CHAPTER I
INTRODUCTION

Background of the Study The papaya, papaw, or pawpaw is the fruit of the plant Carica papaya, the sole species in the genus Carica of the plant family Caricaceae. It is native to the tropics of the Americas, perhaps from southern Mexico and neighbouring Central America. It was first cultivated in Mexico several centuries before the emergence of the Mesoamerican classical civilizations. Papaya leaves contains coumarin, papain and iron which are the three main essentials that increase blood platelets.
Dengue viruses, mosquito-borne members of the Flaviviridae family, are the causative agents of dengue fever. Dengue is the most important emerging viral disease of humans that in recent decades has become a major international public health concern. There is a widespread of dengue cases in every corner of our country. Many people can’t afford to go to hospitals just to get medical assistance especially indigent families. Therefore because of the expensive disbursement in prestigious hospitals such as medicines needed to cure dengue, affected people die without being aided. Most people know about papayas but not many know about the amazing health benefits of Papaya Leaves. Papaya Leaves contain powerful healing compounds that are very important for great health and vitality... and for curing cancer and dengue fever. With the rising number of people catching dengue fever, the demand for papaya leaf juice has soared. The extract of raw papaya leaf helps boost platelets, also known as thrombocytes. Thus, they can formulate an alternative tea out of a simple leaf.
Purpose of the Study The purpose of this study is to inform people that even they suffer from dengue fever, they don’t have to spend for their treatment and this study also inform people about the health benefits that they can get from simple leaves such as C. papaya leaves.
Scope and Limitations This study will determine the effectiveness of C. papaya leaves extract as an alternative tea. The gathering of the materials was selected from the researchers’ backyards in Rosales, Pangasinan. The C. papaya leaves will be mixed in a boiling water. The experimental variables in this study will be the C. papaya leaves extract at different amounts while the control variables will be the salt that will be added while undergoing the decoction process. The amount of water that will be used in the preparation of the tea will be not held constant. The tea won’t be possible if the temperature of the water is not enough to boil the C. papaya leaves and if the researchers don’t have enough equipment such as stove, casserole and water. Additional studies such as the effectiveness of C. papaya leaves extract for dietary means, anti-cancer, removal of intestinal worms and its effectiveness in movement of bowels will not be included. The study will be conducted at the school laboratory in Rosales National High School with the supervision of the Research adviser or at home.

Statement of the Problem This study will aim to lessen the community garbage such as the papaya leaves scattered everywhere. The main objective of the current study is to investigate the potential of C. papaya leaves extracts against Dengue fever. The following questions will be answered: 1. Does the C. papaya leaves effective for increasing the blood platelets of a dengue fever patient? 2. Is there a need to prepare C. papaya leaves extract in the production of an alternative tea for dengue fever? 3. Is it possible that C. papaya leaves can be extracted by decoction process? 4. Is there any content of C. papaya leaves that makes this study possible in producing alternative tea? 5. Are there any advantages that the economy will benefit from this study?
Hypothesis:
Problem No. 1
H₀: The C. papaya leaves are not effective for increasing the blood platelets of a dengue fever patient.
H₁: The C. papaya leaves are effective for increasing the blood platelets of a dengue fever patient.
Problem No. 2
H₀: There is no need to prepare C. papaya leaves extract in the production of an alternative tea for dengue fever.
H₁: The preparation of C. papaya leaves extract in the production of an alternative tea for dengue fever is needed.
Problem No.3
H₀: C. papaya leaves cannot be extracted by decoction process.
H₁: C. papaya leaves can be extracted by decoction process.
Problem No. 4
H₀: The C. papaya leaves do not contain a content that makes this study possible in producing alternative tea.
H₁: There is a content in C. papaya leaves that makes this study possible in producing alternative tea.
Problem No. 5
H₀: There are no advantages that the economy will benefit from this study.
H₁: There are advantages that the economy will benefit from this study.

