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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 Nia 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 peoples 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

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 termsSibuyas; in BisayanCebuyas Bombay and; in IlocanoLasona. 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 substancesBenzidine, guaiac and o-toluidine. The phenolphthalein (KastleMeyer) 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 acidbase 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 onions 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 oxygens p-orbitals, causing a hypsochromic shift, but also leaving a bonding site open. Metal ions such as Mg2+ , Fe2+ , Fe3+, Ca2+,and Al3+ are known to bond with anthocyanin compounds, and the addition of these metal ions could cause a change in color as well. Some of these metals will chelate with multiple anthocyanins, which can produce a very different color than is typically exhibited by the metal ion, or the anthocyanin itself. This also emphasizes the idea that chelation requires a pH above the pKa of the phenolic group, because the H+ ions need to be removed for the metal ion(s) to have an open bonding site. Because the acidified anthocyanins are generally

accepted as red in color, deprotonated anthocyanins must be present either alone, or chelated with certain metal ions to change the color (math.ufl.edu, retrieved 2010). This research aims to investigate on the possible utilization of red onion extract as blood detector.

Statement of the Problem This research aims to investigate on the possible utilization of red onion extract as blood detector. It aims to answer the following questions: 1. What is the anthocyanin extraction procedure of red onions that is suitable for presumptive blood detection? 2. Will there be a color produced by the red onion extract against blood? 3. What is the color produced by the red onion extract against the following :

a. Stained fresh blood on: a.1 wood a.1.1 wiped (dried) blood a.1.2 blood droplets a.2 white fabric a.2.1 wiped (dried) blood a.2.1 blood droplets a.3 metal (knife) a.3.1 wiped (dried) blood a.3.2 blood droplets a.4 concrete (floor) a.4.1 wiped (dried) blood a.4.2 blood droplets a.5 filter paper a.5.1 wiped (dried) blood a.5.2 blood droplets

b. Stained diluted blood on:


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b.1

wood b.1.1 wiped (dried) blood b.1.2 blood droplets

b.2

white fabric b.2.1 wiped (dried) blood b.2.1 blood droplets

b.3

metal (knife) b.3.1 wiped (dried) blood b.3.2 blood droplets

b.4

concrete (floor) b.4.1 wiped (dried) blood b.4.2 blood droplets

b.5

filter paper b.5.1 wiped (dried) blood b.5.2 blood droplets

c. Red Liquid on: c.1 c.2 c.3 c.4 c.5 wood white fabric metal (knife) concrete (floor) filter paper

Hypothesis There is a color reaction produced by the red onion extract when stained on blood samples.

Objectives This research aims to 1. Determine the anthocyanin extraction procedure of red onions that is suitable for presumptive blood detection.
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2. Determine if there will be a color produced by the red onion extract against blood 3. Determine the color produced by the red onion extract against the following :

a. Stained fresh blood on: a.1 wood a.1.1 wiped (dried) blood a.1.2 blood droplets a.2 white fabric a.2.1 wiped (dried) blood a.2.1 blood droplets a.3 metal (knife) a.3.1 wiped (dried) blood a.3.2 blood droplets a.4 concrete (floor) a.4.1 wiped (dried) blood a.4.2 blood droplets a.5 filter paper a.5.1 wiped (dried) blood a.5.2 blood droplets

b. Stained diluted blood on: b.1 wood b.1.1 wiped (dried) blood b.1.2 blood droplets b.2 white fabric b.2.1 wiped (dried) blood b.2.1 blood droplets b.3 metal (knife) b.3.1 wiped (dried) blood b.3.2 blood droplets b.4 concrete (floor)
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b.4.1 wiped (dried) blood b.4.2 blood droplets b.5 filter paper b.5.1 wiped (dried) blood b.5.2 blood droplets

c. Red Liquid on: c.1 c.2 c.3 c.4 c.5 wood white fabric metal (knife) concrete (floor) 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 guaiacs 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 Guaiacs 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 termsSibuyas; in BisayanCebuyas Bombay and; in IlocanoLasona. 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 4.410C 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 onions 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, cyanidins 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 acidbase 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 -20C 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
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hydrochloric acid solution as blank. Total anthocyanin content was determined as cyanin per 100 g fresh tissue using as = 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 methodsby 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 30C. 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 30C. 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 30C. 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.

