EFFECTIVENESS OF CRUDE EXTRACT OF MAYANA LEAVES FOR BLOOD COAGULATION PROCESS

An Undergraduate Thesis Presented to the Faculty of the College of Allied Health Sciences Medical Technology Department Cagayan State University Andrews Campus, Tuguegarao City

In Partial Fulfillment Of the Requirements for the Degree Bachelor of Science in Medical Technology

By Rhoe Anne C. Calabazaron Randolph Ryan G. Gasmin Abigail C. Guinucay Jocelyn P. Oledan Niki Valencia D. Pamittan Rachelle I. Tugad March 2012

Chapter I THE PROBLEM AND ITS BACKGROUND Introduction Use of herbs and medicinal plants as the first medicines is a universal phenomenon. Every culture on Earth, through written or oral tradition, has relied on the vast variety of healing plants for their therapeutic properties. The majority of herbal products available today originated from the same traditional formulas or ingredients. The use of herbal medicines predates human history. Herbal medicine creates a more potent, effective and efficient treatment to ensure quicker responses to re-establish health and balance.

Most people used herbal medicines to treat their wounds, especially in rural areas. Some people use herbal perching for the leaves to produce extract and put the leaves on the wound. They believed that herbal plants are effective in treating wounds especially in minimizing its bleeding. Not knowing that these herbal plants are also effective in minimizing the bleeds on wounds which can be used as an immediate treatment for hemostasis. Herbal medicine can provide a better quality of life

(http://arightpath.com/herbal-medicine/importance.html).

In rural areas, people resorted in using herbal medicine to treat their wound without seeking consultation from a clinic or a hospital. They just get herbal medicine from their backyard because its available locally. Nowadays, people are educated about the usefulness of herbal medicines. All parts of a medicinal plant are used: roots, branches, twigs, stems, leaves, flowers, fruit and seeds. All have separate functions; for instance, the leaves are the most superficial part of a plant and therefore are potent at treating acute and superficial diseases.

Many people believe that herbs are just as effective as drugs, but without the side effects. Herbs do perform many healing functions in the body, but they must be used appropriately and recommended by a trained professional. Although herbal remedies are less likely than most conventional medicines to cause side effects, herbs nevertheless can be very potent.

However, the use of locally available medical plants has been advocated by the Department of Health. Many local plants and herbs in the Philippine backyard and field have been found to be effective in the treatment of common ailments as attested to by the National Science Development Board. The use of locally available medical plants has been advocated by the Department of Health. Many local plants and herbs in the Philippine backyard and field have been found to be effective in the treatment of common ailments as attested to by the National Science Development Board. (Cuevas, 2007)

The folkloric used of Mayana are for carminatives, bruises and sprains, headaches, mild bleeding of wounds, sinusitis, dyspepsia, and eye drops for eye irritations. The use of mayana is reported as an Asian traditional medicine for asthma, angina, bronchitis, epilepsy, insomnia, skin rashes (http://www.stuartxchange.com/ index.html).

Blood is of prime importance in the normal physiologic function of our major organ systems. When you cut yourself, the process of coagulation begins by the formation of a blood clot. This is followed shortly after by digestion or breakdown of the clot. Patients clot and/or bleed because of a variety of identifiable hemostatic abnormalities. The property of the circulation where the circulating fluid is maintained

within the blood vessels is referred to as hemostasis. Logical and effective treatment depends upon the proper identification of the abnormality. (Hemostasis Basics, Dade Behring, 2000). This research entitled Effectual Use of Crude Extracts of Mayana (Coeus blumei benth) leaves for blood coagulation attempts to determine the effective component of the mayana leaves in blood clotting time. Furthermore, this study was conducted to test the effectiveness of cephalin as a thromboplastic agent.

Theoretical Framework

Although the concept of the coagulation cascade represented a significant advance in the understanding of coagulation and served for many years as a useful model, more recent clinical and experimental observations demonstrate that the cascade/waterfall hypothesis does not fully and completely reflect the events of hemostasis in vivo. (Theories of Blood Coagulation; http://aphon.org/files/public/theories_of_coag.pdf)

The Classic Theory of Coagulation as Proposed by Paul Morawitz (1958), in which the presence of calcium and thromboplastin, prothrombin was believed to be converted to thrombin. In turn, the thrombin converted fibrinogen to fibrin, enabling the formation of a fibrin clot. Morawitz posited that all the ingredients of clotting were present in circulating blood and that the fact that such blood did not normally clot was due to the lack of a wettable surface in the blood vessels.

