Hemostatic Effects of Peperomia pellucida Plant Crude Extract, in vitro An Undergraduate Thesis Presented to In Partial Fulfilment Of the Requirement for the Degree Bachelor of Science in Biology PDF

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Hemostatic Effects of Peperomia pellucida Plant Crude Extract, in vitro An Undergraduate Thesis Presented to The Faculty of the Biology Department College of Natural Sciences and Mathematics Mindanao State University Marawi City In Partial Fulfilment Of the Requirement for the Degree Bachelor of Sci...

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Hemostatic Effects of Peperomia pellucida Plant Crude Extract, in vitro An Undergraduate Thesis Presented to In ... Norlainie Omar

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Hemostatic Effects of Peperomia pellucida Plant Crude Extract, in vitro

An Undergraduate Thesis Presented to The Faculty of the Biology Department College of Natural Sciences and Mathematics Mindanao State University Marawi City

In Partial Fulfilment Of the Requirement for the Degree Bachelor of Science in Biology

Norlainie C. Omar

March 2013

Table of Contents Title Page Approval Sheet Acknowledgment Dedication Table of Contents Appendices List of Tables List of Plates Abstract

i ii iii iv v vi vii ix x

CHAPTER I: Introduction Background of the Study Statement of the Problem Research Hypothesis Significance of the Study Objectives of the Study Scope and Limitations

1 2 3 4 4 5

CHAPTER II: Review of Related Literature Experimental Plant Traditional Medicine Hemmorhage Hemostasis Related Studies

6 7 10 11 11

CHAPTER III: Methodology Collection of Plant Preparation of Crude Extract Screening of Blood Donors Blood collection and Preparation Clotting time Determination Experimental Design Statistical Tool

13 13 14 14 14 15 16

CHAPTER IV: Results and Discussion Results and Discussion Two- way ANOVA

17 17

v

Duncan’s Multiple Range Tests Mean Values

18 19

CHAPTER V: Summary, Conclusion, and Recommendation

21

Literature Cited Appendices List of Tables List of Figures List of Plates

39

vi

APPENDICES

Appendices

Title

Page

1

Raw data for blood clotting time on treatments per blocks

26

2

Mean values on effect of P. pellucida on human whole clotting time

27

vii

LIST OF TABLES Table

Title

Page

1

Two-way ANOVA on the effect of P. pellucida on human whole blood clotting time

17

2

DMRT on the effect of P. pellucida on human whole blood clotting time

18

3

Mean values on the effects of P. pellucida on human blood clotting time

19

viii

LIST OF PLATES Figure

Title

Page

1

Experimental plant Pansit-pansitan (Peperomia pellucida)

31

2

Crude extracts of experimental plant.

31

3

Blood collection via Venipuncture Syringe Method

32

4

Blood clots on tubes

32

ix

ABSTRACT Omar, N. March 2013. “Hemostatic effects of Peperomia pellucida plant crude extract, in vitro.” Biology Department, College of Natural Sciences and Mathemetics, Mindanao State University, Marawi City. Adviser: Dr. Apolinario Alicante, DVM

A growing fascination for natural coagulants discoveries stemming from the overwhelming consumer response seeking remedies devoid for unfavorable side effects has prompted the execution of this study. The study was conducted to determine the coagulation properties of Pansit-pansitan (Peperomia pellucida) plant in preventing blood loss which is known for its traditional use. The study is helpful to the people in the community because this served as an affordable and accessible treatment for major bleeding. Blood samples from the donors were tested for coagulation through clotting time assay. To test the coagulation properties of the Pansit-pansitan (Peperomia

pellucida) plant, four

treatments were evaluated. The first treatment serves as the control variable with 0.2ml of whole blood. The second treatment was a mixture of 0.2ml of 50% crude extract and 0.2ml of whole blood. The third treatment was a mixture of 0.2ml of 75% crude extract and 0.2ml of whole blood. And the fourth treatment was a mixture of 0.2ml of 100% crude extract and 0.2ml whole blood. Each treatment had five replicates. The resulting mixtures were scrutinized within a certain period of time to determine the effectiveness of the crude extract for blood coagulation by noting its clotting time. The results of the study showed that Pansit-pansitan (Peperomia pellucida) plant had coagulation properties that can induce blood clotting and augmentation of thrombocytes production. x