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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
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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
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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 Re volutionary War, were dyed with a plant called madder root. Richard Martin Willsttter 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. Willsttter 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 kyneos, 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
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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 grapesused in many red winescan 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 phenolphthaleins 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 ultraviolet), which makes the light it reflects pink. Anthocyanins, because of their hydroxide groups, can act much the same way as phenolphthalein and other weak-acid indicators. The study mentioned that in an alkaline solution, H+ ions from the anthocyanin are removed by excess hydroxide ions. This allows electrons in the anthocyanin to spread out in oxygens p-orbitals, causing a hypsochromic shift, but also leaving a
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bonding site open. Metal ions such as Mg2+ , Fe2+ , Fe3+, Ca2+,and Al3+ are known to bond with anthocyanin compounds, and the addition of these metal ions could cause a change in color as well. Some of these metals will chelate with multiple anthocyanins, which can produce a very different color than is typically exhibited by the metal ion, or the anthocyanin itself. This also emphasizes the idea that chelation requires a pH above the pKa of the phenolic group, because the H+ ions need to be removed for the metal ion(s) to have an open bonding site. Because the acidified anthocyanins are generally accepted as red in color, deprotonated anthocyanins must be present either alone, or chelated with certain metal ions to change the color. 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).


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Guaiacs Test Books Strasinger and Di Lorenzo, 2008 stated that many commercial testing kits are available for occult blood testing with guaiac reagent. The kits contain guaiacimpregnated 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 Turgeons idea. They explained that guaiacs 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.

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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 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 physicians office for development

Internet Guaiacs 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
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suspect bloodstain. The appearance of green color indicates blood. Furthermore, another important presumptive identification test for blood is the luminol test. Unlike the 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
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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 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 bloods 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.
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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 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 animalsincluding humansmake large quantities of proteins called antibodies that protect them against foreign, diseasecausing 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 rabbits blood and added to human blood, they precipitate, forming a clot. A bloodstain from a crime scene that is added to the rabbits 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
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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 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
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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 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. KastleMeyer 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 testsblood will cause a positive test, but
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so do many other substancesand they need to be substantiated by more specific tests.

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 phenolphthaleinare 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).

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Presumptive Blood Detection: Journals 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
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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 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 cells 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
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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 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 Senters 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 Hmase (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
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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 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 65C. 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

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extract. Also water soluble flavonoids (mostly anthocyanins) have no antimicrobial significance and water soluble phenolics only important as antioxidant compound. 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

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used for carrying out the extraction procedures. It is specially used for the selective extraction of only terpenoids. 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
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maceration extraction process. It is used when moderately elevated temperature is not objectionable and the solvent efficiency of the menstrum is increased thereby. h. 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.
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Instability of anthocyanins, especially those with pendant acyl groups, should be considered when mineral acids are used. Another study showed no degradation of 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 compounds 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.

