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The Anthelmintic Property of Methanolic and Ethanolic extract of the Kulitis leaves (Amaranthus viridis) Chapter 1 Introduction The World Health Organization estimates that a staggering two billion people harbor parasitic worm infections. Parasitic worm also infect livestock and crops, affecting food production with a resultant economic impact. Despite this pevelance of parasitic infections, the research o the Anthelmintic drug is sparse. According to the WHO, only a few drugs are used in the treatment of helminthes, treatment of these parasite infections.

In view of this, attempts have been made to studt the anthelmintic activity of traditional medicinal plants. For our studies, we used Kulitis (Amaranthus viridis) that belong to the Amaranthaceae family and are traditionally used as vermifuge drugs (Fajimi anfd 2007), anti-malarial activity (Hilou et al. , 2006), antiadronergic activity (Murgan et al. , 1993), effects on biochemical changes in the epididymis (Murgan et al. , 1993), anti-diabetic, anti-hyperlipidemic and spermatogenic activity (Sangameswaran and Jayakar, 008). The prevention of disease particularly in diminishing intestinal parasites is a major problem especially in developing countries where there is a need for adequate health care and facilities. The use of  these plant Kulitis (Amaranthus viridis) could play a key role for treating people harboring parasitic worm infections. Plants are particularly interesting source of important substances. Plants products have been used in the treatment of diseases for many years and could be considered to be the first drugs. | The medicinal virtues of the plant Kulitis (Amaranthus viridis) are not well known to Filipinos. Most scientist have begun to prove the scientific basis for the use of these plants in medicine. The investigation lead to determine the possible constituent present in Kulitis (Amaranthus viridis) and to determine the wormicidal activity of the methanolic nd ethanolic extract of the Kulitis (Amaranthus viridis) leaves. . Conceptual Framework To perform different processes on the Kulitis (Amaranthus viridis) leaf for the determination of the constituent . Preliminary investigation were conducted to prove the active constituents present in Kulitis (Amaranthus viridis) such as saponin glycoside, cardenolides and bufadienolides, tannins, flavonoids, anthraquinone. The Leaves contains anthraquinone derivatives, cardiac glycosides and saponins. Study yields 18 kinds of amino acids, 8 of which are essential.

Contains 7-p-coumaroyl apigenin 4-O-beta-D-glucopyranoside, a new coumaroyl flavone glycoside called spinoside, xylofuranoxyl uracil, beta-D-ribofuranosyl adenine, beta-sitosterol glucoside, hydroxycinnamates, quercetin and kaempferol glycosides, betalains, betaxanthin, betacyanin; amaranthine and isoamaranthine, gomphrenin, betanin, b-sitosterol, stigmasterol, linoleic acid, rutin and beta- carotene. (http://www. stuartxchange. org/Uray. html) The Research Paradigm Input Process Output 1. Maceration . Phytochemical screening 3. Quantitative analysis a. Moisture content b. ash determination • Efficacy Test Anthel-mintic Kulitis (Amaranthus viridis) plant extract Figure 1 Paradigm of the Study The leaf extract of Amaranthus viridis is prepared by macerating in 80% ethyl alcohol. Then Phytochemical screening was done to determine the presence of alkaloids, saponin glycosides, tannins, fats and oils, cardenolides and bufadienolides, ananthraquinones, cyanogenic glycosides, resin and flavonoids. Quantitative analysis is done by calculating the moisture and ash content of the plant sample.

In Vivo test is then performed to determine the wormicidal effect of the ethanolic and methanolic extract of the Kulitis (Amaranthus viridis) leaf. The test is conducted using earthworms (Phretima posthuma). Statement Of The Problem The study aims to determine the phytochemical constituents presents in Kulitis (Amaranthus viridis) leaves and the anthelmintic property of the methanolic and ethanolic extracts of Kulitis (Amaranthus viridis) leaf. Sub-Problems 1. What are the phytochemical constituents of the Kulitis plant (Amaranthus viridis)? 2.

What was the method of extraction used? 3. What are the solvents used in extraction of the Kulitis (Amaranthus viridis)? 4. Which extract is more effective in causing death to earthworms compared to the control? 5. What kind of parasitic worms is used in testing the wormicidal effect of the extracts? 6. Is Kulitis (Amaranthus viridis) effective in stunning parasitic worms? Hypothesis of the Study The anthelmintic property of Kulitis plant (Amaranthus viridis) is the best herbal medicine in the treatment of intestinal parasites. Assumptions of the Study

AnthelminticHYPERLINK “http://wrongdiagnosis. pubs. righthealth. com/topic/Antiseptics? as=clink&ac=1437&afc=2168586466&p=publisher_scoped_search_wd&dqp. cache. mode=PMBypass” agents are substances that expel parasite worms from the body by either killing them or stunning. Significance of the Study The study will settle a possibility of utilizing Kulitis (Amaranthus viridis) as a source of medicine. It can be a great remedy to our country’s growing population of disease produce by parasite through the utilization of our natural resources.

By means of researches and experimental procedures in the preparation of medicinal agents from our plants-Kulitis (Amaranthus viridis), we can manufacture a good quality drugs which are valuable in stunning intestinal parasites. Scope and Deliminations of the Study The researcher planned and decided that kulitis plant (Amaranthus viridis) will be the center and focus of the study together with its active constituents in preparation of anthelmintic agent. After choosing the plant, collection of leaves, shredding into smaller pieces then macerating in 80% ethyl alcohol for 24-48 hours.