Another related theory is the Coagulation Cascade Model by Macfarlane (1964) appeared in the journal Nature described each clotting factor as a proenzyme that could be converted to an active enzyme. The cascade and waterfall models suggested that the clotting sequences were divided into 2 pathways. Coagulation could be initiated via an intrinsic pathway, so named because all the components were present in blood, or by an extrinsic pathway, in which the subendothelial cell membrane protein, tissue factor (TF), was required in addition to circulating components. The initiation of either pathway resulted in activation of FX and the eventual generation of a fibrin clot through a common pathway (Luchtman-Jones & Broze, 1995).

Furthermore, a major development of Cell-Based Model of Coagulation was the discovery that exposure of blood to cells that express TF on their surface is both necessary and sufficient to initiate blood coagulation in vivo. This finding led to the belief that the intrinsic pathway (the contact system) does not have a true physiological role in hemostasis (Hoffbrand et al., 2005). Very recent evidence suggests that although FXII deficiency does not result in bleeding problems, the absence of FXII does protect against pathological thrombosis (Kleinschnitz et al., 2006).

In the theory of Herbalism, the herbs have been used since the dawn of time as medicines and, in fact, many common drugs are in actual fact, made from herbal extracts. The natural chemical properties of certain herbs have been shown to contain of themselves, medicinal value. However, unlike conventional medicine, herbalists use the 'whole' herb or plant rather than isolating and breaking down chemical compounds and then synthesising it. This is because the plant, being a part of Nature, is said to represent perfect balance; healing requires the natural combination of elements in the plant or herb, not just a single chemical within it.

In relation with the use of Mayana leaves in hemostasis, the crude extract of this herb is also effective in blood clothing time. This is very useful in treating wounds. It can also be used as first aid especially in case of immediate management to prevent blood loss.

Research Paradigm

The Mayana and Cephalin are the Independent variables and the blood coagulation is the dependent variable. But there is a possibility that biological factors such as temperature, different blood types and their concentration as the intervening variables might affect the blood coagulation process.

Figure 1

Independent Variables

Intervening Variables

Dependent Variables

Mayana (Coeus Blumei benth)

- Temperature - Blood type - pH

Blood Coagulation

Cephalin (Positive Control)

Figure 1. Shows the Independent, Intervening and Dependent Variables on the Effectual Use of the Crude Extract of Mayana Leaves (Coeus Blumei benth) for Blood Coagulation Time in Different Blood Types.

Statement of the Problem

The study is designed to find out the effectual use of mayana leaves extract for blood coagulation. Specifically, it answers the following questions: 1. What is the medicinal composition of mayana leaves that is contributory to blood coagulation? 2. What is the extent of effectiveness of mayana leaves extract to hasten blood coagulation time. 3. What health risk possibilities does mayana leaves extract have to human body system? 4. Is there a significant difference in blood coagulation time between the four different blood types using mayana leaves extract?

Assumptions The assumptions of the study are presented in this from: 1.

Significance of the Study

The study is conducted to find out the effectiveness of mayana leaves extract as to the blood coagulation. The results of the study are expected to benefit the following: Remote areas. The researchers findings in this study would help the people inform in remote areas where there is no access to hospitals or clinic that herbal plant is essential to speed up the blood clot of the lesion. Furthermore, this study would provide additional option aside from the cephaline or any other commercially available medicine for wound in cases of emergency situations. Doctors. The results of this research will aid the doctors to advise what kind of herbal plant is efficient in treating an abrasion in case of urgent situation. Public health nurse. The findings of this research will endow additional knowledge regarding health information given or trained by public health nurse about the effectiveness of mayana leaves extract to the blood coagulation process. Researchers. The study would also serve as a proposal and a guide to the future researchers on what variables they would embrace or focus as they carry out another study associated to this subject matter.

Scope and Limitation of the Study

The scope of the study is on the effectiveness of crude extract of mayana leaves in aiding the blood coagulation process. It will be conducted at the laboratory of Cagayan State University, College of Allied Health Science. The blood types to be used in this study are decided to be blood type A, B, AB and O. This is to determine the significant difference of the different blood types on their blood coagulation time when the crude extract of mayana leaves is used in comparable with cephalin known for its thromboplastic agent. Since there is some sort of sensitivity in this experimental study, the accuracy of the result of experimentation varies on the capacity of the researchers, condition of the blood samples and the materials and instruments that are available to use.

Definition of Terms

The following terms are defined to assist a clear understanding of the study: Blood clotting. The conversion of fluid blood into a coagulum that involves shedding of blood, release of thromboplastin, inactivation of heparin, conversion of prothrombin to thrombin, interaction of thrombin with fibrinogen to form an insoluble fibrin network, and contraction of the network to squeeze excess fluid. Blood coagulation. A complex process by which blood forms clots. Cephalin. A thromboplastic substance which initiates the process of blood coagulation. Clotting factor. Any substance in the blood that is essential for blood to coagulate. Crude Extracts. The liquid form of the mayana made by purging of the leaves. Effectiveness. The fulfilling result of the herbal plants used in the clotting time.