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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 infield 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 phenolphthaleins 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
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bathochromically to the blue-green range of the visible spectrum (redder than ultraviolet), which makes the light it reflects pink. Anthocyanins, because of their hydroxide groups, can act much the same way as phenolphthalein and other weak-acid indicators. The study mentioned that in an alkaline solution, H+ ions from the anthocyanin are removed by excess hydroxide ions. This allows electrons in the anthocyanin to spread out in oxygens p-orbitals, causing a hypsochromic shift, but also leaving a bonding site open. Metal ions such as Mg2+ , Fe2+ , Fe3+, Ca2+,and Al3+ are known to bond with anthocyanin compounds, and the addition of these metal ions could cause a change in color as well. Some of these metals will chelate with multiple anthocyanins, which can produce a very different color than is typically exhibited by the metal ion, or the anthocyanin itself. This also emphasizes the idea that chelation requires a pH above the pKa of the phenolic group, because the H+ ions need to be removed for the metal ion(s) to have an open bonding site. Because the acidified anthocyanins are generally accepted as red in color, deprotonated anthocyanins must be present either alone, or chelated with certain metal ions to change the color. However, anthocyanin extraction on different plant samples varies. There were different anthocyanin extractions that are present but anthocyanin extractions involving the red onions were not established. From a thesis made by Wang, 2012 entitled Isolation and Purification of Anthocyanins from Black Bean Wastewater Using Macroporous Resins, a nthocyanins 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.
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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 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 onions outer layer of the flesh. The researchers proposed that the anthocyanin of the red onion extract of its bulbs 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.
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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 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.

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Paradigm of the Study INPUT


1. Extraction of anthocyanin from red onions (Allium cepa). a. Extraction method: Ethanol (solvent) Hot plate apparatus @ 80C a.1. w/ H2O2 b. Extraction method: Ethanol (solvent) Soxhlet apparatus @ 78C b.1. w/ H2O2 b.2. w/o H2O2 c. Extraction method: Ethanol (solvent) Soxhlet apparatus @ 78C and hot plate apparatus @ 80C 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 @ 60C 2. Blood stains from: a. Stained blood a.1 Wood a.1.1 Dried a.1.2 Droplet a.2 Fabric a.2.1 Dried a.2.2 Droplet a.3 Metal(knife) a.3.1 Dried a.3.2 Droplet a.4 Floor a.4.1 Dried Definition of Droplet Terms a.4.2 b. Red Liquid

THROUGHPUT Application of the test extract to the blood stained on different surfaces.

OUTPUT Indicator for stained blood Presence of green color on the surface of blood sample

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Lexical: 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)

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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). 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)
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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 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 layeris the layer after the dry peel of the onion. Blood detectionis the presumptive or initial identification of any red substance, dried or diluted, as blood. Sample refers to the blood specimen for the experiment.

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Chapter III Methodology 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 80C. On the second extraction method, anthocyanins were extracted from red onions soaked in ethanol and was filtered and concentrated using the soxhlet apparatus at 78C. On the third extraction method, anthocyanins were extracted from red onions soaked in ethanol and was filtered and concentrated using the soxhlet apparatus at 78C and hot plate apparatus at 80C. 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 60C 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,
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and SB 312-A for the rotary evaporation. The actual experimentation was performed in 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 80C. The solution obtained was tested with the addition of hydrogen peroxide.

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b. The samples were soaked in 200mL solution (w/v, 1:2) of ethanol for 24-48 hours at room temperature. The mixture was filtered on a Buchner funnel and the flask and the plant material were rinsed with fresh portions of alcohol. The washings and the plant material was transferred to the funnel and combined with the first filtrate. The filtrate was concentrated using the soxhlet apparatus at 78C. The solution obtained was used as indicator. The solution obtained was divided into two. The first one is tested without addition of any solution, while the other one is tested with added hydrogen peroxide.

c. The samples were soaked in 200mL solution (w/v, 1:2) of ethanol for 24-48 hours at room temperature. The mixture was filtered on a Buchner funnel and the flask and the plant material were rinsed with fresh portions of alcohol. The washings and the plant material was transferred to the funnel and combined with the first filtrate. The filtrate was concentrated using the soxhlet apparatus at 78C and hot plate apparatus at 80C. 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 60C. 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.

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II. Preparation of different samples A. Stained blood 10mL of blood sample obtained from a respondent was divided into two separate red top tubes with 5mL each. The first tube was maintained fresh while the other tube was subject to dilution with the ratio of 1:20 (blood is 5ml while distilled water is 95ml). Cell lysis is expected for better reaction. B. Red Liquid Red crepe paper was cut into small pieces and soaked in 5mL of tap water until it produced intense red color similar to blood.