Chapter I INTRODUCTION

Background of the Study Plants have played a significant role in maintaining human health (Craig, 1999) and improving the quality of human life for thousands of years and have served humans well as valuable components of food and medicines (Winston, 1999). Peperomia pellucida is an annual herbaceous plant (Cao, 2012) belonging to family Piperaceae (Ghani, 1998), commonly known as shiny bush (Sio, 2012) or pansit-pansitan (Cao, 2012). It is widely distributed in most South American and Asian countries (Bayma, 2008). According to Ethno-botanical studies, the whole plant has been in medicinal use since long (Majumder, 2011).

It is reported to possess antipyretic, analgesic, anti-inflammatory,

antimicrobial, refrigerant and CNS activity (Majumder, 2011). It is used ethno-medicinally to treat abdominal pain, abscesses, acne, boils, colic, fatigue, gout, headache, renal disorder and rheumatic joint pains (Khan, 2002; Ragasa, 1998; Omoloso, 2002). The roots are used to treat fevers and the aerial parts are used as dressing for wounds (Muñoz, 2000). The whole plant is used to stop hemorrhages (Egwuche, 2011); it is crushed and mixed with water to form a mixture, heated and administered orally or applied topically to arrest wound bleeding or hemorrhage (Majumder, 2011). Hemorrhage is an excessive discharge of blood from blood vessels caused by pathological of the vessels or by traumatic rupture of one or more vessels (Wagman, 2000). The

1

blood has the ability to change from a fluid to a solid and back to a fluid again. The change to a solid is called clotting (Wagman, 2000). Hemostasis, when a small blood vessel is transected or damaged; the injury initiates a series of events that leads to the formation of a clot (Ganong, 2000). It leads to the sealing off blood vessel preventing further blood loss (Ganong, 2000). A wound or cut on blood vessels causes vasoconstriction and thrombin activation which are then accompanied by adhesion and platelet activation, fibrin formation and coagulation activation mechanism. Thrombosis is the pathological formation of hemostatic plug within the vasculature in the absence of bleeding (Rang, 1999). Hemostasis is currently a clinical challenge due to inconsistencies encountered in the blood clot conversion

process. Medical treatment includes administration of drugs either

locally (topical) or systemically (oral or parenteral) with the aim to hasten the time required for blood clotting. Medicinal plants have generated much interest for treatment of wound bleeding as they are affordable and purportedly safe from hypersensitive reactions (Khan, 2010). In the hope of finding a treatment/therapeutic remedies for wound bleeding or hemorrhage, which is cost-efficient, effective, and safe conceived the idea of doing an interventional study to determine the effects of Peperomia pellucida plant extract to the normal blood clotting time of human blood.

2

Statement of the Problem Peperomia pellucida is one of the most studied plants in terms of its ethno-medicinal values (Ytable, 2012). However, studies on the hemostatic activity of this plant are rather limited.

Despite its wide range of folk medicinal use, there is insufficient scientific data

(Majumder, 2011) on the efficacy of this plant in arresting wound bleeding or hemorrhage. Most of the reports are anecdotal therefore, an investigation must be made to verify these claims. The present aim of the study is to provide answer to the following questions: 1. Will Peperomia pellucida plant extract induce an effect on the normal clotting time of human blood? 2. Will Peperomia pellucida plant extract cause to hasten the normal clotting time of human blood? 3. Will Peperomia pellucida plant extract cause to prolong the normal clotting time of human blood?

Research Hypotheses Null:

Induction of Peperomia pellucida plant crude extract has no effect on clotting of

normal human plasma initiated in vitro. Alternative: Induction of Peperomia pellucida plant crude extract has an effect on clotting of normal human plasma initiated in vitro.