Significance of the Study This study will benefit the following: To the students: This study could serve as a source of feedback in the monitoring of curiosity, willingness and dedications for them to create their own experimental set-up and expose students’ oral communications on reasoning skills. This will provide for students to create better research projects out of waste materials. To the future researchers: The findings of the study may provide them insights to further evaluate the use of C. papaya leaves to produce an alternative tea.
Definition of Terms
Coumarin - is a fragrant organic chemical compound in the benzopyrone chemical class, which is a colorless crystalline substance in its standard state. It is a natural substance found in many plants. Coumarin has blood-thinning and anti-fungicidal effects. This medication is a blood thinner used to keep blood flowing smoothly and prevent the formation of blood clots. Dengue Fever - Dengue fever is a painful, debilitating mosquito-borne disease caused by any one of four closely related dengue viruses. These viruses are related to the viruses that cause West Nile infection and yellow fever.
Thrombocytes - are small anuclear fragments of megakaryocytes. They are membrane bound portions of the megakaryocyte cytoplasm and have a finely granular cytoplasm; they are much smaller than other blood cells at 2-3µm and have a lifespan of around 10 days in the circulation.

Tea - is an aromatic beverage commonly prepared by pouring hot or boiling water over cured leaves of the Camellia sinensis, an evergreen shrub native to Asia. After water, it is the most widely consumed beverage in the world.

Iron - is a mineral that our bodies need for many functions. For example, iron is part of hemoglobin, a protein which carries oxygen from our lungs throughout our bodies. It helps our muscles store and use oxygen. Iron is also part of many other proteins and enzymes.
Papain - is a powerful digestive enzyme commonly found and extracted from the papaya fruit (Carica papaya), it is also referred to as papaya proteinase. The papaya-enzyme papain plays a key role in digestive processes involving breaking down tough protein fibers.
Conceptual Framework Independent Variable | Dependent Variable | Papaya leaves extract | Increase of blood platelets |

Chapter 2
REVIEW OF RELATED LITERATURE

Papaya Carica Papaya
Objective
To investigate the potential role of fresh Carica papaya (C. papaya) leaf extract on haematological and biochemical parameters and toxicological changes in a murine model.
Methods
In total 36 mice were used for the trial. Fresh C. papaya leaf extract [0.2 mL (2 g)/mouse] was given only to the test group (18 mice). General behavior, clinical signs and feeding patterns were recorded. Blood and tissue samples were collected at intervals. Haematological parameters including platelet, red blood cell (RBC), white blood cell (WBC), packed cell volume (PCV), serum biochemistry including serum creatinine, serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) were determined. Organs for possible histopathological changes were examined.
Results
Neither group exhibited alteration of behavior or reduction in food and water intake. Similarly, no significant changes in SGOT, SGPT and serum creatinine levels were detected in the test group. Histopathological organ changes were not observed in either group of mice except in three liver samples of the test group which had a mild focal necrosis. The platelet count (11.33±0.35)×105/µL (P=0.000 04) and the RBC count (7.97±0.61)×106/µL (P=0.000 03) were significantly increased in the test group compared to that of the controls. However, WBC count and PCV (%) values were not changed significantly in the test group. The platelet count in the test group started to increase significantly from Day 3 (3.4±0.18×105/µL), reaching almost a fourfold higher at Day 21 (11.3×105/µL), while it was 3.8×105/µL and 5.5×105/µL at Day 3 and Day 21 respectively in the control. Likewise, the RBC count in the test group increased from 6×106/µL to 9×106/ µL at Day 21 while it remained near constant in the control group (6×106/µL).