III. Test for detection of blood The fresh, and diluted blood, and red liquid was applied to the different surfaces. Each surface was tested with 3 trials with equal amount of blood. 3.1 Wood 3.1.1 Dried blood 3.1.2 Blood droplets 3.2 Fabric 3.2.1 Dried blood 3.2.2 Blood droplets 3.3 Metal (knife) 3.3.1 Dried blood 3.3.2 Blood droplets 3.4 Concrete (floor) 3.4.1 Dried blood 3.4.2 Blood droplets 3.5 Filter paper 3.5.1 Dried blood 3.5.2 Blood droplets Two to three drops of the extract were added to each sample. Then, the green color and precipitate production was observed and recorded after 5 minutes.

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A control was run using Guaiacs test. 2-3 drops of guaiacs reagent were added to each sample. Color reaction is observed and recorded after 5 minutes.

Research Flow Chart


Select Topic

Gather Information

Research Methodology

Collection of onion bulb peels

Extraction method a. Extraction method: Ethanol (solvent) Hot plate apparatus @ 80C a.1. w/ H2O2 b. Extraction method: Ethanol (solvent) Soxhlet apparatus @ 78C b.1. w/ H2O2 b.2. w/o H2O2 c. Extraction method: Ethanol (solvent) Soxhlet apparatus @ 78C and hot plate apparatus @ 80C 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 @ 60C

Preparation of Samples

Experimentatio n and Testing of samples

Reading of Results

Interpretation

Summary and Conclusion

Recommendation

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Chapter IV Presentation and Interpretation of Data

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 78C, methanol for 64.70C). 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 Guaics test for positive control test. The hot plate was used by the researchers at 80C 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
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journal named Internationale Pharmaceutica Sciencia, vol. 1 issue 1, 2011 the higher activity of the ethanolic extracts as compared to the aqueous extract can be attributed to the presence of higher amounts of polyphenols as compared to aqueous extracts. It means that they are more efficient in cell walls and seeds degradation which have nonpolar character and cause polyphenols to be released from cells. The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of onions.

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 DILUTED 1 WOOD WHITE CLOTH FILTER PAPER METAL CONCRETE MATERIAL (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DILUTED 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DRY DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

All trials regardless of the specimen (diluted, fresh) produced negative results on wood, white cloth, metal, filter paper and concrete material.

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Table 4.1-A Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 0 0 0 0 0 WEAK POSITIVE 0 0 0 0 0 NEGATIVE 12 12 12 12 12

The researchers conducted a total of 12 trials (3 trials each on the different surfaces). All results (regardless of the specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete all produced negative result. The negative result produced was due to the direct heating of the red onion extract on the hot plate that destroyed the anthocyanin. Also, peroxidase activity of the hydrogen peroxide in the hemoglobin molecule results in the liberation of oxygen which affects the structural stability of anthocyanin structure. This is verified by an article entitled The effect of light, temperature, ph on stability of anthocyanin pigments in Musa acuminata bract (Suganya, 2011) that stated that the stability of anthocyanins and the rate of degradation are notably influenced by temperature. Thermal stability of anthocyanins varies with temperature and pH. The presence of oxygen and interactions with other components, like sugars and ascorbic acid also affect anthocyanin stability.

Method 2: Ethanolic Extraction using Soxhlet apparatus at 78 C

The researchers used the ethanolic extract of onions for the detection of blood and applied a droplet of fresh blood and diluted blood (fresh and dry) on different surfaces to observe the presence of green color which indicates a positive result. The researchers had three trials of blood detection on the following surfaces: wood, white cloth, metal, filter paper and concrete material. A red liquid was acquired for negative test control. Guiacs test was also performed by the researchers as positive control.