Fibrin. A fibrous, non-globular protein involved in the clotting of blood. It is a fibrillar protein that is polymerized to form a "mesh" that forms haemostatic plug or clot over a wound site. Fibrinogen. A clotting factor and is required to prevent major blood loss. Hemophilia. A rare bleeding disorder in which the blood doesn't clot normally. Hemostatic disorder. Conditions that affect the ability of blood to clot properly. Hemostasis. Entire mechanism by which bleeding from an injured blood vessel is spontaneously controlled and stopped; Process by which spontaneous or induced hemorrhage is stopped. Herbal plant. Medicinal herbs to prevent and treat diseases and ailments or to promote health and healing. Mayana leaves. Commonly used as ornamental plant in the country due to its purplish foliage, mayana can grow in the different kinds of habitat. It is one of the traditionally used folklore medicine and it is primarily used for pain, sore, swelling and cuts and in other instances as adjunct medication for delayed menstruation and diarrhea. Prothrombin. A plasma protein that is converted to thrombin during blood clotting. Thromboplastic agent. An agent causing or increasing blood-clot formation. Thromboplastin. Enzyme in blood platelets; A blood-clotting factor in blood platelets that converts prothrombin to thrombin. Time. This word refers to the duration of time the blood will clot if the herbal plants are used during bleeding.

Chapter II REVIEW OF RELATED LITERATURE AND STUDIES MAYANA Mayana (Coeus blumei benth.) is an erect, branched, fleshy annual herb, about 1 m high. The stems are purplish and four-angled. Leaves are blotched or colored, ovate, 5-10 cm long, with toothed margins. Flowers are purplish, numerous, in simple or branched inflorescences, 15-30 cm long. It is one of the traditionally used folklore medicine and is primarily used for pain, sore, swelling and cuts and in other instances as adjunct medication for delayed menstruation and diarrhea. This plant can also make volatile oils and cure bruises and sprains, carminative, headache, mild bleeding of wounds, sinusitis, dyspersia and eye inflammation, (http://www.filipinoherbshealingwonders.filipino vegetarianrecipe.com/mayana.htm).

According to Janzen (2005), Vachellia mayana probably represents a wet-forest edition of V. cornigera. Undoubtedly, the two taxa are very closely related, having many vegetative and floral characteristics in common. However, the large leaflets (more than 10 mm long), the rachis glands between each pinna pair, and the inflorescence, which narrows toward the elongated and pointed apex separate this species from the closely related V. cornigera and V. sphaerocephala. Also, the pair of blade-like longitudinal flanges extending from the spine base to apex separates V. mayana from all other species of ant-acacia. As is typical of most ant-acacias, none of the individuals of V. mayana tested positive for cyanide production. Vachellia mayana is one of the rarer of the antacacias. Collecting data from the few collections observed indicate that it has pinkish

flowers and varies in size from a shrub to a small tree to 10 m tall. Most collections indicate that Vachellia mayana occurs as scattered individuals in moist lowland forests.

Janzen (2005) reported an individual from old second growth cornfield regeneration where the forest was about 15 m tall. Unlike most wet forest antacacias, Beltian body production in Vachellia mayana is extremely high. On developing leaves, nearly all of the leaflets contain Beltian bodies, and these bodies are usually about 2 mm long and up to 0.8 mm wide.

Mayana grows well in open areas with moist, well-drained and friable soil. Occasionally cultivated throughout the Philippines. Common garden plant. It flowers all year round. The plant is deeply rooted. Prefers warm and moist habitat, sensitive to dryness. Soil should be well-drained, and rich in humus to produce higher yields. Use seeds for propagation.

The mature fresh leaves of Mayana are harvested 2 to 3 months after planting. Leaves are picked leaving the branches on the plant to allow it to flower and produce seeds for the next season. The leaves are air-dried until they crumble when crushed with the fingers. Store in amber colored bottles in a cool, dry place.

Chemists from the University of the Philippines isolated sterols and triterpenes from the leaves of mayana and it exhibited analgesic, antiinflammatory and antimicrobial activities. Another interesting component of the plant is its high rosmarinic acid content. This compound was noted for its high biological activities; prominent of those are its anti-inflammatory and anti-oxidant

properties. It may be grown in the garden in bright, indirect light, or in practical shade, and will survive full sun exposure. It is commonly used as ornamental plant in the country due to its purplish foliage; mayana can grow in the different kinds of habitat. This traditional uses of mayana are scientifically supported by studies here and abroad, (http://www.herbanext.com/herbs-mayana).