The alcoholic extract is then filtered and concentrated over a steam bath and subjected for the Phytochemical Screening of its active constituents. Quantitative analysis is done by calculating the moisture and ash content of the plant sample. In vivo anthelmintic activity of Kulitis (Amaranthus viridis) leaves is then performed by the researchers . The anthelmintic assay was conducted using earthworms due to its anatomical and physiological resemblace to the human intestinal roundworm parasite. Definition of Terms:

Antiemetic- drug used to alleviate or prevent nausea and vomiting (Medicine), serving to alleviate or prevent nausea and vomiting. (MedicineNet) Antipyretic- pertaining to a substance which reduces fever, agent which reduces fever. (Pharmacy-Online Dictionary by Babylon) Asthma- chronic inflammation disorder of the bronchiole tubes (airways). (MedicineNet) Diuretic- causing an increase in urination, substance which promotes urine flow  (Pharmacy-Online Dictionary by Babylon) Eczema- particular type of inflammatory reaction of the skin in which there are typically vesicles (tiny blister-like raised areas) MedicineNet) Ethyl alcohol- alcohol, colorless volatile liquid used in liquors and also as a solvent (Pharmacy-Online Dictionary by Babylon) Extract- substance obtained from a plant or other matter (MedicineNet) Intestinal parasite- a parasite (an organism that lives in or on and takes its nourishment from another organism) in the intestinal tract (MedicineNet) Laxative- tending to stimulate evacuation of the bowels, tending to relieve constipation, medication or other substance which relieves constipation. Pharmacy-Online Dictionary by Babylon) Maceration- act or process of softening by soaking; act or process of separating or dissolving; process of becoming thin, wasting away  (MedicineNet) Resin- any of several viscous organic substances derived from plants (used to manufacture medications, paints, plastics, and inks); synthetic substance similar to natural resin  (Pharmacy-Online Dictionary by Babylon) Saponin- group of glycosides found in plants (used in detergents and emulsifiers)  (Pharmacy-Online Dictionary by Babylon)

Tannins- are astringent, bitter plant polyphenols that either bind and precipitate or shrink proteins and various other organic compounds including amino acids and alkaloids. (Wikipedia-The Free Encyclopedia) Water content or moisture content- is the quantity of water contained in a material, such as soil (called soil moisture), rock, ceramics, or wood on a volumetric or gravimetric basis. The property is used in a wide range of scientific and technical areas, and is expressed as a ratio, which can range from 0 (completely dry) to the value of the materials’ porosity at saturation. Wikipedia-The Free Encyclopedia) Chapter 2 Review of Related Literature This chapter includes the historical background, botanical description, chemical composition, uses, and importance of kulitis plant medicinally, pharmaceutically and industrially, as well as different articles, journals, literatures related to the researcher’s plant of study. Common name: Kulitis (Tag. ), Amaranth (Engl. ) Family: Amaranthaceae Scientific name: Amaranthus viridis Other names: Bauan (Bon. ), Halom (Tag. , Bis. ), Nasi (It. ), Halunapa (Sul. ), Siitan (Ilk. ), Kadiapa (Mag. ), Sitan (Ib. ), Kalunai (Ilk. ), Kilitis (Bik. ) Philippine Medicinal Plants (http://www. stuartxchange. org/Kolitis. html) Historical Background Amaranth, common name used for plants with blossoms that do not readily fade when picked, but especially for about 50 species of one genus found in the Tropics and temperate regions where many are widely naturalized. They are herbaceous annuals with simple leaves, and flowers in heads or spikes. The spikes are sometimes several centimetres long. Love-lies-bleeding has dry red bracts that surround the flower which allows them to retain their freshness for some time. For this reason the plant is a symbol of immortality.

The annual tumbleweed of the United States belongs to this genus. The globe amaranth, of the same family but a different genus, has purple flowers that retain their beauty for years. (“Plantnet FloraOnline” (2005) (http://plantnet. rbgsyd. nsw. gov. au/) Botanical Description Kulitis is an erect, smooth, branched unarmed herb, 30-60 cm high. Its leaves are alternate, ovate, long-petioled, 4 to 10 cm long, obtuse tip, usually notched, base truncate or decurrent. The flowers are very small, densely disposed, green, 1 mm long, its sepals are 5 or 1 to 3, ovate to linear, often aristate and has no etals. It has inflorescence terminal axillary, simple or panicled, interrupted spikes and fruits that are compressed, indehiscent or circumcised utricles while seeds are black or brown, orbicular. (Philippine Medicinal Plants (http://www. stuartxchange. org/Kolitis. html) Chemical Composition Fresh Amaranthus spinosus, Amaranthus caudatus, and Amaranthus viridis were collected from Chickballapur and were authenticated by Dr. Rajan from the Department of Botany, Government Arts College, Ootcamund, Tamilnadu. A voucher specimen was deposited in the college herbarium. The whole plant was hade-dried and coarsely powdered. The coarse powder was extracted with methanol using the soxhlet apparatus. The extract was concentrated to dryness in vacuum. The methanol extracts of three plants were screened for the presence of various phytoconstituents such as steroids, alkaloids, terpenoids, glycosides, flavonoids, phenolic compounds, and carbohydrates according to Kokate. (Kokate C. K. (1986) Uses In Nepal, an infusion of powdered seeds of Amaranthus viridis is used for stomach problems and in pregnant women to alleviate labor pains. (Trakia Journal of Science, Vol. 6 (4), 39-51 Mark Turin (2003).