3

Significance of the Study The concern towards the ethno-medicinal use of Peperomia

pellucida against

hemorrhage will be scientifically justified. This study is also a significant attempt on combating major morbidity and mortality rate caused by hemorrhage (Goker, 2008) by obtaining information on the usefulness of Peperomia pellucida plant extract as a supportive therapy in hastening the normal blood clotting or thrombin time. Thus, a positive result of this study will serve as an identification of a novel, effective hemostatic agent that improves the management of bleeding in a wide range of patients from all disciplines of clinical medicine (Goker, 2008). This will be inexpensive and readily available since the test plant is locally found and grown in this country (Mustapha, 2012). This could be a great help in developing countries in utilizing properly their environmental resources. Furthermore, the research work would give an idea to the people to cultivate the commonly found plants such as Pansit-pansitan in our country with valuable medicinal use and would serve as an avenue for further discovery in research and for commercially economic purposes (Ytable, 2012).

Objectives of the Study The main objective of the study is to determine whether the plant extract of Peperomia pellucida contain hemostatic components. Specifically, the researcher aims to accomplish the following: 1. To determine whether or not the plant extract of Peperomia pellucida induce an effect on the normal blood clotting time on human.

4

2. To determine whether or not the plant extract of Peperomia pellucida will cause to hasten the normal clotting time of human blood. 3. To determine whether or not the plant extract of Peperomia pellucida will cause to prolong the normal clotting time of human blood. Scope and Limitation of the Study This study is concerned in knowing the possible effects of Peperomia pellucida plant extract on the normal clotting time of human blood. This study utilized only locally available Peperomia pellucida plant. The entire plant was utilized for extraction by pounding, squeezing and filtration method. The experimental subjects used were human blood drawn from donors via venipuncture syringe method. The experimental design used in this study was Random Complete Block Design (RCBD). There were three blocks with four treatments, each treatment with five replicates (n=60). All Treatments 1 received no crude extract, all Treatments 2 received 50% crude extract, all Treatments 3 received 75% and all Treatments 4 received 100% crude extract. The notable effects of plant crude extract on hemostasis was determined by noting blood clotting time on normal human blood.

Appropriate units of plant extract were added immediately to normal

human plasma respectively, the time it took for blood to clot was noted in minutes. Descriptive statistics of the data results were expressed as Mean ± Standard Deviation (S. D.) using Twoway Analysis of Variance (ANOVA) and Duncan’s Multiple Range Test (DMRT). Phytochemical analyses of the nutrient component of the test plant are beyond the scope of this study. The isolation of active components that may have influence alteration of normal

5

clotting time in human blood were not determined in this study. Other basic mechanisms for action of hemostatic activity of the plant extract are excluded in this study.

6

Chapter II REVIEW OF RELATED LITERATURE A. Experimental Plant TAXONOMIC CLASSIFICATION Kingdom: Plantae Division:

Magnoliophyta

Class:

Magnoliopsida

Order:

Piperales

Family:

Piperaceae

Genus:

Peperomia

Species:

P. pellucida

Peperomia pellucida is an annual, shallow-rooted herb that belongs to the family Piperaceae (Ghani, 1998). It is commonly known as shiny bush. It is found in various shaded, damp habitats all over Asia and America, growing in clumps, thriving in loose, humid soils, tropical and subtropical climate. It usually grows to a height of about 15 to 45 cm and is characterized by succulent stems, shiny, heart-shaped, fleshy leaves and tiny, dot-like seeds attached to several fruiting spikes (Egwuche, 2011 as cited by Dos-Santos, 2001). As ethno-medicinal uses of this plant Peperomia pellucida has been applied for treating abdominal pain, abscesses, acne, boils, colic, fatigue, gout, headache, renal disorders and rheumatic joint pain (Majumder, 2011). The roots are used to treat fevers and the aerial parts are used as dressing for wounds (Muñoz, 2000). The plants have been used as a hypocholesteremic 7

agent (Bayma, 2000). It is a popular cough suppressant, emollient and diuretic as well as effective in the treatment of proteinuria (Theresa, 2012 as cited by Blank, 2002).

The decoction

of the plant is used in the Philippines to decrease uric acid levels and to treat renal problems. It is also used topically for skin disorders such as acne and boils (Egwuche, 2011). The plant is described to passify vitiated cough, pitta, constipation, kidney diseases, urinary retention, dysuria, urinary tract infections, emaciation, edema and general weakness.

Infusion and

decoction of leaves and stems of fresh plant are eaten as salad for the treatment of gout and arthritis (Cao, 2012).