Cris (2008) conducted a study on the Feasibility of Using Mayana (Colleus scutellarioides) as Biological Stain. Results showed that the extract of the mayana leaves can be partly used as biological stain for plant cell. He concluded that almost all the cells in samples can be seen the nucleus and the cell wall of the onion cell. In comparing the mayana extract as biological stain from the commercial ones, many biological stain cant be an alternative for the commercial ones because the mayana extract cant highlight some parts of the plant cells. Cris study is unscientific because the explanation is insufficient.

CEPHALIN

Phosphatidylethanolamine (cephalin, sometimes abbreviated PE) is a lipid found in biological membranes. It is synthesized by the addition of CDP-ethanolamine to diglyceride, releasing CMP. S-adenosyl methionine can subsequently methylate the amine of phosphatidyl ethanolamine to yield phosphatidyl choline.

Cephalin is a phospholipid, which is a lipid derivative. It is not to be confused with the molecule of the same name that is an alkaloid constituent of Ipecac. Cephalin is found in all living cells, although in human physiology it is found particularly in nervous tissue

such as the white matter of brain, nerves, neural tissue, and in spinal cord. Whereas lecithin is the principal phospholipid in animals, cephalin is the principal one in bacteria. As a polar head group, phosphatidylethanolamine (PE) creates a more viscous lipid membrane compared to phosphatidylcholine (PC). For example, the melting temperature of di-oleoyl-PE is -16C while the melting temperature of di-oleoyl-PC is -20C. If the lipids had two palmitoyl chains, PE would melt at 63C while PC would melt already at 41C. Lower melting temperatures correspond, in a simplistic view, to more fluid membranes, (Wan et al. Biochemistry 47 2008).

A study of the activity of purified cephalin and lecithin as antigens in the complement-fixation test and in the animal body is described. The cephalin and lecithin used were both prepared from beef brain and the lecithin from beef liver and beef heart as well. These phosphatides, dispersed by several methods, were inactive, outside the lytic or anticomplementary range, in the in vitro test. Reinforcement with cholesterol had no effect. The lecithins, when preserved under acetone to prevent oxidation, had neither lytic nor anticomplementary properties.

The cephalins were all markedly anticomplementary; only very large amounts, which were unsuitable for testing because of the opacity of the solutions, were lytic. Immunization studies were carried out with three samples of purified cephalin, prepared from brain tissue, and with two samples of purified lecithin, one from liver and one from brain; also, for control, with commercial preparations of both cephalin and lecithin. The sera of rabbits that received mixtures of the purified phosphatides and swine serum did not fix complement with either the purified or the commercial phosphatides, or with beef-

heart extracts. Thus, there was no evidence of the antigenic action of these substances in the tissues. The sera of rabbits inoculated with mixtures of the commercial phosphatides, cephalin and lecithin, with swine serum fixed complement with the commercial preparation used for inoculation, and with the Wassermann antigens but not with the purified phosphatides.

This result strongly suggests that the specific activity of the commerical preparations is associated with some contaminating substance rather than with lecithin or cephalin itself. The advantage of a precise serologic method was demonstrated, since, by its use, reactions which would otherwise have been regarded as significant were shown to be non-specific.

Adsorption of commercial cephalin or lecithin on collodion particles did not confer upon them any demonstrable immunizing properties; it did increase the

anticomplementary action of cephalin, (Related foreign study by Augustus Wadsworth et. al. A Study of the Antigenic Properties of Lecithin and Cephalin).

Study has been made of the rat liver protein catalyzed exchange of both lecithin and cephalin between liposomes and rat liver mitochondria. It has been shown that the exchange activity of these two phospholipids by the protein is almost the same and is apparently not dependent on the nature of donor liposomes. In contrast the spontaneous exchange activity of the above phospholipids strictly depends on the type of donor liposomes. Moreover, the spontaneous exchange of lecithin at any incubation time appears to be almost 100% higher than that of cephalin, (Related foreign study by F M

Ruggiero et. al. Comparative Study of Lecithin and Cephalin Exchange between Liposomes and Mitochondria). In the original Hangers method of cephalin-cholesterol flocculation test (CCF test), crude cephalin from sheep brain was used as a component of the CCF reagent, but in Japan that from ox brain has also been utilized for the same purpose. Crude cephalins from various sources were separate into five fractions by Folch in 1942, the first fraction mainly consisting of inositol phospholoipid, the third of phosphatidyl serine and the fifth of phosphatidyl ethanolamine, respectively. The second and forth fractions gave no additional component other than those described above. Recently plasmalogens were also reported by Klenk and others to be a component of the fifth fraction above mentioned.