The Negritos of the Philippines apply the bruised leaves directly to eczema, psoriasis and rashes. (Eduardo Quisumbing (1951) Other traditional uses are based on their apparent antipyretic, diuretic, antirheumatic, antiulcer, analgesic, antiemetic, laxative, appetite stimulatory, antileprotic properties and for treating respiratory problems, eye ailments and asthma. (Kirtikar, K. R. , and B. D. Basu (1987), (Hassan Sher and Z. D. Khan (2006), (Quershi, S. J. , Khan, M. A. , M. Ahmed (2008) General Considerations Anthelmintic Drugs, medicinal drugs used to rid humans or other host animals f infestations by parasitic worms such as tapeworms, roundworms, pinworms, trichinae, flukes, whipworms, schistosomes, and filariae. These drugs are also known as vermifuges. They act by attacking the worms’ neuromuscular or respiratory systems, interfering with their metabolism, or making them more susceptible to attack by the host’s macrophages. (Microsoft ® Encarta ® 2009. ) Classes of Anthelmintic Drugs Piperazine Piperazine was first used as an anthelmintic in the 1950s and it is still the active constituent of over the counter remedies for thread worm infection in children. (Martin, 1985) Benzamidazoles

The first of this class, thiabendazole, was discovered in 1961 and subsequently a number of further benzamidazoles were introduced as broad spectrum anthelmintics. There is an extensive literature on these compounds reporting a number of different biochemical effects. Nonetheless, it is clear that their anthelmintic efficacy is due to their ability to compromise the cytoskeleton through a selective interaction with ? – tubulin (Borgers and de Nollin, 1975) Levamisole, pyrantel and morantel These anthelmintics are nicotinic receptor agonists and elicit spastic muscle paralysis due to prolonged activation of the excitatory nicotinic cetylcholine (nACh) receptors on body wall muscle. (Aceves et al. , 1970; Aubry et HYPERLINK “http://www. wormbook. org/chapters/www_anthelminticdrugs/anthelminticdrugs. html”al. ,HYPERLINK “http://www. wormbook. org/chapters/www_anthelminticdrugs/anthelminticdrugs. html” 1970) Paraherquamide Paraherquamide A and marcfortine A are both members of the oxindole alkaloid family, originally isolated from Penicillium paraherquei and Penicillium roqueforti, respectively. (Zinser et al. , 2002) Paraherquamide also blocks the actions of other nicotinic agonists, but not equipotently.

Interestingly, this antagonist seems to distinguish nicotinic receptor subtypes on the muscle and has a greater affinity for the receptors mediating the response to levamisole and pyrantel, than the receptors that mediate the response to nicotine. Importantly, the mode of action of this class of anthelmintics differs from the more established drugs that interfere with cholinergic transmission, e. g. , levamisole, in that they act as competitive antagonists rather than cholinomimetics. The use of paraherquamide in forward genetic screens has not yet been reported but could potentially generate interesting new mutants.

As it is a competitive inhibitor of the body wall nACh receptor it would be predicted that mutations that increase transmitter release should confer resistance. Thus a forward genetic screen might reveal further negative regulators of neurotransmitter release. (Zinser et al. , 2002; Robertson et HYPERLINK “http://www. wormbook. org/chapters/www_anthelminticdrugs/anthelminticdrugs. html”al. ,HYPERLINK “http://www. wormbook. org/chapters/www_anthelminticdrugs/anthelminticdrugs. html” 2002) Ivermectin (macrocylic lactones and milbemycins) Ivermectin was introduced as an anthelmintic in the 1980s by Merck.

It is a semi- synthetic derivative of avermectin which is a large macrocyclic lactone fermentation product of the micro-organism Streptomyces avermitilis. It is remarkably potent (~1nM) and persistent in its effect and its discovery enthused other companies to invest in the development of ivermectin analogues which include moxidectin, milbemycin oxime, doramectin, selamectin, abamectin and eprinomectin. (Haber et al. , 1991) Emodepside (cyclodepsipeptides, PF1022A) The cyclodepsipeptide molecule, emodepside, is a semi-synthetic derivative of PF1022A, a fermentation product obtained from the fungus, Mycelia sterilia, of Camelia japonica.

Its discovery and anthelmintic activity has recently been reviewed. It is effective against isolates of parasites that are resistant to benzimidazole, levamisole and ivermectin indicating, importantly, that it has a novel mode of action. The molecule has pore-forming properties in planar lipids, however, this does not appear to be important in conferring its anthelmintic potency as an optical isomer of emodepside, with similar pore forming properties, does not have anthelmintic action. Thus it would appear that it may act through stereospecific binding to a receptor. (Harder, von Samson-HYPERLINK “http://www. wormbook. rg/chapters/www_anthelminticdrugs/anthelminticdrugs. html”HimmelstjernaHYPERLINK “http://www. wormbook. org/chapters/www_anthelminticdrugs/anthelminticdrugs. html”, 2002)  Nitazoxanide Nitazoxanide, a pyruvate ferredoxin oxidoreductase inhibitor, acts against a broad spectrum of protozoa and helminths that occur in the intestinal tract. It is currently used for the treatment of protozoal infections. The site of action of this compound has not been established in nematodes although anaerobic electron transport enzymes may be a potential target. (Gilles and Hoffman 2002) Chapter 3 Research Methodology Experimental Design

The fact that Kulitis (Amaranthus viridis) is common through out the Philippines, the reporters collected their plants in different places. The latex were collected by their friends from Mangaldan, and some are gathered from Pozorrubio, Pangasinan. They gathered the plants the day before the reporters conducted their experiments. The plants picked from Mangaldan are transported to Dagupan for about 30minutes. As the reporters received the Kulitis plant (Amaranthus viridis) they immediately separate the leaves from stems and shredding them into smaller pieces subjected for their experiment. Instrumentation and Data Collection . Preparation of Alcoholic Extract Fifty grams of fresh plant sample was placed in the erlenmayer flask and sufficient amount of 80% ethyl alcohol was added to completely submerge the material. The flask was covered with a stopper and the material was soaked for 24-48 hours. The mixture was filtered and the flask with plant material was rinsed with fresh portions of alcohol. The washings were combined to the washings of the first filtrate. The plant extract was collected and the plant residue was discarded. The extract was evaporated until concentrated. The concentrated extract was kept in a tightly stoppered container.

It was stored with a few drops of chloroform which served as the preservative in order to prevent fungal growth. 2. Preparation of Reagent • 80% Alcohol About 84. 2 ml of 95% ethyl alcohol was measured and added to 15. 8 ml of distilled water. (USP XXII / NF XVII, 1970) • 1% Gelatin One gram of gelatin powder was weighed and sufficient water was added to make 100ml of the solution. (USP XXII / NF XVII, 1970) • 10% Gogo Solution Ten grams of gogo bark were weighed and cut into small pieces. It was placed in the beaker containing 30 ml of distilled water and allowed to boil for 10 minutes.