Conclusions
Fresh C. papaya leaf extract significantly increased the platelet and RBC counts in the test group as compared to controls. Therefore, it is very important to identify those chemicals of C. papaya leaves as it can be recommended to be used as a medication to boost thrombopoiesis and erythropoiesis in humans and in animals in which these cell lineages have been compromised.
Keywords: Carica papaya, Alternative medicine, Erythropoiesis, Thrombopoiesis, Toxicity
Carica papaya (C. papaya, family Caricarceae, papaya) is one of the most popular and economically important plants in the world as its fruit is a common delicacy. It is a soft wooded single-stemmed perennial tree, 2-10 m in height, with a crown of large palmate leaves emerging from the apex of the trunk. The soft, hollow, cylindrical trunk ranges from 30 cm in diameter at the base to about 5 cm in diameter at the crown. Although native to Central America, it has been transported to many parts of the tropics.
The papaya plant is lactiferous as it contains specialized cells known as lactifers that occur in most tissues and secrete latex. Lactifiers secrete latex are dispersed throughout most plant tissues. The papaya-latex is well known for being a rich source of the four cysteine endopeptidases namely papain, chymopapain, glycyl endopeptidase and caricain. Leaves contain an alkaloid called carpaine and a glucoside named carposide.
Different parts of the papaya plants including fruit, dried fruit, leaves, dried leaves, stems, seeds and roots have long been used as ingredients in alternative medicine. For instance, the seeds are used for expelling worms and roots and seeds are used as an abortifacient agent. The leaves (especially fallen ones) are used variously for the treatment of fevers, pyrexia, diabetes, gonorrhoea, syphilis, inflammation and as a dressing for septic wounds.
Untested herbal medicines could be potentially injurious to human health. Many plants used in traditional and folk medicines are potentially toxic, mutagenic, and carcinogenic. Toxicological studies of extracts from different parts of C. papaya plants such as seeds, fruit, roots and leaves have been carried out using several animal models. Acute and chronic toxicities of unripe fruit of the C. papaya have been documented.
Some of the traditional claims of efficacy that have been investigated scientifically using animal models and their efficacy have been proven. Recent studies showed that C. papaya leaf extract has potential anti-sickling (inhibition of sickle cell formation) properties. Indran et al. have shown that there is a protective effect against gastric ulcers in rats. Moreover, C. papaya flowers are known to have antibacterial activities. The contraceptive efficacy of the seeds of C. papaya has been earlier demonstrated in rats, mice and rabbits. Oral administration of C. papaya seed extract could induce reversible male infertility and could be used for pharmaceutical development of a male contraceptive.
Today, many tropical and subtropical countries are engulfed by dengue infection which is caused by viruses belonging to the Flaviviridae family. There is no specific therapy for dengue even though the infection has a significant mortality. Sri Lanka is no exception where, dengue infection is a priority for the national health services for prevention and reducing mortality. In desperation, many people have resorted to use papaya leaf extract covertly. This is applicable even to hospitalized dengue patients. However, recommending C. papaya leaf extract for dengue infection is unethical until it is proven by scientific research.

