Effect of Origanum syriacum Thymus vulgaris …The Islamic University of Gaza Deanship of Postgraduate Studies Faculty of Science Biological Sciences Master Program Effect of Origanum

رارـــــإق

:أنا الموقع أدناه مقدم الرسالة التي تحمل العنوان

Effect of Origanum syriacum, Hibiscus sabdariffa, Thymus vulgaris and Pelargonium graveolens Extracts on

Human Leukemia THP-1 Cells in Vitro

هدي الخاص، باستثناء ما تمت اإلشارة إلیه أقر بأن ما اشتملت علیه هذه الرسالة إنما هي نتاج ج

حیثما ورد، وٕان هذه الرسالة ككل، أو أي جزء منها لم یقدم من قبل لنیل درجة أو لقب علمي أو .بحثي لدى أیة مؤسسة تعلیمیة أو بحثیة أخرى

DECLARATION

The work provided in this thesis, unless otherwise referenced, is the researcher's own work, and has not been submitted elsewhere for any other degree or qualification

Doa'a Mohamad Faris دعاء محمد فارس :اسم الطالب

:Signature :التوقیع

:Date م 11/1/2014 :التاریخ

Student's name:

The Islamic University of Gaza Deanship of Postgraduate Studies

Faculty of Science Biological Sciences Master Program

Effect of Origanum syriacum, Hibiscus sabdariffa, Thymus vulgaris and Pelargonium graveolens Extracts on Human Leukemia THP-1 Cells in Vitro

Prepared by:

Doa'a Faris

Supervisors:

Dr. Abdalla Abed, PhD Human Genetics The Islamic University of Gaza, Faculty of Science, Biology Department

Dr. Basim Ayesh, PhD Molecular Biochemistry Al Aqsa University, Faculty of Science, Medical Technology Department

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master in Biological Sciences/ Medical Technology

2013 A.D/ 1433 A.H.

Gaza, Palestine

II

Abstract

Introduction: Leukemia is an aggressive disease that if untreated it leads to death.

Because the conventional leukemia treatment protocols are not effective in all cases and

result in serious side effects, discovering new safer and more effective anticancer agents

from natural materials is the aim of many medical researches nowadays. Within this

perspective, the present study was constructed to assess the possible anticancer effects

of Origanum syriacum, Hibiscus sabdariffa, Thymus vulgaris, and Pelargonium

graveolens, extracts on human leukemic THP-1 cells and to evaluate the effect of these

extracts toward normal human peripheral blood mononuclear cells (PBMCs).

Methodology: The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide

(MTT) assay was used to assess the antiproliferative effects while cytotoxicity was

evaluated using the lactate dehydrogenase (LDH) assay. Ethanol extracts of the four

plants were prepared at various concentrations then were investigated against both THP-

1 cells and PBMCs by MTT assay. After that, three of the extracts were tested with

LDH assay against THP-1 cells.

Results: All extracts, except P. graveolens extract, exhibited a concentration dependent

antiproliferative effect against THP-1 leukemic cells with IC50 values of 15.47 mg/mL

for H. sabdariffa, 2.126 mg/mL for O. syriacum, and 0.1569 mg/mL for T. vulgaris.

Also they showed inhibition against PBMCs with IC50 values of 3.514 mg/mL for H.

sabdariffa, 0.4247 mg/mL for O. syriacum, and 0.3345 mg/mL for T. vulgaris. Only O.

syriacum extract exerted a cytotoxic effect against leukemic cells, as evaluated by the

LDH assay, with LC50 value of 9.646 mg/mL while T. vulgaris and P. graveolens

extracts did not show any cytotoxicity.

Conclusion: It can be concluded that only T. vulgaris extract, which showed a relative

selective inhibition toward leukemic cells, may be useful in developing successful

treating agents for leukemia.

Key words: Leukemia, THP-1 Cells, Origanum syriacum, Hibiscus sabdariffa, Thymus

vulgaris, Pelargonium graveolens, antiproliferative, cytotoxic.

III

كما أن . الھالكیجب عالجھا وإال أدت الى من األمراض المستعصیة التي ) اللوكیمیا(یعتبر سرطان الدم :مقدمة

ولھذا . ثار جانبیة ضارةمجدي في حاالت كثیرة وینتج عنھ آ العالج الكیمیائي الذي یعطى لمرضى اللوكیمیا غیر

في مجال صناعة األدویة ھو إنتاج مركبات معالجة التي تجرى حالیا ھدف الكثیر من الدراساتالسبب كان

في ھذا النطاق . اتات الطبیة التي تعتبر آمنة وفعالة في آن واحدلسرطان الدم من المواد الطبیعیة وباألخص من النب

)Origanum syriacum( تم تخصیص ھذه الدراسة لبحث التأثیر المثبط أو السام لمستخلصات البردقوش

Pelargonium( والعطرة )Thymus vulgaris( والزعتر )Hibiscus sabdariffa( والكركدیھ

graveolens( على خالیا الدم السرطانیة)THP-1 cells .(ھذه المستخلصات على خالیا الدم یضا لتقییم تأثیرأ

).PBMCs(الطبیعیة

لبحث التأثیر (LDH)لدراسة التأثیر المثبط لنمو الخالیا وفحص ال MTT)(فحص التم استخدام :منھجیة البحث

ومن ثمات الكحولیة للنباتات األربعة بتركیزات مختلفة تم تحضیر المستخلصحیث . السام للمستخلصات على الخالیا

(LDH)فحص التم استخدام ثم .)MTT(بفحص ال لسرطانیة والطبیعیةدراسة تأثیر ھذه التركیزات على الخالیا ا

.مستخلصات على الخالیا السرطانیةال ة منتقییم سمیة ثالثل

تأثیر مثبط لنمو الخالیا السرطانیة والخالیا الطبیعیة فإن كل المستخلصات كان لھا، باستثناء العطرة :جئالنتا

15.47من الخالیا السرطانیة ھو % 50تثبیط أدى إلىیز المستخلصات الذي حیث كان ترك. بدرجات متفاوتة

یز في حین ترك. ملیلیتر للزعتر/ملیجرام 0.1569ملیلیتر للبردقوش و/ملیجرام 2.126ملیلیتر للكركدیھ و/ملیجرام

0.4247ملیلیتر للكركدیھ و/ملیجرام 3.514من الخالیا الطبیعیة كان % 50تثبیط أدى إلىصات الذي المستخل

الذي سبب سمیة مستخلص البردقوش ھو الوحید. ملیلیتر للزعتر/ملیجرام 0.3345ملیلیتر للبردقوش و/ملیجرام

بینما ، من الخالیا السرطانیة% 50ملیلیتر أدى إلى سمیة /ملیجرام 9.646بتركیز حیث أنھ لخالیا اللوكیمیا

.مستخلصي الزعتر والعطرة لم یبدیان أي تأثیر سمي تجاه ھذه الخالیا

نستنتج أن مستخلص الزعتر ھو الوحید الذي ثبط نمو الخالیا السرطانیة أكثر من السابقة النتائج من :الخالصة

لھا من نبات الزعتر إمكانیة العثور على مركباتة وبالتالي ھذه النتیجة تعطي األمل في یتثبیطھ للخالیا الطبیع

.ل فعال وآمن نسبیامرض اللوكیمیا ب–ك القدرة على معالجة

IV

Dedication

To my mother and father.

To my brothers and sister.

To everyone likes science

and works hard to obtain it.

Doa'a M. Faris

V

Acknowledgement

Firstly, all praises and thanks are to Allah, the lord of the worlds. Without his grace and

assistance, nothing can be possible.

I would like to express my profound gratitude and deep regards to my teachers Dr.

Abdalla Abed and Dr. Basim Ayesh, the supervisors of this thesis, for their exemplary

guidance, monitoring, support, patience and constant encouragement throughout the

course of this thesis.

I would like to thank all members of the Faculty of Science in the Islamic University

of Gaza, especially, the biology department's laboratory technicians, as well as the

medical technology department's laboratory technicians, for their help during the

practical stage of the thesis.

My sincere gratitude is to the Scientific Research Affairs in the Islamic University of

Gaza for their financial support.

I would like to thank Eng. Mahmoud Abd El-Al, the area manager of the Arcomed

Medical Supplies Company, as well as all company employees for the ordering of

supplies.

Thank to Eng. Amjad El-Agha and Eng. Raed El-Sakka for their help in plants

collection. In addition, special thanks to Dr. Mohammed Abo Ouda for his kind

assistance in the identification and authentication of the plants used in this work.

I would like to thank all my colleagues in the European Gaza Hospital Laboratory, with

special thanks to the laboratory managers Mr. Rushdi Rasras and Miss Sofia Zourob

for their support, patience, and encouragement during all study stages of master.

Lastly, the biggest thank and the deepest regards are for my parents due to their love,

support, patience and supplications without which the completion of this work would

not be possible. Also, a lot of thanks to my brothers, sister and friends for their

continuous help, and constant encouragement.

VI

Table of Contents

Abstract ………………………………………………………………………….… II

Abstract in Arabic ………………………………………………….……………... III

Dedication ……………………………………………………………….………… IV

Acknowledgement …………………………………………………….………..…. V

List of Figures ………………………………………………………..………….… VIII

List of Tables ……………………………………………………….…………...… IX

List of Abbreviations ……………………………………………………………… X

1. Introduction …………………………………………………… 1 1.1 Overview………………………………………………………………….… 1

1.2 General Objective …………………………………..…………………….… 3

1.3 Specific Objectives ………..…………………………..……………………. 3

1.4 Study Significance …………...………………………..……………………. 3

2. Literature Review ……………………………………………... 4 2.1 Leukemia ………………………………………………………………….... 4

2.1.1 Acute Myeloid Leukemia …..………………………………………………. 5

2.1.2 Management of Acute Myeloid Leukemias …….…………………………. 7

2.2 Medicinal Plants ……………………………..…………………………….. 8

2.2.1 Origanum syriacum ………………..……………………………………….. 9

2.2.2 Hibiscus sabdariffa …………..…………………………………………….. 12

2.2.3 Thymus vulgaris……………………………………………………..……… 16

2.2.4 Pelargonium graveolens …..……………………………………………….. 19

2.2.5 Mechanisms of Medicinal Plants Anticancer Effects ……………………… 21

3. Materials and Methods ……………………………………….. 24 3.1 Materials ……………………………...……………………..……………… 24

3.2 Collection and Preparation of Plant Materials ……………………………… 25

3.3 Extraction of Plant Materials …………………………………..…………… 26

3.4 THP-1 Cell Line Processing and Maintenance …………………..…….....… 27

3.4.1 Cells Receiving and Culturing ……………………………………..……..… 27

3.4.2 Cells Freezing in Stocks ……………………………………………..…...… 28

VII

3.4.3 Determination of THP-1 Cells Growth Characteristics ………...……..….… 28

3.4.4 Routine Maintenance of THP-1 Cells in Culture …...………………..…..… 29

3.5 Determination of MTT and LDH Assays Sensitivity to THP-1 Cells …...…. 29

3.6 Assessment of Plant Extracts Effects on THP-1 Cells by MTT Assay ......… 31

3.7 Assessment of Plant Extracts Effects on Peripheral Blood Mononuclear

Cells (PBMCs) by MTT Assay …………….………………………….……

33

3.8 Determination of Plant Extracts Toxicity on THP-1 Cells by LDH Assay … 34

3.9 Statistical Analysis ……………………………………………………….… 36

4. Results ……………………………………………………….… 37 4.1 Plants Crude Extracts Percentage Yields …………………………………… 37

4.2 Morphology and Growth Characteristics of THP-1 Cells ………………..… 37

4.3 MTT and LDH Assays Sensitivity Studies ………………………………… 39

4.4 Plant Extracts Effects on THP-1 Cells by MTT Assay ……………..……… 39

4.5 Plant Extracts Effects on Peripheral Blood Mononuclear Cells (PBMCs) by

MTT Assay …………………………………………………………….……

43

4.6 Plant Extracts Toxicity on THP-1 Cells by LDH Assay …………………… 47

5. Discussion …………………………………………………….... 50

6. Conclusion and Recommendations …………………………... 57 6.1 Conclusion ………………………………………………………………..… 57

6.2 Recommendations ……………………………………………………......… 57

7. References …….………………………….…………………… 59

VIII

List of Figures

Figure Page

2.1 Origanum syriacum ……………..………………………………….... 10 2.2 Hibiscus sabdariffa ………………...……………………………….... 12 2.3 Thymus vulgaris …………………………………………………….... 16 2.4 Pelargonium graveolens …………………...………………………… 19 3.1 Rotary evaporator ……..……………………..……………………..... 26 3.2 ELISA reader device ………………………………………………..... 30 4.1 THP-1 cells under the inverted microscope ………………………..… 38 4.2 Growth curves of THP-1 cells …………………………………..….... 38 4.3 Sensitivity curves of MTT and LDH assays to THP-1 cells …...…….. 39 4.4 Dose response curves of H. sabdariffa and O. syriacum extracts

effects on THP-1 cells …………………….………..…………………

41 4.5 Dose response curves of T. vulgaris and P. graveolens extracts as

well as DMSO effects on THP-1 cells ……….………..……………...

41 4.6 Dose response curves of H. sabdariffa and O. syriacum extracts

effects on PBMCs …………………………………………..………...

43 4.7 Dose response curves of T. vulgaris and P. graveolens extracts as

well as DMSO effects on PBMCs …………………………................

43 4.8 Comparison between the effects of H. sabdariffa extract on THP-1

cells and on PBMCs ………………………………………………...

46 4.9 Comparison between the effects of O. syriacum extract on THP-1

cells and on PBMCs ……………………………………………..….

46 4.10 Comparison between the effects of T. vulgaris extract on THP-1 cells

and on PBMCs ………………………………………………..…….