In spite of many informations about the chemical nature of cephalin, it is not yet certain whether a single component of cephalin fraction or some mixture of components would participate in CCF reaction. On the other hand, CCF reagents available on the market are known to differ widely in their flocculation activity according to their makers and more or less with bathces of preparation. The inconstancy of reactivities among batches of preparation may lead to confusion of clinical evaluation of CCF test followed by loss of reliance on the test. These facts have hindered the CCF test from its broad application in Japan. The differences in reactivities have not yet been chemically explained. From the knowledge in reactivities have not yet been chemically explained. From the knowledge about cephalin mixture, however, it is assumed that any single fraction of cephalin may be the most potent in reactivity and others non-reactive or inhibitory. The differences in reactivities of reagents above mentioned may be attributed to the relative amount of the active component in cephalin preparation used in the test. If

a single component is reactive, it may be possible properly to control its reactivity by means of admixture of a non-reactive or inhibitory. The differences in reactivities of reagents above mentioned may be attributed to the relative amount of the active component in cephalin preparation used in the test. If a single component is reactive, it may be possible properly to control its reactivity by means of admixture of a non-reactive or an antagonistical lipid matter in its purified state, (Related foreign study Study on Cephalin-Cholesterol-Flocculation Reaction).

Nine partial thromboplastin (cephalin) reagents have been compared in a parallel investigation of groups of patients on 'long-term' anticoagulants, a group with moderate haemophilia, and patients on heparin infusion. Results with the seven commercial reagents and a human cephalin extract have been correlated with those of a specially prepared and standardized reference preparation of human brain origin.

The comparison was similar in principle to that of the prothrombin time thromboplastin standardization using the British Comparative Thromboplastin (BCT). Results, which for comparative purposes were expressed as ratio of patients' cephalin times to control cephalin times, varied greatly in all three groups. In the oral anticoagulant group some of the commercial reagents were particularly insensitive to the 'intrinsic' clotting defect. The correlation between the 'standardized preparation' and the other reagents was not good and the use of a reference cephalin material for quality control of cephalin time tests does not appear promising.

In moderate haemophilia the commercial reagents were either relatively poor 'at picking out the clotting defect compared with the 'standardized preparation' or gave such

a bad endpoint that the results were not dependable. The poor endpoint also limited the dependability of the results of all but the 'standardized preparation' and two of the commercial reagents in controlling heparin administration.

In view of these standardization difficulties, which cannot apparently be resolved.by the use of reference material, there is need for bulk, routine supplies of a sensitive, standardized cephalin reagent giving good reproducible endpoints. The method for the provision of such material in a recently introduced national supply scheme is described. The partial thromboplastin (cephalin) time is employed as an overall measure of 'intrinsic' blood clotting. Its main applications are in the screening for hereditary and acquired 'intrinsic' clotting defects and in assessing the intrinsic defect during oral anticoagulant treatment. The test involves the recalcification of platelet-poor plasma in the presence of a crude phospholipid extract. The origins of the latter vary and different animal tissues are used in their preparation. In addition 'home-made' phospholipid extracts from human brain may be made and used at individual hospitals.

The present report describes the results encountered in clinical practice when a variety of reagents currently available in Britain are used. An attempt has been made to correlate these reagents with a standardized cephalin preparation. The comparison of the cephalin reagents has been on patients on long-term anticoagulants (nicoumalone therapy), a group with moderately severe haemophilia, and patients on heparin therapy. On the basis of the findings a system for standardizing the cephalin time test has been introduced and involving the supply of a sensitive standardized cephalin preparation,

(Related foreign study by L. Poller et.al. The Partial Thromboplastin (cephalin) Time Test).

BLOOD COAGULATION

Theories on the coagulation of blood have existed since antiquity. Physiologist Johannes Mller (1801-1858) described fibrin, the substance of a thrombus. Its soluble precursor, fibrinogen, was thus named by Rudolf Virchow (1821-1902), and isolated chemically by Prosper Sylvain Denis (1799-1863). Alexander Schmidt suggested that the conversion from fibrinogen to fibrin is the result of an enzymatic process, and labeled the hypothetical enzyme thrombin and its precursor prothrombin. Arthus discovered in 1890 that calcium was essential in coagulation. Platelets were identified in 1865, and their function was elucidated by Giulio Bizzozero in 1882(Fogoros, 2003). The theory that thrombin is generated by the presence of tissue factor was consolidated by Paul Morawitz in 1905. At this stage, it was known that thrombokinase/thromboplastin (factor III) is released by damaged tissues, reacting with prothrombin (II), which, together with calcium (IV), forms thrombin, which converts fibrinogen into fibrin (I).

