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 i
Approval Sheet ii
Acknowledgment iii
Dedication iv
Table of Contents v
Appendices vi
List of Tables vii
List of Plates ix
Abstract x

CHAPTER I: Introduction
Background of the Study 1
Statement of the Problem 2
Research Hypothesis 3
Significance of the Study 4
Objectives of the Study 4
Scope and Limitations 5

CHAPTER II: Review of Related Literature

Experimental Plant 6
Traditional Medicine 7
Hemmorhage 10
Hemostasis 11
Related Studies 11

CHAPTER III: Methodology

Collection of Plant 13
Preparation of Crude Extract 13
Screening of Blood Donors 14
Blood collection and Preparation 14
Clotting time Determination 14
Experimental Design 15
Statistical Tool 16

CHAPTER IV: Results and Discussion

Results and Discussion 17
Two- way ANOVA 17

v
 Duncan’s Multiple Range Tests 18
Mean Values 19

CHAPTER V: Summary, Conclusion, and Recommendation 21

Literature Cited 39
Appendices
List of Tables
List of Figures
List of Plates

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 17

clotting time

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 Two-

way 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.com-

herbalism, 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 of the injured tissue, is termed hemostasis consisting of (1) the constriction of blood

vessels (vasoconstrictive phase), (2) the clumping together (aggregation) of platelets (platelet

phase), and (3) blood clotting (coagulation phase) (fhadsfjklsd).

E. Related Studies

The study of Wang (2011) showed that Artemisia annua L. extract and C12 have obvious

pro-coagulant effect in-vitro. C12 is the part of 20% methanol fraction after column

chromatography of MCI gel is the hemostatic active fraction of Artemisia annua L. The crude

extract of Artemisia annua L. has the hemostatic activity, and the R-value is 19.85%, while the

R-value of positive control is 8.54%. Since the ethyl acetate extract isnot completely dissolved

and there is granular which can accelerate the solidification of the plasma in the physic liquor,

the R-value of ethyl acetate extract is a little high than the n-butanol extract. And the result

11
showed that the R-value of n-butanol extract is higher. There was significant effect of the crude

extract and n-butanol extract on PRT.

According to the study of Rajasekaran (2010), the significant reduction in bleeding time

suggest that the Eupatorium ayapana leaf extract and fresh juice have positive effect on

hemostatic phase of wound healing and may possibly act on the integrity of blood vessel or

involvement of platelets forming the hemostatic plug. Platelets are the blood cells involved in

coagulation or it may inhibit the formation of prostaglandin by the vessel walls during injury.

Prostaglandin released during injury is responsible for vessel relaxation, which leads to increase

in bleeding of blood during injury.

Goker (2008) worked on the hemostatic actions of the folkloric medicinal plant extract

Ankaferd Blood Stopper. It was observed that the addition of ABS to normal plasma and serum

resulted in the very rapid (< 1s) formation of a protein network. ABS- induced protein network

was capable of regulating further coagulation and hemostatic reactions. Routine hemostatic and

biochemical tests have revealed that the ABS-induced network formation depended upon

interactions between ABS and blood proteins, mainly fibrinogen and other proteins possibly via

agglutination of these molecules. The ABS-induced network formation isrelated to the functions

of blood proteins and red blood cells.

12
 CHAPTER III

METHODOLOGY

A. Collection of Plant

Experimental plant Peperomia pellucida or Pansit-pansitan (Plate1) was obtained in

Mindanao State University, Marawi City. The whole plant was washed with clean water and

the damaged parts were discarded. Distilled water was used for the final rinse. The plant was

identified using the Flora of Manila book and Pulak Majumder’s Review Article on

medicinal herb Peperomia pellucida plant.

B. Preparation of Plant Crude Extract

The whole plant of Peperomia pellucida was utilized in this study. It was air-dried

for three days, then chopped and pounded using wooden mortar and pestle. The extraction

was done through squeezing method using sterilized cheese cloth. The collected extract was

then filtered twice using filter paper. The brownish liquid filtrate was transferred into

sterilized glass vials and stored in the refrigerator, to prevent contamination, until required.