47 4.11 LDH toxicity dose response curves of O. syriacum, T. vulgaris, and

P. graveolens extracts as well as DMSO on THP-1 cells ……..……...

48

IX

List of Tables

Table Page 4.1 Percentage yields (%) of all dried extracts obtained after solvent

evaporation …………………………………………………………. 37

4.2 THP-1 cells viability results by MTT test ………………………….. 40 4.3 IC50 values of the extracts on THP-1 cells ………………………….. 42 4.4 Peripheral blood mononuclear cells viability results by MTT test …. 44 4.5 IC50 values of the extracts on PBMCs ……………………………… 45 4.6 THP-1 cells viability results by LDH test ……………………...…… 48

X

List of Abbreviations

ALL Acute Lymphoblastic Leukemia

AML Acute Myeloid Leukemia

AMoL Acute Monocytic Leukemia

ANLL Acute Non-Lymphocytic Leukemia

BM Bone Marrow

C3b the large element of complement 3

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

ECACC European Collection of Cell Cultures

ELISA Enzyme Linked Immuno-Sorbent Assay

FAB French–American–British

FBS Fetal Bovine Serum

Fc Fragment, crystallizable

H. sabdariffa Hibiscus sabdariffa Linn.

HDL High Density Lipoprotein

HEPES Hydroxyethyl Piperazineethanesulfonic Acid

IC50 Median Inhibition Concentration

LC50 Median Lethal Concentration

LDH Lactate Dehydrogenase

LDL Low Density Lipoprotein

MDS Myelodysplastic Syndromes

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAD Nicotinamide Adenine Dinucleotide

O. syriacum Origanum syriacum L.

P. graveolens Pelargonium graveolens

PBMCs Peripheral Blood Mononuclear Cells

PBS Phosphate Buffered Saline

PHA Phytohemagglutinin

R2 Coefficient of correlation

ROS Reactive Oxygen Species

RPMI Roswell Park Memorial Institute

XI

SD Standard Deviation

T. vulgaris Thymus vulgaris L.

UV Ultraviolet

WHO World Health Organization

x g gravity at the Earth’s surface (relative centrifugal force unit)

1

Chapter 1 Introduction

1.1 Overview

Cancer is one of the most severe health problems in both developing and

developed countries. It is a general term applied to malignant diseases characterized by

rapid and uncontrolled proliferation of abnormal cells which may mass together to form

a solid tumor or proliferate throughout the body, and when progress it causes death.

Cancer cells are able to grow, invade neighboring tissues and may also affect other

organs through the lymphatic system or bloodstream. Carcinogens such as viruses, some

chemicals, tobacco use and radiation cause aberration of the genetic material of cells.

This leads to the up-regulation of oncogenes which promote cell growth and/or the

down-regulation of tumor suppressor genes which inhibit cell division. Changes in

many genes are required to transform a normal cell into a cancer cell (Knudson, 2001).

In Palestine, the cancer incidence rate was 74.0 per 100,000 of population in

2012, while in the previous year, 2011, it was 64.2 per 100,000 of population. In these

two years, cancer was considered as the second leading cause of death among

Palestinians after cardiovascular diseases (Ministry of Health, 2011, 2013). Also there

was a remarkable increase in cancer mortality compared with 2007 and 2010, from

10.3% in 2007 to 10.8% in 2010 then increases to reach 13.6% from the total deaths in

2012 (Ministry of Health, 2013).

There are many types of cancer and each type is named according to the tissue

or organ in which it was originated. Leukemia is cancer of the blood or bone marrow

that was first recognized by the German pathologist Rudolf Virchow in 1845 (Virchow,

1845). Leukemia refers to a group of malignant diseases characterized by an abnormal

proliferation and differentiation of blood cells and their precursors in the bone marrow.

Leukemias cases in 2012 comprised 6.1% of cancer cases in Palestine (Ministry of

Health, 2013). Anemia, neutropenia and thrombocytopenia are important consequences

of infiltration of the bone marrow, which in turn can lead to infection and hemorrhage

(Bain, 2003). There are both acute and chronic forms of leukemia, each with many

subtypes that vary in their response to treatment. Leukemia is treated mainly with

2

chemotherapy in addition to medical radiation therapy, hormone treatments, or bone

marrow transplantation.

The use of chemotherapeutic drugs in leukemia treatment although is effective

in some cases, it has diverse side effects. These agents are highly toxic to all body cells,

and they destroy normal cells as well as cancerous cells. This leads to disturbances in

normal cells function where fatigue, infection, anemia, hair loss and bleeding problems

result after each chemotherapy dose, also heart problems and lung tissue damage may

appear later after treatment has ended (Limper, 2004; De et al., 2012). In addition, in

some cases chemotherapy resistance develops against the used chemotherapeutic agents

and other drugs must be used in the treatment (Marie & Legrand, 2003).

These severe side effects of chemotherapy have highlighted the importance of

natural products, especially medicinal plants, and its usage as alternative agents in

cancer treatment. Plants are natural reservoir of medicinal agents almost free from the

side effects normally caused by synthetic chemicals (Fennel et al., 2004). The World

Health Organization estimates that herbal medicine is still the main stay of about 75-

80% of the world population, mainly in the developing countries for primary health care

because of better cultural acceptability, better compatibility with the human body, and

lesser side effects (Kamboj, 2000; Yadav & Dixit, 2008).

Plants have been used for treating various diseases of human beings and animals

since time immemorial. More than 50% of all modern drugs in clinical use are of

natural products, many of which have the ability to control cancer cells (Rosangkima

and Prasad, 2004). Medicinal plants possess immunomodulatory and antioxidant

properties, leading to anticancer activities. They are known to have versatile

immunomodulatory activity by stimulating both non-specific and specific immunity

(Agrawala et al., 2001; Pandey and Madhuri, 2006). The antioxidants may prevent and

cure cancer and other diseases by protecting the cells from damage caused by free

radicals. Many plant-derived products have been reported to exhibit potent antitumor

activity against several rodent and human cancer cell lines (Rao et al., 2004).

In the Mediterranean region there are about 2,600 species of plants, many of

which are considered to have medicinal effects (Saad et al., 2005). However, there is

relatively limited research on the medicinal plants and herbs in this region. Therefore,

any researches in this field are encouraged to discover the medicinal effects of these

3

plants and to develop new effective drugs from these plants to prevent and treat the

various diseases including cancer.

1.2 General Objective

The general objective of the study is to assess the effects of Origanum syriacum,

Hibiscus sabdariffa, Thymus vulgaris, and Pelargonium graveolens extracts on human

leukemia THP-1 cells in vitro.

1.3 Specific Objectives:

• To assess the anti-proliferative effects of Origanum syriacum, Hibiscus

sabdariffa, Thymus vulgaris and Pelargonium graveolens extracts each alone

against the leukemia cell line THP-1 by means of MTT assay.

• To assess the cytotoxic effects of Origanum syriacum, Hibiscus sabdariffa,

Thymus vulgaris and Pelargonium graveolens extracts each alone on THP-1 cell

line by the means of LDH assay.

• To evaluate the possible inhibitory effect of the four extracts toward normal

peripheral blood mononuclear cells (PBMCs).

• To compare between the effects of the four extracts on the leukemia cell line.

1.4 Study Significance

Cancer is the leading cause of death worldwide and finding a cure for this

disease is always an important objective for human. The assessment of medicinal plants

cytotoxicity on leukemic cells may aid in the introduction of the selective, tolerable and

safe plant constituents in the treatment protocols for leukemia patients therefore,

avoiding the side effects of chemotherapeutic agents and maximizing the therapeutic

effect of such protocols. Also herbal constituents are available and cheap in comparison

to chemotherapeutic agents.

To our knowledge, this study is the first to deal with the effect of Origanum

syriacum on leukemia cell lines in general and the first to deal with the effect of Thymus

vulgaris, Pelargonium graveolens and Hibiscus sabdariffa on monoblastic leukemia

cell lines.

4

Chapter 2 Literature Review

2.1 Leukemia

Leukemia is a type of hematopoietic neoplasms that characterized by abnormal

proliferation and increase of immature blood cells in bone marrow and peripheral blood.

The cause of leukemia is a mutation in a single stem cell, the progeny of which form a

clone of leukemic cells. Often there is a series of genetic alterations rather than a single

event. Genetic events contributing to malignant transformation include inappropriate

expression of oncogenes and/or loss of function of tumor suppressor genes. The cell in

which the leukemic transformation occurs may be a lymphoid precursor, a myeloid

precursor or a pluripotent stem cell capable of differentiating into both myeloid and

lymphoid cells. Myeloid leukemias can arise in a lineage-restricted cell or in a

multipotent stem cell capable of differentiating into cells of erythroid, granulocytic,

monocytic and megakaryocytic lineages.

Leukemias are divided into acute leukemias, which, if untreated, lead to death in

weeks or months; and chronic leukemias, which, if untreated, lead to death in months or

years. They are further subdivided into lymphoid, myeloid and biphenotypic leukemias,

the latter showing both lymphoid and myeloid differentiation. In acute leukemias there

is a defect in maturation, leading to an imbalance between proliferation and maturation;

since cells of the leukemic clone continue to proliferate without maturing to end cells,

as a result, there is continued expansion of the leukemic clone and immature cells

predominate. Chronic leukemias are characterized by an expanded pool of proliferating

cells that keep their capacity to differentiate to end cells.

The lone detailed Palestinian cancer report published by the Palestinian Ministry

of Health was covering the period 1995-2000. In that period, acute lymphoid leukemia

accounted for 36.5% of leukemia cases in Gaza Strip, acute myeloid leukemia

accounted for 20.7%, plasma cell multiple myeloma accounted for 15.2% of cases,

chronic lymphocytic leukemia accounted for 12.5% of cases and chronic myelocytic

leukemia accounted for 15.2% of cases (Al Najar et al., 2002).

5

2.1.1 Acute Myeloid Leukemia

The terms acute myeloid leukemia (AML), acute myelogenous leukemia, and

acute nonlymphocytic leukemia (ANLL) refer to a group of marrow-based neoplasms

that have clinical similarities but distinct morphologic, immunophenotypic, and

cytogenetic features (Greer et al., 2003). AML is one of the acute leukemias' major

types in which there is a clonal growth of any one of several non-lymphoid

hematopoietic progenitors which preserve the capacity of self-renewal, but are severely

limited in their ability to differentiate into functional mature cells. These various

progenitors include cells of granulocytic, monocyte/macrophage, erythroid, and

megakaryocytic lineages. The other major type of acute leukemias is called acute

lymphoblastic leukemia (ALL), in which the abnormal proliferation is in the lymphoid

progenitor cells (that is, immature lymphocytes). The distinction between the two types

is based on morphological, cytochemical, immunological and cytogenetic differences

and is of paramount importance as the treatment and prognosis are usually different

(Provan, 2003).

In AML, a myeloid stem cell (or an early progenitor cell) is transformed. This

stem cell expands and proliferates in the blood and bone marrow and suppresses normal

hematopoiesis. A characteristic finding in AML is the leukemic bulge (the appearance

of immature leukemic cells in blood and bone marrow without terminal maturation).

Blasts of myeloid leukemias proliferate under the influence of myeloid growth factors,

some of which are produced by the leukemic cells themselves (autocrine growth of

tumor cells) (Munker et al., 2007).

According to French–American–British (FAB) classification (Bennett et al.,

1976), AML is categorized as acute myeloblastic leukemia without (M1) and with (M2)

maturation, acute hypergranular promyelocytic leukemia and its variant (M3 and M3V),

acute myelomonocytic leukemia (M4), acute monoblastic (M5a) and monocytic (M5b)

leukemia, acute erythroleukemia (M6) and acute megakaryoblastic leukemia (M7)

(Bennett et al., 1985). M0 is AML without maturation and with minimal evidence of

myeloid differentiation. In addition to the above categories there are several very rare

types of AML, which are not included in the FAB classification. These include mast

cell leukemia and Langerhans’ cell leukemia. In addition, the diagnosis of hypoplastic

6

AML requires consideration. Transient abnormal myelopoiesis of Down’s syndrome

may also be regarded as a variant of AML (Bain, 2003).

The WHO classification of acute leukemias and myelodysplastic syndromes

(MDS) has evolved away from the FAB classification, which is based on morphology,

to include not only morphology, but also clinical, immunophenotypic, and cytogenetic

features (Brunning et al., 2001; Vardiman et al., 2002). According to this classification,

AMLs are recognized as one of the three main categories of myeloid neoplasms, along

with MDS and myeloproliferative disorders. There are five major categories recognized

by the WHO: (a) AML with recurrent genetic abnormalities; (b) AML with multilineage

dysplasia; (c) AML and MDS, therapy related; (d) AML not otherwise categorized; and

(e) acute leukemia of ambiguous lineage.

Acute myeloid leukemia accounts for 10-15% of childhood leukemia, but it is

the commonest leukemia of adulthood, particularly as chronic myeloproliferative

disorders and preleukemic conditions such as myelodysplasia usually progress to acute

myeloid leukemia rather than acute lymphoblastic leukemia. The incidence increases

with age, and the median age at presentation is 60 years (Provan, 2003).

The acute monocytic/monoblastic leukemia (AMoL, FAB-M5) is an AML

subtype where the monocytic component of bone marrow (BM) non-erythroid cells is

greater than 80%. In acute monoblastic leukemia (FAB-M5a), the majority of

monocytic cells are monoblasts (in the BM and/or peripheral blood), while in acute

monocytic leukemia (FAB-M5b), the majority of monocytic cells are promonocytes

(≥80%) (Jaffe et al., 2001). AMoL is considered a distinct type of AML with

characteristic biological and clinical features; it is associated with hyperleukocytosis

(Cuttner et al., 1980), extramedullary involvement (Peterson et al., 1981), and

coagulation abnormalities including disseminated intravascular coagulation (Mangal et

al., 1984).

AMoL is frequently associated with specific chromosomal translocations

including t(8;16)(p11;p13) (Heim et al., 1987) and various translocations involving the

MLL locus at 11q23 such as t(9;11)(p22;q23), t(11;19)(q23;p13.1), and others (Baer et

al., 1998). Many patients with AMoL have mutations in the Flt3 gene (approximately

40%) either internal tandem duplications of the juxtamembrane region or point

7

mutations in the second tyrosine kinase domain, and such mutations are associated with

an unfavorable outcome (Rombouts et al., 2000; Thiede et al., 2002).

2.1.2 Management of Acute Myeloid Leukemias

The common therapeutic strategy for most patients with AML has been divided

into two general phases: remission induction and postremission (or consolidation)

therapy.