Ten milliliters of gogo extract were filtered and measured and it was added in 90 ml of distilled water to make 100 ml. (USP XXII / NF XVII, 1970) d. Gelatin Salt Solution Fifty milliliters of 1% gelatin solution and 50 ml of 10% sodium chloride were dissolved to make a 100 ml of the reagent. (USP XXII / NF XVII, 1970) e. Mayer’s Reagent A solution of 1. 358 grams of mercuric chloride in 60 ml of water was mixed with 5 grams of potassium iodide in 10 ml of water and the mixture was diluted to 100 ml of water. (USP XXII / NF XVII, 1970) f. 10% Sodium Chloride

Ten grams of NaCl were dissolved in 100 ml of water. (USP XXII / NF XVII, 1970) g. Ferric Chloride Reagent About 0. 3 ml of ferric chloride was added to glacial acetic acid to make 50 ml. (USP XXII / NF XVII, 1970) h. Wagner’s Reagent Sufficient water was added to 1. 27 grams of iodide and 3. 6 grams of potassium iodide to make 100 ml of the solution. (Knevel and Digangi, 1977) i. Ammonia Solution Prepared by diluting 40ml of ammonia with sufficient amount of distilled water to make 100 ml of the solution. (USP XXII / NF XVII, 1970) j. 1% Ferric Chloride Solution

Nine grams of ferric chloride were dissolved in distilled water to make 100 ml of the solution. (USP XXII / NF XVII, 1970) k. Phloroglucinol Test Solution About 500 mg of phloroglucinol was dissolved in 25 ml of water. (USP XXII / NF XVII, 1970) l. Preparation and Standardization of 1N KOH About 68 grams of KOH were added in 950 ml of water. Freshly prepared saturated solution of Barium hydroxide was added into the solution until no more precipitate forms. The mixture was shaken thoroughly and allowed to stand overnight in a stopper bottle and was standardized.

About 5 grams of potassium biphthalate were accurately weighed, previously crushed lightly and dried at 120 degrees for 2 hours and dissolved in 75 ml of carbon dioxide free water. Added into the solution was 2 drops of phenolphthalein TS and titrated with the KOH solution until the production of a permanent pink color. (Knevel and Digangi, 1977) m. Preparation and Standardization of 2M HCl About 170mL of HCl was diluted with distilled water to make 500 ml of the solution and was standardized against 1. 5 grams of primary standard anhydrous sodium carbonate, which has been heated for a temperature of 270?

C for one hour, dissolved in 120 ml of distilled water with an indicator of methyl red TS. A faintly pink color served as an end point of the titration until it no longer disappeared when heated on a steam bath. (Knevel and Digangi, 1977) n. Preparation and Standardization of 1N NaOH About 162g of NaOH was dissolved in 150mL of carbon dioxide free water. It was allowed to cool and filtered. About 54. 5mL of the clear filtrate was transferred to a tight, polyolefin container and diluted with water up to 1000mL. It was standardized by weighing 5g of potassium biphthalate, previously crushed and and dried at 120?

C for 2 hours, dissolved in 75mL of water. About 2 drops of phenolphthalein TS was added and titrated with NaOH solution to the production of permanent pink color. o. Kedde reagent About 1 g of 35 Nitrobenzoic acid was dissolved in a mixture of 50 ml methyl alcohol and 50 ml 2 M KOH. 3. Test procedures for phytochemical screening A. Test for alkaloids a. Preliminary Test An equivalent of 2 g of plant sample was taken and evaporated to syrupy consistency. About 5ml of 2 M HCL was added to the extract. It was stirred while heating for 5 minutes and then allowed to cool. About 0. g powdered NaCI was added while heating for two minutes. Then the mixture was cooled and filtered. Enough fresh 2M HCI was added to wash the residue and brought the volume of the filtrate to 5 ml. About 1ml of the filtrate was placed in two test tubes and tested as follows: to 1 ml of the filtrate few drops of Mayer’s reagent was added. To another 1 ml of the filtrate, few drops of Wagner’s reagent was added. b. Confirmatory Test To the remaining 3mL of the filtrate, enough 28% ammonia was added drop wise until the solution was alkaline to litmus. The alkaline solution was extracted three times with 10mL portions of chloroform.

The lower chloroform extracts were combined and the upper aqueous layer was reserved. The chloroform extract was evaporated to dryness under the hood and over the steam bath. The residue was taken up with 5mL of 2M HCl and stirred over a steam bath for about 2 minutes, then allowed to cool. The filtrate was filtered and divided into two equal portions and tested as in preliminary test. Positive results indicate the presence of secondary or tertiary alkaloids. c. Test for Quaternary and Amine Oxide Bases The alkaline aqueous layer obtained above was acidified with 2 M HCl.

The filtrate was filtered and divided into two equal portions and tested as above. Positive results indicate the presence of quaternary and amine oxide bases. B. Test for saponin glycosides a. Froth Test A volume of the alcohol extract equivalent to 2 g plant material was taken and placed in a test tube. For control, 2 ml of 10% gogo extract was placed in a separate tube. About 10 ml of distilled water was placed to each test tube. The test tubes were covered with stopper and shaken vigorously for 30 seconds. They were allowed to stand and observed over a period of 30 minutes. b .

Hemolysis Test after removing interfering tannins and other plant polyphenolic compounds A blood agar plate was obtained and using a small test tube, mini cups of blood agar were removed from three areas of the plate which are equidistant from one another. Each agar cup was numbered at the bottom of the inverted plate with a marking pencil. With a small pipette, enough plant extract was added to one of the agar cups to fill it. The second agar cup was filled with the same volume of gogo extract as positive control. The third agar cup was filled with distilled water as negative control. The covered plates were allowed to stand undisturbed.