Appropriate units of solution extracts were made. Distilled water was used as medium

for dilution. There were three solution extracts prepared, 50%, 75% and 100% crude extracts.

Fifty percent solution extract was prepared by dilution of 0.5ml pure extract into 1ml distilled

water. Seventy-five percent solution extract was obtained by diluting 0.75ml pure extract

into 1ml distilled water. One hundred percent extract was a pure concentrated extract. Final

solution extracts were kept in pre-labeled sterilized vials and stored in the refrigerator (Plate

2).
13
C. Screening of Blood Donors

Blood donors were drawn from healthy volunteer donors (n=15) of both genders

(ages 18- 25 years old). The donors were told the purpose of the work and written donor

consent forms (Appendix 4) for blood collection were given to all respondents and signed.

They then bled for the experiment.

D. Blood Collection and Preparation

Blood sample was drawn from donor via venipuncture (Appendix 6) method using

syringe at the antecubital area of the forearm (Plate 3). Only approximate 1mL of blood was

drawn from the donor using 1ml sterile disposable syringe with 25-gauge needle size. The

blood drawn was then transferred into pre-warmed glass tubes immediately.

For blood transfer, glass tubes used were sterilized and pre-warmed in a 37o C water

bath. There were four glass tubes designated accordingly for normal plasma, for 50%

extract, for 75% extract and for 100% extract; tubes were pre-labeled as A, B, C, and D

respectively. The test for blood clotting determination instantaneously followed.

E. Clotting Time Determination

From the collected blood of 1ml, about 0.2ml aliquots of blood were distributed into

four prepared glass tubes. Immediately, appropriate crude extract solutions were added into

respective tubes. Upon immediate addition of crude extracts, stopwatch was started. Clotting

time determination on glass tubes were observed simultaneously by tilting test tubes to a 45o

angle at intervals of 30 seconds until the test tube can be completely inverted without spilling

the contents, which is blood, was completely clotted (Plate 4). Glass tube A contained 0.2ml

14
whole blood (control) and observed for any trace of blood clot. Glass tube B contained 0.2ml

whole blood and received 0.2ml of 50% crude extract and observed for any trace of blood

clot. Glass tube C contained 0.2ml whole blood and received 0.2ml 75% crude extract and

observed for any trace of blood clot. Glass tube D contained 0.2ml whole blood and received

0.2ml 100% crude extract and observed for any trace of blood clot. Observed clotting time

was noted in minutes (Appendix 1).

F. Experimental Design

This study used Randomized Complete Block Design (RCBD). The experimental

treatments were basically whole blood distributed into glass tubes. The design had three

Blocks (I, II, and III) with four Treatments (A, B, C, and D) each; and each Treatment had

five replicates totaling to 60 treatments (n=60). All Treatments A were treatments with no

crude extract received (control). All Treatments B received 50% solution extract. All

Treatments C received 75% solution extract. All Treatments D received 100% crude

extract.

Treatment A Treatment B Treatment C Treatment D

(control) (50% crude extract) (75% crude extract) (100% crude extract)
BLOCK I

T1 T1 T1 T1

T2 T2 T2 T2

T3 T3 T3 T3

T4 T4 T4 T4

T5 T5 T5 T5

Table1. Sample table for raw data

15
H. Statistical Tool

Results will be expressed as Mean ± Standard Deviation (S. D.). Statistical analysis

involved are two-way Analysis of Variance (ANOVA) followed by Duncan’s Multiple

Range Test (DMRT) for determination of differences in mean. A value P less than 0.05

(p<0.05) was considered statistically significant.

16
 Chapter IV

RESULTS AND DISCUSSIONS

The potential hemostasis-induced properties of the Peperomia pellucida plant was

evaluated in vitro on human blood as being mixed with its crude extract. This study involved 15

respondents and treatments totaling to 60 (n= 60). There were three appropriate units of crude

extract tested on treatments: 50%, 75% and 100% crude extracts. Raw data on the blood clotting

time at different blocks and treatments are shown in Appendix I.

Table 1 shows the hemostatic effects of the Peperomia pellucida plant crude extracts on

the normal blood clotting time of human plasma using two-way ANOVA.