The remission induction therapy in leukemia is designed to produce the rapid

restoration of normal bone marrow function. The term complete remission is reserved

for patients who have full recovery of normal peripheral blood counts with recovery of

normal bone marrow cellularity; less than 5% blast cells are present in the bone marrow,

and none can have a leukemic phenotype or cytogenetic abnormality (Rathnasabapathy

& Lancet, 2003). The standard induction protocol is the combination of an

anthracycline with cytosine arabinoside (3 + 7 protocol). The common regimen

combines 3 days of daunorubicin with 7 days of continuous infusion of cytosine

arabinoside. With minor variations, this protocol has been the basis for the treatment of

AML for the last 25 years and has been able to induce remission in 60–80% of patients

with newly diagnosed AML. After the induction treatment, the patients remain

granulocytopenic for at least 15–20 days and are susceptible to bacterial, fungal, and

viral infections. The disturbances of coagulation and the thrombocytopenia predispose

the patients to bleeding and need support with platelet concentrates and other blood

products (Munker et al., 2007).

A consolidation or postremission treatment is administered to patients who have

reached complete remission. Two (to four) courses of consolidation are considered as

standard. Depending on the risk factors for AML, 20–25% of the patients will survive

for more than 4 year (Munker et al., 2007). Postremission therapy is directed toward

further reduction in the residual leukemic cell number, which may be as high as 108 to

109 cells at initial complete remission. The elimination of these residual leukemic cells

may be accomplished by either cytotoxic chemotherapy, causing significant

myelosuppression and even myeloablation (e.g. requiring autologous stem cell rescue)

or by replacement of a patient’s stem cells through allogeneic transplantation, a

procedure combining myeloablation and immunotherapy (Rathnasabapathy & Lancet,

2003).

8

Despite the hopeful steps in leukemias treatment researches, considerable

challenges remain (Lucas et al., 2010). Successful treatment of patients with

hematologic malignancies varies widely not just by disease category but by subset

(genetic or otherwise) within each disease, and reasons for this are generally unclear.

Second, scientific advances made in recent years have a long trek to clinical application,

and improving testing strategies to streamline the process and eliminate poor drug

candidates early will be invaluable. Third, the dissemination of tumor cells in

hematologic malignancies precludes localized surgery and radiation approaches, and the

sheltered microenvironment of bone marrow limits the efficacy of many treatments.

Advances in understanding the protective role of the microenvironment will be essential

for successful treatment of leukemias and lymphomas. Finally, novel and potent agents

must be identified that target tumor cell survival pathways while sparing normal cells.

This is a significant challenge, as there are undoubtedly targetable survival mechanisms

yet to be discovered. To make advances toward curative therapy, identification of new

tumor survival pathways and protective mechanisms must be continued; we cannot rely

only on what is currently known.

This last challenge highlights a key strength of natural product investigation.

Using cytotoxic activity-guided screening strategies, agents with potent anti-tumor

activity can be identified in an equitable way, increasing the probability of revealing

novel tumoricidal pathways and survival mechanisms. Once an effective molecule is

identified, the mode of action can be investigated and additional or alternative agents

that impact the target can be synthesized. Thus even when an active natural product

does not reach clinical use, its investigation can provide critical clues for the

development of new targeted cancer drugs (Lucas et al., 2010).

2.2 Medicinal Plants

Natural products, obtained to date mainly from fungi, higher plants, and soil

microorganisms, have a long history of helpful use by human for the treatment of

various diseases. These substances may be useful in their structurally original form or

may be derivatized by chemical synthesis to enhance potency or pharmacologic

properties such as water solubility or thermostability (Kinghorn, 2008). The term

“natural product” is generally taken to mean a compound that has no known primary

biochemical role in an organism. Such compounds are also called “secondary

9

metabolites”, and apparently are produced by the organism for ecological or defensive

purposes, thus promoting its survival (Williams et al., 1989).

Terrestrial plants have been used as medicines in Egypt, China, India and

Greece from ancient time and a significant number of modern drugs have been

developed from them (Shoeb, 2006). The first written records on the medicinal uses of

plants appeared in about 2600 BC from the Sumerians and Akkaidians (Samuelsson,

1999). The “Ebers Papyrus”, the best known Egyptian pharmaceutical record, which

documented over 700 drugs, represents the history of Egyptian medicine dated from

1500 BC (Shoeb, 2006). The Chinese Materia Medica, which describes more than 600

medicinal plants, has been well documented with the first record dating from about

1100 BC (Cragg et al., 1997). Documentation of the Ayurvedic system recorded in

Susruta and Charaka dates from about 1000 BC (Kappor, 1990). The Greeks also

contributed significantly to the logical development of the herbal drugs. Dioscorides,

the Greek physician (100 AD), described in his work “De Materia Medica” more than

600 medicinal plants (Samuelsson, 1999).

The National Cancer Institute collected about 35,000 plant samples from 20

countries and has screened around 114,000 extracts for anticancer activity (Shoeb,

2005). There are some plant derived natural products which currently used in the

treatment of hematological malignancies such as vinca alkaloids, which used in

lymphomas and acute lymphoblastic leukemias treatments (Karon et al., 1966; Dancey

& Steward, 1995), and podophyllotoxin derivatives, which have a potential usefulness

in the treatment of acute myeloid leukemias, Hodgkin’s and non-Hodgkin’s lymphomas

(Hande, 1998). Other plant natural products have been under clinical trials where they

showed promise for treating several hematological malignancies as flavopiridol (Byrd et

al., 2007) and meisoindigo (Wang et al., 2005; Weng et al., 2005), while there are plant

natural products which are yet under preclinical investigations for hematological

malignancies treatment as combretastatins (Fang et al., 2007; Petit et al., 2008) and

honokiol (Battle et al., 2005).

2.2.1 Origanum syriacum (Syrian Oregano)

The genus Origanum (oregano) is significant in the family Lamiaceae and

comprises of around 900 species of annual, perennial and shrubby herbs, widespread

throughout the world (Bayder et al., 2004; Kordali et al., 2008

arranged in groups and sections

Majorana, together with Origanum majorana

1980).

O. syriacum inhabits a large area in the eastern Mediterranean. It can be found in

southern Turkey, on Cyprus, in Syria,

Peninsula and grows from nearly sea level up to at least 2000 m in rocky soils,

limestone (Ietswaart, 1980).

traditional medicine, flavor and fragrance, and for aromatherapy in the form of bath,

massage, steam inhalation and vaporization. It is used in teas and cooked or baked foods

(Alma et al., 2003).

2.2.1.1 Botanic Description

Origanum syriacum L. is a perennial

herb, 60–90 cm high, with creeping woody

roots, branched woody, hairy stems. Leaves are

opposite, shortly or subsessile (petiolate to 8

mm), ovate, 5–35 mm × 4–23 mm and hairy,

margins are entire or remotely serrate, the apex

is obtuse. The upper leaf surface is darker; the

lower leaf surface is brighter with secretory

glands. Flowers are shortly petiolate and hairy.

Bracts are obovate or elliptic, 2

3.5 mm, acute or obtuse, entire or denticulate.

A two lipped pale purple corolla 4.5

and a five toothed tubular campanulas calyx (

2.2.1.2 Chemical Composition

The green leaves of the

characteristic and fragrance. The extraction product can vary in quality, quantity and

composition according to climate, soil composition, geographical location, seasonal

variation, plant organ, age and

al., 2007, 2008). Origanum essential oils were found to contain mainly thymol and

10

Bayder et al., 2004; Kordali et al., 2008). These species are

s and sections where Origanum syriacum L. is placed in the section

Origanum majorana L. and Origanum onites L (

inhabits a large area in the eastern Mediterranean. It can be found in

southern Turkey, on Cyprus, in Syria, Lebanon, Palestine, Jordan, and on the Sinai

Peninsula and grows from nearly sea level up to at least 2000 m in rocky soils,

The leaves of O. syriacum have been used



L. is a perennial

with creeping woody

roots, branched woody, hairy stems. Leaves are

opposite, shortly or subsessile (petiolate to 8

23 mm and hairy,

margins are entire or remotely serrate, the apex

is obtuse. The upper leaf surface is darker; the

wer leaf surface is brighter with secretory

glands. Flowers are shortly petiolate and hairy.

Bracts are obovate or elliptic, 2–5 mm × 1.5–

3.5 mm, acute or obtuse, entire or denticulate.

A two lipped pale purple corolla 4.5–7.5 mm

campanulas calyx (Alma et al., 2003; Kintzios, 2004


The green leaves of the Origanum herb are rich in essential oil which confers its



and vegetative cycle stage, and harvesting time (

essential oils were found to contain mainly thymol and

Figure 2.1: Origanum syriacum(http://www.wildflowers.co.il/english/plant.asp?ID=129

These species are

L. is placed in the section

L (Ietswaart,

inhabits a large area in the eastern Mediterranean. It can be found in

, Jordan, and on the Sinai

Peninsula and grows from nearly sea level up to at least 2000 m in rocky soils, often on

have been used in herbal



Alma et al., 2003; Kintzios, 2004).

herb are rich in essential oil which confers its



harvesting time (Abu Lafi et

essential oils were found to contain mainly thymol and

Origanum syriacum http://www.wildflowers.co.il/english/plant.asp?ID=129).

http://www.wildflowers.co.il/english/plant.asp?ID=129


11

carvacrol, monoterpene glycosides, phenols including gallic acid, rosmarinic acid,

caffeic acid, apigenin, naringenin and luteolin-7-O-glucoside (Zein et al., 2011).

The detailed investigation of the methanol extract of the aerial parts of O.

syriacum led to the isolation of eleven flavonoid compounds: acacetin-7-O-[2"-O-α-L-

rhamnopyranosyl-6"-O-β -D-glucopyranosyl]-β-D-glucopyranoside luteolin, apigenin,

luteolin-6-C-glucoside, luteolin-3'-methylether-6-C-glucoside, luteolin-7,4'-

dimethylether-6-C-glucoside, apigenin-7-methylether-6-C-glucoside, apigenin-7-O-

glucoside, diosmetin-7-O-glucoside, acacetin-7-O-glucoside, and acacetin-7-O-

rutinoside (El-Desouky et al., 2009).

2.2.1.3 Medicinal Uses

In folk medicine Origanum syriacum is used for treating gastrointestinal

problems and respiratory diseases (Ali-Shtayeh et al., 2000; Aburjai et al., 2007). In

Jordan the plant is reported to be additionally used as a carminative, pectoral,

antitussive, aperitif, and anti-stomachache and against arthritis (Lev & Amar, 2002;

Aburjai et al., 2007). In different historical records from the area of Bilad Al-Sham,

Origanum sp. was used against internal diseases, hemorrhoids, sexual diseases, pains,

animals' bites, and poison (Lev, 2002).

In recent years, O. syriacum has drawn attention for its antioxidant activity and

acetylcholinesterase inhibition (Alzheimer’s disease) (Loizzo et al., 2009; Zein et al.,

2011), antifungal and antibacterial activity (Kintzios, 2004), analgesic activity,

antiflogistic activity (atherosclerosis, Alzheimer’s disease), antirheumatic, expectorant,

sedative, antiparasitic and antihelminthic activities (Zein et al., 2011).

2.2.1.4 Anticancer Studies

Several studies were carried on the composition, antioxidant and antibacterial

effects of Origanum syriacum but few researches which were concerned with the

antiproliferative and antitumor activities of this plant.

El-Desouky et al. in 2009 have investigated the cytotoxicity of the methanol

extract of O. syriacum against human cervical adenocarcinoma, Hela cells, by the MTT

assay and the results indicated that the extract induced growth inhibition activity in a

time and dose dependent manner on the Hela cells. The IC

extract on Hela cells was 474.92

Another study by Al-Kalaldeh et al.

have investigated O. syriacum

breast cell line (MCF7). The antiproliferative activities of the hydrodistilled volatile oils

and the crude ethanol and water extracts were evaluated using the sulphorhodamine B

assay. The ethanol crude extracts of

toward MCF7 with IC50 value of

essential oils nor aqueous extracts demonstrated cytotoxic activity.

2.2.2 Hibiscus sabdariffa (Roselle

Hibiscus belongs to the plant family Malvaceae and it is one of the most flower

plants grown worldwide. There are more than 300 species of

(Ismail et al., 2008). One of them is roselle (

commonly used to make jellies, jams and beverages. The brilliant red color of its calyx

makes it a valuable food product, apart from its multitude of traditional medicinal uses.


Hibiscus sabdariffa

branched, annual shrub. Stems are

and up to 3.5 m tall. Leaves are dark

alternate, glabrous, long-petiolate,

divided into 3–7 lobes, with serrate margins.

Flowers are red to yellow with a dark center

containing short-peduncles. The flowers have both

male and female organs. Seedpods are enclosed in

their red, fleshy calices which

for making food and tea (Qi et al., 2005


Hibiscus sabdariffa calices

eugenol), flavonoid-type polyphenol

anthocyanidins; flavonol quercetin), organic acids and their derivatives, vitamin

12

manner on the Hela cells. The IC50 value of the

extract on Hela cells was 474.92 μg/mL after treatment for 12 h.

Kalaldeh et al. (2010) was performed in Jordan where they

O. syriacum antiproliferative activity toward the adenocarcinoma of

The antiproliferative activities of the hydrodistilled volatile oils

thanol and water extracts were evaluated using the sulphorhodamine B

assay. The ethanol crude extracts of O. syriacum showed antiproliferative activity

value of 6.40 μg/mL. However, neither the hydrodistilled

aqueous extracts demonstrated cytotoxic activity.

Roselle)

belongs to the plant family Malvaceae and it is one of the most flower

plants grown worldwide. There are more than 300 species of Hibiscus around the world

One of them is roselle (Hibiscus sabdariffa Linn.



Hibiscus sabdariffa is an erect, mostly

annual shrub. Stems are reddish in color

m tall. Leaves are dark green to red,

petiolate, and palmately

lobes, with serrate margins.

yellow with a dark center

The flowers have both

male and female organs. Seedpods are enclosed in

ces which are commonly used

Qi et al., 2005).


calices have phenolic compounds (protocatechuic

type polyphenol compounds (3-glucoside anthocyanins,

flavonol quercetin), organic acids and their derivatives, vitamin

Figure 2.2: Hibiscus sabdariffa(Qi et al., 2005)

of the methanol

was performed in Jordan where they

the adenocarcinoma of

The antiproliferative activities of the hydrodistilled volatile oils

thanol and water extracts were evaluated using the sulphorhodamine B

showed antiproliferative activity

the hydrodistilled

belongs to the plant family Malvaceae and it is one of the most flower

around the world

Linn.) which is



rotocatechuic acid and

glucoside anthocyanins,

flavonol quercetin), organic acids and their derivatives, vitamin C

Hibiscus sabdariffa )

13

(ascorbic acid), B1 (thiamin), B2 (riboflavin), and a carotenoid (β-carotene) (Carvajal-

Zarrabal et al., 2012).