After an hour, the three agar plates were observed for any zone of inhibition. The diameters of the halo were measured in millimeter. c. Liebermann-Burchard Test An equivalent of 10 g of plant sample was evaporated to dryness on a waterbath. About10 ml of hexane was added to the cooled residue, stirred for a few min it was decanted into a test , allowed to settle and the supernatant was decanted. Then 10 ml of chloroform is added to the residue and stirred for 5 min. It was decanted into a test tube containing 100 mg of anhydrous Sodium sulfate, shaked and passed through a dry filter paper.

The filtrate is divided into 2 clean dry test tubes. To one portion, 3 drops of Acetic anhydride was added and mixed gently. Afterwards another 1 drop of concentrated Sulfuric acid was mixed gently. Then color change was observed after a period of 1 hour. The other test tube was used as a reference. C. Test for cardenolides and bufadienolides a. Keller-killiani Test About 80% alcohol extract equivalent to 10 g of plant sample was taken and evaporated to incipient dryness over a water bath. The extract was defatted by trituration with hexane to remove as much of colored pigments as possible.

The hexane solution was discarded. The defatted residue was warmed over a water bath to remove the residual hexane solvent. About 3ml of FeC13 reagent was added. It was stirred and transferred to a test tube. The test tube was held in an incline position and carefully 1 ml of concentrated sulfuric acid was added and allowed to run along the walls of the test tube. The liquid mixture was allowed to stand undisturbed. Any coloration at the interface was observed. The result reddish brown color which may gradually become blue or purple is indicative of the presence of 2 deoxysugars. b.

Salkwoski Test An equivalent of 10 g of plant extract was evaporated to dryness using a water bath. About 10 ml hexane or petroleum ether was added to the cool residue. It was stirred for a few minutes and allowed to settle. The supernatant liquid was decanted. It was repeated until all the color has been removed. About 10 ml of chloroform was added to the residue and stirred for 5 minutes. It was decanted into a test tube containing about 100 mg of anhydrous Na2SO4. Then it was shaken and passed through a dried filter paper. The filtrate was divided and placed into two clean dry test tubes.

To one portion, 1 ml of concentrated sulfuric acid was added and allowed to run down inside of the test tube. The test tube was held at 45 degree angle. Any immediate color changed at the junction of the extract and the sulfuric acid was noted. The sulfuric acid and extract were gently mixed and observed for color changes over a period of one hour. The other tube was used as reference. c. Kedde reaction To 5 ml of 80% ethanolic extract in an evaporating dish was added 5 ml of Kedde reagent and mixed well with a stirring rod. 2 ml of 1 N Sodium hydroxide was added to the mixture and mixed. The color development was observed.

A purple color is a positive indication of the presence of the unsaturated ring. D. Test for Flavonoids a. Preparation of the Defatted Plant-Extract The alcohol plant extract equivalent to 10g was evaporated to incipient dryness over a water bath. It was allowed to cool at room temperature. The residue was triturated with 10 ml of petroleum ether or hexane. Then it was decanted and the trituration of the residue was repeated with fresh volumes until the equivalent was almost colorless. The petroleum ether or hexane extract was discarded. The defatted residue was dissolved in 20 ml of 80% ethyl alcohol. Any insoluble residue was filtered.

It was divided into 4 test tubes labeled A, B, C, D. Test tube A was kept blank. b. Bate-Smith and Metcalf Test for Leucoanthrocyanins To test tube B, about 0. 5 ml of concentrated HCI was added. Any color change was observed and recorded. The test tube was warmed on a water bath for 15 minutes. Any color change was observed within an hour. Gradual development of a strong red or violet color is indicative of the presence of leucoanthocyanins. Color formation may be slow. If the color is not immediately apparent, the test solution is allowed to stand at room temperature for 1 hour before recording the result as negative. . Wilstatter “cyanidin” test To test tube C, 0. 5 ml of concentrated HCI and 3 or 4 pieces of magnesium turnings were added. Any color change was noted (green, red etc. ) within 10 minutes. It was compared with test tube serving as blanked. NOTE: Should definite coloration occur, it is allowed to cool and diluted with an equal volume of water and 1 ml of octyl alcohol. It is shaken and allowed to separate. The color in each layer is noted. E. Test for Tannins and Polyphenols The ethanol extract equivalent to 10 g of plant materials was taken and evaporated to incipient dryness over a water bath and cooled.

About 25 ml of hot distilled water was added to the residue. It was mixed well with stirring rod and allowed to cool at room temperature spontaneously. About 5 drops of 10% NaCI solution were added to salt out undesirable constituents. It was filtered and the filtrate was divided into 4 test tubes labeled A, B, C, and D. The contents of test tube A was reserved as blank. a. Gelatin test To the test tube B, about 1% gelatin solution was added drop wise. To test tube C, 5 drops of gelatin-salt reagent were added. Any formation of precipitate was observed. The gelatin precipitates indicate the presence of tannins. . Ferric chloride Test To test tube D, about 5 drops of ferric chloride solution were added. Any color changed for formation of precipitate was observed. A blue-black color may indicate the presence of hydrolysable tannins, while a brownish-green color, greenish blue or greenish black may indicate condensed tannins if the gelatin is positive. Polyphenolic compounds give a negative gelatin test. F. Test for Anthraquinone a. Borntrager Test An equivalent of 1 g of the alcoholic plant extract was evaporated to incipient dryness using a water bath. The residue was taken up with 10 ml of distilled water and filtered.

The filtrate was extracted with 5 ml of benzene, twice. The combined benzene extract was divided into two portions. One portion was reserved as blank. To the second portion, about 5 ml of ammonia solution was added. The alkaline layer was observed for color change. b. Modified Borntrager Test An equivalent of 1 g of the plant extract was evaporated to incipient dryness using a water bath. The residue was taken up with 10 ml of 0. 5 N KOH and 1 ml diluted H2O2. It was heated over the water bath for 10 minutes. It was cooled and filtered. Glacial acetic acid was added drop wise until acidic to litmus paper.