Coagulation factors the remainder of the biochemical factors in the process of coagulation were largely discovered in the 20th century. A first clue as to the actual complexity of the system of coagulation was the discovery of proaccelerin (initially and later called Factor V) by Paul Owren (1905-1990) in 1947. He also postulated its function to be the generation of accelerin (Factor VI), which later turned out to be the activated form of V (or Va); hence, VI is not now in active use. Factor VII (also known as serum prothrombin conversion accelerator or proconvertin, precipitated by barium sulfate) was

discovered in a young female patient in 1949 and 1951 by different groups. Factor VIII turned out to be deficient in the clinically recognized but etiologically elusive hemophilia A; it was identified in the 1950s and is alternatively called antihemophilic globulin due to its capability to correct hemophilia A.

Factor IX was discovered in 1952 in a young patient with hemophilia B named Stephen Christmas (1947-1993). His deficiency was described by Dr. Rosemary Biggs and Professor R.G. MacFarlane in Oxford, UK. The factor is, hence, called Christmas Factor or Christmas Eve Factor. Christmas lived in Canada, and campaigned for blood transfusion safety until succumbing to transfusionrelated AIDS at age 46. An alternative name for the factor is plasma thromboplastin component, given by an independent group in California.

Hageman factor, now known as factor XII, was identified in 1955 in an asymptomatic patient with a prolonged bleeding time named of John Hageman. Factor X, or Stuart-Prower factor, followed, in 1956. This protein was identified in a Ms. Audrey Prower of London, who had a lifelong bleeding tendency. In 1957, an American group identified the same factor in a Mr. Rufus Stuart. Factors XI and XIII were identified in 1953 and 1961, respectively. The view that the coagulation process is a cascade or waterfall was enunciated almost simultaneously by MacFarlane in the UK and by Davie and Ratnoff in the USA, respectively (Hara, 2005).

BLOOD COAGULATION MECHANISM

Blood Clotting is one of the three mechanisms that reduce the loss of blood from broken blood vessels. These three mechanisms are Vascular Spasm, platelet plug formation and blood clotting. In vascular spasm the smooth muscle in blood vessel walls contracts immediately the blood vessel is broken. This response reduces blood loss for some time, while the other haemostatic mechanisms become active. The clotting

mechanism is one of the most important and complex of physiologic systems. Blood must flow freely through the blood vessels in order to sustain life. But if a blood vessel is traumatized, the blood must clot to prevent life from flowing away. Thus, the blood must provide a system that can be activated instantaneously and that can be contained locally to stop the flow of blood. This system is called the clotting mechanism (Fogoros, 2003).

When blood platelets encounter a damaged blood vessel, platelet plug formation will occur to help close the gap in the broken blood vessel. The key stages of this process are called platelet adhesion, platelet release reaction, and platelet aggregation. Following damage to a blood vessel, vascular spasm occurs to reduce blood loss while other mechanisms also take effect. Blood platelets congregate at the site of damage and a mass to form a platelet plug. This is the beginning of the process of the blood breaking down from its usual liquid form in such a way that its constituents play their own parts in processes to minimize blood loss. Blood normally remains in its liquid state while it is within the blood vessels but when it leaves them the blood may thicken and form a gel (coagulation). Blood clotting technically blood coagulation is the process by which

(liquid) blood is transformed into a solid state. This blood clotting is a complex process involving many clotting factors (incl. calcium ions, enzymes, platelets, damaged tissues) activating each other. (Kozier, 2006)

The three stages of this process are Formation of Prothrombinase, prothrombin converted into the enzyme thrombin, and fibrinogen (soluble) converted to fibrin (insoluble). Prothrombinase which is the stage one can be formed in two ways, depending of which of two systems or pathways apply. These are Intrinsic System and extrinsic System. Intrinsic System is initiated by liquid blood making contact with a foreign surface, i.e. something that is not part of the body. The Extrinsic System is initiated by liquid blood making contact with damaged tissue. Both the intrinsic and the extrinsic systems involve interactions between coagulation factors.

These coagulation factors have individual names but are often referred to by a standardised set of Roman Numerals, e.g. Factor VIII (antihaemophilic factor), Factor IX (Christmas factor). In stage two, Prothrombin converted into the enzyme Thrombin; Prothrombinase formed in stage one converts prothrombin, which is a plasma protein that is formed in the liver, into the enzyme thrombin; and in stage tree Fibrinogen (soluble) will be converted to Fibrin (insoluble). In turn, thrombin converts fibrinogen (which is also a plasma protein synthesized in the liver) into fibrin. Fibrin is insoluble and forms the threads that bind the clot (Hara, 2005).

MEDICINAL PLANTS

In 2005, the National Policy on Traditional Medicine and Regulation of Herbal Medicines explained that various types of traditional medicine (TM) and medical practices referred to as complementary or alternative medicine (CAM) have been increasingly used in both developing and developed countries. One of the major components of the WHO Traditional Medicine Strategy is to promote the integration of such medicine within a national health care system.