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The researchers divided the extract into 2 (one tested with hydrogen peroxide; one tested without hydrogen peroxide). The hydrogen peroxide acts as the major reactant to blood wherein its oxidation product will be measured by the ph indicator. Soxhlet apparatus was used by the researchers at 78 C to separate the ethanol from the extract to get a pure anthocyanin from the red onions. Ethanol was used as the solvent because according to a journal named Internationale Pharmaceutica Sciencia, vol. 1 issue 1, 2011 the higher activity of the ethanolic extracts as compared to the aqueous extract can be attributed to the presence of higher amounts of polyphenols as compared to aqueous extracts. It means that they are more efficient in cell walls and seeds degradation which have nonpolar character and cause polyphenols to be released from cells. The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of onions.

Table 4.2-A Ethanolic Extraction using Soxhlet apparatus at 78 C (with hydrogen peroxide) FRESH DILUTED 1 WOOD WHITE CLOTH FILTER PAPER METAL CONCRETE MATERIAL (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DILUTED 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DRY DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

All trials regardless of the specimen (diluted, fresh) produced negative results on wood, white cloth, metal, filter paper and concrete material.

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Table 4.2-A Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 0 0 0 0 0 WEAK POSITIVE 0 0 0 0 0 NEGATIVE 12 12 12 12 12

The researchers conducted a total of 12 trials (3 trials each on the different surfaces). All results (regardless of the specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete all produced negative result.

Table 4.2-B Ethanolic Extraction using Soxhlet apparatus at 78 C (without hydrogen peroxide) FRESH DILUTED 1 WOOD WHITE CLOTH FILTER PAPER METAL CONCRETE MATERIAL (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DILUTED 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DRY DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

All trials regardless of the specimen (diluted, fresh) produced negative results on wood, white cloth, metal, filter paper and concrete material.

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Table 4.2-B Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 0 0 0 0 0 WEAK POSITIVE 0 0 0 0 0 NEGATIVE 12 12 12 12 12

The researchers conducted a total of 12 trials (3 trials each on the different surfaces). All results (regardless of the specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete all produced negative result.

The negative results obtained from this method upon addition of Hydrogen Peroxide was due to peroxidase activity of the hydrogen peroxide in the hemoglobin molecule which results in the liberation of oxygen which affects the structural stability of anthocyanin structure. This is verified by an article entitled The effect of light, temperature, ph on stability of anthocyanin pigments in Musa acuminata bract (Suganya, 2011) that stated that the stability of anthocyanins and the rate of degradation are notably influenced by temperature. Thermal stability of anthocyanins varies with temperature and pH. The presence of oxygen and interactions with other components, like sugars and ascorbic acid also affect anthocyanin stability.

Negative results obtained can be due to destruction of the anthocyanin structure since it is a thermolabile compound therefore this method cannot be used for thermolabile compounds as prolonged heating may lead to degradation of compounds (Internationale Pharmaceutica Sciencia, vol. 1 issue 1, 2011).

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Method 3: Ethanolic Extraction using Soxhlet apparatus at 78C and hot plate at 80C

The researchers used the ethanolic extract of onions for the detection of blood and applied a droplet of fresh blood and diluted blood (fresh and dry) on different surfaces to observe the presence of green color which indicates a positive result. The researchers had three trials of blood detection on the following surfaces: wood, white cloth, metal, filter paper and concrete material. A red liquid was acquired for negative test control. Guiacs test was also performed by the researchers as positive control. The researchers divided the extract into 2 (one tested with hydrogen peroxide; one tested without hydrogen peroxide). The hydrogen peroxide acts as the major reactant to blood wherein its oxidation product will be measured by the ph indicator. Soxhlet apparatus was used by the researchers at 78 C to separate the ethanol from the extract to get a pure anthocyanin from the red onions. Ethanol was used as the solvent because according to a journal named Internationale Pharmaceutica Sciencia, vol. 1 issue 1, 2011 the higher activity of the ethanolic extracts as compared to the aqueous extract can be attributed to the presence of higher amounts of polyphenols as compared to aqueous extracts. It means that they are more efficient in cell walls and seeds degradation which have nonpolar character and cause polyphenols to be released from cells. The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of onions.