It was extracted with 5 ml of benzene, two times. The combined benzene extract was divided into two portions. One portion was reserved as control. To the second portion about 5 ml of ammonia solution was used to alkalinify the sample. The alkaline layer was observed for color change. The red or pink color indicates the presence of anthraquinone. G. Test For Resin Phloroglucinol Test The equivalent 1 g of the plant extract was evaporated to incipient dryness using a water bath. Fews drops of phloroglucinol and hydrochloric acid were added to the residue and observed for any formation of brick red precipitate. H.

Test for Cyanogenic Glycosides Guignard Test About 2-5 g of the crushed plant sample was placed in a test tube. It was moistened with water and few drops of chloroform were added to enhanced enzyme activity. For a firm stopper on the tube, a cork from which a piece of picrate paper was suspended was used. The paper strip did not touch the inner sides of the test tube. The tube was warmed at 35-40? C or it was kept at room temperature for 3 hours. Any change in color of the paper was observed. The appearance of various shades of red within 15 minutes is a measure of relative concentration of cyanogenic glycosides.

If no color is observed after 3 hours, absence of glycoside is indicated. I. Test for Fixed oils Stain Test About 2 g of the shredded fresh plant sample was weighed, and boiled in a beaker containing 10 ml of petroleum ether. About 3 drops of the of the extract was dropped in the filter paper and watch glass. Any stain produced was noted. Quantitative Analysis A. Moisture Content Determination The plant sample was prepared by cutting or shredding so that the parts are about 3 mm in thickness. About 10 g of the plant sample was accurately weighed in a tared evaporating dish and then dried at 105 degree centigrade for 5 hours.

Cooled and weighed. The heating, drying and weighing was continued at 1 hour interval until the loss is not more 0. 25% or until the weight was constant. The moisture content was determined from the weight of the plant sample taken. • M. Knevel, F. E. Digandi, Jenkin’s Quantitative Pharm. Chem. ) B. Ash Content Determination About 10 g of the ground plant sample was accurately weighed in a tared crucible and incinerated at low temperature not to exceed very dull redness (500 to 550 degree) until free from carbon and the weight of the ash was determined. A. M. Knevel, F. E. Digandi, Jenkin’s Quantitative Pharm. Chem. C. Acid Insoluble Ash Accurately weigh quantity of Pakong –alagdan representing 4 grams of oven dried sample in a tarred crucible and incinerate at low temperature, not to exceed very dull redness until free from carbon and determined the weight of ash. Boil the ash obtained with 25 ml dilute HCl for 5 minutes. Collect all the insoluble in in asl less filter paper , wash with hot water, ignite and weigh. Chapter IV Results and Discussion Phytochemical Screening Table 1 Test for Alkaloid Tests Perform |Reagents |Results | | | | | | |Wagner’s |Light brown solution | |Preliminary test | | | | | | | | |Mayer’s |Light brown solution | | | | | | |Wagner’s |Light Yellow solution | |Confirmatory test | | | | | | | | |Mayer’s |Light Yellow solution | | | | |Quaternaryand Amine Oxide |Wagner’s |brown solution | | | | | |Bases test | | | | | | | | |Mayer’s |brown solution | Table I presents as findings of the absence of alkaloids for the two test reagents used namely Mayers and Wagners the results indicates absence of Alkaloids due to non- formation of precipitate on the leaves of the sample. Table 2 Test for Saponin Glycoside | | |Tests perform |Results | | | | |Froth test |After 3 min: 1. 3cm | | | | | |After 30 min: 1 cm | | | |Hemolysis test |1 mm halo | | | | |Liebermann-burchard test |Formation of green solution | Table 2: The formation of froth that does not change after 3 and 30 minutes interval with 1. 8 cm measurement, formation of halo in hemolysis test and of color change in the Liebermann-burchard test indicates the presence of saponin glycoside. Table 3 Test for Cardenolides and Bufadienolides | | |Tests perform |Results | | | | |Keller-killiani Test |formation of greenish black solution | | | | | |Formation of 2 layers; upper layer is aqua | |Salkwoski Test | | | |green in color, while the lower layer is yellow | | | | | |in color | | | |Kedde reaction |formation of yellow brown solution | Table 3: In the Keller-killiani Test the The formation of greenish black solution indicates the absence of 2 deoxysugars. In the Salkwoski Test the formation of aqua green color in the upper layer and yellow in color in the lower layer indicates the presence of cholesterol. While in the Kedde reaction there is no formation of blue violet color hence it indicates the absence of unsaturated lactone ring. Table 4 Test for Flavonoids | | |Tests Perform |Results | | | | |Bate-Smith and Metcalf Test for |formation of dark green solution | | | | |Leucoanthrocyanins | | | | | |Wilstatter “cyanidin” test |formation of brown solution | Table 4: In the Bate-Smith and Metcalf Test, there is no formation of strong red or violet color therefore indicates the absence of Leucoanthocyanins. While in the Wilstater “cyaniding” Test, there is no color change of Wilstatter Cyanidin test, indicates the absence of y- benzopyrone nucleus. Table 5 Test for Tannins and Polyphenols | | |Tests Perform |Results | | | | | |1% gelatin solution- formation of | | | | |Gelatin test |yellowish green solution with few precipitate | | | | | Gelatin salt reagent- formation of yellowish | | | | | |green solution with few precipitate | | | | |Ferric chloride Test |formation of brownish green solution | Table 5: The formation of precipitates in the gelatin test and formation of brownish green solution in the ferric chloride indicates the presence of tannins specifically condensed tannins. Table 6 Test For Anthraquinone | | |Tests perform |Results | | | | |Borntrager Test |Light yellow solution | | | | |Modified Borntrager Test |formation of 2 layers, the upper layer is clear | | | | | |while the lower layer is yellow in color | Table 6: There is no formation of pink or red color in both tests therefore indicates the absence of Flavonoids. Table 7 Test for Resin | | |Test Perform |Results | | | | |Phloroglucinol Test |blue green residue | Table 7: There is no formation of bloody red color therefore indicates the absence of Resin Table 8 Test for Cyanogenic Glycoside | | |Test perform |Result | | | | |Guignard Test |Formation of slight reddish brown color at the | | | | | |tip of the yellow picrate paper. | Table 8: The slight reddish brown color on yellow picrate paper indicates that there is a trace of cyanogenic glycoside. Table 9 Test for Fixed oil | | |Test Perform |Result | | | | |Stain Test |Filter paper- No oily stain produced | | | | | |Watch glass- colorless residue | Table 9: There is no oily stain in the filter paper and greasy appearance in the watch glass therefore indicates the absence of fixed oil. Appendix Preparation of Alcoholic Extract weight of the beaker ________ 50. 3 g___ weight of the beaker + plant sample _______ 100. 3 g__ weight of the plant sample ______ 50g_____ volume of the plant extract _________32mL __