The use of medicinal plants is the most common form of traditional medication worldwide. Regulation of herbal medicines is a key means of ensuring safety, efficacy and quality of herbal medicinal products. WHO has been receiving an increasing number of requests from governments for guidance on how to regulate herbal medicines.

During the last four years, many countries have established, or initiated the process of establishing national regulations regarding herbal medicines. WHO has been conducting a global survey on national policy on traditional medicine and on the regulation of herbal medicines aiming to: (1) Collect updated and comprehensive information on TM/CAM policies and regulations of herbal medicines; (2) Clarify the current situation, in each country, on the TM/CAM policies and regulations of herbal medicines, and their major challenges on these particular area; (3) Identify the specific needs on capacity building for TM/CAM policy development including establishment of regulations of herbal medicines, and the type of direct support WHO should provide to member states; and (4) Monitor the impact of the WHO Strategy for Traditional Medicine in relation to present national policy and regulation on TM/CAM/herbal medicines.

WHO had received completed survey return from 141 countries. The raw data of the survey results were fed into a database specifically designed for this project, to create basic country profiles. Government clearance has been obtained for each country profile; the manuscript of the draft summary report was finalized in English. The present document provides a summary of the results of the WHO global survey with information from 141 member states. The baseline information gathered in the first of its kind, will be valuable not only to help countries compare and learn each others experiences in strengthen their current TM/CAM system, but also for guiding WHO on provision of support to member states. National policy on traditional medicine and regulation of herbal medicines: Report of a WHO global survey.

BIBLIOGRAPHY

Books Cuevas, Frances Prescilla L., Editor in chief (2007). Public Health Nursing in the Philippines; Publication Committee, National League of Philippine Government Nurses, Incorporated 11th Ed. Govoni, Laura E. & Janice E. Hayes (2002). Drugs and Nursing Implications;Meredith Publishing Company. Gutierez, Kathleen & Sherry F. Queener (2005). Pharmacology for Nursing; Mosby Inc. Kozier, Barbara, et. Al.(2006). Fundamentals of Nursing: Concepts, Process, and Practice 8th ed. Pearson Education Inc. Ramont, Robeta Pavy, Maldonado, Dolores and Towle, Mary Ann. (2006). Comprehensive Nursing Care. United States of America. Pearson Education, Inc.. Venzon, Lydia M. (2004). Introduction to Nursing Research; Quest for Quality Nursing. Quezon Avenue. Wan et al. Biochemistry 47 2008

Electronic Sources: Internet Fogoros,Richard N., M.D., (2003) Retrieved from http://heartdisease.about.com/cs/heartattacks/a/ clotting.htm

Hara,

Hari C., (2005) Retrieved from http://www.shvoong.com/exactsciences/biology/1757270-blood-clotting-mechanism/ (2005) Retrieved from http://www.filipinoherbshealingwonders. filipinovegetarian-recipe.com/ balanoy.htm

Janzen,

Jesse Cris. (2008) Retrieved from The Feasibility of Using Mayana (Colleus scutellarioides) as Biological Stain L. et.al.(1972) Retrieved from http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC477616/ The Partial Thromboplastin (cephalin) Time Test

Poller

Ruggiero F M et. al. (1981)- Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/7259878 Comparative Study of Lecithin and Cephalin Exchange between Liposomes and Mitochondria Samuel A. Guttman (1944) Retrieved from http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC435458/pdf/jcinvest00592-0046.pdf Study on CephalinCholesterol-Flocculation Reaction Wadsworth, Augustus et. al (1934) Retrieved from http://www.jimmunol.org/ content/26/1/25.abstract Study of the Antigenic Properties of Lecithin and Cephalin

http://www.filipinoherbshealingwonders.filipinovegetarianrecipe.com/mayana.htm http://www.herbanext.com/herbs-mayana

CHAPTER III MATERIALS AND METHODS

This chapter will present the research methodology of the study. It includes the research methodology, materials, equipment and apparatus, procedure training of the panelists, evaluation of the products and statistical treatment of data.

Research Method This study uses the descriptive-comparative as experimental design. It describes the effectiveness of the crude extract of mayana leaves when used in blood coagulation process. It also correlates the accurate blood clotting time using cephalin as positive control, the crude extract of mayana leaves as the experimental specimen and the normal blood clotting time as the negative control. The experimental research controls the condition of the study. Parallel-group design is the type of experimental design used. Wherein the normal coagulation time of blood serves as control in comparing the effectiveness of the crude extract of mayana leaves with the effect of cephalin when used by different blood types which are A, B, AB and O in coagulation process.