Papaya Leaves Papaya leaves have medicinal values. In folk medicines, papaya leaves extract are used as dietary means. They are also used in wounds and in snake bites. The papaya benefits are attributed to the nutrients in the papaya leaf extract such as minerals magnesium, potassium, iron, most aminoacids, coumarins and vitamins A, C, and B. Some of the key phytonutrients include quinic acid, rutin, malic acid and its derivatives, and the papaya enzyme papain. The potent antioxidant activity of papaya leaf extract is due to the array of phenolic compounds such as caffeic acid, chlorogenic acid, quercetin and kaempferol which are the chief constituents. The key papaya benefits include immune boosting and antioxidant properties. Papaya leaf extract is derived from the leaves of the papaya tree. Papaya leaf extract are one of the popular dietary supplements in Europe and fermented papaya products are commonly used in Japan for many decades. The papaya leaf extract is prepared by a natural fermentation process, after which the papaya is dried and ground into a powder.

Papain Papain, also known as papaya proteinase I, is a cysteine protease (EC 3.4.22.2) enzyme present in papaya (Carica papaya) andmountain papaya (Vasconcellea cundinamarcensis).
Papain belongs to a family of related proteins with a wide variety of activities, including endopeptidases, aminopeptidases, dipeptidyl peptidases and enzymes with both exo- and endo-peptidase activity. Members of the papain family are widespread, found in baculovirus eubacteria, yeast, and practically all protozoa, plants and mammals. The proteins are typically lysosomal or secreted, and proteolytic cleavage of the propeptide is required for enzyme activation, although bleomycin hydrolase is cytosolic in fungi and mammals. Papain-like cysteine proteinases are essentially synthesised as inactive proenzymes (zymogens) with N-terminal propeptide regions. The activation process of these enzymes includes the removal of propeptide regions, which serve a variety of functions in vivo and in vitro. The pro-region is required for the proper folding of the newly synthesised enzyme, the inactivation of the peptidase domain and stabilisation of the enzyme against denaturing at neutral to alkaline pH conditions. Amino acid residues within the pro-region mediate their membrane association, and play a role in the transport of the proenzyme to lysosomes. Among the most notable features of propeptides is their ability to inhibit the activity of their cognate enzymes and that certain propeptides exhibit high selectivity for inhibition of the peptidases from which they originate.
The papain precursor protein contains 345 amino acid residues, and consists of a signal sequence (1-18), a propeptide (19-133) and the mature peptide (134-345). The amino acid numbers are based on the mature peptide. The protein is stabilised by threedisulfide bridges. Its three-dimensional structure consists of two distinct structural domains with a cleft between them. This cleft contains the active site, which contains a catalytic diad that has been likened to the catalytic triad of chymotrypsin. The catalytic diad is made up of the amino acids - cysteine-25 (from which it gets its classification) and histidine-159. Aspartate-158 was thought to play a role analogous to the role of aspartate in the serine protease catalytic triad, but that has since then been disproved. Nonetheless, it does appear to play a role.
The mechanism by which papain breaks peptide bonds involves the use of a catalytic triad with a deprotonated cystine. Asp-175 helps to orient the imidazole ring of His-159 to allow it to deprotonate the catalytic Cys-25. This cysteine then performs a nucleophilic attack on the carbonyl carbon of a peptide backbone. This frees the amino terminal of the peptide, and forms a covalent acyl-enzyme intermediate. The enzyme is then deacylated by a water molecule, and releases the carboxy terminal portion of the peptide. In immunology, papain is known to cleave the Fc (crystallisable) portion of immunoglobulins (antibodies) from the Fab (antigen-binding) portion.
Papain is a relatively heat resistant enzyme, with a temperature optimal range of 60-70°C. Papain prefers to cleave at: (hydrophobic)-(Arg or Lys)- cleaves here -(not Val). Hydrophobic is Ala, Val, Leu, Ile, Phe, Trp, or Tyr.[8]Papain breaks down tough meat fibres, and has been used for thousands of years to tenderise meat eaten in its native South America. Meat tenderisers in powder form with papain as an active component are widely sold.
Papain can be used to dissociate cells in the first step of cell culture preparations. A ten-minute treatment of small tissue pieces (less than 1 mm cubed) will allow papain to begin breaking down the extracellular matrix molecules holding the cells together. After ten minutes, the tissue should be treated with a protease inhibitor solution to stop the protease action (if left untreated, papain's activity will lead to complete lysis of the cells). The tissue must then be triturated (passed quickly up and down through a Pasteur pipette) to break up the pieces of tissue into a single cell suspension.
It is also used as an ingredient in various enzymatic debriding preparations, notably Accuzyme. These are used in the care of some chronic wounds to clean up dead tissue.
Papain is also used in some toothpastes and mint sweets as a tooth whitener; however, its whitening effect is minimal, because the papain is present in low concentrations and is quickly diluted by saliva. It would take several months of use to have a noticeable effect.
It is the main ingredient of Papacarie, a gel used for chemomechanical dental caries removal. It does not require drilling and does not interfere in the bond strength of restorative materials to dentin.
Papain has been known to interfere with urine drug tests for cannabinoids. It is found in some drug detox products.
An antibody digested by papain yields three fragments: two 50 kDa Fab fragments and one 50kDa Fc fragment. The papain-digested antibody is unable to promote agglutination, precipitation, opsonization, and lysis.
Papain is usually produced as a crude, dried material by collecting the latex from the fruit of the papaya tree. The latex is collected after scoring the neck of the fruit, where it may either dry on the fruit or drip into a container. This latex is then further dried. It is now classified as a dried, crude material. A purification step is necessary to remove contaminating substances. This purification consists of the solubilization and extraction of the active papain enzyme system through a government-registered process. This purified papain may be supplied as powder or as liquid.

