EFFECTIVENESS OF CRUDE EXTRACT OF MAYANA LEAVES FOR BLOOD COAGULATION PROCESS

An Undergraduate ThesisPresented to the Faculty of the

College of Allied Health SciencesMedical Technology Department

Cagayan State UniversityAndrew’s Campus, Tuguegarao City

In Partial Fulfillment Of the Requirements for the Degree

Bachelor of Science in Medical Technology

ByRhoe Anne C. CalabazaronRandolph Ryan G. Gasmin

Abigail C. GuinucayJocelyn P. Oledan

Niki Valencia D. PamittanRachelle 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 it’s 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

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.

Mayana (Coeus Blumei benth)

- Temperature- Blood type- pH

Blood Coagulation

Cephalin(Positive Control)

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 researcher’s 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 ant-

acacias.  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 ant-acacias,

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, anti-inflammatory 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 can’t

be an alternative for the commercial ones because the mayana extract can’t 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 Hanger’s 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 Müller (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 transfusion-related 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 other’s 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/exact-sciences/biology/1757270-blood-clotting-mechanism/

Janzen, (2005) – Retrieved from http://www.filipinoherbshealingwonders. filipinovegetarian-recipe.com/ balanoy.htm

Jesse Cris. (2008) – Retrieved from “The Feasibility of Using Mayana (Colleus scutellarioides) as Biological Stain”

Poller L. et.al.(1972) – Retrieved from http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC477616/ “The Partial Thromboplastin (cephalin) Time Test

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 Cephalin-Cholesterol-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 Quantity

70% alcohol

crude extract of mayana

cephalin

Blood types:

A (3 samples)

B (3 samples)

AB (3 samples)

O (3 samples)

60ml

240μL

240μL

0.18cc

0.18cc

0.18cc

0.18cc

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 (2-

4) 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 10μL 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

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.

Wash the mayana leaves with clean water

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

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

Finely chop the leaves with the knife.

Place the crude extract in the beaker.

Figure 2.2

Blood Type A Blood Type B Blood Type AB Blood Type O

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.

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

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

Conclusion

Get a 10μL of crude extract of mayana

leaves/cephalin and mix it with the specimen