with bread, grilled,
or used for flavoring beans and sauces. Sprouted seeds used in salads and on
sandwiches.
Onions are very useful, Willey and Sons, 2002 give emphasis on the two
flavonoid subgroups are found in the onion, the anthocyanins, which impart a red/
purple color to some varieties and flavonols such as quercitin and its derivatives
responsible for the yellow and brown skins of many varieties.
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Journals
According to Asis, 2001, onion is biennial plant which may persist vegetative as a
perennial by means of bulbs. The root system is relative shallow and fibrous. The
underground stem is short and subconal; from it hallow cylindrical leaves are diverged
in one-half phyllotaxy (leaf arrangement). The sheathing base of each leaf completely
encircles the short stem, and it is this development of the fleshy leaf bases, together
with the absence of intermodal elongation, that results in the formation of the
commercial bulb. Onion is grown locally on a limited scale and gives good returns
although much labor is required in the cultivation. Crops produced in the Philippines are
relatively poor in quality and rot more quickly and storage. This may be related to the
short natural light period in the tropics during the time of bulbing. The water requirement
increases during the bulbing time. The bulbs need about two months storage at 4.4°-
10°C before planting. Red Bermuda or sibuyas Bombay is better adapted to our
conditions than the granex or excel variety but is off poorer keeping quality and more
pungent. Commercial production of onions is restricted to definite seasons. The cooler
months (Nov.-Jan.) are the planting months; the harvest takes place during the dry and
warm months (March- May). Furthermore, on the study conducted by Lanzotti, 2006,
onion is characterized by polar compounds of phenolic and steroidal origin, often
glycosilated, showing interesting pharmacological properties. The flavonoids in onion
tend to be more concentrated in the outer layers of the flesh.
Anthocyanin, which is comparable to phenolphthalein when used as pH indicator,
is responsible for the various colors of plant leaves, fruits and flowers. Red onion (Allium
cepa) has that substance in its outer layers of the flesh. Onion bulbs itself contains
anthocyanins, organosulfur compounds and quercetin. Onion bulbs are also said to be
aphrodisiac, diuretic, expectorant, emmenagogue, hypoglycemic, and stimulant. The
scales outside the onion bulb are one of the richer sources of quercetin. This flavonoid
is said to be an antioxidant, deactivating molecules that are injurious to cells in the body
(National Onion Association).
Agric, 2007, mentioned that the qualitative anthocyanin content of red onion
cultivars includes a wide structural assortment including several unique flavonoid
structure. Red onion is a rich source of anthocyanin. The index onion cultivars
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according to their content of flavonoids measured as quercetin. The only compound
belonging to flavonols, anthocyanins, and the dihydroflavonols have been report to
occur in onion bulb. Red onion contains 415-1917 mg of flavonols per kilogram of fresh
weight. Flavonols are the predominant pigments of onion. The anthocyanin of red onion
is mainly cyaniding glycosides acylated with malonic acid or non-acylated. The
quantities content of anthocyanin in some red onion cultivars has been reported to be
approximately 10% of the total flavonoid content or 39-240 mg.
In the article entitled, “Concentrations of Anthocyanins in Common Foods in the
United States and Estimation of Normal Consumption” (Wu. et al, 2006) various fruits
and vegetables were screened for their anthocyanin content. There are a wide range of
samples tested and yielded varying total anthocyanin per 100 grams (mg/100g). In the
case of red onion, the anthocyanin content present is 38.8 mg per 100 grams. The
quantification of anthocyanin is mainly achieved either by spectrophotometry or by
HPLC.
Moreover in the article entitled, “Onions: A Source of Unique Dietary Flavonoids”
(Slimestad. Et al, 2007), a review on the qualitative and quantitative information in red
and yellow onions was conducted. It stated that onion in general is one good source of
dietary flavonoids. This includes flavonols, anthocyanins, and dihydroflavonols. The
quantitative content of anthocyanins in some red onion cultivars has been reported to
be approximately 10% of the total flavonoid content or 39-240mg per kg of fresh weight.
Internet
Onion is an olfactory indicator. The onion odor isn't detectable in strongly basic
solutions. Red onion can act as a visual indicator at the same time. It changes from pale
red in acid solution to green in basic solution (antoine.frostburg.edu retrieved: 2/10/13).
On the other hand, World Health Foods states that onions are members of the
Allium family that are rich in sulfur-containing compounds that are responsible for their
pungent odor and for many of their health-promoting effects. Onions are an outstanding
source of polyphenols, including the flavonoid polyphenols which makes it a standout
source of quercetin. The flavonoids in onion tend to be more concentrated in the outer
layers of the flesh. In animal studies, there is evidence that onion’s sulfur compound
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may work in an anti-clotting capacity and help prevent the unwanted clumping together
of blood platelet cells. It can also lower blood levels of cholesterol and triglycerides and
improve cell membrane function in red blood cells. In human studies, onion provides
protection for the heart and blood vessels when consumed in a diet that is rich in other
vegetables and fruits – especially flavonoid-containing vegetables and fruits. It can also
increase our bone density and provide direct benefits to our connective tissue due to its
high sulfur content (whfoods.com retrieved Jan.2013).
Anthocyanins
Books
Harborne, 2000, mentioned that lycopene, saponins, tannins, anthocyanin, and
flavonoids are the usual components commonly seen in the of leaves, fruits, trunks,
peels, roots of plants like guava, tomato, garlic, onions, atis and cabbage. Anthocyanins
are all based chemically on a single aromatic structure, that of cyaniding, and all are
derived from this pigment by addition or subtraction of hydroxyl groups or by
methylation or by glycosylation. Orange-red colors are due to pelargonidin with one less
hydroxyl group than cyaniding, while mauve, purple and blue colors are generally due to
delphinidin, which has one more hydroxyl group than cyaniding.
According to Willstatter, 2003, nearly any fruit or flower that is bright red, blue or
purple contains pigment molecules that are based on cyanidin. The molecular structure
is responsible for all these colors. Like phenolphthalein, cyanidin’s structure changes
with pH. In acidic solution, there is a high formal charge on the oxygen in the structure
which makes it bright red. In basic solution, removal of hydrogen from the OH group on
the right outmost ring and forms a blue or violet color. In natural forms of the molecule,
the hydrogen on at least one of the -OH groups are replaced with more sugar
molecules. A cyanidin with attached sugars is called an anthocyan or anthocyanin.
Guevarra, 2005 added that anthocyanins also make up the most important and
widespread group of coloring matter in plants. It is one of the subclass of the phenolic
compound named flavonoids.
15
Furthermore, Bhowmik, 2009 added that the addition of citric acid in anthocyanin
extraction stabilizes the anthocyanin by creating an acidic environment which facilitates
the release of anthocyanin from the vacuoles of the plant.
Journals
From a study entitled “Anthocyanins in Wild Blueberries of Quebec: Extraction
and Identification” by Strack and Wray, 2000 it was stated that the use of acid stabilizes
anthocyanins in the flavylium cation form, which is red at low pH .To avoid or at least
minimize the breakdown of acylated anthocyanins, organic acids such as acetic, citric,
or tartaric acids, which are easier to eliminate during anthocyanin concentration, have
been preferred.
According to a research article written by Fera Amelia, et al.2000, it was stated
that the greatest yield of anthocyanins extraction from buni fruits was obtained from
ethanol 70% acidified with citric acid solvent rather than HCl. This can be explained that
the use of HCl may cause pigment degradation during concentration, especially the
occurrence of acid hydrolysis of labile acyl and sugar residues. This degradation was
reduced with the use of weaker organic acids such as citric acid. In a research article
entitled, “Use Of Anthocyanin Extracted From Natural Plant Materials To Develop A Ph
Test Kit For Measuring Effluent From Animal Farms”, Suppadit et al., 2011, discussed
that Anthocyanins (comes from the Greek anthos which means flower and kianos which
means blue) are the most important pigments of the vascular plants; they are harmless
and can be easily incorporated into aqueous media which makes them interesting for
use as natural water soluble colorants. These pigments are responsible for the shiny
orange, pink, red, violet and blue colors in the flowers and fruits of some plants.
Anthocyanins can be found in different chemical forms depending on the pH of the
solution. In the research, two factors were considered in optimal extraction of
anthocyanin. First, the type of natural plant materials is considered. On the research
conducted, they utilized butterfly pea (Clitoriaternatea L.) flower roselle red (Hibiscus
sabdariffa L.) flower and dragon fruit (Hylocereusundatus (Haw) Britt. andRose.) peel.
Then they consider the type of solvent to be used. This includes consisting of distilled
water, 1% Hydrochloric acid/95% methanol, 0.1 N acetic acid, 0.5% vinegar and 20%
16
white liquor. The butterfly pea flower yielded the highest amount of anthocyanins at
541mg/100 g dry weight followed by roselle red flower (280 mg/100 g) and dragon fruit
peel. For solvents, the distilled water used for the extraction yielded the highest amount
of anthocyanins (394 mg/100 g dry weight) followed by white liquor (388 mg/100 g), 1%
Hydrochloric acid/95% methanol (303 mg/100g), acetic acid (288 mg/100 g) and vinegar
(282 mg/100 g) (P<0.05).
From a journal written by Frosburg, 2001 he mentioned that any substance that
undergoes a reversible chemical change when pH changes can be used as an acid-
base indicator. In practice, a sharp change in some easily detectable property of the
substance is required. Usually, the property is color; but other properties such as odor
can also change with pH. Almost any flower, fruit, or plant part that is red, blue, or
purple contains a class of chemical compounds called anthocyanins that change color
with pH. The color of a flower or fruit depends on which anthocyanins are present, the
pH of the pigment-bearing tissues, and the presence of other pigments, like yellow
flavones. Red cabbage contains a mixture of anthocyanins and other pigments that
indicate a wide range of pH.
On a study published from Medwell Journals, 2010, argue that synthetic pigment
despite their advantages with respect to heat, light, ph ability and purity, compared with
natural colorants such as anthocyanins, they are increasingly rejected by consumers
owing to health concerns, thus there is worldwide interest in additional use of
anthocyanins as consequence of perceived consumer preferences as well as legislative
action which has continued the delisting of approved artificial dyes. The study shows the
use of onion solid waste which was frozen with liquid nitrogen and ground with a pestle
and a mortar. An amount of approximately 500 milligrams of ground tissue was place in
a 30 ml glass vials and 10 ml of solvent was added. Extraction was carried under
magnetic stirring at 400 rpm at room temperature for predetermined time periods. Upon
completion of extraction, the extract were filtered through paper filter and stored at
-20°C until analyzed. All extracts were also filtered through 0.45 micrometer syringe
filters prior to determinations. Briefly an aliquot of extract was combined with methanolic
hydrochloric acid solution (0.25M) to give a dilution 1:10. The solution was mixed
thoroughly and the absorbance at 520 nm was read after 5 mins using the methanolic
17
hydrochloric acid solution as blank. Total anthocyanin content was determined as
cyanin per 100 g fresh tissue using as ɛ= 26900 and MW=449.2.
On the other hand anthocyanin was also extracted from rose petals to be used
as acid-base indicators as describe by Vankar and Majpai, 2010, anthocyanin extraction
were carried out by three methods—by 1) Hydrochloric acid, 2) Citric acid 3) Tartaric
acid. On the first method, anthocyanins were extracted from flowers with 0.1%
hydrochloric acid in methanol for two to three hours at room temperature in darkness.