Materials The materials to be used in this proposed study will be 70% alcohol, crude extract of mayana, cephalin, A, B, AB, O blood types. Table 1 presents the materials used in the extraction of crude extract from the mayana leaves (Coeus Blumei benth) and used in the coagulation time.

Table 1 Materials 70% alcohol crude extract of mayana cephalin Blood types: A (3 samples) B (3 samples) AB (3 samples) O (3 samples) 0.18cc 0.18cc 0.18cc 0.18cc 60ml 240L 240L Quantity

Table 1. Materials used in the extraction of crude extract from the mayana leaves (Coeus Blumei benth) and used in the coagulation time.

Apparatus The apparatuses to be used in this proposed study will be clean glass slides, cotton, disposable lancet, needle, sterile gauze, stopwatch, mortar and pestle, beaker and knife. Table 2 presents the apparatus used in the extraction of crude extract from the mayana leaves (Coeus Blumei benth) and used in the coagulation time.

Table 2 Apparatus Quantity

Clean glass slides Cotton Disposable lancet (sterile) Needle Sterile gauze Stopwatch Mortar and pestle Beaker Knife

36 pieces 1 pack 36 pieces 36 pieces 1 piece 3 units 1 set 1 piece 1 piece

Table 2. Apparatus used in the extraction of crude extract from the mayana leaves (Coeus Blumei benth) and used in the coagulation time.

Procedures The procedure that will be used in conducting the study for coagulation time or clotting time when the crude extract of mayana leaves is used in different blood types is called the Slide Method or Drop Method. The steps or procedures in this study will be done following in this order: (1) Disinfect site of puncture with 70% alcohol sponge and dry. (2) Puncture to a depth of 3mm. (3) Wipe off first drop of blood. (4) Start the timer as soon as the second drop of blood appears. (5) Transfer the second drop of blood onto the center of glass slide. (6) Pass the tip of needle through the drop of blood every thirty seconds and note for the formation of fibrin strands. (7) Stop the timer as soon as fibrin strands are seen clinging at the tip of the needle.

The normal value of blood coagulation time in this test is said to be two to four (24) minutes. After conducting the test to the four different blood types, the result will be noted weather there are differences in the coagulation time of blood types A, B, AB and O. The time noted will be used as a negative control on the next procedure where testing the effectivity of mayana leaves in coagulation process while cephalin will be used as positive control.

Repeat procedure one to five. (6) When the second drop of blood is onto the center of the glass slide, get a 10L of crude extract of mayana leaves and mix it with the specimen. (7) Pass the tip of needle through the drop of blood with a crude extract every thirty seconds and note for the formation of fibrin strands. (8) Stop the timer as soon as fibrin strands are seen clinging at the tip of the needle. Then repeat the same procedure for test of cephalin. After noting all results in every procedures of this experiment. Tabulate the data for better comparison. The researchers will write their conclusion on the effectiveness of crude extract of mayana leaves in blood coagulation time based from the experiment they conducted.

To obtain a crude extract from mayana leaves. The researchers will do these following procedures. (1) Collect the mayana leaves early in the morning, before the heat of the day. Choose mature leaves that are fully formed and mature but are not aged or damaged. (2) Wash the mayana leaves with clean water. (3) Finely chop the leaves with the knife to easily release all the components from the plant. (4) Pound the leaves using a mortar and pestle to make extract. (5) Place the crude extract in the beaker. The container should be cleaned, dried and sterilized with boiling water before use for a pure extract.

Figure 2.1 Crude extract of mayana leaves

Collect the mayana leaves early in the morning. Choose mature leaves that are fully formed.

Wash the mayana leaves with clean water

Finely chop the leaves with the knife.

Pound the leaves using a mortar and pestle to make extract.

Place the crude extract in the beaker.

Figure 2.1 Process flow sheet for the extraction of crude extract from the mayana leaves (Coeus Blumei benth) that will be used as experimental material for the study.

Figure 2.2 Blood Type A Blood Type B Blood Type AB Blood Type O

Natural Blood Clotting time (- control)

Crude extract of mayana leaves (Experimental)

Cephalin (+ control)

Disinfect site of puncture with 70% alcohol sponge and dry

Puncture to a depth of 3mm

Wipe off first drop of blood

Start the timer as soon as the second drop of blood appears Transfer the second drop of blood onto the center of glass slide Get a 10L of crude extract of mayana leaves/cephalin and mix it with the specimen

Pass the tip of needle through the drop of blood every thirty seconds and note for the formation of fibrin strands

Stop the timer as soon as fibrin strands are seen clinging at the tip of the needle

Figure 2.2 Process flow sheet for the comparison of the four different blood types (A, B, AB, O) as the crude extract of mayana leaves and cephalin are used in the coagulation time, comparing the result with the natural clotting time of the blood.