According to ethno-botanical studies, the whole plant has been in

medicinal use since long. It is crushed and mixed with water to form a mixture, heated and administered orally to cure hemorrhage (Egwuche, 2011; Majumder, 2011; Theresa, 2012). Literature of many researchers prove that the plant contain alkaloids, saponins, tannins and cardenolides (Khan, 2010) flavonoids, essential oils and carotol (Khan, 2002). B. Traditional Medicine as an Alternative Source of Treatment Plants have long played a significant role in maintaining human health and have served as food for humans. WHO estimated that over 80% of the earth’s inhabitants rely on traditional medicine for their primary health care needs, and most of this therapy involves the use of plant extracts or their active components (Winston, 1999). Plants evolved the ability to synthesize chemical compounds that help them defend against attack from a wide variety of predators. By chance, some of these compounds, while being toxic to plant predators, turn out to have beneficial effects when used to treat human diseases (Ytable, 2012).

Traditional use of medicines is recognized as a way to learn about

8

potential future medicine. In 2001, researchers identified 122 compounds used in mainstream medicine that were derived from “ethno-medical” plant sources; 80% of these compounds were in the same or related manner as the traditional ethno-medical use (www.wikipedia.comherbalism, September 20, 2012). Both in modern and traditional medicine, medicinal plants continue to provide valuable therapeutic agents in the cure of disease and aliments (Davison, 2006). Doubts about the efficacy and safety of the oral hypoglycemic agents have prompted a search for safer and more effective drugs in the treatment of many diseases (Davison, 2006 as cited by Bannerman, 1993). Many herbs have remained as an alternative to conventional therapy especially in poor areas where insulin is not readily available; due to the high cost and the lack of medical aid (Davison, 2006 as cited by El-Demerdash, 2005). Medicinal plants are the oldest known health-care products, where renewed interest is growing based on the ethnological, medical and historical background of each country (Davison, 2006). Medicinal plants have always been important for pharmacological research and drug development, where the plant constituents are used directly as therapeutic agents, for the synthesis of drugs or as models for pharmacologically active compounds (Davison, 2006 as cited by Levetin, 1999). The plant based on indigenous knowledge was passed down from generations in various parts of the world throughout its history and has significantly contributed to the development of different traditional systems of medicine. More recently, drug discovery techniques have been applied to the standardization of herbal medicines, to elucidate analytical marker compounds (Jachak, 2007). 9

The use traditional medicine has increased in developed countries also, mainly due to the failure of modern medicine to provide effective treatment for chronic diseases and emergence of multi-drug resistant bacteria and parasites.

The current emphasis of new drug discovery

processes from plants is the development of products with new pharmacological modes of actions (Jachak, 2007). C. Hemorrhage Blood loss, while minor in every day cuts and bruises, is one of the main causes of mortality. Hemorrhage threatens the life safety of patients and the wounded in trauma care and surgical intervention. Hemorrhage is the main reason in the causes of death in 48 h after trauma, which accounts for 80% in all trauma accident (Wang, 2011). Early control of hemorrhage remains the most effective strategy for treating combat casualties. Catastrophic blood loss often results in hemorrhagic shock as demonstrated in animal models (Vallejo, 2005), resembling human outcomes (Jarrar, 1999). Therefore, development of compounds to improve hemostasis and save patient’s life in the trauma is of medical importance (Wang, 2011). Controlling hemorrhage will always remain a top priority in trauma care, and the development of materials to achieve this goal more effectively is of obvious benefit. In response to the changing combat and trauma casualty care, there has been an increase in efforts to develop better hemostatic agents. An ideal agent should be effective, easy to use, safe, logistically superior, and durable (Wang, 2011).

10

D. Hemostasis The ability of the body to control the flow of blood following vascular injury is paramount to continued survival (unknown). Hemostasis is the process of forming clots in the walls of damaged blood vessels and preventing blood loss while maintain blood in a fluid state within the vascular system. A collection of complex interrelated systemic mechanisms operates to maintain a balance between coagulation and anticoagulation (Barma, 2010). Events in hemostasis includes (1) contraction of the smooth muscles in the blood vessel wall. This reduces the flow of blood and loss from the defect in the vessel wall. The term for this reduction in the diameter of a vessel is vasoconstriction; (2) The process of blood clotting and then the subsequent dissolution of the clot, following repair...