The mixture was filtered on a Buchner funnel and the remaining solids were washed
with 0.1% Hydrochloric acid in methanol until a clear solution was obtained. The
combined filtrates were dried using a rotary evaporator at 30°C. The concentrate was
dissolved in 0.01% hydrochloric acid in distilled water and in the solution was used as
indicator. On the second method anthocyanins were extracted from flowers with 4.0%
citric acid in methanol for two to three hours at room temperature in darkness. The
mixture was filtered in a Buchner funnel and the remaining solids were washed with
4.0% citric acid in methanol until a clear solution was obtained. The combined filtrates
were dried using a rotary evaporator at 30°C. The concentrate was dissolved in 4.0%
citric acid in distilled water and in the solution was used as indicator. On the third
method anthocyanins were extracted from flowers with 4.0% tartaric acid in methanol
for two to three hours at room temperature in darkness. The mixture was filtered in a
Buchner funnel and the remaining solids were washed with 4.0% tartaric acid in
methanol until a clear solution was obtained. The combined filtrates were dried using a
rotary evaporator at 30°C. The concentrate was dissolved in 4.0% tartaric acid in
distilled water and in the solution was used as indicator. As a result, a convenient
method of extraction of anthocyanin from Rose flowers has been develop using
methanolic solution of 4.0% citric acid which gave better yield of anthocyanins than the
methanolic solution of 0.1% hydrochloric acid.
Electronic Journal of Environmental, Agriculture and Food Chemistry, 2010,
added that anthocyanin changes color when the ph is 2-9 and the color will be from dark
pink to mehdi green. Phenolphthalein which has similar molecular structure with
anthocyanin is an organic compound (C20H14O4) used as an acid-base indicator.
18
Moreover on a study entitled “Determining the Acid/Alkali and Color Properties of
the Anthocyanin Delphinidin-3-Monoglucoside in Hydrangea Macrophylla“ by Hurwitz,
2008, he indicated that the study identified 43 different types of anthocyanins in four
wild forms of garden iris, and concluded, “There was no particular relationship between
the type of pigment present, and the flower color,” (Tsutomu Yabuya, 1990). This
suggests that plant colors (and changes) are the result of structural changes the
anthocyanins undergo. He added that anthocyanins, because of their hydroxide groups,
can act much the same way as phenolphthalein and other weak-acid indicators.
According to the research conducted by Barnes, 2010, entitled “Analytical
Characterization of Anthocyanins from Natural Products by Reverse-Phase Liquid
Chromatography-Photodiode Array-Electrospray Ionization-ion Trap-time of Flight Mass
Spectrometry”, he said that extraction of anthocyanins from natural sources may be
more favourable than laboratory synthesis because of the labile nature of the
compound. Anthocyanin structure can exist in many different forms depending on the
acidity of the environment to which it is subjected, which may significantly affect the
extraction of it from a solid or liquid. When the pH of a solution is below 2.5, the
anthocyanin is in the flavylium cation state and a red color can be observed. In weakly
acidic solutions, where the pH is between 4 and 6, the compound favors a secondary
structure, a mixture of anhydrobases and pseudobases. The purple anhydrobases are
formed first, and then they decolorize rapidly to form colorless pseudo bases, caused by
nucleophilic attack from water on the pyran ring. Above a pH of 8, the pyran ring opens,
creating a colorless chalcone structure. In vivo, pH levels may not be lower than 4, but
complexation of the anthocyanin can be present, which may alter its characteristics, due
to stabilization of the anthocyanins by forming “tertiary structures”, through self-
association, inter- and intramolecular co-pigmentation, and metal complex formation.
Co-pigmentation has been observed over a wide range of pH conditions.
From a research conducted by Wang, 2012 entitled “Isolation and Purification of
Anthocyanins from Black Bean Wastewater Using Macroporous Resins”, Anthocyanins
are polar molecules, thus using solvents like aqueous mixtures of ethanol, methanol or
acetone is better for extraction (Kahkonen, et.al, 2001). Because anthocyanins are not
stable in neutral or alkaline solutions, acidic aqueous solvents have been used as
19
extraction solvents in order to disrupt cell membranes and at the same time dissolve the
water-soluble pigments. The most common methods are those which use acidified
methanol or ethanol as extractants. HCl (usually <1%) is chosen for acidulating the
extraction solvent (Rodriguez-Saona and Wrolstad 2001; Amr and Al-Tamimi 2007).
Ethyl acetate, methanol and aqueous mixtures (50%-90%,v/v), and ethanol and
aqueous mixtures (10-90%) have been investigated (Ignat, et.al.,2011). He added that
methanol is the most effective of these solvents.
According to the study on Isolation and Purification of Anthocyanins and its
application as pH indicator, it was said that recovery of the flavylium form cannot be
achieved by simple re-acidification. As is discussed before, the colour of Anthocyanin
pigments changes drastically with change in pH value. The color of anthocyanins
depends on the acidity of the medium. At acidic pH = 1-3, anthocyanins exist
predominantly in the form of the red flavylium cation (2-phenylchromenylium cation).
Increasing the pH leads to a decrease in the color intensity and the concentration of the
flavylium cation which undergoes hydration to produce the colorless carbinol
pseudobase (hemiacetal or chromenol). The conjugated 2-benzopyrilium system is
disrupted due to a nucleophilic attack of water at the 2-position of the anthocyanin
skeleton. A rapid proton loss of the flavylium cation takes place as the pH shifts higher.
Now the equilibrium is shifted toward a purple quinoidal anhydrobase at pH < 7 and a
deep blue ionized anhydrobase at pH < 8. When pH increases further the carbinol form
yields, through opening of the central pyran ring, the light yellow chalcone. The color of
the alkaline solutions can be reverted by changing the pH back to acidic. The
anthocyanin equilibrium forms shift back to the equilibrium where the red colored
flavylium cation predominates.
Additionally according from an article entitled “The effect of light, temperature, ph
on stability of anthocyanin pigments in Musa acuminata bract” (Suganya, 2011) the
stability of anthocyanins and the rate of degradation are notably influenced by
temperature. Thermal stability of anthocyanins varies with temperature and pH. The
presence of oxygen and interactions with other components, like sugars and ascorbic
acid also affect anthocyanin stability. The main cause of pigment color loss seems to be
related to anthocyanin hydrolysis due to the observed proportionality between the speed
20
of red color disappearance from anthocyanins and the velocity of free sugar formation.
Heat causes anthocyanins, which are found at pH 2.0 to 4.0, to undergo hydrolysis at
glycoside linkages to produce chalcone and, later, alpha-diketones (Adams, 1973).
Internet
According to math.ufl.edu, retrieved 2010, Anthocyanins have played a
prominent role in the enrichment of human lives for thousands of years. Historians and
scientists believe that cave paintings from as far back as 15,000 B.C.E. were colored
using various plant pigments, and in Egypt and China, dyed fabrics have been found
and dated back to 2,000 B.C.E. The ancient Britons used a blue plant dye to color their
bodies in an attempt to frighten enemies in battle, and more recently in history, the
famous “red coats,” worn by British soldiers in the American Revolutionary War, were
dyed with a plant called ‘madder root’. Richard Martin Willstätter was the first scientist to
identify anthocyanins as the primary red/blue pigmentation in some plants and fruits. He
received the Nobel Prize in Chemistry in 1915 for his work with chlorophyll in connection
to anthocyanins and plant coloring. Specifically, he isolated the characteristic pigment in
cornflowers, roses, pelargonias, larkspurs, and hollyhock, and showed that
anthocyanins attached to glucoses produced an anthocyanin. Willstätter also explained
how the same anthocyanin can have blue or red color properties, and proposed that in
roses the anthocyanin is bonded to a plant acid, which makes it red. Conversely, he
claimed, in cornflower, the anthocyanin is bonded to a plant alkali, which is why it is
distinctly blue. The word anthocyanin is derived from two Greek words, “anthos”, which
means ‘flower’, and “kyáneos”, which means ‘purple’. Nearly three hundred different
anthocyanins have been discovered, and different fruits and vegetables have their own
signature mix of pigments. Red wine, for example, contains over fifteen different
anthocyanin compounds, depending on the amount and type of grapes with which it is
made. The differing concentrations and types of compounds are what give wine its
different color shades. Anthocyanins are also thought to play an important role in the
high antioxidant levels in fruits and vegetables. Blueberries, for example, contain a very
high concentration of antioxidant compounds, which guard the cell walls of the berry
21
from harmful free radicals existing inside the plant. When people ingest blueberries,
they obtain the same protection from free radicals, which can be just as harmful to cell
membranes as cell walls. Blueberries, cranberries, and cherries can contain up to
400mg of antioxidants per 100g of berry, and concord grapes—used in many red wines
—can contain up to 750mg per 100g of grape (Sriram, 2004). In the twelfth century,
bilberry (Vacciniummyrtillus) was used as an herbal medicine to induce menstruation,
and during World War II, British pilots took the same drug before nighttime missions to
enhance their night-vision. Now, researchers know that although anthocyanins probably
cannot increase nighttime awareness, nor encourage menstruation, they can prevent
oxidation damage in both large and small blood vessels because of their anti-oxidant
properties. Anthocyanins are also believed to inhibit degenerative nerve damage, and in
laboratory conditions, delphinidin and cyanidin compounds have been found to inhibit
the epidermal growth factor receptor in cancer cells, which could potentially stunt the
growth of tumor cells in humans. Also under study are anthocyanins’ abilities to reduce
low density lipoprotein (the “bad”) cholesterol, and prevent blood clotting.
In organic compounds, conjugated (alternating double) bonds primarily affect the
color the compound absorbs. In phenolphthalein, every carbon except for the central
carbon has overlapping p-orbitals, which create pi bonds between these carbon atoms.
The light absorbed by this structure is actually in the ultraviolet range, reflecting in the
infrared range, which is why phenolphthalein in a pH below 8.2 appears clear. When
phenolphthalein is in the presence of an alkali, the hydrogen atoms in phenolphthalein’s
Hydroxide ions are removed first. In a solution with a pH higher than 8.2, the structure
opens up, and the central carbon acquires a pi bond. Because electrons are less
confined in pi bonds than sigma bonds, the absorption for this molecule shifts
bathochromically to the blue-green range of the visible spectrum (redder than ultra-
violet), which makes the light it reflects pink. Anthocyanins, because of their hydroxide
groups, can act much the same way as phenolphthalein and other weak-acid indicators.
The study mentioned that in an alkaline solution, H+ ions from the anthocyanin
are removed by excess hydroxide ions. This allows electrons in the anthocyanin to
spread out in oxygen’s p-orbitals, causing a hypsochromic shift, but also leaving a
bonding site open. Metal ions such as Mg2+ , Fe2+ , Fe3+, Ca2+,and Al3+ are known
22
to bond with anthocyanin compounds, and the addition of these metal ions could cause
a change in color as well. Some of these metals will chelate with multiple anthocyanins,
which can produce a very different color than is typically exhibited by the metal ion, or
the anthocyanin itself. This also emphasizes the idea that chelation requires a pH above
the pKa of the phenolic group, because the H+ ions need to be removed for the metal
ion(s) to have an open bonding site. Because the acidified anthocyanins are generally
accepted as red in color, deprotonated anthocyanins must be present either alone, or
chelated with certain metal ions to change the color.
Anthocyanins are water soluble pigment that provides color to plants. The cyan
part of the name comes from the Greek word for blue, and cyan is also a
complementary color of red. Some good food sources of anthocyanins are eggplant,
checkberries, cherries, elderberries, red grapes, blueberries, oranges, red onion, red
wine, strawberries, radishes and purple cabbage (thirdplanetfood.com, retrieved 2013).
Anthocyanins are contained in red, purple, and blue colored flowers, fruits,
leaves, and roots of higher plants. They mainly exist as glycosides in plants and their
aglycon. Anthocyanin is a chromophore in pigments. It changes the color with pH like
litmus form flavylium ions strongly acidic solutions resulting in a very stable orange to
red. In weakly acidic or neutral solutions they first begin to form anhydrobases, so that
the color is reddish violet to violet. The blue they produce in alkaline media is the
predominance of anhydrobase anions. However, anhydrobases and anhydrobase
anions are unstable and are easily hydrated at the 2-position of the anthocyanin
nucleus, resulting in a rapid change to the colorless pseudobase (crcnetbase.com,
retrieved 2011).
