Final Paper

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

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(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

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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

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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

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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

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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.3 metal (knife)



a.4 concrete (floor)



a.5 filter paper



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.3 metal (knife)



b.4 concrete (floor)



b.5 filter paper



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.2 white fabric



a.3 metal (knife)



a.4 concrete (floor)



a.5 filter paper



b. Stained diluted blood on:

b.1 wood



b.2 white fabric



b.3 metal (knife)



b.4 concrete (floor)

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b.5 filter paper



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

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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.

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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%

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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




22








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











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


49



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




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


3.3 Metal (knife)

3.3.1 Dried blood


3.4 Concrete (floor)

3.4.1 Dried blood


3.5 Filter paper

3.5.1 Dried blood


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






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.



onions.

Table 4.2-A Ethanolic Extraction using Soxhlet apparatus at 78 °C (with hydrogen

peroxide)

FRESH DRYRED LIQUID


1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

CONCRETE

MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)





56

WOOD 0 0 12

WHITE CLOTH 0 0 12

METAL 0 0 12

FILTER PAPER 0 0 12

CONCRETE

MATERIAL0 0 12




Table 4.2-B Ethanolic Extraction using Soxhlet apparatus at 78 °C (without hydrogen

peroxide)

FRESH DRYRED LIQUID


1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

CONCRETE

MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)



Table 4.2-B Summary of Onion Extract Reactions


WOOD 0 0 12

57

WHITE CLOTH 0 0 12

METAL 0 0 12

FILTER PAPER 0 0 12

CONCRETE

MATERIAL0 0 12




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 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.



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


1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

CONCRETE

MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)





WOOD 0 0 12

WHITE CLOTH 0 0 12

METAL 0 0 12

FILTER PAPER 0 0 12

CONCRETE

MATERIAL0 0 12




Table 4.3-B Ethanolic Extraction using Soxhlet apparatus at 78 °C and hot plate at 80°C

(without hydrogen peroxide)



60

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

CONCRETE

MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)





WOOD 0 0 12

WHITE CLOTH 0 0 12

METAL 0 0 12

FILTER PAPER 0 0 12

CONCRETE

MATERIAL0 0 12


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






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.



onions.

Table 4.4-A Aqueous Extract (with hydrogen peroxide)



62

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

CONCRETE

MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)





WOOD 0 0 12

WHITE CLOTH 0 0 12

METAL 0 0 12

FILTER PAPER 0 0 12

CONCRETE

MATERIAL0 0 12





Table 4.4-B Aqueous Extract (without hydrogen peroxide)



63

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

WOOD (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

WHITE CLOTH (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

FILTER PAPER (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

METAL (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)

CONCRETE

MATERIAL(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)





WOOD 0 0 12

WHITE CLOTH 0 0 12

METAL 0 0 12

FILTER PAPER 0 0 12

CONCRETE

MATERIAL0 0 12





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).


diluted blood (fresh and dry) is proportional to the droplet of the methanolic (with 4%

citric acid) extract of onions.

65

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


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).

66

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


WOOD 12 0 0

WHITE CLOTH 11 1 0

METAL 11 0 1

FILTER PAPER 11 1 0

CONCRETE

MATERIAL11 0 1




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

67

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

68

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

69

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

70

5. To use different concentration of the pure anthocyanin extract

71

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