Coumarin Coumarin is a fragrant organic chemical compound in the benzopyrone chemical class, which is a colorless crystalline substance in its standard state. It is a natural substance found in many plants.
The name comes from a French term for the tonka bean, coumarou, one of the sources from which coumarin was first isolated as a natural product in 1820. It has a sweet odor, readily recognised as the scent of new-mown hay, and has been used in perfumes since 1882. Sweet woodruff, Hierochloe odorata (sweet grass) and sweet-clover in particular are named for their sweet smell, which in turn is due to their high coumarin content. When it occurs in high concentrations in forage plants, coumarin is a somewhat bitter-tasting appetite suppressant, and is presumed to be produced by plants as a defense chemical to discourage predation.
Coumarin is used in certain perfumes and fabric conditioners. Coumarin has been used as an aroma enhancer in pipe tobaccos and certain alcoholic drinks, although in general it is banned as a flavorant food additive, due to concerns regarding its hepatotoxicity in animal models.
Coumarin was first synthesized in 1868. It is used in the pharmaceutical industry as a precursor reagent in the synthesis of a number of synthetic anticoagulant pharmaceuticals similar to dicoumarol, the notable ones being warfarin (brand name Coumadin) and some even more potent rodenticides that work by the same anticoagulant mechanism. Coumarins are a type of vitamin K antagonists. Pharmaceutical coumarins were all developed from the study of sweet clover disease; see warfarin for this history.
Coumarin has clinical medical value by itself, as an edema modifier. Coumarin and other benzopyrones, such as 5,6-benzopyrone, 1,2-benzopyrone, diosmin, and others, are known to stimulate macrophages to degrade extracellular albumen, allowing faster resorption of edematous fluids. Other biological activities that may lead to other medical uses have been suggested, with varying degrees of evidence.

Iron Iron is a chemical element with the symbol Fe (from Latin: ferrum) and atomic number 26. It is a metal in the first transition series.[3] It is by mass the most common element on Earth, forming much of Earth's outer and inner core. It is the fourth most common element in the Earth's crust. Its abundance in rocky planets like Earth is due to its abundant production by fusion in high-mass stars, where the production of nickel-56 (which decays to the most common isotope of iron) is the last nuclear fusion reaction that is exothermic. Consequently, radioactive nickel is the last element to be produced before the violent collapse of a supernova scatters precursorradionuclide of iron into space. Iron plays an important role in biology, forming complexes with molecular oxygen in hemoglobin and myoglobin; these two compounds are common oxygen transport proteins in vertebrates. Iron is also the metal used at the active site of many important redox enzymesdealing with cellular respiration and oxidation and reduction in plants and animals. Iron is an essential mineral contained in your red blood cells and is what allows them to carry oxygen to every cell in your body, the University of Maryland Medical Center notes. Iron is also stored in your liver, muscles and bone marrow. Iron deficiency is a typical cause of anemia. Iron deficiency anemia causes your platelet count to increase, according to a study published in a 2009 issue of "Platelets." This suggests that iron levels play a role in platelet production.

Other Benefits of the C. papaya leaves Indigestion-Heartburn - Papaya Leaves are great for getting rid of invading bacteria that cause upset stomach problems because they contain karpain which kills bacteria. And it contains lots of papain, protease enzyme, and amylase enzyme which help to breaks down proteins, carbs, and sooth the GI tract... and helps with acid reflux.
Papaya Leaves reduce inflammation of the stomach lining and heal gastric ulcers by killing H. pylori bacteria. And Papaya Leaf tea soothes away colon inflammation from IBS and other inflammatory bowel diseases.

Break Down Wheat Gluten - Papaya Leaves have the ability to break down wheat gluten making it easy to digest.

Cancer - Papaya Leaves have a milky sap that’s great for preventing and killing cancer cells because it contains acetogenin.
There are many studies including studies in Japan showing that Papaya Leaves are effective for curing cancer. And there are people who have cured their cancer by drinking Papaya Leaf Tea for extended periods of time.
The University of Florida did studies showing that Papaya Leaves contain agents that kill cancer, especially cervix, prostate, liver, breast, and lung cancer. The more concentrated the tea the better the results... and there are no side effects of any kind.
Down through history in Australia the native aboriginal people have talked about Papaya Leaf Tea as a great cancer healing agent, also in Asia and other places.
Most recommend taking 10 leaves, cutting them up and boiling them in a 1/2 gallon water until it boils down to quart... then let it cool. The tea will keep in the frig for 2 days in a glass jar with a tight fitting lid... drink as much as possible.
Plus Papaya Leaf tea enhances chemotherapy... and helps with chemotherapy side effects.
Prostate Enlargement - Papaya Leaf tea can help with benign prostate enlargement and also help with rectal lesions problems associated with prostate enlargement.