For some metals, especially heavy metals, it has been demostrated that
anthocyanins can bind with these heavy metals reducing consequently their toxicity (see
for molibdenum). Otherwise, pH is important because it influences the
protonation/deprotonation of anthocyanins and consequently their colour and properties
(both optical and biological)
(http://www.researchgate.net/post/What_is_the_relationship_between_pH_and_metal_i
on_in_pigment_anthocyanin, retrieved Jul 31 2013).
Guaiac’s Test
23
Books
Strasinger and Di Lorenzo, 2008 stated that many commercial testing kits are
available for occult blood testing with guaiac reagent. The kits contain guaiac-
impregnated filter paper, to which the fecal specimen and hydrogen peroxide are added.
Two or three filter paper areas are provided for application of material taken from
different areas of stool, and positive and negative controls are also included. Obtaining
samples from the center of the stool avoids false-positive from external contamination.
Addition of hydrogen peroxide to the back of the filter paper that contains the stool
produces a blue color with guaiac reagent when pseudoperoxidase activity is present.
However, Turgeon, 2008 wrote that various interfering substances may give false
positive results. These are mostly enzyme peroxidase from hemoglobin and myoglobin
found in red meat, vegetables like horseradish, turnips and brocoli, and fruits like
bananas, black grapes, pears, plums and melons. In addition, White blood cells and
bateria also have peroxidase activity.
Likewise, according to Harborne, 2000 most test for occult blood in feces is gum
guiac, a phenolic compound that produces a blue color when oxidized. The test requires
the presence of hydrogen peroxide or a suitable precursor. The peroxidase activity of
the hemoglobin molecule results in the liberation of oxygen.
Journals
Dr. Winchester and Dr. Wansbrough 2003, supported Turgeon’s idea. They
explained that guaiac’s tests rely on the fact that heme can catalyze the breakdown of
hydrogen peroxide. As the hydrogen peroxide breaks down, another substance in the
reaction mixture is oxidized, producing a color change. It is important to note that a
positive test does not mean that a given stain is blood, let alone that it is human blood,
as various enzymes and certain metals can also give positive results.
Alliso, 2004 added that heme is present in red meat and peroxidase activity is
present in fresh fruits and vegetables such as radishes, turnips and broccoli. These
foods, therefore, have the potential to produce false-positive results.
In an article by Allison, 2007, it was mentioned that the guaiac test (GT) detects
the peroxidase activity of heme either as intact hemoglobin or free heme. In the
24
presence of heme and a developer (hydrogen peroxide) guaiac acid is oxidized
producing a blue color. Heme is present in red meat and peroxidase activity is present
in fresh fruits and vegetables such as radishes, turnips and broccoli. These foods,
therefore, have the potential to produce false-positive results. Although there are
several available GTs, there were only three tests namely Hemoccult II, Hemoccult
Sensa, and Hema-screen have been extensively evaluated in large screening
populations. The Hemoccult test first became available around 1970 and was in use
until modifications in 1977 led to the Hemoccult II test. Each Hemoccult II and
Hemoccult Sensa slide has two windows of guaiac impregnated paper, on which a small
amount of stool is smeared. This is repeated with two subsequent bowel movements.
The three-slide package is then returned to the laboratory or physician’s office for
development
Internet
Guaiac’s Test is a test for blood in urine or feces using a reagent containing
guaiacum that yields a blue color when blood is present (http://en.wikipedia.org, ret.
2013).
Forensic detection of blood
Books
Saferstein, 2011 in the book “Forensic Science: An Introduction”, discusses the
different forensic characterization of blood. These are color test, Luminol and Bluestar,
and Microcrystalline tests. The determination of blood is best made by means of
preliminary color tests, like Kastle-Meyer test; luminol test; and precipitin test. The color
tests are based on the observation that blood hemoglobin possesses peroxidase-like
activity. Peroxidases are enzymes that accelerate the oxidation of several classes of
organic compounds when combined with peroxides. Field investigators have found
Hemastix strip a useful presumptive field test for blood. Designed as a urine dipstick test
for blood, the strip can be moistened with distilled water and placed in contact with a
suspect bloodstain. The appearance of green color indicates blood. Furthermore,
another important presumptive identification test for blood is the luminol test. Unlike the
25
benzidine and Kastle-Meyer tets, the reaction of luminol with blood produces light rather
than color. By spraying luminol reagent onto a suspect item, investigators can quickly
screen large area for bloodstains. The sprayed objects must be located in a darkened
area while being viewed for the emission of light (luminescence); any bloodstains
produce a faint blue glow. In addition, a relatively new product, Bluestar, is now
available to be used in place of luminol. Its reaction with blood can be observed readily
without having to create complete darkness. It is capable of detecting blood stains
diluted to as little as 1 in 100,000. Another test for detecting blood is microcrystalline
test. This is more specific and two most popular tests under this test are Takayama and
Teichmann tests. Both depend on the addition of specific chemicals to the blood to form
characteristic crystals containing hemoglobin derivatives. These are far less sensitive
than color tests for blood identification and are more susceptible to interference from
contaminants that may be present in the stain. Once the stain has been characterized
as blood, the serologist determines whether the blood is of human or animal origin. The
standard test is the precipitin test. The principle is based on the fact that when the
animals like rabbit are injected with human blood, antibodies form that react with the
invading human blood to neutralize its presence.
In the book, entitled “World of Forensic Science, vol.1” (Lerner and Lerner,
2006), the presumptive test of blood is stated as critical because an investigator can be
confronted by a variety of fluids at the crime or accident scene. While a detailed
examination of a suspect bloodstain requires the equipment and technical expertise of
an analysis laboratory, a fluid suspected of being blood can be examined at the scene
to determine if it indeed could be blood. This examination is called blood presumptive
test. This can rule out the possibility that the fluid is blood if it is properly done. The test
relies on chemicals that will change color when in the presence of blood. When a blood
presumptive test is done at a crime or accident scene, an investigator must include the
use of controls to ensure the accuracy of the result. This is because blood presumptive
test is susceptible to false positives and false negatives. Standard procedures can rule
out the possibility of false positive or false negative results. However, if these controls
are not run, then the accuracy of the presumptive test can be questioned and would not
be admissible in a court of law. Presumptive blood tests are commercially available in a
26
convenient form that is easily transportable to the crime or accident scene. Typically, a
sample is placed in a sterile plastic bag or box to which are added the chemicals. Upon
mixing, the solution is visually observed for the development of the target color. Other
containers contain the positive and negative controls.
Journals
Until 1967, police investigators assumed that what looked like blood on a crime
scene was probably blood. Several tests have since been developed to confirm that a
red liquid or stain is actually blood. After a homicide or an assault has been committed,
police investigators usually find blood at the scene of the crime, giving them clues as to
what happened. The blood’s texture and shape and how it is distributed around the
victim often help investigators determine when the crime was committed, whether the
crime was preceded by a fight between individuals, and which weapon was used—say,
a knife, a gun, or an object used to hit a person. But criminals have tried many ways to
hide, clean up, and remove blood evidence. One of these tests consists of spraying a
suspected sample with a solution of luminol (C8H7N3O2), a chemical popularized by
the TV series “CS I” (short for “Crime Scene Investigation”), and hydrogen peroxide
(H2O2). If blood is present, the sample glows with a bluish color in the dark. The luminol
is first activated with an oxidant, usually a solution of hydrogen peroxide and a
hydroxide salt in water. Then, in the presence of a protein present in blood called
hemoglobin, the hydrogen peroxide is decomposed to form oxygen and water. When
luminol reacts with the hydroxide salt, a dianion is formed. The oxygen produced from
the hydrogen peroxide then reacts with the luminol dianion. The product of this reaction,
organic peroxide, is very unstable and immediately decomposes with loss of nitrogen to
produce 3-aminophthalic acid (3- APA) in an excited state. As 3-APA relaxes, it
releases a visible blue light. Luminol is sensitive to the presence of extremely small
amounts of blood. It can detect bloodstains that have been diluted up to 300,000 times.
Since it is nearly impossible to clean up every trace of blood at a crime scene, luminol is
especially effective at detecting blood after the scene has been cleaned or washed.
Additionally according from a journal named Chemmatters, 2008, once
investigators suspect that a stain is blood, they need to find out whether it is from an
27
animal or a human being. The reason they ask such a question is that pets are
sometimes present at a crime scene and can be either victims of a crime or involved in
it. To confirm whether a pet was present, investigators use various tests that
differentiate between animal and human blood. One of the most widely used tests is
called the precipitin test, in which the presence of human blood is revealed by making it
clot. The precipitin test is based on the fact that animals—including humans—make
large quantities of proteins called antibodies that protect them against foreign, disease-
causing substances. In this test, human blood is injected into a rabbit, which develops
antibodies against human blood. When these “anti-human” antibodies are extracted
from the rabbit’s blood and added to human blood, they precipitate, forming a clot. A
bloodstain from a crime scene that is added to the rabbit’s anti-human antibodies will
precipitate if it is of human origin.
Internet
According to "Physical Evidence in Forensic Science" by Dr. Henry C. Lee, a
forensics expert who has assisted law enforcement in more than 6,000 major criminal
investigations, and Howard Harris, a lawyer and forensic scientist, blood evidence is
found most often in crimes of violence such as homicide, assault and sexual assault.
Blood specimens can be found in a variety of forms, such as liquid, dried or coagulated,
and different testing methods can be performed based on the blood evidence. Methods
can range from use of Luminol, which is sprayed at the crime scene and reacts to blood
allowing criminalists to detect blood, to DNA testing
(http://www.ehow.com/about_5669371_forensic-blood_testing-methods.html - retrieved
March 2013)
Blood analysis is a simple test which can be useful for many cases involving a
blood stained crime scene and in the verification/identification of an unknown victim's
identity. When a stain is found at the scene of a crime, the first thing that has to be
determined is whether the stain is blood or some other bodily fluid. This is done using a
simple test involving a solution that changes color when it comes into contact with
hemoglobin or peroxidase in the blood. Another type of test commonly used involves
luminal spray, which makes any residue containing blood, glow in the dark as well as
28
picking up on traces of blood that may have been scrubbed away. The next step is
confirming whether the bloodstain belongs to a human. Serologists, people who study
blood, place the sample and a testing solution into small wells on a gel-coated glass
plate, and the two will defuse towards each other. If the sample is human blood, it will
contain human antigens and where the two solutions meet on the gel-coated plate, a
noticeable band forms (library.thinkquest.org/04oct/00206/text_pti_blood_analysis.htm,
- retrieved March 2013)
. Forensic investigations refer to the use of science and technology in the
investigation and establishment of facts or evidence to be used in criminal justice or
other proceedings (utb.edu, retrieved March 2013). . It takes just a swab on the surface
suspected with presence of blood and the swab will instantly change color if blood is
positive (chemistry.about.com).
We all have about ten pints of blood getting pumped throughout our bodies,
when wounded bodies leak or spray blood, and the behavior of blood in flight tends to
be unaffected by such things as temperature, humidity, or atmospheric pressure. In
other words, it's uniform. Despite how well the crime scene may get cleaned up, even
the finest trace of blood can often be detected and further tested. It is often the case
that while the perpetrator may scrub down the obvious places, he can still miss between
floorboards, under pipes, and inside drains. Merely by pouring water on some tiles at a
murder scene and pulling them up wherever the water flowed beneath them, one
detective found the only existing trace of the crime--blood. His discovery so surprised
the killer, who felt certain he'd done a thorough job of cleaning up, that he instantly
confessed (http://www.trutv.com/library/crime/criminal_mind/forensics/serology/3.html, -
retrieved March 2013).