Equivalent of plant material/ml of the extract _____ 1. 56g/mL__ Calculation: Equivalent of plant material/ml = weight of sample volume of sample = ____50g___ 32mL = 1. 56g/mL A. Moisture Content Determination DATA T1 T2 wt. of tared evaporating dish 43. 7g 43. 3g wt. of fresh plant sample10g 10g wt. of evap. dish + plant sample 53. 7g 53. 3g wt. of evap. dish + dried plant sample 50. 2g 50. 1g wt. of dried plant sample 6. 5g 6. 8g loss of wt. f the plant sample 3. 5g 3. 2g Computation: Trial 1 loss in wt. of plant sample = wt. of fresh sample – wt. of dried plant sample = 10g – 6. 5g = 3. 5g % Moisture = loss in wt. of plant sample wt. of fresh plant sampleX 100 = 3. 5g X 100 10g = 35 % Trial 2 loss in wt. of plant sample = wt. of fresh sample – wt. of dried plant sample = 10g – 6. 8g =3. 2g % Moisture = loss in wt. of plant sample wt. of fresh plant sample x 100 = 3. 2g X 100 10g = 32% Average % Moisture Average % Moisture = T1 + T2 2 = 35% + 32% 2 = 33. 5% B.

Ash Content Determination Data: T1 T2 wt of crucible 35. 7g 35. 2g crucible + dried plant sample 38. 7g38. 2g wt of dried plant sample 3g 3g wt of crucible + ash 36. 3g35. 8g wt of ash 0. 6g 0. 5g Computation: Trial 1 % Ash Content = __wt of ash _ x 100 Wt of dried plant sample = 0. 6g _ x 100 3g = 20% Trial 2 Ash Content = _______wt of ash ___ _ x 100 Wt of dried plant sample = ____0. 5g_ x 100 3g = 16. 67% Average % Ash Content Ash Content = T1 + T2 2 = 20% + 16. 67% x 100 2 = 18. 33% ACID INSOLUBLE ASH Acid-insoluble Ash Trial 1 % Acid Insoluble Ash= wt. of acid insoluble ash x 100 Ws = _0. 2g_ X 100 3 g = 6. 67% Acid-insoluble Ash Trial 2 % Acid Insoluble Ash= wt. of acid insoluble ash x 100 Ws = _0. 1 g_ x 100 3 g = 3. 33% Average % Acid Insoluble Ash Acid Ash Content = T1 + T2 2 = 6. 67% + 3. 33% x 100 2 = 8. 34%

Efficacy test: CONCENTRATION OF METHANOLIC, ETHANOLIC EXTRACT AND CONTROL: Control used: Combantrin 125mg/5ml 125mg X 1g = 0. 125g 1000 0. 500g X 100ml = 10% 5ml 1g X 100ml = 20% 5ml 1. 5g X 100ml = 30% 5ml Weight of earthworms (Poristima pustuma) used: | | |Weight | | |% Concentration | | |Control (Combantrin) 125mg/5mL |10% |0. g | | |20% | | | |30% | | |Methanolic extract |10% |0. 7 g | | |20% | | | |30% | | |Ethanolic extract |10% |0. g | | |20% | | | |30% | | Time of Death after introducing 3drops: | |10% |20% |30% | |Control (Combantrin) | 2hrs and 2mins |1hr and 1min. | 56 mins. | |Methanolic extract |2hrs and 6mins |1hr and 15mins. |1hr and 4mins. | |Ethanolic extract |2hrs and 22mins |2hrs and 1min. |1hr and 32 min. | CHAPTER 5 Summary:

The research was done for the purpose of determining the phytochemical constituents presents in Kulitus (Amaranthus viridis) leaves as well as to determine the wormicidal activity of the ethanolic and methanolic extracts of the Kulitis (Amaranthus viridis) leaves that is effective in stunning intestinal parasites. The leaves of the plant were macerated with 100ml of 80% ethyl alcohol. The ethanolic extracts of Kulitis (Amaranthus viridis) leaves were screnned for the presence of various phytochemical constituents such as glycosides, flavonoids, and phenolic compounds . The leaves was macerated with methanol and ethanol extract into two separated erlenmeyer flasks and it was extracted using achless filter paper. This extract was subjected to test to be able to determine the Anthelmintic Property of Ethanolic and Methanolic extract of the Kulitis leaves (Amaranthus viridis). Findings:

Based on the preliminary phytochemical screening it was found that the leaves of Kulitis (Amaranthus viridis) extracts contains saponin, tannin espicifically condensed tannins and cyanogenic glycosides which give a positive results. The moisture of the dried leaves of of Kulitis (Amaranthus viridis) was determined using the digital balance, which yield an Average of 33. 5%. The ash determination was also performed using digital balance, which yield an average of 34. 9%. The ethanolic and methanolic extracts of of Kulitis (Amaranthus viridis) leaves exhibited dose-dependent paralysis ranging from loss of motility to loss of response to external stimuli which eventually progressed to death. At 10, 20, 30mg/ml concentrations of ethanolic extracts death was observed espectively at 2hrs and 22mins, 2hrs and 1minute, 1hour and 32minutes. At 10, 20, 30mg/ml concentrations of methanolic extracts death was observed respectively at 2hrs and 6mins, 1hour and 15minutes, 1hour and 4minutes. While the control Combantrin at 10, 20, 30mg/ml concentrations death was observed repectively at 2hrs and 2mins, 1hour and 1minute, 56 minutes. Conclusion: In conclusion , the Kulitis (Amaranthus viridis) leaves have different constituents present s such as saponin, tannins specifically condensed tannins and cyanogenic glycosides. The authors also concluded that methanolic and ethanolic extract are effective in causing death to the parasites (earthworms).

The wormicidal activity of methanolic extracts of the Kulitis (Amaranthus viridis) leaves suggests that it could be more effective against parasitic infections of humans compared to ethanolic extract of the Kulitis (Amaranthus viridis) leaves. Recommendations: A need for further experimental procedures to test specific constituents present in the plant Kulitis (Amaranthus viridis) which possesess anthelmintic property that could be very effective in causing death to parasites which could lead to the discovery of new anthelmintic products in the market. The researchers also recommended to use a higher concentrations of the ethanolic and methanolic extract in experiment. Bibliography (Borgers and de Nollin, 1975) (Philippine Medicinal Plants (http://www. stuartxchange. org/Kolitis. html) (Kokate C. K. 1986) (Eduardo Quisumbing (1951) (Harder, von Samson-HYPERLINK “http://www. wormbook. org/chapters/www_anthelminticdrugs/anthelminticdrugs. html”HimmelstjernaHYPERLINK “http://www. wormbook. org/chapters/www_anthelminticdrugs/anthelminticdrugs. html”, 2002) (Gilles and Hoffman 2002) (Kirtikar, K. R. , and B. D. Basu (1987), (Hassan Sher and Z. D. Khan (2006), (Quershi, S. J. , Khan, M. A. , M. Ahmed (2008) (Microsoft ® Encarta ® 2009. ) (Martin, 1985) Quisumbing, Dr. Eduardo Medicinal Plants of the Philippines Quezon City: Katha Publishing Co. 1978 (Trakia Journal of Science, Vol. 6 (4), 39-51 Mark Turin (2003). Tyler, Varro E. Brady, Lynn R.

Robbers, James E. Pharmacognosy. Lea ; Febiger. Philadelphia,1988 USP XXII/NF XVII,1970 (Knevel and Digangi, 1977) Abstract The Anthelmintic Property of Methanolic and Ethanolic extract of the Kulitis leaves (Amaranthus viridis) College of Pharmacy , Lyceum- Northwestern University ,Tapuac District, Dagupan City. This research was conducted to determine the anthelmintic property of methanolic and ethanolic extract of the Kulitis leaves (Amaranthus viridis) using different concentrations. The research made used of phytochemical screening and quantitative analysis. The leaf extract of Amaranthus viridis is prepared by macerating in 80% ethyl alcohol.

Then phytochemical screening was done to determine the presence of saponin glycosides, tannins, cardenolides and bufadienolides, anthraquinones, cyanogenic glycosides, and flavonoids. After which Quantitative analysis is done by calculating its moisture, ash content and acid insoluble ash content. Results show the presence of saponin glycoside, flavonoids, tannins espicifically condensed which give a positive results in preliminary test . Indian adult earthworms (Poristima pustuma) were used to study anthelmintic activity due to its anatomical and physiological resemblace to the human intestinal roundworm parasite. The earthworms were collected from moist soil and washed with Normal saline to remove all fecal matter. Earthworms weighed 0. grams were used for all experimental protocol. The earthworms were divided into three different groups, each group containing 3 worms. Five mL concentrations of methanolic, ethanolic extracts of Kulitis (Amaranthus viridis) leaves and Combantrin used as a control (10,20,30mg/mL distilled water) were prepared. Three drops of the prepared concentrations was used to introduce into the earthworms. The time of death was noted when no movement of any sort was observed. Appendices Figure 1. Picture of the whole plant of Kulitis (Amaranthus Viridis) Figure 2. Picture of the leaves of Kulitis (Amaranthus viridis) used in the expirement In vivo test Figure 5.

Picture of the earthworm before dropping 10% ethanolic extract. Figure 6. Picture of the dead earthworm after droping a 10% ethanolic extract. Figure 7. Picture of the earthworm before droping a 10% methanolic extract. Figure 8. Picture of the dead earthworm after droping a 10% methanolic extract. Figure 9. Picture of the earthworm before droping a 10% Combantrin. Figure 10. Picture of the dead earthworm after droping a 10% Combantrin. Figure 11. Picture of the earthworm before droping a 20% ethanolic extract. Figure 12. Picture of the dead earthworm after droping a 20% ethanolic extract. Figure 13. Picture of the earthworm before droping a 20% methanolic extract. Figure 14.

Picture of the dead earthworm after droping a 20% methanolic extract. Figure 15. Picture of the earthworm before droping a 20% Combantrin. Figure 16. Picture of the dead earthworm after droping a 20% Combantrin. Figure 17. Picture of the earthworm before droping a 30% ethanolic extract. Figure 18. Picture of the dead earthworm after droping a 30% ethanolic extract. Figure 19. Picture of the earthworm before droping a 30% methanolic extract. Figure 20. Picture of the dead earthworm after droping a 30% methanolic extract. Figure 21. Picture of the earthworm before droping a 30% Combantrin. Figure 22. Picture of the dead earthworm after droping a 30% Combantrin.