The composition of the essential oil includes benzoic acid, nonanoic acid,

citrnellic acid, benzyl benzoate, eugenol, and eicosane (Zhang & Wang, 2007).


Hibiscus sabdariffa has several pharmacological effects as antihypertensive,

hepatoprotective, antihyperlipidemic, antioxidant, anticancer, anticonvulsant,

anxiogenic, analgesic, central nervous system depressant, antipyretic, anti-

inflammatory, and antimicrobial activities (Mahadevan et al., 2009; Khatun et al.,

2011).

Tea of H. sabdariffa calices showed 11.2 % reduction in the systolic blood

pressure and 10.7 % decrease in diastolic pressure (Haji Faraji & Haji Tarkhani, 1999).

Effectiveness and tolerability of a standardized extract was studied in patients with mild

to moderate hypertension which revealed a reduction in systolic and diastolic blood

pressure by more than 10% (Herrera-Arellano et al., 2004).

Protocatechuic acid, a simple phenolic compound isolated from H. sabdariffa

showed protective effects against cytotoxicity and genotoxicity of hepatocytes induced

by tert-butylhydroperoxide. One of mechanisms may be associated with its property of

scavenging free radicals (Tseng et al., 1996). H. sabdariffa extract offers

hepatoprotection by influencing the levels of lipid peroxidation products and liver

marker enzymes in experimental hyperammonemia and this could be due to the free

radical scavenging property of natural antioxidants present in the plant (Essa et al.,

2006).

Inhibitory effects of the plant extract on low density lipoprotein (LDL) oxidation

and anti-hyperlipidemia in fructose and cholesterol fed rats was demonstrated (Chen et

al., 2004). It revealed that the extract reduced the level of LDL and the ratio of LDL-

cholesterol to HDL-cholesterol (Carvajal-Zarrabal et al., 2005). Antioxidant effects of

the aqueous extracts of dried calyx using rat low density lipoprotein was investigated

and the study demonstrated protective effect of roselle on LDL oxidation (Hirunpanich

et al., 2005).

14

The antioxidant and free radical scavenging effects of two fractions of the

ethanol extract (chloroform soluble fraction and ethylacetate soluble fraction) obtained

from its dried flowers were investigated and found that both fractions scavenge

hydrogen peroxide and inhibit superoxide anions radicals (Farombi & Fakoya, 2005).


Several studies were performed on the apoptotic and antitumor activities of

Hibiscus sabdariffa extracts and its constituents as anthocyanins and protocatechuic

acid. Also the apoptotic pathway was examined in most of these studies to assess the

mechanism which is followed by H. sabdariffa to act as antiproliferative and anticancer

plant.

Treatment with Hibiscus protocatechuic acid (PCA) (0.2 to 2 mm) isolated from

H. sabdariffa was carried out on HL-60 and Bcl-2 overexpressed human leukemia cells

(HL60/Bcl-2-350) (Tseng et al., 2000). Inhibition of HL-60 cell survival was dependant

on PCA concentration and time (2 mM; 9 hours). Further studies showed that Hibiscus

PCA application reduced Bcl-2 protein expression to 47%, and increased Bax protein

expression to 181% after 1.5 hr as compared with time 0. Overexpression of Bcl-2 in

HL-60 cells delayed the occurrence of Hibiscus PCA-induced apoptosis. These data

suggest that Hibiscus PCA is an apoptosis inducer in human leukemia cells, and that RB

phosphorylation and Bcl-2 protein may play a crucial role in the early stage.

In 2005, a study performed in Japan by Hou et al., where Delphinidin 3-

sambubioside (Dp3-Sam), an anthocyanin which was isolated from the dried calices of

Hibiscus sabdariffa L. induced a dose-dependent apoptosis in human leukemia cells

(HL-60) as characterized by cell morphology, DNA fragmentation, activation of

caspase-3, -8, and -9, and inactivation of poly(ADP)ribose polymerase (PARP).

Molecular data showed that Dp3-Sam induced Bid truncation, mitochondrial membrane

potential loss, and cytochrome c release from mitochondria to cytosol. Moreover, Dp3-

Sam caused a time- and dose-dependent elevation of intracellular reactive oxygen

species (ROS) level in HL-60 cells, and antioxidants such as N-acetyl-L-cysteine

(NAC) and catalase could effectively block Dp3-Sam-induced ROS generation,

caspase-3 activity, and DNA fragmentation. These data indicate that Dp3-Sam might

15

induce apoptosis in HL-60 cells through a ROS-mediated mitochondrial dysfunction

pathway.

Another study in 2005 was performed in Taiwan by Chang et al. where they

explored the effect of Hibiscus anthocyanins (HAs) on human cancer cells. The results

showed that HAs could cause cancer cell apoptosis, especially in HL-60 cells. Using

flow cytometry, they found that HAs treatment (0–4 mg/ml) markedly induced

apoptosis in HL-60 cells in a dose- and time-dependent manner.

In the same year, a study by Lin et al. was published. Treatment with methanolic

H. sabdariffa extract was carried out on seven cancer cell lines: human gastric

adenocarcinoma (AGS), human promyelocytic leukemia (HL-60), hepatocellular

carcinoma (Hep3B), colorectal adenocarcinoma (Caco-2), hepatoblastoma (HepG2),

adenocarcinoma (MCF-7), human oral epidermoid carcinoma (KB), and one control

cellular line: mouse fibroblast cells. Evaluation of implied apoptotic pathway(s) was

made. Methanolic H. sabdariffa extract induced apoptosis in all cellular lines in a

concentration-dependent form. The AGS cells were the most susceptible with a drug

concentration resulting in 50% in vitro inhibition (IC50) of 0.95 mg/mL compared to

control cells (IC50 of 2.98 mg/mL). Evidence that methanolic H. sabdariffa extract

promoted both apoptotic routes (intrinsic: phosphorylation by kinase p53; extrinsic:

phosphorylation by kinase p-38) was observed.

Akim et al. (2011) in Malaysia investigated Hibiscus sabdariffa antiproliferative

effect on breast (MCF-7 and MDA-MB-231), ovarian (Caov-3) and cervical (HeLa)

cancer cell lines. Commercialized roselle juice (RJ) at three storage periods, 1 week

(WRJ), 1 month (MRJ) and 1 year (YRJ), were tested using the MTT (3-[4,5-

dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide) assay on the four cell lines to

obtain the percentage viability of the cells. The cells were incubated for 72 h after

inoculation with RJ and the control group was without treatment. The IC50 was found to

be highest for Caov-3 cells (2.267±1.193% (v/v)) whereas MCF-7 cells exhibited the

lowest (0.432±0.278% (v/v)) IC50 value after treatment with MRJ. Increasing

concentrations of sample corresponded to lower percentage viability of cells for all

samples, however the interaction within and between cell type and storage period was

not significant (p>0.05).

2.2.3 Thymus vulgaris (Common

The genus Thymus comprising of

herbs and subshrubs is predominantly

Europe and North Africa (Maksimovic et al., 2008

locally known “zaatar”, a member of the family Lamiaceae,

which grows in several regions in the world (

the composition of the essential oils determines the specific a

of condiments (Martins et al, 1999


Thyme is a small perennial subshrub, a

semievergreen groundcover that rarely grows

more than 40 cm tall. It has both horizontal

and upright habits. The stems become

with age. Thyme leaves are very small, usually

2.5 to 5 mm in length and vary considerably in

shape and hair covering, depending on the

cultivar, with each species having a slightly

different scent. T. vulgaris leaves are

oblong in shape and somewhat fleshy. Leaves

are almost stalkless with

inwards. The flowers terminate the branches in whorls. The calyx is tubular, striated,

closed at the mouth with small hairs and divided into two lips, the uppermost cut

three teeth and the lower into two. The corolla consists of a tube about the

calyx, spreading at the top into

turned back and notched at the end, the under lip longer and

segments. The seeds are round and very small and retain their germinating power for 3

years (Directorate Plant Production


The main constituents of thyme include thymol, carvacrol and flavonoids.

chemicals of thyme are essential oil

principle, tannin, saponins and triterpenic acids

16

Common Thyme)

comprising of more than 300 species of perennial, aromatic

herbs and subshrubs is predominantly found in Mediterranean region, Asia, Southern

Maksimovic et al., 2008). Thymus vulgaris L.

“zaatar”, a member of the family Lamiaceae, is a pleasant smelling

which grows in several regions in the world (Davis, 1982). It is commonly known that

essential oils determines the specific aroma of plants and flavor

Martins et al, 1999).

Thyme is a small perennial subshrub, a

groundcover that rarely grows

than 40 cm tall. It has both horizontal

habits. The stems become woody

eaves are very small, usually

length and vary considerably in

depending on the

having a slightly

leaves are oval to

somewhat fleshy. Leaves

margins curved


small hairs and divided into two lips, the uppermost cut

the lower into two. The corolla consists of a tube about the length of the

calyx, spreading at the top into two lips of a pale purple color, the upper lip erect or

turned back and notched at the end, the under lip longer and divided into three

e seeds are round and very small and retain their germinating power for 3

Directorate Plant Production, 2012).

Composition

The main constituents of thyme include thymol, carvacrol and flavonoids.

essential oil (borneol, carvacrol, linalool, and thymol

principle, tannin, saponins and triterpenic acids (Shabnum & Wagay, 2011).

Figure 2.3: Thymus vulgaris

of perennial, aromatic

found in Mediterranean region, Asia, Southern

L. (thyme),

is a pleasant smelling herb,

). It is commonly known that

roma of plants and flavor


small hairs and divided into two lips, the uppermost cut into

length of the

upper lip erect or

divided into three

e seeds are round and very small and retain their germinating power for 3

The main constituents of thyme include thymol, carvacrol and flavonoids. Major

and thymol), bitter

Thymus vulgaris

17

Thymus vulgaris shows a polymorphic variation in monoterpene production, the

presence of intraspecific chemotype variation being common in the genus Thymus. Each

of the six chemotypes, geraniol (G), α-terpineol (A), thuyanol-4 (U), linalool (L),

carvacrol (C), and thymol (T), is named after its dominant monoterpene (Thompson et

al., 2003).


Extracts of T. vulgaris are useful in traditional medicine because of their anti-

asthmatic, bronchodilator, antiseptic, antispasmodic, antitussive, antibacterial,

antifungal and antiviral activities (Marino et al., 1999; Pina-Vaz et al., 2004). Also,

these extracts have shown immunomodulating properties (Bukovska et al., 2007; Ocaña

& Reglero, 2012).

T. vulgaris is also quoted by various authors for its polyphenol and flavonoid

contents and its potential antioxidant and free radical scavenging, anti-inflammatory,

vasorelaxant, anti-platelet, antithrombin, anti-hyperlipidemic and anti-diabetic

properties (Miura et al., 2002; Vigo et al., 2004; El-Nekeety et al., 2011). A recent study

(Kensara1 et al., 2013) also shows that supplementation with T. vulgaris as an herbal

remedy has shown remarkable antihypertensive effect and marked improvement on

hypertension-related biochemical changes and aortic vascular damage in rats.

In aromatherapy, the distinct types, thymol, ‘red thyme oil’, linalol type are used

for its very gentle soft action and thuyanol for antiviral properties. A rectified product,

‘white thyme oil’ is also used, and it is milder on the skin. Applied to the skin, thyme

relieves bites and stings, and relieves sciatica and rheumatic aches and pains. Thyme is

useful for ringworm, athlete’s foot, thrush, and other fungal infections, as well as

scabies and lice (Directorate Plant Production, 2012).


Some recent studies that concerned with the cytotoxic effect of Thymus vulgaris

are mentioned here.

In Moroco, Jaafari et al. (2007) published a paper where T. vulgaris essential

oils as well as two pure products (carvacrol and thymol) were tested for their antitumor

18

activity against P815 mastocytoma cell line using colorimetric MTT assay. While all

these products showed a dose dependent cytotoxic effect, the carvacrol (with IC50 less

than 0.004% (v/v)) was the most cytotoxic one compared to the others. The IC50 of

thymol was 0.015% (v/v). Interestingly, when these products were tested against the

normal human peripheral blood mononuclear cells (PBMC), they (except thymol)

showed a proliferative effect instead of a cytotoxic one. Thymol had cytotoxic effect on

the PBMC.

Zu et al. (2010) in China, tested the essential oil of T. vulgaris for its in vitro

toxicology against three human cancer cell lines, PC-3, A-549 and MCF-7 cancer cells.

T. vulgaris essential oil exhibited cytotoxicity towards three human cancer cells. Its IC50

values on PC-3, A549 and MCF-7 tumor cell lines were 0.010% (v/v), 0.011% (v/v) and

0.030% (v/v), respectively.

Another study in Germany was performed by Sertel et al. (2011), where

cytotoxicity of thyme essential oil was investigated on the head and neck squamous cell

carcinoma (HNSCC) cell line. They found that the IC50 of thyme essential oil extract

was 369 μg/ mL.

Berrington and Lall (2012) in South Africa published a paper where acetone

extract of T. vulgaris L. was evaluated for its in vitro cytotoxicity against a

noncancerous African green monkey kidney (Vero) cell line and an adenocarcinoma

cervical cancer (HeLa) cell line. Cytotoxicity was measured using XTT (Sodium 3'-[1-

(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic

acid hydrate) colorimetric assay, and low cytotoxic effect was exhibited by the extract

on the studied cell lines. IC50 on Hela cell line was >200 μg/mL and on Vero cell line

was 138.4±2.60 μg/mL.

In Poland, Berdowska et al. (2013) evaluated the cytotoxicity of dried aqueous

extracts from T. vulgaris on two human breast cancer cell lines: Adriamycin-resistant

MCF-7/Adr and wild-type MCF-7/wt by the MTT assay, and found that T. vulgaris

exhibited cytotoxicity against both cell lines with higher toxicity against MCF-7/Adr.