Kastle-Meyer Test
Books
According to Saferstein, 2011, for many years, the most common test was
benzidine color test. However, because it is carcinogenic, benzidine use has been
discontinued, and phenolphthalein (phph) has taken its place. This is now known to be
the Kastle-Meyer test. Both these color tests are based on the observation that blood
29
hemoglobin possesses peroxidase-like activity. Peroxidases are enzymes that
accelerate the oxidation of several classes of organic compounds when combined with
peroxides. For example, when a bloodstain, phph, and hydrogen peroxide are mixed
together, oxidation of the hemoglobin in the blood produces a deep pink color. Kastle-
Meyer test is not specific for blood; some vegetable materials, for instance, may turn the
reagent pink. These substances include potatoes and horseradish. However, such
materials will probably not be encountered in criminal situations, and thus from a
practical point of view, a positive Kastle-Meyer test is highly presumptive field test for
blood.
Journals
According from a journal named Chemmatters, 2008, Luminol is especially
effective at detecting minute traces of blood that may not be visible to the naked eye.
But this technique has some limitations, since the light can be produced in the presence
not only of blood but also of other substances, such as copper ions, horseradish, and
bleach. To positively identify a substance as blood, it is often sent to a laboratory for
further analysis. Another important test is the Kastle-Meyer test. In many crime shows
on television, a blood sample is collected on a cotton swab, and then a clear solution is
applied, turning the swab bright pink to confirm the presence of blood. This is most likely
a demonstration of the Kastle-Meyer test. The clear solution in this test consists of a
reduced form of phenolphthalein and hydrogen peroxide, which react with each other to
produce a pink solution made of water and a phenolphthalein ion. In this test, the
phenolphthalein has been modified from its conventional form by being reduced and pre
dissolved in alkaline solution, giving it a faint yellow color. Then, in the presence of
hydrogen peroxide in alkaline solution, the hemoglobin in the blood catalyzes the
oxidation of this form of phenolphthalein to its normal form, which generates an intense
pink color. Like the luminol test, the Kastle-Meyer test is very sensitive because it relies
on a reaction catalyzed by hemoglobin, but other substances can also catalyze the
reaction. Both tests are called presumptive tests—blood will cause a positive test, but
so do many other substances—and they need to be substantiated by more specific
tests.
30
Internet
According to Helmenstine, the Kastle-Meyer test is an inexpensive, easy and
reliable forensic method to detect the presence of blood. The Kastle-Meyer blood test is
an extremely sensitive test, capable of detecting blood dilutions as low as 1:107. If the
test result is negative, it is reasonable proof that heme is absent in the sample,
however, the test will give a false positive result in the presence of any oxidizing agent
in the sample. Examples include peroxidases naturally found in cauliflower or broccoli.
Also, it is important to note that the test does not differentiate between heme molecules
of different species. A separate test is required to determine whether blood is of human
or animal origin. The Kastle-Meyer solution is a phenolphthalein indication solution
which has been reduced, usually by reacting it with powdered zinc. The basis of the test
is that the peroxidase-like activity of the hemoglobin in blood catalyzes the oxidation of
the colorless reduced phenolphthalein into bright pink phenolphthalein
(chemistry.about.com).
On the other hand, according to Whyte, the Kastle-Meyer test relies on
the iron in hemoglobin, which is the iron-containing portion of a red blood cell, to
promote the oxidation of phenolphthalin to phenolphthalein. Phenolphthalin is colorless,
but in the presence of blood and hydrogen peroxide, it changes to
phenolphthalein, which makes the solution pink. The names of the two chemicals—
phenolphthalin and phenolphthalein—are very similar, but they are structurally different.
Phenolphthalin is a special form of the common indicator phenolphthalein. It is made by
treating phenolphthalein with zinc, which is a reducing agent. In other words,
phenolphthalin is made by reducing phenolphthalein. Phenolphthalein, on the other
hand, can be made by oxidizing phenolphthalein (sciencebuddies.org).
Presumptive Blood Detection:
Journals
31
An article published in American Journal of Pathology mentioned regarding the
efficacy of phenolphthalein as blood detector compared to other three substances—
Benzidine, guaiac and o-toluidine. The phenolphthalein (Kastle-Meyer) test, first
described in 1903, if properly carried out is much more reliable and specific for
hemoglobin than the three tests in general use. In the chemical laboratories of Bellevue
Hospital have made a comparative study of all four tests for several years and are
convinced that the phenolphthalin test is the most specific for hemoglobin (Gettler and
Kaye, nd).
Furthermore according to the article entitled “Chemical Enhancement for the
Detection of Bloodstains” by Asghar, 2012, the substrate surface texture upon which
stain is located plays an important role in chemical enhancement and successful
detection of blood. An absorbent material consists of substrates with irregular porous
surfaces such as wood-finish panelling, walls, and interstitial spaces between tiles or
wood objects which, due to the grooves or cracks onto the surface. These materials
show superficial absorbent properties and they are able to have blood remains, even
after thorough scrubbing of the surface, for a long time. In this way, they are often able
to retain significant amounts of blood, maintaining it in relatively undegraded form even
for a long period of time, thus giving intense reaction with presumptive tests for blood.
Porous surfaces not only help in prevention of degradation by environmental biological
agents such as bacterial hydrolytic enzymes but it also protects blood from physical or
chemical environmental agents such as solar rays, moisture, water, or cleaning
attempts after the crime has been committed. Luminol, Bluestar and Fluorescein have
been found to be suitable chemical enhancement reagents for blood for porous
surfaces.
Furthermore, he added that non-porous surfaces are non-absorbent substrates
such as non-textured linoleum, vinyl, tile, glass, metal etc. They present difficulty in both
reagent application and in the quality of chemiluminescence. They don't have the
capacity to retain and store blood and, moreover, cannot prevent degradation of blood
by physical and chemical agents. These surfaces can be cleaned completely with mild
washing by water and soap. This results in negative response of presumptive tests of
blood. The surface can also complicate analysis as it can lead to the bloodstain pattern
32
running, due to the limited retention of the resulting solution by the smooth surface. This
can lead to complete loss of the bloodstain pattern. It is recommended that minimum
amount of reagent should be used when applying reagent on nonporous surfaces.
Luminol on such surfaces can be used with along with nebulizer, whereas Fluorescein
should be used along with a second application of commercial thickener, Keltrol RD,or
xanthan gum, an exo-cellular hetero-polysaccharide, which can reduce the issue of run
down pattern of bloodstain on non-porous surfaces. Methanol and titanium oxide are
found to be a better alternate solution for the detection of blood on non-porous surface.
Moreover amido black is very sensitive and works well on non-porous surfaces.
Peroxidase and Hydrogen peroxide
Books
Peroxidases are enzymes that belong to class I of enzymes—the
oxidoreductases. These enzymes catalyze oxidation-reduction reactions. Oxidation
means the loss of electrons, and reduction means the addition of electrons. Many
different electron acceptors are used in biological systems. Similar mechanism is seen
in the cell’s peroxisomes which have catalase, a heme enzyme, which catalyzes the
conversion of hydrogen peroxide to water and oxygen (Delvin, 2011).
As defined by Funk and Wagnalls of the New World Encyclopedia (2002),
hydrogen peroxide is a chemical compound of hydrogen and oxygen with the formula
H2O2. Pure, anhydrous hydrogen peroxide is a colorless, syrupy liquid with a specific
gravity of 1.44. It blisters the skin and has a metallic taste. The liquid solidifies at –0.41°
C (31.4° F). Concentrated solutions are unstable, and the pure liquid may explode
violently if heated to a temperature above 100° C (302.4° F). It is soluble in water in all
proportions, and the usual commercial forms are a 3% and a 30% aqueous solution. To
retard the decomposition of the peroxide into water and oxygen, organic substances,
such as acetanilide, are added to the solutions, and they are kept in dark bottles at low
temperature. Hydrogen peroxide is manufactured in large amounts by the electrolysis of
aqueous solutions of sulfuric acid or of potassium bisulfate or ammonium bisulfate. It is
also prepared by the action of acid on other peroxides, such as those of sodium and
barium. Hydrogen peroxide acts as both an oxidizing and a reducing agent. Its oxidizing
33
properties are used in the bleaching of substances, such as hair, ivory, feathers, and
delicate fabrics, which would be destroyed by other agents. It is also used medicinally,
in the form of a 3% aqueous solution, as an antiseptic and throat wash. Hydrogen
peroxide is used in restoring the original colors to paintings that have darkened through
the conversion of the white lead used in the paintings to lead sulfide. The hydrogen
peroxide oxidizes the black lead sulfide to white lead sulfate. It is also used as a source
of oxygen in the fuel mixture for many rockets and torpedoes. As a reducing agent it
reacts only with such easily reduced chemicals as silver oxide and potassium
permanganate.
Journals
In an article entitled, “The Decomposition of Hydrogen Peroxide by Blood.
George Senter’s Discovery of the Enzyme Involved” (Stock and Stuart, 2005), various
experiments were done to determine the enzyme that catalyze the oxidation of
hydrogen peroxide. Enzymes in substances that caused the decomposition of hydrogen
peroxide were provisionally termed superoxidases. Preliminary experiments on the rate
of decomposition, carried out with diluted blood, implied that the rate of reaction was of
first order with respect to the concentration of hydrogen peroxide. The research was
conducted by George Senter by the 1900s. Senter concluded that, in dilute solutions,
the rate of decomposition of hydrogen peroxide was proportional to the product of the
respective concentrations of peroxide and of Hämase (now termed catalase).
Internet
The enzyme catalase in blood speeds up the decomposition of hydrogen
peroxide. Catalase is very efficient at decomposing hydrogen peroxide; one molecule of
the enzyme can catalyze the conversion of over 6000,000 hydrogen peroxide molecules
into water and oxygen every second. The enzyme occurs widely in tissues such as the
liver and prevents accumulation of and tissue damage by, hydrogen peroxide that is
produced during metabolism. Catalase found in human red blood cells is a complicated
chemical consisting of four polypeptide chains with 500 amino acids in each chain. Each
peptide chain includes a porphyrin heme group. These are the active components which
34
allow the enzyme to catalyze the decomposition of hydrogen peroxide. Hydrogen
peroxide is a powerful oxidizing agent but is unusual in that it can act as reducing agent
under certain conditions. The oxidation number of oxygen in hydrogen peroxide is -1,
intermediate between 0 in oxygen and -2 in water, and this allows the oxygen to act as
both a reductant and oxidant in either acid (H2O2)hydrogen peroxide or alkali (HO2-)
hydroxide solution (www.eic.com, ret, February 2013).
Plant Extraction
Books
According to Hein, et. al., 2005 the physical properties of alcohols are related to
those both water and alkane hydrocarbons. One striking property of alcohols is their
relatively high boiling points. The simplest alcohol, methanol, boils at 65°C. The boiling
points of the normal alcohols increase in a regular fashion with increasing number of
carbon atoms. The hydroxyl group on the alcohol molecule is responsible for both the
water solubility and the relatively high boiling points of the low-molecular-mass alcohols.
Each polar alcohol group attracts water molecules and increases the solubility of
organic compounds in water. Methanol and ethanol have approximately the same acid
strength as water, while the larger alcohols are weaker acids than water, reflecting the
properties of the longer alkenelike carbon chains. Both water and alcohols react with
alkali metals to release hydrogen gas and an anion.
Journals
Additionally from a journal named “Internationale Pharmaceutica Sciencia, vol. 1
issue 1, 2011”, they conducted a review on phytochemical screening and extraction; it
showed that the various solvents that are used in the extraction procedures are: 1.
Water – is a universal solvent, used to extract plant products with antimicrobial activity.
Though traditional healers use primarily water but plant extracts from organic solvents
have been found to give more consistent antimicrobial activity compared to water
extract. Also water soluble flavonoids (mostly anthocyanins) have no antimicrobial
significance and water soluble phenolics only important as antioxidant compound.
35
2. Acetone - dissolves many hydrophilic and lipophilic components from the two plants
used, is miscible with water, is volatile and has a low toxicity to the bioassay used, it is a
very useful extractant, especially for antimicrobial studies where more phenolic
compounds are required to be extracted. A study reported that extraction of tannins and
other phenolics was better in aqueous acetone than in aqueous methanol. Both acetone
and methanol were found to extract saponins which have antimicrobial activity. 3.