Table 1. Two-way ANOVA on the effect of P. pellucida on human whole blood clotting time

Source Sum of df Mean Square F Sig.

Squares

Block 33.013 2 16.506 7.625* 0.001

Treatment 51.492 3 17.164 7.928* 0.000

Block*treatment 6.879 6 1.146 0.530ns 0.783

Error 103.916 48 2.165

Total 195.300 59

* Significant
ns-Not Significant

17
 Results reveal that the change in the mean scores between different blocks and treatments

on human blood clotting time is significant that not only due to chance (P> 0.05). This shows

that the P. pellucida plant crude extract did induce an effect on the normal clotting time of the

human blood treatments. However, the interaction between the block and the treatment was not

significant (P> 0.05).

Table 2. DMRT on the effect of P. pellucida on human whole blood clotting time

Subset*

Treatment N 1 2 3

15 a
C- 75% Crude extract 5.9867

15 ab bc
B- 50% Crude extract 7.0067 7.0067

15 c
A- Control Group 7.2200

15 d
D- 100% Crude extract 8.5867

Sig. 0.064 0.693 1.000

* Means for groups in homogenous subsets are displayed

Based on observed means, critical value is 0.05
Means having the same letter/s are not significantly different

DMRT was used to determine which pairs of treatment means on the clotting time of

human blood were different. Table 2 shows that the 75% Crude extract treatment and the 50%

Crude extract treatment were in the same subset. That is, the clotting time rate of 75% Crude

extract was not significantly different from 50% Crude extract. This subset had the fastest rate

18
of clotting time based on observed means. Similarly, 50% Crude extract was followed by the

Control group in the next subset having the same clotting time range. Significant interval in the

clotting time range in this subset can be attributed to the diluted concentration of 50% Crude

extract that gives a clotting time near equivalent with the Control group. Also, this suggests that

even the lesser concentration of the Crude extract at 75% can still induce blood clotting.

On the other hand, only the 100% Crude extract belongs to the last subset. This implies

that 100% Crude extract was significantly different from the other three treatments. This subset

has the slowest rate of clotting time, in other words it prolongs the normal clotting time human

blood usually takes. Probably, there are chemical components in this P. peperomia plant that

when used at higher concentrations can cause prolongation on the clotting mechanism.

Table 3. Mean values on the effects of P. pellucida on human blood clotting time

Treatment Mean N Std. Deviation

Control group 7.2200 15 2.39141

50% Crude Extract 7.0067 15 0.95728

75% Crude Extract 5.9867 15 1.18132

100% Crude Extract 8.5867 15 1.49708

Total 7.2000 60 1.81939

Table 3 illustrates the mean values of blood samples between different treatments.

Control standard samples were run parallel and comparatively with the test samples on blood

19
human volunteers as shown in Appendix 2. Control group clotting time was 7.22 minutes. It

was distant to P. pellucida that showed 6.00 minutes for 75%Crude extract/0.2ml of blood; and

near equivalent of 7.00 minutes for 50%Crude extract/0.2ml of blood. Meanwhile, 100%Crude

extract showed anticoagulation for 8.59 minutes before it clotted. The normal average clotting

time established locally in this study is 7.20 minutes.

Results showed imply that the greater the value of the mean, the more clotting time is

prolonged. Thus, it demonstrates that 75% crude extract established appropriate concentration

for coagulation. On the other hand, pure concentration of extract slows down blood coagulation.

20
 Chapter V

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

The hemostasis-inducing efficacy of Peperomia pellucida plant crude extract on human

blood was investigated in vitro. There were three different crude extract concentrations tested,

50%, 75% and 100%. Initially, blood was drawn from fifteen volunteer donors. Hemostatic

effects were measured by determination of blood clotting time using glass tubes noted in

minutes. Blood samples were grouped into three blocks and then divided into four treatments.

Treatment A is the standard control of whole blood and no crude extract received. Treatment B

received 50% crude extract. Treatment C received 75% crude extract and Treatment D received

100% crude extract. All experiments in the control were replicated five times with one blood

donor per replicate. A novel average clotting time of human blood was established locally.

Blood clotting time under different blocks and treatments were compared and analyzed using

two-way ANOVA and Duncan’s multiple range tests.