2.2.4 Pelargonium graveolens

Pelargonium graveolens

belongs to the Geraniaceae family (

Africa (Lis-Balchin, 2004) as well as reunion Madagascar, Egypt and Moroco (

al., 2006). There are over 700

grown for ornamental purposes (



much-branched shrub that can reach a height of

up to 1.3 m and a spread of 1 m. The hairy stems

are herbaceous when young, becoming woody

with age. The deeply incised leaves are velvety

and soft to the touch due to the presence of

numerous glandular hairs. The leaves are

strongly rose-scented. The showy white to

pinkish flowers are borne in an umbel

inflorescence and are present from

summer peaking in spring (http://www.plantzafrica.com/plantnop/


The P. graveolens whole plant extract yield

oils (Kolodziej, 2000; Butles, 2004

geranium essential oil have been identified. The major components were citronellol

(29.90%), trans-geraniol (18.03%), 10

linalool (5.13%), geranyl acetate (4.52%),

(2.53%), geranyl tiglate (2.50%) and gemacrene D (2.05%).

Geranium essential oil is rich in oxygenated components and commercial

rhodinol (linalool + citronellol + geraniol) fraction (

2009). Galloyl C-glycosidic flavones, non galloyl flavones, phenolics (flavonoids and

tannins), benzoic and cinnamic acid derivatives were reported in aerial parts (

et al., 2005; Kolodziej & Kiderlen, 2007

19

Pelargonium graveolens (Rose-Scented Geranium)

Pelargonium graveolens is a perennial aromatic and medicinal herb/shrub that

belongs to the Geraniaceae family (Verma et al., 2011). It was originated in

as well as reunion Madagascar, Egypt and Moroco (

). There are over 700 varieties of cultivated geraniums; however, most are

for ornamental purposes (Lis-Balchin, 2002; Shawl et al., 2006).

Pelargonium graveolens is an erect,

can reach a height of

3 m and a spread of 1 m. The hairy stems

are herbaceous when young, becoming woody

with age. The deeply incised leaves are velvety

and soft to the touch due to the presence of

numerous glandular hairs. The leaves are

scented. The showy white to

pinkish flowers are borne in an umbel-like

inflorescence and are present from late winter to

http://www.plantzafrica.com/plantnop/pelarggrav.htm


whole plant extract yield contains high quantity

Butles, 2004). Thirty two compounds constituting 99.23% of


geraniol (18.03%), 10-epi-γ-eudesmol (8.27%), isomenthone (5.44%),

linalool (5.13%), geranyl acetate (4.52%), γ- cadinene (2.89%), geranyl butyrate

(2.53%), geranyl tiglate (2.50%) and gemacrene D (2.05%).


rhodinol (linalool + citronellol + geraniol) fraction (Rajeswara Rao et al., 2002; Fayed

glycosidic flavones, non galloyl flavones, phenolics (flavonoids and

tannins), benzoic and cinnamic acid derivatives were reported in aerial parts (

Kiderlen, 2007).

Figure 2.4: Pelargonium graveolens

is a perennial aromatic and medicinal herb/shrub that

). It was originated in South

as well as reunion Madagascar, Egypt and Moroco (Shawl et

of cultivated geraniums; however, most are

pelarggrav.htm).

of essential

). Thirty two compounds constituting 99.23% of


eudesmol (8.27%), isomenthone (5.44%),

adinene (2.89%), geranyl butyrate


et al., 2002; Fayed,

glycosidic flavones, non galloyl flavones, phenolics (flavonoids and

tannins), benzoic and cinnamic acid derivatives were reported in aerial parts (Goedecke


http://www.plantzafrica.com/plantnop/

http://www.plantzafrica.com/plantnop/

20


The geranium essential oil has historically been used in the treatment of

dysentery, hemorrhoids, inflammation, heavy menstrual flows and even cancer (Kang et

al., 2010). The French medicinal community currently treats diabetes, diarrhea,

gallbladder problems, gastric ulcers, liver problems, sterility and urinary stones with

this oil (Amabeoku, 2009; Elmann et al., 2010). In Chinese homeopathy, the geranium

essential oil is known to open up the liver charka and promote the expulsion of toxins

that prohibit the achievement of balance within the body (Higley & Higley, 2001).

Geranium essential oil is also utilized in the perfumery, cosmetic and

aromatherapy industries all over the world. It has since become indispensable

aromatherapy oil. It is one of the best skincare oils because it is good in opening skin

pores and cleaning oily complexions (Weiss, 1997; Miller, 2002; Peterson et al., 2005).

The geranium leaves are used as a form of herbal tea to de-stress, fight anxiety, ease

tension, improve circulation and cure tonsillitis (Peterson et al., 2005).


Some of the major chemical compounds (citronellol, citronellyl formate,

geraniol, and citronellyl acetate) of P. graveolens oil possess marginal antitumor

activities (Fang et al., 1989). ). Geraniol inhibited the growth of leukemia and

melanoma cells (Shoff et al., 1991), hepatoma cells (Yu et al., 1995), and pancreatic

cancer cells (Burke et al., 1997).

Analysis of geranium essential oil showed citronellol and transgeraniol as the

major constituents which are known to possess antioxidant and anticancer properties

(Haag et al., 1992). The antiproliferative effects of geraniol on hepatoma and melanoma

cell growth have been ascribed to inhibition of 3-hydroxy-3-methylglutaryl-CoA

(HMG-CoA) reductase, a key enzyme of mevalonate biosynthesis (Elson, 1995).

Burke et al. (1997) assessed the anticancer activity of geraniol and found that it

has significant (60-90%) inhibition of the anchorage-independent growth of human

MIA PaCa-2 pancreatic tumor cells. Also, many studies showed that P. graveolens has

potential antitumor activity against uterine cervical neoplasia (Duke & Ayensu, 1985;

De Moura et al., 2002).

21

Geraniol sensitizes colonic cancer cells to 5-FU treatment, by increasing the

cytotoxicity of the drug, resulting from the facilitated transport of 5-FU and the

blockade of the morphological and functional differentiation of the cancer cells

(Carnesecchi et al., 2002). This essential oil caused a 50% decrease of ornithine

decarboxylase activity, a key enzyme of polyamine biosynthesis, which is enhanced in

cancer growth. Geraniol also activated the intracellular catabolism of polyamines,

indicating that polyamine metabolism is presumably a target in the antiproliferative

properties of geraniol. Geraniol has no cytotoxic effect, is mainly cytostatic, and inhibits

DNA synthesis, leading to the accumulation of Caco-2 cells in the S phase (Carnesecchi

et al., 2004).

The anticancer activity of the geranium essential oil on two human

promyelocytic leukemia cell lines (HL-60 and NB4) using trypan blue assay showed the

anticancer activity with the LC50: 62.50 and 86.5 μg/ml in NB4 and HL-60 cell lines

respectively, demonstrating the potential of the essential oils for cancer treatment

(Fayed, 2009). Also, Zhuang et al. (2009) reported that citronellol, an oil soluble

compound derived from the geranium, has anticancer and anti-inflammatory properties.

2.2.5 Mechanisms of Medicinal Plants' Anticancer Effects

The medicinal plants experiments on cell lines as well as experiments in animals

demonstrated that these plants may display an anticancer effect by different

mechanisms, including: suppressing the initiation or reversing the promotion stage in

multistep carcinogenesis, blocking the progression of precancerous cells into malignant

ones, inducing apoptosis and differentiation of cancer cells, enhancing the immune

system of the body, inhibiting angiogenesis and reversing multidrug resistance of

chemotherapeutic agents (Surh, 2003; Romero-Jimenez et al., 2005).

The initiation of carcinogenesis can be prevented by natural products which act

as potent antioxidants and free radical scavengers and which are supposed to minimize

DNA damage by reacting with free radicals. Some of the phytochemical antioxidants of

folk medicine are inhibitors of lipoxygenase and urokinase. Inhibition of these enzymes

by folk medicines could prevent or reduce cancer growth and in this way, their

mechanism of action can be established (Rao et al., 2008).

22

Various medicinal plants, which are called cytotoxic, have been documented in

the literature to induce apoptosis in cancer cells. Apoptosis (programmed cell death) is

the principal mechanism through which unwanted or injured cells are safely eliminated

from the body. This programmed cell death is mediated via either an extrinsic apoptotic

pathway or an intrinsic apoptotic pathway (Elmore, 2007). These two apoptosis

signaling pathways differ in the origin of their apoptosis signal, but converge upon a

common pathway (Elmorea et al., 2005). The extrinsic pathway is initiated by the

stimulation of the cell surface ‘death receptor’ due to the binding of death ligand and the

intrinsic pathway is also known as the mitochondrial pathway in which an intracellular

apoptotic signal initiates the process (Deep et al., 2010).

Cytostasis, at the cellular level, may be defined as the inhibition of cell growth

and/or proliferation, and this initial event can be followed by cell death if the cytostasis

is prolonged and profound; alternatively, it could also be followed by cellular escape

and regrowth (Rixe & Fojo, 2007). Inhibition of cells proliferation is tightly linked to

mechanisms that regulate cell cycle progression or mechanisms that induce metabolic

suppression in the cells. Cell cycle can be arrested while cells are metabolically active,

but when metabolism is suppressed, the cell cycle and consequently the proliferation are

stopped.

During the cell cycle, mammalian cells coordinate cell growth, genome

replication, and division. Two irreversible events subdivide the cell cycle into distinct

phases: the onset of DNA replication defines S phase; and cell division defines M

phase. Cells grow and carry out additional functions during the gap phases G1 and G2.

The changing activity states of cyclin-dependent kinases (Cdks) regulate the transition

between different stages of the cell cycle (Murray, 2004). Prolonged cell cycle arrest in

a phase other than G0 is intolerable to a cell and must be resolved by either initiating a

path to cell death or escaping the block, a decision that may depend in part on the

cellular context in which the arrest occurs. It is well known that the propensity to

apoptosis varies among cell types, with lymphoid cells most prone to undergo

apoptosis. In cells more prone to apoptosis, one can often see cytostatic agents inducing

apoptosis. In an apoptosis-prone cellular context, the cellular arrest, weak though it may

be, is sufficient to trigger an apoptotic response (Rixe & Fojo, 2007).

23

Metabolic suppression in cancer cells which can be induced by some anticancer

agents leads to proliferation arrest. Autophagy is a central metabolic stress response

conserved throughout evolution from yeast to man and represents a key pathway in

metabolic stress adaptation. Macroautophagy involves the sequestration of internal

components and organelles into double-membrane structures known as autophagic

vesicles, and subsequent degradation by the lysosome. Autophagy performs an

important physiological role as a waste disposal system for the removal of aged or

damaged organelles, particularly mitochondria. However, when faced with metabolic

stress such as nutrient depletion (amino acids, glucose) or hypoxia, cells activate the

autophagic pathway (Lum et al. 2005).

In this study, two kinds of in vitro anticancer effects of the extracts were studied

by two assays, MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)

assay and LDH (lactate dehydrogenase) assay, to discriminate between cytostatic and

cytotoxic effects of the studied medicinal plants.

24

Chapter 3 Materials and Methods

All procedures of this study were performed in the laboratories of Biology

Department in the Islamic University of Gaza in the period from September 2012 to

June 2013.

3.1 Materials

Items Company

Cells

THP 1 cell line (human leukemic monocyte) ECACC

Kits

In vitro toxicology assay kit MTT based Sigma

In vitro toxicology assay kit, lactic dehydrogenase based Sigma-Aldrich

Reagents and Consumables

RPMI 1640 medium modified with 2.05 mM L-glutamine and

25 mM HEPES

Sigma-Aldrich

Fetal bovine serum Sigma

Histopaque-1077 Sigma-Aldrich

Penicillin-Streptomycin solution stabilized, with 10,000 units

penicillin and 10 mg streptomycin/mL.

Sigma

Trypan blue solution cell culture tested Sigma-Aldrich

Phosphate buffered saline tablets Sigma-Aldrich

Dimethylsulfoxide (DMSO) Sigma-Aldrich

Triton X-100 solution

Phytohemagglutinin PHA-P, lyophilized powder (contains

buffer salts and NaCl)

Sigma-Aldrich

Disposables

Millex-GP syringe filter units, pore size 0.22 μm, filter diam.

33 mm, sterile; γ-irradiated

Sigma-Aldrich

Suspension culture flask with filter cap (75cm2) Greiner Bio- One

24 well suspension plate with lid, sterile Greiner Bio- One

25

96 well suspension culture plate with lid, clear, sterile (flat

bottom)

Greiner Bio- One

Sterile Tube Test PP Conical Graduated 15 ml, 50 ml Greiner Bio- One

Sterile serological pipettes/ Sterile pipette tips with barrier/

Sterile Petri dishes

Labcon

Equipments

Rotary Evaporator Hahn shin Scientific

Bio Safety Cabinet N-Biotek

CO2 Incubator N-Biotek

Centrifuge Hanil BioMed Inc.

ELISA reader Thermo Scientific

Autoclave Cristofoli Biosseguranca

37°C Water Bath N-Biotek

4 °C Refrigerator and -20°C, -80°C Freezers Selecta

Oven Boxun

Balances Adam

Compound Light Microscopes: Upright and Inverted LW Scientific

Pipettes 10, 50, 200, and 1000 µL, Multichannel Pipette, and

Motorized Pipetting Device

Labmed, Boeco

3.2 Collection and Preparation of Plant Materials

Thymus vulgaris seeds were purchased from PlantiCo.(Poland) and implanted in

Al-Breem plantation (Khanyounus) in January 2012.

In July 2012, aerial parts of both Origanum syriacum and Thymus vulgaris and

leaves of Pelargonium graveolens plants were collected from Al-Breem plantation after

kindly performing taxonomy authentication of the plants' samples by Dr. Mohammed

Abo Ouda (PhD plant taxonomy, Alaqsa University, Gaza). The collected plants parts

were washed with water in order to remove dust, soil and insects then dried by tissue

paper, after that they were air dried under shade for 3 weeks, and then they were

completely dried by oven at 40°C for 1 to 1.5 hour.

Hibiscus sabdariffa plant calices were collected in September 2012 from

Ministry of Agriculture plantations and after washing with water, they were ai

under shade for 25 day then were completely dried by oven at 50°C for 1 hour.

The dried parts for each plant were gri

stored in nylon bags until extraction

3.3 Extraction of Plant M

Origanum syriacum extract was prepared by soaking the plant

parts in 40% ethanol in a weight/solvent volume ratio of 1/10 at room temperature. The

plant powder was soaked for 7 days with occasional mixing through this period.