Alcohol - the higher activity of the ethanolic extracts as compared to the aqueous
extract can be attributed to the presence of higher amounts of polyphenols as compared
to aqueous extracts. It means that they are more efficient in cell walls and seeds
degradation which have nonpolar character and cause polyphenols to be released from
cells. More useful explanation for the decrease in activity of aqueous extract can be
ascribed to the enzyme polyphenol oxidase, which degrade polyphenols in water
extracts, whereas in methanol and ethanol they are inactive. Moreover, water is a better
medium for the occurrence of the micro-organisms as compared to ethanol. The higher
concentrations of more bioactive flavonoid compounds were detected with ethanol 70%
due to its higher polarity than pure ethanol. By adding water to the pure ethanol up to
30% for preparing ethanol 70% the polarity of solvent was increased. Additionally,
ethanol was found easier to penetrate the cellular membrane to extract the intracellular
ingredients from the plant material. Since nearly all of the identified components from
plants active against microorganisms are aromatic or saturated organic compounds,
they are most often obtained through initial ethanol or methanol extraction. Methanol is
more polar than ethanol but due to its cytotoxic nature, it is unsuitable for extraction in
certain kind of studies as it may lead to incorrect results. 4. Chloroform - Terpenoid
lactones have been obtained by successive extractions of dried barks with hexane,
chloroform and methanol with activity concentrating in chloroform fraction. Occasionally
tannins and terpenoids will be found in the aqueous phase, but they are more often
obtained by treatment with less polar solvents. 5. Ether - is commonly used selectively
for the extraction of coumarins and fatty acids. 6. Dichloromethanol - is another solvent
used for carrying out the extraction procedures. It is specially used for the selective
extraction of only terpenoids.
36
Furthermore the article mentioned about the Extraction procedures: a. Plant
tissue homogenization: Plant tissue homogenization in solvent has been widely used by
researchers. Dried or wet, fresh plant parts are grinded in a blender to fine particles, put
in a certain quantity of solvent and shaken vigorously for 5 - 10 min or left for 24 h after
which the extract is filtered. The filtrate then may be dried under reduced pressure and
re-dissolved in the solvent to determine the concentration. Some researchers however
centrifuged the filtrate for clarification of the extract. b. Serial exhaustive extraction: It is
another common method of extraction which involves successive extraction with
solvents of increasing polarity from a non polar (hexane) to a more polar solvent
(methanol) to ensure that a wide polarity range of compound could be extracted. Some
researchers employ soxhlet extraction of dried plant material using organic solvent. This
method cannot be used for thermolabile compounds as prolonged heating may lead to
degradation of compounds. c. Soxhlet extraction: Soxhlet extraction is only required
where the desired compound has a limited solubility in a solvent, and the impurity is
insoluble in that solvent. If the desired compound has a high solubility in a solvent then
a simple filtration can be used to separate the compound from the insoluble substance.
The advantage of this system is that instead of many portions of warm solvent being
passed through the sample, just one batch of solvent is recycled. This method cannot
be used for thermolabile compounds as prolonged heating may lead to degradation of
compounds. d. Maceration: In maceration (for fluid extract), whole or coarsely powdered
plant-drug is kept in contact with the solvent in a stoppered container for a defined
period with frequent agitation until soluble matter is dissolved. This method is best
suitable for use in case of the thermolabile drugs. e. Decoction: this method is used for
the extraction of the water soluble and heat stable constituents from crude drug by
boiling it in water for 15 minutes, cooling, straining and 102 passing sufficient cold water
through the drug to produce the required volume. f. Infusion: It is a dilute solution of the
readily soluble components of the crude drugs. Fresh infusions are prepared by
macerating the solids for a short period of time with either cold or boiling water. g.
Digestion: This is a kind of maceration in which gentle heat is applied during the
maceration extraction process. It is used when moderately elevated temperature is not
objectionable and the solvent efficiency of the menstrum is increased thereby. h.
37
Percolation: This is the procedure used most frequently to extract active ingredients in
the preparation of tinctures and fluid extracts. A percolator (a narrow, cone-shaped
vessel open at both ends) is generally used. The solid ingredients are moistened with
an appropriate amount of the specified menstrum and allowed to stand for
approximately 4 hrs. in a well closed container, after which the mass is packed and the
top of the percolator is closed. Additional menstrum is added to form a shallow layer
above the mass, and the mixture is allowed to macerate in the closed percolator for 24
hrs. The outlet of the percolator then is opened and the liquid contained therein is
allowed to drip slowly. Additional menstrum is added as required, until the percolate
measures about three-quarters of the required volume of the finished product. The marc
is then pressed and the expressed liquid is added to the percolate. Sufficient menstrum
is added to produce the required volume, and the mixed liquid is clarified by filtration or
by standing followed by decanting. i. Sonication: The procedure involves the use of
ultrasound with frequencies ranging from 20 kHz to 2000 kHz; this increases the
permeability of cell walls and produces cavitation. Although the process is useful in
some cases, like extraction of rauwolfi a root, its large-scale application is limited due to
the higher costs. One disadvantage of the procedure is the occasional but known
deleterious effect of ultrasound energy (more than 20 kHz) on the active constituents of
medicinal plants through formation of free radicals and consequently undesirable
changes in the drug molecules.
Whereas according to Barnes, 2010, although the flavylium cation is stabilized at
low pH when using acidified extraction solvents, instability of the anthocyanin may occur
in these conditions. Typically, mildly acidified solvents are used in solid-liquid
extractions, showing increased extraction efficiency with some acidified solvents. A
study on the effects of acids (1% v/v) used in a variety of extract solvents of
anthocyanins from red grapes reported hydrolytic degradation of acetylated
anthocyanins, resulting in noticeable changes in the anthocyanin profiles detected
which may falsely demonstrated improved extraction of some modified anthocyanins.
Neutral solvent extraction was demonstrated to be just as efficient in this study.
Instability of anthocyanins, especially those with pendant acyl groups, should be
considered when mineral acids are used. Another study showed no degradation of
38
anthocyanins when extracting with 0.1% 12HCl in methanol. In addition, the acidic
environment promotes denaturing of the cell tissue membrane, which allows for
improved extraction rates of natural products. Therefore, very low amounts of acids
(~0.1%) in the extraction solvents may be justifiable in stabilizing the anthocyanin
compound in solution without degradation.
He added that due to the large number of hydroxyl groups and the compound’s
formal charge, the water soluble compound has a fair degree of polarity. The
polyphenolic structure adds a measure of hydrophobic character which gives the
anthocyanin solubility in organic solvents. The combination of this polar and
hydrophobic nature makes aqueous/organic solvent mixtures the ideal solvent.
Typically, the organic solvent content varies from 50% to 100% of the mixture. The
organic solvent is usually methanol but many other solvents have also been used such
as acetone, ethanol or acetonitrile. One study on the extraction of wine grape pomace,
which compared the extraction efficiency of ethanol, methanol and water, determined
that methanol was 23% more efficient than ethanol and 73% more efficient than water.
In a study on isoflavone extraction in methanol, which has a structure closely related to
that of an anthocyanin, evidence suggested that the alcohol group of the solvent
provided strong hydrogen bonding with the isoflavone.
Theoretical Framework
The researchers found out the screening test for the fecal occult blood is the
Guaiac test. It is used by most laboratories because of its cost-effectiveness. The
components of the test are gum guaiac and hydrogen peroxide. This test applies the
peroxidase-like activity of the heme portion of the erythrocytes. However, guaiac test is
susceptible to having false positive result, meaning the test will turn positive even when
blood is not present (http://en.wikipedia.org, ret. 2013). The test result can be confirmed
using immunochemical test. Nevertheless, guaiac test for fecal occult blood is still
reliable and sensitive for the initial detection of blood.
The researchers as well found out that another test that is used to detect blood is
the Kastle-Meyer test. Phenolphthalein is the principal color indicator and hydrogen
peroxide is the main reactant. This is used more in forensic science for an initial and in-
39
field detection of blood. This may be subjected to false positives but it is assumed that
on the scene of the crime, such false reactants will not be present (Saferstein, 2011).
Like the principle in guaiac test, this test also utilizes the pseudoperoxidase activity of
the heme to hydrogen peroxide. There are no health hazards in using phenolphthalein
in minute amounts but controversies arise on the carcinogenicity of the compound
(http://digipac.ca, ret. 2013).
The key chemical compound responsible for the ability of phenolphthalein to
change color is from the change of structure depending on the pH
(antoine.frostburg.edu, ret. 2013). So the researchers thought of finding a substitute for
phenolphthalein wherein it can also change structure depending on the ph. According to
the National Onion Association, anthocyanin, which is comparable to phenolphthalein
when used as pH indicator, is responsible for the various colors of plant leaves, fruits
and flowers. Red onion (Allium cepa) has that substance in its outer layers of the flesh.
Onion bulbs itself contains anthocyanins, organosulfur compounds and quercetin. The
scales outside the onion bulb are one of the richer sources of quercetin. This flavonoid
is said to be an antioxidant, deactivating molecules that are injurious to cells in the
body. Anthocyanins are intensely colored water-soluble pigments responsible for nearly
all the pink, scarlet, red, mauve, violet and blue colors in petals, leaves, and fruits of
higher plants. These are based chemically on a single aromatic structure, cyanidin
(Harborne, 2000). Anthocyanins also make up the most important and widespread
group of coloring matter in plants (Guevarra, 2005). It is one of the subclass of the
phenolic compound named flavonoids.
According to math.ufl.edu, retrieved 2010, Anthocyanins have played a
prominent role in the enrichment of human lives for thousands of years. When
phenolphthalein is in the presence of an alkali, the hydrogen atoms in phenolphthalein’s
Hydroxide ions are removed first. In a solution with a pH higher than 8.2, the structure
opens up, and the central carbon acquires a pi bond. Because electrons are less
confined in pi bonds than sigma bonds, the absorption for this molecule shifts
bathochromically to the blue-green range of the visible spectrum (redder than ultra-
violet), which makes the light it reflects pink. Anthocyanins, because of their hydroxide
groups, can act much the same way as phenolphthalein and other weak-acid indicators.
40
The study mentioned that in an alkaline solution, H+ ions from the anthocyanin
are removed by excess hydroxide ions. This allows electrons in the anthocyanin to
spread out in oxygen’s p-orbitals, causing a hypsochromic shift, but also leaving a
bonding site open. Metal ions such as Mg2+ , Fe2+ , Fe3+, Ca2+,and Al3+ are known
to bond with anthocyanin compounds, and the addition of these metal ions could cause
a change in color as well. Some of these metals will chelate with multiple anthocyanins,
which can produce a very different color than is typically exhibited by the metal ion, or
the anthocyanin itself. This also emphasizes the idea that chelation requires a pH above
the pKa of the phenolic group, because the H+ ions need to be removed for the metal
ion(s) to have an open bonding site. Because the acidified anthocyanins are generally
accepted as red in color, deprotonated anthocyanins must be present either alone, or
chelated with certain metal ions to change the color.
However, anthocyanin extraction on different plant samples varies. There were
different anthocyanin extractions that are present but anthocyanin extractions involving
the red onions were not established.
From a thesis made by Wang, 2012 entitled “Isolation and Purification of
Anthocyanins from Black Bean Wastewater Using Macroporous Resins”, anthocyanins
are polar molecules, thus using solvents like aqueous mixtures of ethanol, methanol or
acetone is better for extraction (Kahkonen, et.al, 2001). Because anthocyanins are not
stable in neutral or alkaline solutions, acidic aqueous solvents have been used as
extraction solvents in order to disrupt cell membranes and at the same time dissolve the
water-soluble pigments.