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Table 4.3-A Ethanolic Extraction using Soxhlet apparatus at 78 C and hot plate at 80C (with hydrogen peroxide) FRESH DILUTED 1 WOOD WHITE CLOTH FILTER PAPER METAL CONCRETE MATERIAL (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DILUTED 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DRY DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

All trials regardless of the specimen (diluted, fresh) produced negative results on wood, white cloth, metal, filter paper and concrete material.

Table 4.3-A Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 0 0 0 0 0 WEAK POSITIVE 0 0 0 0 0 NEGATIVE 12 12 12 12 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.

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Table 4.3-B Ethanolic Extraction using Soxhlet apparatus at 78 C and hot plate at 80C (without hydrogen peroxide) FRESH DILUTED 1 WOOD WHITE CLOTH FILTER PAPER METAL CONCRETE MATERIAL (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DILUTED 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DRY DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

All trials regardless of the specimen (diluted, fresh) produced negative results on wood, white cloth, metal, filter paper and concrete material.

Table 4.3-B Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 0 0 0 0 0 WEAK POSITIVE 0 0 0 0 0 NEGATIVE 12 12 12 12 12

The researchers conducted a total of 12 trials (3 trials each on the different surfaces) using Guiacs 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
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onion extract. The utilization of hydrogen peroxide liberate oxygen which affects the structural stability of anthocyanin.

The negative result produced in method without Hydrogen Peroxide was due to the double heating process which alters the stability of anthocyanin structure affecting its thermolabile property.

Method 4: Aqueous Extract of red onion

The researchers used the aqueous extract of onions for the detection of blood and applied a droplet of fresh blood and diluted blood (fresh and dry) on different surfaces to observe the presence of green color which indicates a positive result. The researchers had three trials of blood detection on the following surfaces: wood, white cloth, metal, filter paper and concrete material. A red liquid was acquired for negative test control. Guiacs test was also performed by the researchers as positive control. The researchers divided the extract into 2 (one tested with hydrogen peroxide; one tested without hydrogen peroxide). The hydrogen peroxide acts as the major reactant to blood wherein its oxidation product will be measured by the ph indicator. Distilled Water is used because according from a journal named Internationale Pharmaceutica Sciencia, vol. 1 issue 1, 2011 it is a universal solvent, used to extract plant products with antimicrobial activity. Though traditional healers use primarily water but plant extracts from organic solvents have been found to give more consistent antimicrobial activity compared to water extract. Also water soluble flavonoids (mostly anthocyanins) have no antimicrobial significance and water soluble phenolics only important as antioxidant compound. The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and diluted blood (fresh and dry) is proportional to the droplet of the ethanolic extract of onions.

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Table 4.4-A Aqueous Extract (with hydrogen peroxide) FRESH DILUTED 1 WOOD WHITE CLOTH FILTER PAPER METAL CONCRETE MATERIAL (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DILUTED 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DRY DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

All trials regardless of the specimen (diluted, fresh) produced negative results on wood, white cloth, metal, filter paper and concrete material.

Table 4.4-A Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 0 0 0 0 0 WEAK POSITIVE 0 0 0 0 0 NEGATIVE 12 12 12 12 12

The researchers conducted a total of 12 trials (3 trials each on the different surfaces) using Guiacs 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.

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Table 4.4-B Aqueous Extract (without hydrogen peroxide) FRESH DILUTED 1 WOOD WHITE CLOTH FILTER PAPER METAL CONCRETE MATERIAL (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DILUTED 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) DRY DROPLET 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

All trials regardless of the specimen (diluted, fresh) produced negative results on wood, white cloth, metal, filter paper and concrete material.