Hibiscus sabdariffa extract was prepared by adding

plant dried and grinded calices

percentage in the solvent. Plant

plant was allowed to soak in the

Thymus vulgaris and Pelargonium graveolens

the plant dried and grinded

weight/solvent volume ratio of 1/10 at room temperature. The plant powder was soaked

for 4 days with occasional mixing

After the soaking periods, the extracts were filtered

ensure removing any remaining debris, then all extracts were dried by rotary evaporator

(see figure 3.1) at 40°C for

Figure 3.1: Rotary evaporator

26

plant calices were collected in September 2012 from

Ministry of Agriculture plantations and after washing with water, they were ai

then were completely dried by oven at 50°C for 1 hour.

The dried parts for each plant were grinded by a blender mixer and

s until extraction has been made.

Materials

extract was prepared by soaking the plant dried and grinded

in 40% ethanol in a weight/solvent volume ratio of 1/10 at room temperature. The

soaked for 7 days with occasional mixing through this period.

extract was prepared by adding boiling distilled water on

calices and after cooling, ethanol was added to reach 10%

. Plant weight/solvent volume ratio was 1/10, and then

the 10% ethanol solvent for 6 hours at room temperature

Pelargonium graveolens extracts were prepared by

dried and grinded parts in 90% ethanol (Bayoub et al., 2010


days with occasional mixing through this period.

After the soaking periods, the extracts were filtered under vacuum three times to


(see figure 3.1) at 40°C for T. vulgaris and P. graveolens extracts and 45°C for

Rotary evaporator

plant calices were collected in September 2012 from

Ministry of Agriculture plantations and after washing with water, they were air dried

then were completely dried by oven at 50°C for 1 hour.

and directly

dried and grinded

in 40% ethanol in a weight/solvent volume ratio of 1/10 at room temperature. The

soaked for 7 days with occasional mixing through this period.

boiling distilled water on the

ethanol was added to reach 10%

and then the

at room temperature.

extracts were prepared by soaking

Bayoub et al., 2010) in a


three times to


extracts and 45°C for O.

27

syriacum extract while H. sabdariffa extract was dried at 50°C. Evaporating flask

rotation speed was 100 rpm through drying of all extracts.

The dried extracts were either stored directly at -20 °C freezer or dissolved in

appropriate solvent at concentration of 200 mg/ml (20% extract dry weight/volume)

(Freidberg, 2009) to prepare stock solutions which also were stored at -20 °C freezer

until they were used. T. vulgaris and P. graveolens extracts stock solutions were

prepared in Dimethyl Sulfoxide (DMSO) solvent while O. syriacum and H. sabdariffa

extracts stock solutions were prepared in RPMI 1640 cell culture medium with 10%

fetal bovine serum (FBS).

Throughout cell culture experiments, stock solutions were diluted with RPMI

1640 medium with 10% fetal bovine serum to achieve the working concentrations. The

first extracts working solutions were sterilized by filtration with 0.22 µm syringe filters

and the next working solutions have been prepared aseptically from these sterile

concentrations.

3.4 THP-1 Cell Line Processing and Maintenance

All cell culture procedures and cells handling processes were performed under

strict aseptic technique guidelines to avoid any contamination may be occur. Safety

cabinet was sterilized by 70% ethanol and UV radiation before each use and all

equipments which were used in cells handling either were already purchased sterile or

sterilized by autoclaving before use. Anything or equipment had been entered to the

hood was sprayed with 70% ethanol before entering.

3.4.1 Cells Receiving and Culturing

THP-1 cell line (human monocytic leukemia cell line) was received in March

2013 from European Collection of Cell Cultures (ECACC, UK) via Sigma-Aldrich.

THP-1 cell line was derived from the peripheral blood of a 1 year old male with acute

monocytic leukemia. THP-1 cells have Fc and C3b receptors and lack surface and

cytoplasmic immunoglobulins. These cells also stain positive for alpha-napthhyl

butyrate esterase, produce lysozymes and are phagocytic (both latex beads and

sensitized erythrocytes). THP-1 cells can also restore the response of purified T

28

lymphocytes to Concanavlin A, show increased CO2 production on phagocytosis and

can be differentiated into macrophage-like cells using for example DMSO.

The THP-1 cell line was shipped in frozen ampoule surrounded with dry ice

templates and upon receiving, the cells were immediately processed as recommended

by ECACC to avoid any delay that may cause cell stress and death. The media used

firstly for culturing cells was RPMI 1640 cell culture media with 2.05 mM L-glutamine,

100 µg/mL streptomycin, 100 U/mL penicillin and 20 % fetal bovine serum (FBS). FBS

was previously heat inactivated in water bath at 56°C for 30 minutes; this high FBS

concentration was used to recover cells' growth by providing growth factors. Cells were

cultured in Greiner Bio- One, DNase- and RNase- free tissue culture flasks, incubated in

the CO2 incubator with 5% CO2 at 37°C and were checked daily until cells growth was

established.

3.4.2 Cells Freezing in Stocks

As soon as cell line propagation was initiated and cell number had been

increasing there was necessity for making cell line stocks. Freezing of cells at -85°C

was performed in cryovials at high cell concentration (6-8X106 cells/mL) in freezing

medium which was heat inactivated fetal bovine serum with 10-15% glycerol. Freezing

was made while cells were in the log phase of growth (Freshney, 2005). Cell stocks

provide a reservoir if any unexpected sudden cell loss in culture has occurred, also cells

in culture must be substituted periodically from stocks because it is not recommended to

maintain cells in continuous culture for long periods of time. Through the progress of

this study, every three months of continuous cell culture, cells were substituted from

new cell line stock to conserve cells characteristics.

3.4.3 Determination of THP-1 Cells Growth Characteristics

Growth characteristics were defined by following cells growth for nine days.

Three cell concentrations, 1X105, 3X104 and 1X104 cells/mL, were prepared in

modified RPMI 1640 culture medium with 2.05 mM L-glutamine and 25 mM HEPES,

supplemented with 20% heat inactivated FBS. Nine 24-well plates' wells were seeded

from each concentration in triplicates and the plates were incubated in the CO2

incubator at 5% CO2 and 37°C. Every day (except the fifth day), one plate was taken

out from the incubator and cells in all seeded wells were counted by hemocytometer and

29

cells number in each milliliter was calculated. The cells/mL concentration was plotted

against days to construct a curve representing cell growth through this period.

3.4.4 Routine Maintenance of THP-1 Cells in Culture

Cells which were used in MTT and LDH assays, were routinely maintained by

subculturing in Greiner Bio- One, DNase- and Rnase- free tissue culture flasks in

Modified RPMI-1640 complete medium, with 2.05 mM L-glutamine and 25 mM

HEPES and without antibiotics, the media was supplemented with 10% heat inactivated

FBS. The flasks were incubated in CO2 incubator with 5% CO2 at 37°C and were

regularly screened by naked eye as well as by inverted microscope to observe any

morphology change or any contamination have been occurred. When, rarely,

contamination has been seen, the contaminated flasks were immediately discarded to

avoid the spread of this contamination to other flasks.

Media addition was performed periodically (often every 2-4 days), firstly, to

feed cells and secondly, to conserve cells at most times in the log phase of growth.

Media and flasks were changed usually at intervals to remove any remained toxic

metabolic byproducts. Media change was made by centrifuging cells at 150 x g for 5

minutes in sterile centrifuge tubes (Falcon tubes) then discarding the supernatant and

resuspending in appropriate volume of fresh warm cell culture media.

Cells' counts were performed regularly to determine the media dilution factor

needed to preserve cells concentration between 105-106 cells/mL. Also cells viability

estimation by trypan blue exclusion test was performed concurrently with cells count to

confirm cells health and wellness. Before each MTT or LDH assay, cells count and

viability estimation were performed and only the cells which were in the exponential

phase of growth and with viability of at least 95% were used in the assays.

3.5 Determination of MTT and LDH Assays Sensitivity to THP-1 Cells

To determine the range of THP-1 cells number that MTT and LDH assays can

detect in a linear manner, different concentrations of THP-1 cells (cells/mL medium)

were prepared and assayed by both MTT and LDH kits. Sensitivity curves were

constructed by plotting the kit absorbance against cell concentrations.

Serial dilutions of cell concentrations rang

prepared in RPMI 1640 medium supple

wells of 96-well plates were seeded with 100 µL from each concentration

in addition to blank wells which were filled with media alone.

The MTT plate was incubated in 5% CO

from MTT reagent were added to each well. After incubation of the plate in the CO

incubator for 4 hours, 100 µL from MTT solubilization solution were added to each

well and mixing was performed by Pipetting up and down t

crystals. The plate absorbance

(figure 3.2) at 550 nm and the reference absorbance

absorbance) at 620 nm. (The procedure was performed following the

provided by MTT kit from Sigma

wavelengths).

After seeding the various cell concentrations to the LDH plate, 10 µL from the

cell lysis solution provided in

incubated in the CO2 incubator for 45 minutes. After incubation, the plate was

centrifuged at 250 x g for 4 minutes, and 50 µL from each well supernatant were

transferred to new plate wells. Reaction m

of LDH assay substrate solution, LDH

preparation. From this mixture,

incubation was made at room temperature for 20 minute

with aluminum foil). Ten microliters from 1N HCl solution were added to each well to

Figure 3.2:

30

ell concentrations ranging from 103 to 107 cells/mL

prepared in RPMI 1640 medium supplemented with 10% heat inactivated FBS, and

well plates were seeded with 100 µL from each concentration in triplicates,

in addition to blank wells which were filled with media alone.

The MTT plate was incubated in 5% CO2 at 37°C for 30 minutes then 10 µL

from MTT reagent were added to each well. After incubation of the plate in the CO


well and mixing was performed by Pipetting up and down to dissolve any formazan

absorbance was read by microplate reader device (or ELISA reader

at 550 nm and the reference absorbance (non specific background

(The procedure was performed following the instructions sheet

MTT kit from Sigma-Aldrich with slight alteration of the reading


cell lysis solution provided in the LDH kit were added to each well and the plate was

incubator for 45 minutes. After incubation, the plate was


transferred to new plate wells. Reaction mixture was prepared by mixing equal volumes

olution, LDH assay dye solution, and LDH assay

From this mixture, 100 µL were added to the 50 µL supernatant and

incubation was made at room temperature for 20 minutes in dark (the plate was foiled


Figure 3.2: ELISA reader device

cells/mL were

mented with 10% heat inactivated FBS, and

in triplicates,

at 37°C for 30 minutes then 10 µL

from MTT reagent were added to each well. After incubation of the plate in the CO2


o dissolve any formazan

ELISA reader)

non specific background

tructions sheet

with slight alteration of the reading


LDH kit were added to each well and the plate was

incubator for 45 minutes. After incubation, the plate was


mixing equal volumes

ssay cofactor

100 µL were added to the 50 µL supernatant and

s in dark (the plate was foiled


31

stop the reaction, and then the absorbance of the plates was read by ELISA reader at

450 nm and the reference absorbance was read at 620 nm (The procedure was

performed following the instructions sheet provided by LDH kit from Sigma-Aldrich

with slight alteration of the reading wavelengths).

The MTT and LDH absorbance was plotted against cell concentration and the

linear part of both sensitivity curves was determined.

3.6 Assessment of Plant Extracts Effects on THP-1 Cells by MTT

Assay

The THP-1 cells at constant initial number were exposed to various

concentrations prepared from the four tested plant extracts for 48±1 hours in the CO2

incubator and then were assayed by MTT assay to determine cells' activity after treating

with each extract concentration as well as non treated cells' activity (control). The

results were expressed as % of control.

Various extracts concentrations were prepared from stock extracts solutions in

RPMI 1640 cell culture medium supplemented with 10% heat inactivated FBS in sterile

centrifuge or microcentrifuge tubes. The concentrations range for O. syriacum extract

was from 16 to 0.2 mg/mL, for H. sabdariffa extract was from 40 to 0.15625 mg/mL,

for T. vulgaris extract was from 4.0 to 0.04 mg/mL, and for P. graveolens extract was

from 10 to 0.15625 mg/mL. DMSO concentrations were also prepared to eliminate any

DMSO toxicity in T. vulgaris and P. graveolens extract solutions because their stock

solutions were prepared in DMSO. Experimented DMSO concentrations were in the

range from 2.5 to 0.2% (v/v).

The working extracts concentrations (in the above) were determined by initial

extracts toxicity experiments with 5-fold or 3-fold serial dilution primary concentrations

(the highest experimented concentration was 40 mg/mL for all extracts), then according

to the resulting curves, the concentrations were widen in the range of extract

effectiveness.

THP-1 cell suspension of 4X105 cells/mL was prepared in RPMI 1640 cell

culture medium supplemented with 10% heat inactivated FBS and 100 µL from this

suspension were seeded by a multichannel pipette to the test and control wells of sterile

32

96-well plates, then 100 µL from each concentration of the proper extract or DMSO

were added in triplicates to the corresponding wells and 100 µL RPMI 1640 cell culture

medium with 10% heat inactivated FBS and without extracts were added to the control

wells (untreated cells). Blank wells were filled with 200 µL RPMI 1640 cell culture

medium with 10% heat inactivated FBS only. Peripheral plates' columns were filled

with either culture medium or phosphate buffered saline (PBS) to provide humidity to

the other wells then the plates were capped and put in plastic container containing

distilled water dish to confirm high humidity environment and this container was put in

the CO2 incubator at 37°C and 5% CO2. The final cells concentration was 2X105

cells/mL (4X104 cells/ well) and the above extracts concentrations were halved.

After incubation for 48±1 hours, the plates were taken out from the incubator,

inspected for any signs of contamination by naked eye as well as by inverted

microscope scanning. Contamination free plates were centrifuged at 250 x g for 4

minutes in plate centrifuge, and then 150 µL from supernatants were discarded, after

that the wells were washed with 150 µL PBS until extracts color disappeared (2-5

washing cycles according to extract concentration). Washing centrifugation was at 250

x g for 4 minutes. After removing the last supernatant, the cells sediment in each well

was resuspended in 100 µL fresh RPMI 1640 cell culture medium with 10% heat

inactivated FBS. Ten microliters from MTT solution (5 mg/mL RPMI 1640 culture

medium) were added to each well in the plate (test, control, and blank wells), then the

plates were incubated in the CO2 incubator at 37°C and 5% CO2 for 4 hours.