Another study according from an article entitled “The effect of light, temperature,
ph on stability of anthocyanin pigments in Musa acuminata bract” (Suganya, 2011) the
stability of anthocyanins and the rate of degradation are notably influenced by
temperature. Thermal stability of anthocyanins varies with temperature and pH. The
presence of oxygen and interactions with other components, like sugars and ascorbic
acid also affect anthocyanin stability.
From the researches that the researchers found out, these have led the
researchers to investigate on the possible utilization of red onion extract as blood
detector. Both phenolphthalein and the anthocyanin component of red onion have
41
structures that exhibit change in structure upon exposure to a given pH and may
possess the ability of the substance to detect blood. Different findings on anthocyanin
extraction as well led the researchers to investigate what extraction method will best
isolate great amount of anthocyanin from red onion’s outer layer of the flesh. The
researchers proposed that the anthocyanin of the red onion extract of its bulb’s outer
layer of the flesh could be utilized for blood detection.
Conceptual Framework
This study will utilize the outer layer of the flesh of the red onions (Allium cepa)
and different anthocyanin extraction methods and tested separately. Anthocyanin
extraction was carried out by three methods by ethanol, distilled water, and methanol
with 4% citric acid.
On the first extraction method, anthocyanins were extracted from red onions
soaked in ethanol. The mixture was filtered then concentrated using the hot plate
apparatus. On the second extraction method, anthocyanins were extracted from red
onions soaked in ethanol. The mixture was filtered then concentrated using the soxhlet
apparatus. On the third extraction method, anthocyanins were extracted from red onions
soaked in ethanol. The mixture was filtered then concentrated using the soxhlet
apparatus and hot plate apparatus. On the fourth extraction method, anthocyanins were
extracted from red onions soaked in distilled water. Lastly, on the fifth extraction
method, anthocyanins were extracted from red onions soaked in 4.0% citric acid in
methanol. The mixture was filtered then concentrated using the rotary evaporator and
the concentrate was dissolved in 0.4% citric acid in distilled water. The solution obtained
from the first extraction method was tested with hydrogen peroxide. While each solution
obtained from the second to fourth extraction methods were divided into two. The first
one is tested without addition of any solution, while the other one is tested with added
hydrogen peroxide. However, the solution obtained from the fifth extraction method was
used alone as an indicator.
Two types of blood were used for the test, one diluted with distilled water, and
the other maintained as a fresh blood sample. Both diluted and fresh blood was tested
as a dried sample and as a droplet. Two to three drops of the extract were added on
42
different surfaces such as wood, white cloth, metal, filter paper and concrete material.
On each extraction method, each sample was tested for three trials wherein for every
trial there is an equal amount of blood. A green color formation was observed which
indicates a positive result of the presence of blood in the different surfaces. A red liquid
was acquired for negative test control.
Paradigm of the Study
43
OUTPUT
Indicator for stained blood
Presence of green color on the surface of blood sample
THROUGHPUT
Application of the test extract to the blood stained on different surfaces.
INPUT
1. Extraction of anthocyanin from red onions (Allium cepa).
a. Extraction method: Ethanol (solvent) – Hot plate apparatus @ 80°C
Definition of Terms
Lexical:
44
OUTPUT
Indicator for stained blood
Presence of green color on the surface of blood sample
THROUGHPUT
Application of the test extract to the blood stained on different surfaces.
INPUT
1. Extraction of anthocyanin from red onions (Allium cepa).
a. Extraction method: Ethanol (solvent) – Hot plate apparatus @ 80°C
Allium cepa – scientific name of red onions; Sibuyas-tagalog is a low herb, 15 to 50 cm
high, with red, ovoid, subterranean bulbs, 1.5 to 4 cm long, 1 to 4 cm in diameter,
with accessory bulbs (Duke, 2003).
Anthocyanin - are water soluble pigments that provide color to plants, mainly shades of
red, purple and blue. They may be found in the roots, stems, leaves, flowers or
fruits of the plant (Quinn, 2008).
Buchner Funnel – a cylindrical often porcelain filtering funnel that has a perforated plate
on which the filter paper is placed and that is used usually with a vacuum
(Merriam-webster dictionary, 2012).
Chromogen – a precursor of a biochemical pigment (Merriam-webster dictionary, 2012).
Fecal Occult Blood test - used to detect the hidden blood in the stool and a positive
result suggests a blood loss or bleeding in the gastrointestinal tract
(webmedicaldictionary.com, retrieved 02/10/13)
Flavonoids – any group of oxygen-containing aromatic antioxidant compounds that
includes many common pigments (as the anthocyanins and flavones)
(Guevarra, 2005).
Extracts – these are products obtained from plants that are relatively complex mixtures
of metabolites and are intended for oral or external use (Merriam-webster
dictionary, 2012).
Guaiac Test - a test for blood in urine or feces using a reagent containing guaiacum that
yields a blue color when blood is present (http://en.wikipedia.org, ret. 2013).
Hemoglobin – an iron containing respiratory pigment of vertebrae red blood cell that
consists of a globins’ composed of four subunits each of which is linked to
a heme molecule (sciencebuddies.org, retrieved 2/8/2013).
Hot Plate Stirrer – a device which uses magnetic forces to drive a shaft and spin it
around very rapidly into a vessel of liquid, in order to stir the liquid efficiently.
Meanwhile, a hotplate at the bottom provides heating to catalyze the process
when necessary. (http://www.newstarenvironmental.com, 2011)
Indicator – a substance used to show visually (as by change of color) the condition of a
solution with respect to the presence of a particular material (Merriam-
webster dictionary, 2012).
45
Kastle-Meyer Test - is a presumptive blood test, first described in 1903, in which
the chemical indicator phenolphthalein is used to detect the possible
presence of hemoglobin. It relies on the peroxidase-like activity of
hemoglobin in blood to catalyze the oxidation of phenolphthalin (the colorless
reduced form of phenolphthalein) into phenolphthalein, which is
visible as a bright pink color (Saferstein, 2011).
Pathological - relating to, involving, or caused by disease (Merriam-webster dictionary,
2012).
Peel – the skin or rind of a fruit (Merriam-webster dictionary, 2012).
Pharmacological – the science of drugs; properties and reaction of drugs (Merriam
-webster dictionary, 2012).
Phenol – a corrosive poisonous crystalline acidic compound C6H5OH present in the tars
of coal and wood that in dilute solution is used as a disinfectant. (Merriam -
webster dictionary, 2012).
Phenolic compounds – are the most important single group of phenolics in food and
consist mainly of the catechins, proanthocyanins, anthocyanins, and the
flavones, flavonols and their glycosides (Merriam-webster
dictionary, 2012).
Phenolphthalein - is an organic compound (C20H14O4) used as an acid-base indicator.
The compound is colorless in acidic solution and pinkish in basic solution.
It does not dissolve very well in water, so for titrations, it is usually prepared
in alcohol solution (digipac.ca. retrieved 1/22/13).
Polyphenol – a polyhydroxyphenol; especially an antioxidant phytochemical (Merriam
-webster dictionary, 2012).
Pseudoperoxidase reaction – peroxidase like activity; an enzyme that catalyzes the
oxidation of various substances by peroxides (Saferstein, 2011)
Rotary Evaporation – is a technique which employs a rotary evaporator (also called a
“rotavap”) in order to remove excess solvents from samples by applying heat to
a rotating vessel at a reduced pressure. (http://webapps.utsc.utoronto.ca, 2010)
Soxhlet apparatus– the apparatus, first described in 1879, is a versatile tool that can be
used to separate a single gram to hundreds of grams with near 100% recovery. The
46
basic procedure calls for a solid sample to be placed in a porous container and
allowing condensed solvent to extract continuously.
(www.erowid.org/archive/rhodium/pdf/soxhlet4dummies.pdf, 2013)
Swab – A small piece of absorbent material attached to the end of a stick or wire and
used for cleansing or applying medicine on infected site of the body
(Merriam -webster dictionary, 2012).
Operational:
Outer flesh or layer—is the layer after the dry peel of the onion.
Blood detection—is the presumptive or initial identification of any red substance, dried
or diluted, as blood.
Sample – refers to the blood specimen for the experiment.
Chapter III
Methodology
47
This chapter discusses the locale of the study, the methods and procedures used
in performing the study, the research design, and research instrument/methods.
Research design
The study is basically a qualitative research for the results of the experiment only
to detect presence of blood in the samples. The study also covers the different
anthocyanin extraction methods. However, the study does not focus on the differences
on each extraction method. Also, the study does not cover the relationships of the
different amounts of variables and therefore a descriptive design is utilized.
The method used in the obtaining the extract of red onion is as described by
Guevarra, 2005, and Vankar and Bajpai, 2010 and modified by the researchers.
Anthocyanin extraction was carried out by three methods by ethanol, distilled water, and
methanol with 4% citric acid. The chopped outer flesh of red onion is weighed about 0.1
kilograms. On the first extraction method, anthocyanins were extracted from red onions
soaked in ethanol and was filtered and concentrated using the hot plate apparatus at
80°C. On the second extraction method, anthocyanins were extracted from red onions
soaked in ethanol and was filtered and concentrated using the soxhlet apparatus at
78°C. On the third extraction method, anthocyanins were extracted from red onions
soaked in ethanol and was filtered and concentrated using the soxhlet apparatus at
78°C and hot plate apparatus at 80°C. On the fourth extraction method, anthocyanins
were extracted from red onions soaked in distilled water. Lastly, on the fifth extraction
method, anthocyanins were extracted from red onions soaked in 4.0% citric acid in
methanol and was filtered then concentrated using the rotary evaporator at 60°C and
the concentrate was dissolved in 0.4% citric acid in distilled water.
Research locale
The process of extraction was performed in rooms 215, 302-B and 312-A of
Science Building (SB) at Far Eastern University (FEU). Buchner funnel filtration was
done in SB 215, Soxhlet extraction process in SB 302-B, hot plate process in SB 214,
and SB 312-A for the rotary evaporation. The actual experimentation was performed in
48
the Clinical Chemistry Laboratory located at SB 214. FEU is situated at Nicanor Reyes
Street, Sampaloc Manila, Philippines.
Data Collection
The respondents are selected by means of purposive non-probability sampling.
The researchers handpicked the subject based on age and health status that will best fit
the study. The respondents are of the age within 18-30 years old and are in good
health. In addition, blood samples are obtained from healthy individuals through
venipuncture.
I. Collection of red onion bulb peels.
0.5 kilograms of red onion bulbs were obtained from Central Market in Manila.
The plant materials are properly identified and authenticated by Wilfredo F. Vendivil, a
curator from the Botany Section of the National Museum.
Research Methodology
I. Preparation and extraction of anthocyanin from red onion peels.
The red onion bulbs were washed peeled and the peelings including the outer
flesh were collected. It will yield approximately 0.1 kilograms peels and flesh of red
onions. Fresh red onions peels and outer flesh were weighed 0.1 kilograms in a beaker.
The samples were placed in an osterizer to finely chop. Finely chopped samples were
subjected to different extraction methods.
a. The samples were soaked in 200mL solution (w/v, 1:2) of ethanol for 24-48 hours at
room temperature. The mixture was filtered on a Buchner funnel and the flask and the
plant material were rinsed with fresh portions of alcohol. The washings and the plant
material was transferred to the funnel and combined with the first filtrate. The filtrate
was concentrated using the hot plate apparatus at 80°C. The solution obtained was
tested with the addition of hydrogen peroxide.
b. The samples were soaked in 200mL solution (w/v, 1:2) of ethanol for 24-48 hours at
room temperature. The mixture was filtered on a Buchner funnel and the flask and the
49
plant material were rinsed with fresh portions of alcohol. The washings and the plant
material was transferred to the funnel and combined with the first filtrate. The filtrate
was concentrated using the soxhlet apparatus at 78°C. The solution obtained was used
as indicator. The solution obtained was divided into two. The first one is tested without
addition of any solution, while the other one is tested with added hydrogen peroxide.
c. The samples were soaked in 200mL solution (w/v, 1:2) of ethanol for 24-48 hours at
room temperature. The mixture was filtered on a Buchner funnel and the flask and the
plant material were rinsed with fresh portions of alcohol. The washings and the plant
material was transferred to the funnel and combined with the first filtrate. The filtrate
was concentrated using the soxhlet apparatus at 78°C and hot plate apparatus at 80°C.