Results showed that Peperomia pellucida plant crude extract induced an effect on the

clotting time of human blood at 50% and 75% crude extract concentrations. Moreover, the 75%

crude extract revealed to hasten the normal clotting time while 50% crude extract showed near

equal results with the control group. However, 100% crude extract induced prolongation on the

normal average clotting time.

The following are recommendations for further studies:

21
1. Investigate and isolate the potential components of the plant that induces hemostatic

mechanisms in blood.

2. Utilize other blood clotting assay methods such as Dale’s method and Duke’s method.

3. Apply other hemostasis measurement assays like bleeding time assay, prothrombin time (PT)

assay and partial thromboplastin time (aPTT) assay

4. Investigate its Median lethal dose for recommended dosage.

5. Increase number of replicates in the study to rule out variations caused by genetic and

individual differences of the respondents.

22
 LITERATURE CITED

BOOKS

Rang, H.P., Dale, M.M., Ritter, J.M., (1999). Pharmacology (4th ed.). Churchill Livingstone,
UK:Churchill Livingstone Publishing Company.

Ganong, W.F., (2000). Review of Medical Physiology (10th ed.).Kuala Lumpur, Malaysia:
Khapu Publishing Company.

Ghani A. (1998). Medicinal Plants of Bangladesh (1st ed.). Dhaka, Bangladesh: Asiatic Society
of Bangladesh.

Wagman, R.J. (2000). The New Complete Medical and Health Encyclopedia (Vol. 2, pp. 408
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Egwuche, R.U., Odetola, A., Erukrainure, O. (2011). Preliminary investigation into the chemical
properties of Peperomia pellucida. Research J. of Phytochemistry, 5, 48-53.

Majumder, P., Priya, A., Satya, V. (2011). Ethno-medicinal, Phytochemical and

Pharmacological review of an amazing medicinal herb Peperomia pellucida (L.) HBK.
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bioactive compunds in Bolivia through a multidisciplinary approach: Part III. Evaluation

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 of the antimalarial activity of plants used by Altenos Indians. Journal
Ethnopharmacology, 71, 123-131.

Goker, H., Haznedaroglu, I.C., Ercetin, S., Kirazli, S., Akman, U., Ozturk, Y., Firat, H.C. (2008).
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from Peperomia pellucida. Life Sciences and Medicine Research, 1, 1-10.

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Unpublished Undergraduate Theses

Mustapha, R. (2012). Thrombocyte increasing activity of orally administered decoction of

Euphorbia hirta, Ipomea batatas, Durio zibethinus and Moringa oleifera on white mice
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25
26
Appendix 1. Raw data for blood clotting time on treatments per blocks

Clotting time
Treatment A Treatment B Treatment C Treatment D
(control) (50% crude extract) (75% crude extract) (100% crude extract)
mins. mins. mins. mins.
BLOCK I

R1 6.30 R1 7.00 R1 6.00 R1 8.00

R2 7.30 R2 8.00 R2 6.30 R2 9.30

R3 6.00 R3 7.00 R3 5.30 R3 10.00

R4 6.30 R4 6.30 R4 4.00 R4 7.00

R5 7.00 R5 7.00 R5 6.00 R5 10.30

Clotting time
Treatment A Treatment B Treatment C Treatment D
(control) (50% crude extract) (75% crude extract) (100% crude extract)
mins. mins. mins. mins.
BLOCK II

R1 15.00 R1 7.0 R1 6.30 R1 9.30

R2 5.30 R2 7.30 R2 7.00 R2 10.30

R3 9.30 R3 9.30 R3 9.00 R3 10.00

R4 6.30 R4 7.00 R4 6.00 R4 6.00

R5 8.30 R5 8.00 R5 7.00 R5 10.00

Clotting time
Treatment A Treatment B Treatment C Treatment D
(control) (50% crude extract) (75% crude extract) (100% crude extract)
mins. mins. mins. mins.
BLOCK II