After 4 hours incubation, 100 µL from the MTT solubilization solution were

added to each well and mixing was performed by Pipetting up and down to dissolve any

formazan crystals. The plates were entered to the ELISA reader device and mixing was

repeated at medium speed for 15 minutes to ensure dissolving all crystals, and then the

plates' absorbance was read at 550 nm and the reference absorbance at 620 nm.

Percentage viability at each extract concentration was calculated as following:

Cell viability (%) = Extract concentration reading - Blank reading

Control reading - Blank reading × 100

33

3.7 Assessment of Plant Extracts Effects on Peripheral Blood

Mononuclear Cells (PBMCs) by MTT Assay

The aim of modern cancer chemotherapy is to selectively destroy cancerous

cells while having as little effect as possible on the patient’s normal cell populations and

general homeostatic balance. A plant extract or compound isolated from such an extract

exhibiting anticancer activity is not only required to induce apoptosis in malignant cells;

it also needs to do so selectively in malignant cells without affecting normal healthy

cells.

The degree of toxicity expressed by extracts of O. syriacum, H. sabdariffa, T.

vulgaris and P. graveolens on THP-1 cells by means of MTT assay have been

investigated as seen in the previous section. Investigation of the possible expression of

toxicity of the extracts in normal, healthy human cells was judged as the next logical

step.

In order to obtain PBMCs, venous blood was collected from a healthy,

consenting adult in tubes containing sodium heparin as anticoagulant and processed

within 2 hours of collection. 5.0 ± 0.5 mL from this blood were layered on

approximately equal volume of Histopaque-1077 previously prepared in two or three 15

mL conical centrifuge tubes (according to the total blood volume; roughly 10-15 mL in

each experiment). The layered blood was centrifuged at 400 x g for exactly 30 minutes

at room temperature, after that the upper layer (plasma) was carefully aspirated and

discarded except for approximately 0.5 mL and the opaque interface which contains

PBMCs was transferred with a pipette to clean 15 mL conical centrifuge tube. The cells

were washed with 10 mL PBS then washed twice with 5 mL PBS or culture medium.

Washing centrifugation was at 250 x g for 10 minutes. PBMCs were resuspended in

modified RPMI 1640 complete medium, with 2.05 mM L-glutamine and 25 mM

HEPES supplemented with 10% FBS, 5 µg/ mL phytohemagglutinin (PHA), 100

µg/mL streptomycin, and 100 U/mL penicillin. The cells were firstly resuspended in 5

mL medium, counted by hemocytometer and diluted by medium to reach concentration

of 1X106 cells/mL (The procedure was performed following the instructions sheet

provided by Histopaque-1077 from Sigma-Aldrich with slight alteration of the starting

volumes). Concurrently with cell counting and by trypan blue exclusion test, viability

was estimated and in all experiments, PBMCs viability was at least 98%.

34

Extracts concentrations ranges tested on PBMCs were as following: from 10 to

0.15625 mg/mL for O. syriacum; from 20 to 0.3125 mg/mL for H. sabdariffa; from 2.5

to 0.078125 mg/mL for T. vulgaris and from 5.0 to 0.15625 mg/mL for P. graveolens.

DMSO concentrations were from 5.0 to 0.15625% (v/v).

From 1X106 cells/mL PBMCs suspension, 100 µL were seeded by a

multichannel pipette to the test and control wells of sterile 96-well plates, then 100 µL

from each extract or DMSO concentration were added in triplicates (except otherwise

indicated) to the corresponding wells and 100 µL RPMI 1640 cell culture medium with

10% heat inactivated FBS and without extracts were added to the control wells. The

final cells concentration was 5X105 cells/mL (1X105 cells/ well) and the above extracts

concentrations were halved also PHA and antibiotics concentrations were halved. Blank

wells were filled with 200 µL RPMI 1640 cell culture medium with 10% heat

inactivated FBS only. As in leukemia cells experiments, peripheral plates' columns were

filled with either culture medium or phosphate buffered saline (PBS) then the plates

were put in plastic container containing distilled water dish and this container was put in

the CO2 incubator at 37°C and 5% CO2.

After 48 ±1 hours of incubation, contamination free plates were processed by

MTT assay which was performed typically as with THP-1 cells. Percentage viability at

each extract concentration was calculated and plotted against extract concentrations.

3.8 Determination of Plant Extracts Toxicity on THP-1 Cells by LDH

Assay

To discriminate between the cytotoxic effect of the plant extract and the growth

inhibitory effect, LDH assay was proposed because it investigates the lysis degree of the

cells under study, while MTT assay investigates the degree of metabolic activity and

proliferation of the cells under study.

Three plants' extracts only (O. syriacum, T. vulgaris, and P. graveolens) which

their cytotoxic effects were investigated by LDH toxicity assay. H. sabdariffa effective

concentrations presented by MTT assay were very high (~10-20 mg/mL) and to process

LDH assay on this extract, higher concentrations (>20 mg/mL) than that used in MTT

assay is required (as happened with the other extracts) and this is difficult because the

35

0.22 µm filter is plugged with high viscous concentrations, also the H. sabdariffa

extract was mostly consumed in MTT assays on THP-1 and on PBMCs.

Various extracts concentrations were prepared from stock extracts solutions in

RPMI 1640 cell culture medium supplemented with 10% heat inactivated FBS in sterile

centrifuge or microcentrifuge tubes. The concentrations for the three extracts, O.

syriacum, T. vulgaris, and P. graveolens were from 20 to 0.3125 mg/mL. DMSO

concentrations were also prepared, to eliminate any DMSO toxicity in T. vulgaris and

P. graveolens extract solutions, and were from 10 to 0.15625% (v/v).

THP-1 cell suspension of 4X105 cells/mL was prepared and 100 µL from this

suspension were seeded to the test and control wells of sterile 96-well plates, then 100

µL from each extract or DMSO concentration were added in triplicates to the

corresponding wells. Also 100 µL from each extract or DMSO concentration were

added to one well of the 96-well plates previously seeded with 100 µL RPMI 1640 cell

culture medium with 10% heat inactivated FBS without cells; these wells were serve as

blanks for the corresponding test wells. 100 µL RPMI 1640 cell culture medium with

10% heat inactivated FBS and without extracts were added in triplicates to the negative

control wells (untreated cells) and to blank wells with 100 µL RPMI 1640 cell culture

medium with 10% heat inactivated FBS. 100 µL of 2% Triton X-100 solution

(previously prepared in the same culture medium) were added in triplicates to the

positive control wells previously seeded with cells (100% cell lysis) and to blank wells

with 100 µL RPMI 1640 cell culture medium with 10% heat inactivated FBS.

Peripheral plates' columns were filled with either culture medium or phosphate buffered

saline (PBS) then the plates were put in plastic container containing distilled water dish

and this container was put in the CO2 incubator at 37°C and 5% CO2.

After incubation for 48±1 hours, the plates were taken out from the incubator,

inspected for any signs of contamination by naked eye as well as by inverted

microscope scanning. Contamination free plates were centrifuged at 250 x g for 4

minutes in plate centrifuge, and then 50 µL from each well supernatant were transferred

to new wells of clean 96-well plates. 100 µL from freshly prepared LDH reaction

mixture were added to each well and the plates were incubated for 25 minutes at room

temperature foiled with aluminum foil to provide darkness for LDH reaction to take

place. After the incubation period, 15 µL from previously prepared 1N HCl solution

36

were added to each well to stop the reaction, and then the plates' absorbance was read

on ELISA reader at 450 nm and the reference absorbance at 620 nm. Percent

cytotoxicity was calculated, after all blanks subtractions from all readings, as in the

following:

Cell Cytotoxicity (%) = Extract concentration reading – Negative control reading

Positive control reading - Negative control reading × 100

Cell viability (%) was expressed as (100 – cell cytotoxicity)

3.9 Statistical Analysis

All experiments involving the application of the extracts on the leukemia THP-1

cells and peripheral blood mononuclear cells (PBMCs) were performed in triplicates

and each experiment was repeated for an additional time independently and the average

of the six replicates was expressed as a mean viability percentage ± standard deviation

(SD) (three concentrations in PBMCs results have only 4 or 5 replicates only). Growth

curves, kits' sensitivity curves, dose response curves and non linear regression analysis

all were processed by means of the GraphPad Prism software, version 6.03 for

Windows in order to determine IC50 values, the concentration of each extract at which

50% of the cells are inhibited, and to compare between extract effects on leukemia and

normal cells.

37

Chapter 4 Results

4.1 Plants Crude Extracts Percentage Yields

After evaporation of the extracts solvent by rotary evaporator, the pre-weighed

evaporating flasks containing the crude extracts of H. sabdariffa, O. syriacum, T.

vulgaris and P. graveolens were weighed again in order to determine the weight of

crude extracts obtained. Table 4.1 shows the percentage yields of the dry extracts

obtained (w/w).

Table 4.1: Percentage yields (%) of all dried extracts obtained after solvent evaporation.

Plant Weight of dried plant material

(gram)

Weight of extracts after solvent

evaporation (gram)

Percentage yields (%) w/w

H. sabdariffa 17.7 2.8 15.8

O. syriacum 20.0 3.4 17.0

T. vulgaris 20.0 2.4 12.0

P. graveolens 20.0 1.3 6.5

The firstly prepared O. syriacum and P. graveolens extracts have been

consumed during this study and a second round of extraction was needed. The

percentage yield of the dried extracts in the second round was different where O.

syriacum extract percentage yield was reduced to 9.5% and P. graveolens extract

percentage yield was reduced to 5.5%.

4.2 Morphology and Growth Characteristics of THP-1 Cells

THP-1 cells are monoblastic, leukemic cells and when culturing them, they grow

in suspension either as single cells or in small aggregates floating in the medium

without attaching to the container surface. These cells were spherical in shape and have

shown size variation. Some cells had protrusions and this is a common feature for

monocytic lineage. Figure 4.1 shows the morphology of THP-1 cells in culture as seen

under the inverted microscope.

38

Figure 4.1: THP-1 cells under the inverted microscope. A: at low power, B: at high power, and C: enlarged view of cells.

Growth characteristics of THP-1 cells were determined by daily counting cells

with various initial cell concentrations. The wells were seeded in triplicates for each cell

concentration, and the average cells number in the milliliter was plotted against days to

construct the three growth curves. Figure 4.2 shows the growth pattern for each THP-1

cell concentration.

Figure 4.2: Growth curves of THP-1 cells. The curves were constructed by GraphPad Prism software, where the third order polynomial (cubic) equation fit was the preferred model (R2 = 0.9933) for the curve with 105 cells/mL initial concentration (the blue curve), while the other two concentrations (3X104 and 1X104 cells/mL) followed the exponential growth equation with R2 values of 0.9709 and 0.9854, respectively.

The highest concentration has reached the plateau phase after the seventh day

while the other two concentrations were still in the exponential phase of growth

throughout the nine days. This means that the exponential growth of THP-1 cells ends

when cells concentration reaches 2X106 cells/mL. Throughout the cells maintenance,

this concentration was not exceeded and throughout the toxicity studies the cells

concentration was kept between 2 to 8 X105 cells/mL.

C A B

39

4.3 MTT and LDH Assays Sensitivity Studies

Various concentrations of THP-1 cells ranging from 103 to 107 cells/mL were

assayed by both MTT and LDH kits to determine the range of THP-1 cells

concentrations that these assays identify them in linear manner. Sensitivity curves were

constructed by plotting kit absorbance against cell concentration. Figure 4.3 shows the

sensitivity curves for MTT and LDH kits.

Figure 4.3: Sensitivity curves of MTT and LDH assays to THP-1 cells. The curves were constructed by GraphPad Prism software and for both MTT (the red curve) and LDH readings (green curve), hyperbola model was the chosen fit model where R2 was 0.9995 for MTT curve and 0.9902 for LDH curve.

It was clear that the linear part of MTT and LDH curves has resulted by the cell

concentration range of 105 to 106 cells/mL. So, the initial cell concentration used in

these assays was 2X105 cells/mL.

4.4 Plant Extracts Effects on THP-1 Cells by MTT Assay

The THP-1 cells were incubated with various concentrations of the four tested

plant extracts as well as DMSO respective concentrations for 48±1 hours and then were

assayed by MTT assay to determine the extracts and DMSO toxicity to the cells as

indicated in the materials and methods chapter. The results were expressed as a mean

percentage ± standard deviation (SD) of the mean negative control (untreated cells)

absorbance (see table 4.2). These data were analyzed using GraphPad Prism software

which used to construct dose response curves of the various extracts on the cells.

40

Table 4.2: THP-1 cells viability results by MTT test

H. sabdariffa O. syriacum T. vulgaris P. graveolens DMSO

Ext. Con. Cell Viability ± SD Ext. Con. Cell Viability ± SD Ext. Con. Cell Viability ± SD Ext. Con. Cell Viability ± SD Conc. Cell Viability ± SD

20 4.855837 ± 2.144999 8 2.736752 ± 1.074961 2 1.8547 ± 0.907174 5 3.088908 ± 0.677149 1.25 49.73694 ± 13.19307

15 55.26018 ± 4.429925 4 5.4888 ± 1.256352 1 1.77851 ± 0.828674 2.5 56.0178 ± 8.515269 0.5 81.98459 ± 12.5284

10 87.58607 ± 3.889591 2 53.69078 ± 5.803497 0.6 3.512489 ± 0.369931 1.5 100.8334 ± 11.00955 0.375 96.83851 ± 13.81734

5 82.98313 ± 6.372505 1.5 78.74228 ± 6.008436 0.4 11.77499 ± 2.022615 1.25 102.6689 ± 14.45633 0.3125 98.86212 ± 7.99779

2.5 100.0763 ± 13.11831 0.9 91.95539 ± 17.80762 0.2 48.26649 ± 7.536668 0.625 102.1776 ± 7.772026 0.25 112.3425 ± 17.32835

1.25 95.52168 ± 10.46768 0.7 92.25451 ± 18.00967 0.1 83.81131 ± 14.24472 0.3125 106.6482 ± 21.08935 0.15625 119.737 ± 6.406995

0.625 96.69471 ± 6.036152 0.5 93.27058 ± 13.71432 0.07 106.663 ± 20.01096 0.078125 116.401 ± 14.18109 0.1 107.6259 ± 20.48886

0.3125 100.8167 ± 9.708884 0.3 96.64085 ± 12.18187 0.02 119.5942 ± 28.42441

Ext. Conc. = Extract concentration in mg/mL Conc. = Concentration of DMSO (%)

SD = Standard deviation Cell Viability unit is percentage (%) 0.078125 96.32957 ± 5.106631 0.1 98.97904 ± 18.73132

41

The three extracts, H. sabdariffa, O. syriacum, and T. vulgaris, significantly

inhibited cell growth or induced cell death (see figures 4.4 and 4.5) in concentration

dependent manner, while the fourth extract, P. graveolens, has not shown any activity

against the THP-1 cells compared to the DMSO used as solvent in the extract stock

solution preparation.