The solution obtained was divided into two. The first one is tested without addition of
any solution, while the other one is tested with added hydrogen peroxide.
d. The samples were soaked in 200mL solution (w/v, 1:2) of distilled water for 2-3 hours
at room temperature in darkness. The mixture was filtered on a Buchner funnel and the
flask and the plant material were rinsed with fresh portions of water. The washings and
the plant material was transferred to the funnel and combined with the first filtrate. The
solution obtained was divided into two. The first one is tested without addition of any
solution, while the other one is tested with added hydrogen peroxide.
e. The samples were soaked in 200mL solution (w/v, 1:2) of methanol with 4% citric
acid for 2-3 hours at room temperature in darkness. The mixture was filtered on a
Buchner funnel and the remaining solids were washed with 4.0% citric acid in methanol
until a clear solution was obtained. The combined filtrates were concentrated using
rotary evaporator at 60°C. The extract, about 55mL, was dissolved in a solution (v/v,
1:1) of 0.4% citric acid (w/v) in distilled water and the solution obtained was used as a
blood detector.
II. Preparation of different samples
A. Stained blood
50
10mL of blood sample obtained from a respondent was divided into two separate
red top tubes with 5mL each. The first tube was maintained fresh while the other tube
was subject to dilution with the ratio of 1:20 (blood is 5ml while distilled water is 95ml).
Cell lysis is expected for better reaction.
B. Red Liquid
Red crepe paper was cut into small pieces and soaked in 5mL of tap water until it
produced intense red color similar to blood.
III. Test for detection of blood
The fresh, and diluted blood, and red liquid was applied to the different surfaces.
Each surface was tested with 3 trials with equal amount of blood.
3.1 Wood
3.1.1 Dried blood
3.1.2 Blood droplets
3.2 Fabric
3.2.1 Dried blood
3.2.2 Blood droplets
3.3 Metal (knife)
3.3.1 Dried blood
3.3.2 Blood droplets
3.4 Concrete (floor)
3.4.1 Dried blood
3.4.2 Blood droplets
3.5 Filter paper
3.5.1 Dried blood
3.5.2 Blood droplets
Two to three drops of the extract were added to each sample. Then, the green
color and precipitate production was observed and recorded after 5 minutes.
A control was run using Guaiac’s test. 2-3 drops of guaiac’s reagent were added
to each sample. Color reaction is observed and recorded after 5 minutes.
51
Select Topic
Gather Information
Research Methodology
Collection of onion bulb
peels
Extraction methodPreparation of Samples
Experimentation and Testing
of samples
Reading of Results
Interpretation
Summary and Conclusion
Recommendation
Research Flow Chart
Chapter IV
Presentation and Interpretation of Data
52
a. Extraction method: Ethanol (solvent) – Hot plate apparatus @ 80°C a.1. w/ H2O2
b. Extraction method: Ethanol (solvent) – Soxhlet apparatus @ 78°C b.1. w/ H2O2
b.2. w/o H2O2
c. Extraction method: Ethanol (solvent) – Soxhlet apparatus @ 78°C and hot plate apparatus @ 80°C c.1. w/ H2O2
c.2. w/o H2O2
d. Extraction method: Distilled water (solvent) d.1. w/ H2O2
d.2. w/o H2O2
e. Extraction method: Methanol w/ 4% citric acid (solvent) - Rotary evaporator @ 60°C
This chapter presents all the analyzed and interpreted data collected from the
experiments using different extraction procedures. The results are reported as (-) for
negative; no color change and (+) for positive; presence of green color. Positive results
that are italicize and bold (+) are considered weak positive.
One of the objectives of the researchers is to determine the anthocyanin extraction
procedure of red onions that is suitable for presumptive blood detection. The
researchers utilized five extraction methods and tested the red onion extract obtained
from each procedure for the detection of blood. The researchers used different solvent
and some of the methods done required heating at different temperature. The
temperature used in hot plate, soxhlet apparatus and rotary evaporator depends on the
boling point of the solvent used (ethanol for 78°C, methanol for 64.70°C). Among the
five different extraction methods performed, the fifth extraction method gave the suitable
anthocyanin extraction procedure for presumptive blood detection. This observation is
supported by the following data below. The results of each experiment in sequence are
also shown below.
Method 1: Hot Plate of Ethanolic Extract at 80 °C
The researchers used the ethanolic extract of onions for the detection of blood
and applied a droplet of fresh blood and diluted blood (fresh and dry) on different
surfaces to observe the presence of green color which indicates a positive result. The
researchers had three trials of blood detection on the following surfaces: wood, white
cloth, metal, filter paper and concrete material. A red liquid was acquired for negative
control test and Guaic’s test for positive control test.
The hot plate was used by the researchers at 80°C to evaporate the ethanol from
the extract to get pure anthocyanin from the red onions. The test used hydrogen
peroxide, it acts as the major reactant to blood wherein its oxidation product will be
measured by the ph indicator. Ethanol was used as the solvent because according to a
journal named “Internationale Pharmaceutica Sciencia, vol. 1 issue 1, 2011” the higher
activity of the ethanolic extracts as compared to the aqueous extract can be attributed to
the presence of higher amounts of polyphenols as compared to aqueous extracts. It
53
means that they are more efficient in cell walls and seeds degradation which have
nonpolar character and cause polyphenols to be released from cells.
The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and
diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of
onions.
The table presentations of the result are the following:
Table 4.1-A Hot Plate of Ethanolic Extract at 80 °C (with hydrogen peroxide)
FRESH DRYRED LIQUID
DILUTED DROPLET DILUTED DROPLET
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
CONCRETE
MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
All trials regardless of the specimen (diluted, fresh) produced negative results on
wood, white cloth, metal, filter paper and concrete material.
Table 4.1-A Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 0 0 12
54
WHITE CLOTH 0 0 12
METAL 0 0 12
FILTER PAPER 0 0 12
CONCRETE
MATERIAL0 0 12
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces). All results (regardless of the specimen used; diluted and fresh) in wood, white
cloth, metal, filter paper and concrete all produced negative result.
The negative result produced was due to the direct heating of the red onion
extract on the hot plate that destroyed the anthocyanin. Also, peroxidase activity of the
hydrogen peroxide in the hemoglobin molecule results in the liberation of oxygen which
affects the structural stability of anthocyanin structure. This is verified by an article
entitled “The effect of light, temperature, ph on stability of anthocyanin pigments in
Musa acuminata bract” (Suganya, 2011) that stated that the stability of anthocyanins
and the rate of degradation are notably influenced by temperature. Thermal stability of
anthocyanins varies with temperature and pH. The presence of oxygen and interactions
with other components, like sugars and ascorbic acid also affect anthocyanin stability.
Method 2: Ethanolic Extraction using Soxhlet apparatus at 78 °C
The researchers used the ethanolic extract of onions for the detection of blood
and applied a droplet of fresh blood and diluted blood (fresh and dry) on different
surfaces to observe the presence of green color which indicates a positive result. The
researchers had three trials of blood detection on the following surfaces: wood, white
cloth, metal, filter paper and concrete material. A red liquid was acquired for negative
test control. Guiac’s test was also performed by the researchers as positive control.
The researchers divided the extract into 2 (one tested with hydrogen peroxide;
one tested without hydrogen peroxide). The hydrogen peroxide acts as the major
reactant to blood wherein its oxidation product will be measured by the ph indicator.
55
Soxhlet apparatus was used by the researchers at 78 °C to separate the ethanol
from the extract to get a pure anthocyanin from the red onions. Ethanol was used as the
solvent because according to a journal named “Internationale Pharmaceutica Sciencia,
vol. 1 issue 1, 2011” the higher activity of the ethanolic extracts as compared to the
aqueous extract can be attributed to the presence of higher amounts of polyphenols as
compared to aqueous extracts. It means that they are more efficient in cell walls and
seeds degradation which have nonpolar character and cause polyphenols to be
released from cells.
The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and
diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of
onions.
Table 4.2-A Ethanolic Extraction using Soxhlet apparatus at 78 °C (with hydrogen
peroxide)
FRESH DRYRED LIQUID
DILUTED DROPLET DILUTED DROPLET
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
CONCRETE
MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
All trials regardless of the specimen (diluted, fresh) produced negative results on
wood, white cloth, metal, filter paper and concrete material.
Table 4.2-A Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
56
WOOD 0 0 12
WHITE CLOTH 0 0 12
METAL 0 0 12
FILTER PAPER 0 0 12
CONCRETE
MATERIAL0 0 12
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces). All results (regardless of the specimen used; diluted and fresh) in wood, white
cloth, metal, filter paper and concrete all produced negative result.
Table 4.2-B Ethanolic Extraction using Soxhlet apparatus at 78 °C (without hydrogen
peroxide)
FRESH DRYRED LIQUID
DILUTED DROPLET DILUTED DROPLET
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
CONCRETE
MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
All trials regardless of the specimen (diluted, fresh) produced negative results on
wood, white cloth, metal, filter paper and concrete material.
Table 4.2-B Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 0 0 12
57
WHITE CLOTH 0 0 12
METAL 0 0 12
FILTER PAPER 0 0 12
CONCRETE
MATERIAL0 0 12
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces). All results (regardless of the specimen used; diluted and fresh) in wood, white
cloth, metal, filter paper and concrete all produced negative result.
The negative results obtained from this method upon addition of Hydrogen
Peroxide was due to peroxidase activity of the hydrogen peroxide in the hemoglobin
molecule which results in the liberation of oxygen which affects the structural stability of
anthocyanin structure. This is verified by an article entitled “The effect of light,
temperature, ph on stability of anthocyanin pigments in Musa acuminata bract”
(Suganya, 2011) that stated that the stability of anthocyanins and the rate of
degradation are notably influenced by temperature. Thermal stability of anthocyanins
varies with temperature and pH. The presence of oxygen and interactions with other
components, like sugars and ascorbic acid also affect anthocyanin stability.
Negative results obtained can be due to destruction of the anthocyanin structure
since it is a thermolabile compound therefore this method cannot be used for
thermolabile compounds as prolonged heating may lead to degradation of compounds
(“Internationale Pharmaceutica Sciencia, vol. 1 issue 1, 2011”).
Method 3: Ethanolic Extraction using Soxhlet apparatus at 78°C and hot plate at 80°C
58
The researchers used the ethanolic extract of onions for the detection of blood
and applied a droplet of fresh blood and diluted blood (fresh and dry) on different
surfaces to observe the presence of green color which indicates a positive result. The
researchers had three trials of blood detection on the following surfaces: wood, white
cloth, metal, filter paper and concrete material. A red liquid was acquired for negative
test control. Guiac’s test was also performed by the researchers as positive control.
The researchers divided the extract into 2 (one tested with hydrogen peroxide;
one tested without hydrogen peroxide). The hydrogen peroxide acts as the major
reactant to blood wherein its oxidation product will be measured by the ph indicator.
Soxhlet apparatus was used by the researchers at 78 °C to separate the ethanol
from the extract to get a pure anthocyanin from the red onions. Ethanol was used as the
solvent because according to a journal named “Internationale Pharmaceutica Sciencia,
vol. 1 issue 1, 2011” the higher activity of the ethanolic extracts as compared to the
aqueous extract can be attributed to the presence of higher amounts of polyphenols as
compared to aqueous extracts. It means that they are more efficient in cell walls and
seeds degradation which have nonpolar character and cause polyphenols to be
released from cells.
The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and
diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of
onions.