Table 4.4-B Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 0 0 0 0 0 WEAK POSITIVE 0 0 0 0 0 NEGATIVE 12 12 12 12 12

The researchers conducted a total of 12 trials (3 trials each on the different surfaces) using Guiacs reagent as positive control test. All results (regardless of the specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete all produced negative result.

The negative result obtained from the method upon addition of Hyrogen Peroxide was due to peroxidase activity of the hydrogen peroxide in the hemoglobin molecule which results in the liberation of oxygen which affects the structural stability of
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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.

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 60C 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. Guiacs 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).

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The different surfaces were labeled 1, 2 and 3. The droplet of fresh blood and diluted blood (fresh and dry) is proportional to the droplet of the methanolic (with 4% citric acid) extract of onions.

Table 4.5 Rotary Evaporation at 60C of 4% Citric Acid in Methanol DRY DILUTED 1 WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL (+) (+) (+) (+) (+) 2 (+) (+) (+) (+) (+) 3 (+) (+) (+) (+) (+) 1 (+) (+) (+) (+) (+) FRESH 2 (+) (+) (+) (+) (+) 3 (+) (+) (+) (+) (+) DROPLET DILUTED 1 (+) (+) (+) (+) (+) 2 (+) (+) (+) (+) (+) 3 (+) (+) (+) (+) (+) 1 (+) (+) (+) (+) (+) FRESH 2 (+) (+) (+) (+) (+) 3 (+) (+) (+) (+) (+) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

Positive results that are italicize and bold (+) are considered weak positive.

Table 4.5 Summary of Onion Extract Reactions POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 11 12 10 12 4 WEAK POSITIVE 1 0 2 0 8 NEGATIVE 0 0 0 0 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.
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The researchers performed Guiacs test as control to each method. The table below shows the result of guiacs reagent on blood (diluted, fresh) and on red liquid which acts as the negative control. The drop of guiacsregeant on wood, white cloth, metal, filter paper and concrete floor is proportional to the drop of blood (fresh, diluted). Table 4.6 Guiacs Test DRY DILUTED 1 WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL (+) (+) (+) (+) (+) 2 (+) (+) (+) (+) (+) 3 (+) (+) (+) (+) (+) 1 (+) (+) (+) (+) (+) FRESH 2 (+) (+) (+) (+) (+) 3 (+) (+) (+) (+) (+) DROPLET DILUTED 1 (+) (+) (+) (+) (+) 2 (+) (+) (+) (+) (+) 3 (+) (+) (+) (+) (+) 1 (+) (+) (+) (+) (-) FRESH 2 (+) (+) (+) (+) (+) 3 (+) (+) (-) (+) (+) RED LIQUID 1 (-) (-) (-) (-) (-) 2 (-) (-) (-) (-) (-) 3 (-) (-) (-) (-) (-)

Positive results that are italicize and bold (+) are considered weak positive. Table 4.6 Summary of Guiacs Test POSITIVE WOOD WHITE CLOTH METAL FILTER PAPER CONCRETE MATERIAL 12 11 11 11 11 WEAK POSITIVE 0 1 0 1 0 NEGATIVE 0 0 1 0 1

The researchers conducted a total of 12 trials (3 trials each on the different surfaces) using Guiacs reagent as positive control test. All results (regardless of the specimen used; diluted and fresh) in wood, white cloth, metal, filter paper and concrete floor is summarized in the table above.

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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 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).

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CHAPTER V Summary, Conclusion, and Recommendation

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 Guiacs 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 Guiacs reagent resulted positive; blue color was observed in all trials

Using method 3: Ethanolic Extraction using Soxhlet apparatus at 78 C and Hotplate at 80C- all 12 trials (with or without hydrogen peroxide) on the surface of
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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 Guiacs reagent resulted positive; blue color was observed in all trials

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 Guiacs 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 Guiacs 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
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2. To try other extraction method to extract anthocyanin 3. To try other sources of anthocyanin 4. To device method to extract pure anthocyanin 5. To use different concentration of the pure anthocyanin extract

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