R1 5.30 R1 5.30 R1 4.30 R1 6.00

R2 6.30 R2 6.30 R2 5.30 R2 7.30

R3 7.00 R3 7.30 R3 6.00 R3 8.30

R4 6.30 R4 6.30 R4 5.30 R4 9.00

R5 6.30 R5 6.00 R5 6.00 R5 8.00

26
Appendix 2. Mean values on effect of P. pellucida on human whole clotting time

Block Treatment Mean Std. Deviation N

A 6.5800 0.54498 5
B 7.0600 0.60663 5
C 5.5200 0.92574 5
I

D 8.9200 1.3976 5
Total 7.0200 1.52371 20

Block Treatment Mean Std. Deviation N

A 8.8400 3.78920 5
B 7.7200 0.97314 5
C 7.0600 1.16962 5
II

D 9.1200 1.78241 5
Total 8.1850 2.21627 20

Block Treatment Mean Std. Deviation N

A 6.2400 0.60663 5
B 6.2400 0.71972 5
C 5.3800 0.69785 5
III

D 7.7200 1.13886 5
Total 6.3950 1.14362 20

27
 MINDANAO STATE UNIVERSITY
College of Natural Sciences and Mathematics
DEPARTMENT OF BIOLOGY
Marawi City

January 2013

Ma’am/Sir,

Greetings!

I, Norlainie Castro Omar, a proponent of the research study “Hemostasis effects of Peperomia pellucida
plant extract, in vitro”, would like to indicate my interest on requesting for your participation as a
respondent (Blood donor) in this study. The study concerns the investigation on the usefulness of the
said plant extract against hemorrhage and its effects on the management of blood clotting during
hemostasis.

In line with this, the study shall utilize human blood samples as its experimental variables, which will be
drawn from donors using Syringe Method Venipuncture. Attached herein are the details on how the
proponent shall perform the procedures on blood collection as based from non-surgical procedure
descriptions and guidance. An informed written consent will be given for you to affix your insignia of
volunteer participation.

Rest assured that the proponent would be liable on any unfortunate circumstances during the blood
collection.

Thank you.

Yours truly,

NORLAINIE C. OMAR
Thesis Proponent

Recommended by:

PROF. APOLINARIO ALICANTE, DVM

Thesis Adviser

28
 INFORMED WRITTEN CONSENT

Date

I, , agreed to participate as respondent (Blood donor) in

the research study entitled “Hemostatic effects of Peperomia pellucida plant crude extract, in vitro”.

The study has been well explained to my understanding by the proponent, affixed
herein is my signature that may serve as my voluntary willingness to participate.

Witness Respondent

INFORMED WRITTEN CONSENT

Date

I, , agreed to participate as respondent (Blood donor) in

the research study entitled “Hemostatic effects of Peperomia pellucida plant crude extract, in vitro”.

The study has been well explained to my understanding by the proponent, affixed
herein is my signature that may serve as my voluntary willingness to participate.

Witness Respondent

29
 MINDANAO STATE UNIVERSITY
College of Natural Sciences and Mathematics
DEPARTMENT OF BIOLOGY
Marawi City

October 9, 2012

PROF. APOLINARIO ALICANTE, DVM

Faculty
Department of Biology
CNSM

Sir:

Greetings!

The undersigned senior student from the Department of Biology is a proponent of the proposed thesis
study entitled “Hemostatic Effects of Peperomia pellucida Plant Crude Extract, in vitro”. She would like
to indicate her interest on requesting you to be the proponent’s thesis adviser. Thus, you shall be
notified for consultations needed and undertakings of the proponent regarding to the said thesis study.

The proponent would be appreciative to have your honor supervising her study. Your expertise and
knowledge in Medical Physiology studies would be of great assistance on the success of this study.

The proponent of the study is hoping for a positive response on this matter.

Thank you.

Respectfully yours,

NORLAINIE C. OMAR
IV BS Biology

30
Plate1. Experimental plant Pansit-pansitan (Peperomia pellucida)

Plate 2. Crude extracts of experimental plant. (A) 50% crude extract; (B) 75% Crude extract;
(100%) Crude extract.

31
Plate 3. Blood collection via Venipuncture Syringe Method

Plate 4. Blood clots on tubes. (A) Control; (B) 50% crude extract; (C) 75% crude extract; (D)
100% crude extract.

32