Acne Remedy - If you have acne then take dried Papaya Leaves and putting them in a blender with just enough water to make a paste. Then take the paste and used it as a mask, let it dry, and then wash it off... it works great for acne!

Increase Platelet Count - Papaya Leaf Tea cures thrombocytopenia or low platelet counts which keeps a person from clotting. There have been many studies showing that Papaya Leaf Tea can increase platelet counts in cases of vitamin deficiencies, chemotherapy, dengue fever and more.

Menstrual Pain - Take 6 Papaya Leaves and boil them in a liter of water for 15 minutes. Then add any kind of juice that you like... let cool and drink one large glass 3 times a day.

Laxative - Papaya Leaves made into a strong tea also work well as a good laxative... for those who suffer from constipation.

Dengue Fever - Papaya Leaves made into a tea lower fevers and helps with colds, flues, and even dengue fever... and some even call it a cure for dengue fever.

Skin Problems - Papaya Leaves can help with fungal infections of the skin, warts, scars... and get rid of freckles.

Increase Appetite - Papaya Leaf tea increases appetite bringing vigor and vitality back to the body.

Cataracts - Papaya Leaf tea can prevent cataracts.

Emphysema - Papaya leaves contain lots of Vitamin D which prevents emphysema.

Immune Booster - Papaya Leaves contains lots of powerful antioxidants that boost your immune system and stave off cardiovascular disease, strokes, and cancer. Papaya Leaves create “Th1” a cytokines which boost the immune system and fights cancer.
Anti-Aging - Papaya Leaf also contains 50 or more different amino acids such as: threonine, glutamate acid, glycine, valine, leucine, phenylalanine, lysine, tritophan, cysteine, histidine, tyrosine, alanine, proline, aspartate, and more... and some of these agents are used in cosmetics for creating young healthy looking skin, and have been used as anti-aging compounds.

Contains - Papaya Leaf contains lots of great nutrients such as B-Vitamins for vitality and enhanced mood, Vitamins A and C which are powerful antioxidants, calcium and Vitamin D which are important for strong bones, Vitamin E which is important for sexual and cardiovascular health. Flavonoids that are powerful antioxidants that keep away cancer and cardiovascular disease, tannins that boost the immune system, and betacarotene for immune enhancement and eye health.

Chapter 3
METHODOLOGY

Acquisition of papaya leaves Papaya leaves will be gathered from the researcher’s backyard
In order to produce a tea out of C. papaya leaves extract, the researchers must have to gather materials that are capable of producing the certain tea. These will be the materials to be used:
Materials and Equipments
The materials to be used will be: * Water * Salt * boiling pot * cup * filter paper
Cleaning and Washing of the Leaves To remove all possible microbes or bacteria that may harm people, the researches should make sure that the leaves and the other materials that they will use are clean and safe.
Decoction of the leaves Coumarin is essential that extract of papaya leaves possesses so the researcher should extract the leaves by decoction.
Filtration of the Extract The researchers must filter the extract using the filter paper so that the unnecessary contents will be removed especially the microbes and bacteria.
General Procedure in Preparing the Tea
Take 2 to 5 pieces of C. papaya leaves plant. Cut off and exclude the roots. Wash the plant with running water. Clean it and make sure there are no foreign objects or dirt. Fill a boiling pot with clean water. Boil the papaya leaves for 1 (one) minute in a medium fire. While boiling, put 1 teaspoon of salt to reduce the bitterness. Wait for it to cool down. Place the strainer on top of the mouth of the pitcher and slowly pour the tea into the pitcher. Drink nothing but the papaya leaves tea for the next 12 to 24 hours. Sip 1 to 1.5 glasses of C. papaya water every hour for best results.
Flowchart
Gathering of Papaya Leaves.

Decoction of the leaves.
Cleaning of the leaves.

Filtration of the Extract.