Table 4.3-A Ethanolic Extraction using Soxhlet apparatus at 78 °C and hot plate at 80°C
(with hydrogen peroxide)
FRESH DRY RED LIQUID
59
DILUTED DROPLET DILUTED DROPLET
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
CONCRETE
MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
All trials regardless of the specimen (diluted, fresh) produced negative results on
wood, white cloth, metal, filter paper and concrete material.
Table 4.3-A Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 0 0 12
WHITE CLOTH 0 0 12
METAL 0 0 12
FILTER PAPER 0 0 12
CONCRETE
MATERIAL0 0 12
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces). All results (regardless of the specimen used; diluted and fresh) in wood, white
cloth, metal, filter paper and concrete all produced negative result.
Table 4.3-B Ethanolic Extraction using Soxhlet apparatus at 78 °C and hot plate at 80°C
(without hydrogen peroxide)
FRESH DRY RED LIQUID
DILUTED DROPLET DILUTED DROPLET
60
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
CONCRETE
MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
All trials regardless of the specimen (diluted, fresh) produced negative results on
wood, white cloth, metal, filter paper and concrete material.
Table 4.3-B Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 0 0 12
WHITE CLOTH 0 0 12
METAL 0 0 12
FILTER PAPER 0 0 12
CONCRETE
MATERIAL0 0 12
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces) using Guiac’s reagent as positive control test. All results (regardless of the
specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete
all produced negative result.
The negative result produced in method with addition of Hydrogen Peroxide was
due to the evaporation of the ethanol which leaches out the anthocyanin from the red
onion extract. The utilization of hydrogen peroxide liberate oxygen which affects the
structural stability of anthocyanin.
61
The negative result produced in method without Hydrogen Peroxide was due to
the double heating process which alters the stability of anthocyanin structure affecting
its thermolabile property.
Method 4: Aqueous Extract of red onion
The researchers used the aqueous extract of onions for the detection of blood
and applied a droplet of fresh blood and diluted blood (fresh and dry) on different
surfaces to observe the presence of green color which indicates a positive result. The
researchers had three trials of blood detection on the following surfaces: wood, white
cloth, metal, filter paper and concrete material. A red liquid was acquired for negative
test control. Guiac’s test was also performed by the researchers as positive control.
The researchers divided the extract into 2 (one tested with hydrogen peroxide; one
tested without hydrogen peroxide). The hydrogen peroxide acts as the major reactant to
blood wherein its oxidation product will be measured by the ph indicator.
Distilled Water is used because according from a journal named “Internationale
Pharmaceutica Sciencia, vol. 1 issue 1, 2011” it is a universal solvent, used to extract
plant products with antimicrobial activity. Though traditional healers use primarily water
but plant extracts from organic solvents have been found to give more consistent
antimicrobial activity compared to water extract. Also water soluble flavonoids (mostly
anthocyanins) have no antimicrobial significance and water soluble phenolics only
important as antioxidant compound.
The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and
diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of
onions.
Table 4.4-A Aqueous Extract (with hydrogen peroxide)
FRESH DRY RED LIQUID
DILUTED DROPLET DILUTED DROPLET
62
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
CONCRETE
MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
All trials regardless of the specimen (diluted, fresh) produced negative results on
wood, white cloth, metal, filter paper and concrete material.
Table 4.4-A Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 0 0 12
WHITE CLOTH 0 0 12
METAL 0 0 12
FILTER PAPER 0 0 12
CONCRETE
MATERIAL0 0 12
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces) using Guiac’s reagent as positive control test. All results (regardless of the
specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete
all produced negative result.
Table 4.4-B Aqueous Extract (without hydrogen peroxide)
FRESH DRY RED LIQUID
DILUTED DROPLET DILUTED DROPLET
63
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
CONCRETE
MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
All trials regardless of the specimen (diluted, fresh) produced negative results on
wood, white cloth, metal, filter paper and concrete material.
Table 4.4-B Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 0 0 12
WHITE CLOTH 0 0 12
METAL 0 0 12
FILTER PAPER 0 0 12
CONCRETE
MATERIAL0 0 12
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces) using Guiac’s reagent as positive control test. All results (regardless of the
specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete
all produced negative result.
The negative result obtained from the method upon addition of Hyrogen Peroxide
was due to peroxidase activity of the hydrogen peroxide in the hemoglobin molecule
which results in the liberation of oxygen which affects the structural stability of
anthocyanin structure. This is verified by an article entitled “The effect of light,
temperature, ph on stability of anthocyanin pigments in Musa acuminata bract”
(Suganya, 2011) that stated that the stability of anthocyanins and the rate of
64
degradation are notably influenced by temperature. Thermal stability of anthocyanins
varies with temperature and pH. The presence of oxygen and interactions with other
components, like sugars and ascorbic acid also affect anthocyanin stability.
The negative result obtained from the method without addition of Hydrogen
Peroxide was because the polyphenol oxidase is active which degrades the
polyphenols. This is according from the journal named “Internationale Pharmaceutica
Sciencia, vol. 1 issue 1, 2011” which stated that more useful explanation for the
decrease in activity of aqueous extract can be ascribed to the enzyme polyphenol
oxidase, which degrade polyphenols in water extracts, whereas in methanol and ethanol
they are inactive.
Method 5: Rotary Evaporation at 60°C of 4% Citric Acid in Methanol
The researchers used the methanolic (with 4% citric acid) extract of onions for
the detection of blood and applied a droplet of fresh blood and diluted blood (fresh and
dry) on different surfaces to observe the presence of green color which indicates a
positive result. The researchers had three trials of blood detection on the following
surfaces: wood, white cloth, metal, filter paper and concrete material. The researchers
had three trials of experimentation. A red liquid was acquired for negative test control.
Guiac’s test was also performed by the researchers as positive control.
Rotary evaporation was used because the evaporation of the solvent is more
rapid at low temperature without destroying the integrity of anthocyanin. Methanol was
the used solvent because it does not activate the polyphenol oxidase, which is an
enzyme that degrades polyphenol compounds. The addition of citric acid stabilizes the
anthocyanin by creating an acidic environment which facilitates the release of
anthocyanin from the red onions (Bhowmik, 2009).
The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and
diluted blood (fresh and dry) is proportional to the droplet of the methanolic (with 4%
citric acid) extract of onions.
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Table 4.5 Rotary Evaporation at 60°C of 4% Citric Acid in Methanol
DRY DROPLETRED LIQUID
DILUTED FRESH DILUTED FRESH
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
WHITE CLOTH (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
METAL (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
FILTER PAPER (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
CONCRETE
MATERIAL(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
Positive results that are italicize and bold (+) are considered weak positive.
Table 4.5 Summary of Onion Extract Reactions
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 11 1 0
WHITE CLOTH 12 0 0
METAL 10 2 0
FILTER PAPER 12 0 0
CONCRETE
MATERIAL4 8 0
On the surface of white cloth and filter paper, all 12 trials resulted positive. On
the wood surface, there were 11 positive results and 1 weak positive result obtained.
However, on the metal surface, only 10 positive results were obtained and 2 weak
positive results. Lastly 4 positive and 8 weak positive results were detected on the
concrete surface.
The researchers performed Guiac’s test as control to each method. The table
below shows the result of guiac’s reagent on blood (diluted, fresh) and on red liquid
which acts as the negative control. The drop of guiac’sregeant on wood, white cloth,
metal, filter paper and concrete floor is proportional to the drop of blood (fresh, diluted).
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Table 4.6 Guiac’s Test
DRY DROPLETRED LIQUID
DILUTED FRESH DILUTED FRESH
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WOOD (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
WHITE CLOTH (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
METAL (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-) (-)
FILTER PAPER (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-)
CONCRETE
MATERIAL(+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (+) (+) (-) (-) (-)
Positive results that are italicize and bold (+) are considered weak positive.
Table 4.6 Summary of Guiac’s Test
POSITIVE WEAK POSITIVE NEGATIVE
WOOD 12 0 0
WHITE CLOTH 11 1 0
METAL 11 0 1
FILTER PAPER 11 1 0
CONCRETE
MATERIAL11 0 1
The researchers conducted a total of 12 trials (3 trials each on the different
surfaces) using Guiac’s reagent as positive control test. All results (regardless of the
specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete
floor is summarized in the table above.
The researchers interpreted the weak positive result when the green color was
slightly not visible. The particles formed from the blood upon the addition of the
methanolic extract of the onions were also a basis of positivity. It was observed by the
researchers that there were no particles formed upon the addition of the methanolic
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extract of the onions in red liquid. The surfaces of metal and concrete material showed
some weak positive results, one possible reason was because generally, blood (diluted
and fresh) does not have the capacity to retain blood on non-porous surfaces (Asghar,
2012).
CHAPTER V
Summary, Conclusion, and Recommendation
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This chapter outlines the general results obtained from the experiments
conducted by the researchers. In addition, it also includes supposition and
recommendations of the researchers.
Summary
Based on the number of experiments done by the researcher using onion extract
as blood detector, the researchers observed that the extract yielded only a positive
result on majority of the samples when using method 5 on different surfaces, and the
extract yielded negative results on majority of the samples when using the other
extraction methods. Three blood detection trials were performed on various surfaces—
wood, white cloth, metal, filter paper, and concrete.
Regardless of the sample used, whether dry or droplet; fresh or diluted, the
results are summarized as follows:
Using method 1: Ethanolic extraction using Hotplate at 80 °C- all 12 trials on the
surface of wood, white cloth, knife, filter paper and concrete material resulted negative.
The red liquid acquired all yielded negative results; no green color was observed in all
trials. All trials on Guiac’s reagent resulted positive; blue color was observed in all trials
Using method 2: Ethanolic extraction using Soxhlet apparatus at 78 °C- all 12
trials (with or without hydrogen peroxide) on the surface of wood, white cloth, knife, filter
paper and concrete material resulted negative. The red liquid acquired all yielded
negative results; no green color was observed in all trials. All trials on Guiac’s reagent
resulted positive; blue color was observed in all trials
Using method 3: Ethanolic Extraction using Soxhlet apparatus at 78 °C and
Hotplate at 80°C- all 12 trials (with or without hydrogen peroxide) on the surface of
wood, white cloth, knife, filter paper and concrete material resulted negative. The red
liquid acquired all yielded negative results; no green color was observed in all trials. All
trials on Guiac’s reagent resulted positive; blue color was observed in all trials
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Using method 4: Aqueous Extraction at 80 °C- all 12 trials (with or without) on the
surface of wood, white cloth, knife, filter paper and concrete material resulted negative.
The red liquid acquired all yielded negative results; no green color was observed in all
trials. All trials on Guiac’s reagent resulted positive; blue color was observed in all trials
Using method 5: Methanolic extraction with 4% citric acid using Rotary
Evaporator at 60 °C - all 12 trials on the surface of white cloth and filter paper resulted
positive. On the wood surface, there were 11 positive results and 1 weak positive result
obtained. However, on the metal surface, only 10 positive results were obtained and 2
weak positive results. Lastly 4 positive and 8 weak positive results were detected on the
concrete surface. The red liquid acquired all yielded negative results; no green color
was observed in any trials.
The Guiac’s test on wood produced 12 positive results. However, on the surface
of the white cloth and filter paper, there were 11 positive results and 1 weak positive
result obtained on each surfaces. Lastly, on the surface of metal and concrete material,
11 resulted positive and 1 resulted negative on each surfaces.
Conclusion
The researchers therefore conclude that methanolic extract can be used best on
paper and cloth surfaces followed by wood and metal surfaces in the presence of blood.
Recommendation
With the effective result of the research on the detection of blood using the onion
extract containing anthocyanin, the researchers would recommend for the future
researchers the following:
1. To try blood detection on other surfaces
2. To try other extraction method to extract anthocyanin
3. To try other sources of anthocyanin
4. To device method to extract pure anthocyanin
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5. To use different concentration of the pure anthocyanin extract
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