It is clear from figure 4.4 that the toxicity of O. syriacum is stronger than that of

H. sabdariffa regardless of the difference in plant extraction method.

Figure 4.4: Dose response curves of H. sabdariffa and O. syriacum extracts effects on THP-1 cells. The curves were constructed by GraphPad Prism software, where the red curve represents H. sabdariffa extract effect and the blue curve represents O. syriacum extract effect.

Figure 4.5: Dose response curves of T. vulgaris and P. graveolens extracts as well as DMSO effects on THP-1 cells. The curves were constructed by GraphPad Prism software, where the green curve represents T. vulgaris extract effect, the blue curve represents P. graveolens extract effect, and the red curve represents DMSO effect. In order to fit the curves of the extracts' concentrations with the equivalent DMSO concentrations, the actual experimented DMSO concentrations were duplicated before they were blotted on the same graph with the extracts. Each unit of DMSO concentration (% v/v) was equivalent for two units of extract concentration (mg/mL).

42

The T. vulgaris and P. graveolens extracts have been dissolved in DMSO

solvent and by comparison to the DMSO effect (see figure 4.5), it is clear that in the

range of the inhibitory concentrations of T. vulgaris extract, the DMSO had no activity

on the cells, so the dose dependent inhibition seen was attributed to the T. vulgaris

extract alone. In contrast, P. graveolens extract cell inhibition which appeared in figure

4.5 was attributed to the DMSO solvent effect because the two curves behaved roughly

similar.

GraphPad Prism software was used to perform non linear regression analysis for

each of the extracts as well as DMSO control on the THP-1 cells in order to determine

IC50 values, the concentration of each extract as well as DMSO, at which 50% of the

cells were inhibited. Table 4.3 shows the IC50 values for all extracts and DMSO.

Table 4.3: IC50 values of the extracts on THP-1 cells.

Plant Extract IC50 value (mg/mL) R2 value 95% Confidence Interval

H. sabdariffa 15.47 0.9187 14.99 to 15.96

O. syriacum 2.126 0.9094 1.934 to 2.339

T. vulgaris 0.1569 0.9312 0.1326 to 0.1857

P. graveolens 2.535 0.9041 2.334 to 2.753

DMSO 1.919 0.7018 1.470 to 2.505

All values were determined by GraphPad Prism nonlinear regression analysis. Log (inhibitor) vs. response - Variable slope (four parameters) equation was the best fit model for all the extracts and DMSO curves when compared by the software to the other dose response inhibition models. The bottom of all curves was constrained to zero value. R2 values and 95% confidence intervals for each IC50 are also indicated.

T. vulgaris extract had the highest cell growth inhibitory effect or toxicity on

THP-1 cells with lowest IC50 value followed by O. syriacum then H. sabdariffa extracts.

IC50 value for DMSO was less than that of P. graveolens which considered having no

toxic effect on THP-1 cells.

43

4.5 Plant Extracts Effects on Peripheral Blood Mononuclear Cells

(PBMCs) by MTT Assay

PBMCs were separated by Histopaque-1077 and the effect of the plant extracts

on them was assessed by MTT assay after exposing the cells to various extract

concentrations as with THP-1 cells (PBMCs MTT viability results are detailed in table

4.4). Dose response curves of the extracts effects on PBMCs are shown in figures 4.6

and 4.7.

Figure 4.6: Dose response curves of H. sabdariffa and O. syriacum extracts effects on PBMCs. The curves were constructed by GraphPad Prism software, where the red curve represents H. sabdariffa extract effect and the blue curve represents O. syriacum extract effect.

As in toxicity studies on THP-1 cells, the toxicity of O. syriacum on PBMCs is

stronger than that of H. sabdariffa.

Figure 4.7: Dose response curves of T. vulgaris and P. graveolens extracts as well as DMSO effects on PBMCs. The curves were constructed by GraphPad Prism software, where the green curve represents T. vulgaris extract effect, the blue curve represents P. graveolens extract effect, and the red curve represents DMSO effect. In order to fit the curves of the extracts' concentrations with the equivalent DMSO concentrations, the actual experimented DMSO concentrations were duplicated before they were blotted on the same graph with the extracts. Each unit of DMSO concentration (% v/v) was equivalent for two units of extract concentration (mg/mL).

44

Table 4.4: Peripheral blood mononuclear cells viability results by MTT test

H. sabdariffa O. syriacum T. vulgaris P. graveolens DMSO

Ext. Con. Cell Viability ± SD Ext. Con. Cell Viability ± SD Ext. Con. Cell Viability ± SD Ext. Con. Cell Viability ± SD Con. Cell Viability ± SD

20 20.4263 ± 7.783533 10 19.18277 ± 4.965453 2.5 6.211406 ± 2.103669 5 5.080394 ± 4.728847 5 16.65396 ± 3.636308

10 14.13379 ± 2.146318 5 8.580562 ± 2.505742 1.25 3.735115 ± 1.682673 2.5 9.028989 ± 3.42996 2.5 58.1178 ± 4.478077

5 16.86796 ± 4.816498 2.5 7.429661 ± 4.544233 0.625 9.731462 ± 2.897807 1.25 19.33511 ± 4.078954 1.25 82.17661 ± 5.649995

2.5 62.82931 ± 7.711574 1.25 10.23557 ± 5.335506 0.3125 49.67798 ± 4.579114 0.625 47.33645 ± 5.625008 0.625 97.45749 ± 7.848894

1.25 71.9416 ± 8.730852 0.625 18.32953 ± 7.645902 0.15625 78.28277 ± 4.515461 0.3125 69.78986 ± 4.080817 0.3125 91.04869 ± 10.13889

0.625 81.74278 ± 5.671631 0.3125 63.85361 ± 7.829562 0.078125 88.10691 ± 6.401121 0.15625 88.17717 ± 12.14388 0.15625 102.3971 ± 5.811382

0.3125 84.10846 ± 4.269885 0.15625 81.1585 ± 6.287436

Ext. Conc. = Extract concentration in mg/mL Conc. = Concentration of DMSO (%) SD = Standard deviation Cell Viability unit is percentage (%)

45

Visually (figure 4.7), it appears that in the range of the inhibitory concentrations

of T. vulgaris and P. graveolens extracts, the DMSO had little activity on the PBMCs,

so the dose dependent inhibition of these cells was mainly attributed to the T. vulgaris

and P. graveolens extracts. P. graveolens extract has inhibitory effect on PBMCs while

on THP-1 cells it has not had any inhibitory activity.

The IC50 values for the four extracts and DMSO on PBMCs are listed in table

4.5. Figures from 4.8 to 4.10 show comparison between the effect of each extract on

both THP-1 cells and PBMCs.

Table 4.5: IC50 values of the extracts on PBMCs.

Plant Extract IC50 value (mg/mL) R2 value 95% Confidence Interval

H. sabdariffa 3.514 0.8975 2.908 to 4.246

O. syriacum 0.4247 0.8663 0.3385 to 0.5327

T. vulgaris 0.3345 0.9820 0.3114 to 0.3593

P. graveolens 0.5534 0.9635 0.4323 to 0.7085

DMSO 5.676 0.9386 5.140 to 6.268

All values were determined by GraphPad Prism nonlinear regression analysis. Log (inhibitor) vs. response - Variable slope (four parameters) equation was the best fit model for all extracts, except H. sabdariffa, curves. Also it was the best fit for DMSO curve. H. sabdariffa extract curve was fitted by asymmetric sigmoidal, 5 PL, equation. In all of these extracts curves the bottom was constrained to zero value. R2 values and 95% confidence intervals for each IC50 are also indicated.

T. vulgaris extract had the highest toxicity on PBMCs with lowest IC50 value

followed by O. syriacum then P. graveolens and H. sabdariffa extracts. IC50 value for

DMSO was high in comparison to T. vulgaris and P. graveolens extracts.

46

Cel

l Via

bilit

y (%

)

Figure 4.8: Comparison between the effects of H. sabdariffa extract on THP-1 cells and on PBMCs. The curves were constructed by GraphPad Prism software, where the blue curve represents the H. sabdariffa extract effect on THP-1 cells and the red curve represents its effect on PBMCs.

By scrutinizing in figure 4.8, it is obvious that the inhibitory effect of H.

sabdariffa extract was much higher on PBMCs than on THP-1 cells. After comparison

of the IC50 values of H. sabdariffa extract on both cell types by GraphPad Prism F test,

it was concluded that IC50 of H. sabdariffa extract on THP-1 cells (15.47 mg/mL) is

significantly higher than that on PBMCs (3.514 mg/mL) where P-value was less than

0.0001.

Figure 4.9: Comparison between the effects of O. syriacum extract on THP-1 cells and on PBMCs. The curves were constructed by GraphPad Prism software, where the blue curve represents the O. syriacum extract effect on THP-1 cells and the red curve represents its effect on PBMCs.

It is clear from figure 4.9 that the toxic effect of O. syriacum extract was much

higher on PBMCs than on THP-1 cells. After comparison of the IC50 values of O.

syriacum extract on both cell types by GraphPad Prism F test, it was concluded that IC50

of O. syriacum extract on THP-1 cells (2.126 mg/mL) is significantly higher than that

on PBMCs (0.4247 mg/mL) where P-value was less than 0.0001.

47

Figure 4.10: Comparison between the effects of T. vulgaris extract on THP-1 cells and on PBMCs. The curves were constructed by GraphPad Prism software, where the blue curve represents the T. vulgaris extract effect on THP-1 cells and the red curve represents its effect on PBMCs.

It is obvious from figure 4.10 that the toxic effect of T. vulgaris extract was

higher on THP-1 cells than on PBMCs. After comparison of the IC50 values of T.

vulgaris extract on both cell types by GraphPad Prism F test, it was concluded that IC50

of T. vulgaris extract on THP-1 cells (0.1569 mg/mL) is significantly less than that on

PBMCs (0.3345 mg/mL) where P-value was less than 0.0001.

Because it was concluded that there was no significant inhibitory effect of P.

graveolens extract on THP-1 cells, it is not considerable to compare the effects of this

extract on THP-1 cells and PBMCs.

From the above comparisons, it seems that only T. vulgaris extract which can

inhibit the leukemic cells more than normal cells while other extracts exerted more

effect on normal cells than on leukemic cells.

4.6 Plant Extracts Toxicity on THP-1 Cells by LDH Assay

To distinguish between the cytotoxic effect of the plant extract and the growth

inhibitory effect, LDH assay was used. After exposing THP-1 cells to various

concentrations of O. syriacum, T. vulgaris, and P. graveolens extracts as well as

DMSO, LDH assay was performed and the percent cytotoxicity of the positive control

(treated with Triton X-100) was calculated as explained in the materials and methods

chapter. Percent viability was calculated as (100 – percent cytotoxicity) (the results are

48

listed in table 4.6) then data analysis and dose response curves were processed by

GraphPad Prism software.

Table 4.6: THP-1 cells viability results by LDH test Ext.

Conc. Cell Viability ± Standard Deviation (%)

O. syriacum T. vulgaris P. graveolens DMSO

20 -5.11004 ± 33.28134 69.27668 ± 11.2473 64.47616 ± 6.810537 69.50853 ± 6.001513

10 49.73109 ± 6.13207 73.98201 ± 8.411463 72.82904 ± 3.853286 72.42552 ± 4.153989

5 77.34154 ± 3.874169 82.31145 ± 5.650866 77.26892 ± 8.014075 82.7447 ± 1.265742

2.5 93.27701 ± 2.722283 87.86831 ± 10.33276 92.48398 ± 6.372939 94.08492 ± 4.1521

0.625 96.55623 ± 3.565946 95.42127 ± 4.289322 94.49315 ± 3.338085 96.71546 ± 3.253288

0.3125 100.1726 ± 3.416478 95.75146 ± 5.786234 97.01363 ± 2.562171 97.63185 ± 3.140129

Ext. Conc. = Extract concentration in mg/mL

5 10 15 20 25

-50

0

50

100

150

Extract Concentration (mg/mL)

Cel

l Via

bilit

y (%

)

O. syriacumT. vulgarisP. graveolensDMSO Effect

Figure 4.11: LDH toxicity dose response curves of O. syriacum, T. vulgaris, and P. graveolens extracts as well as DMSO on THP-1 cells. These curves were constructed by GraphPad Prism software. In order to fit the curves of the T. vulgaris and P graveolens extracts' concentrations with the equivalent DMSO concentrations, the actual experimented DMSO concentrations were duplicated before they were blotted on the same graph with the extracts. Each unit of DMSO concentration (% v/v) was equivalent for two units of extract concentration (mg/mL).

As seen in figure 4.11, both T. vulgaris and P. graveolens had no cytotoxic

activity on THP-1 cells and the slight inhibition which appears in the figure was not

different from their solvent (DMSO) inhibition. By comparing the three curves (T.

vulgaris, P. graveolens and DMSO curves) with GraphPad Prism F test through non

49

linear regression analysis, the P-value was 0.5649 and this indicates that there was no

significant difference between the three curves. These findings means that while P.

graveolens extract has not had any inhibitory effect on THP-1 cells, the T. vulgaris

extract expressed only growth and proliferation inhibition activity and not cytotoxic

activity against THP-1 cells at the applied concentrations and exposure time.

O. syriacum extract exerted cytotoxic effect against THP-1 cells, as it appears in

figure 4.11, by relatively high concentrations. LC50 value for O. syriacum extract was

9.646 mg/mL (R2 = 0.8798) as calculated by non linear regression analysis using

asymmetric sigmoidal, 5 PL equation model with constraining the bottom to zero value.

50

Chapter 5 Discussion

In the recent years, a remarkable interest in the investigation of anticancer

properties of natural products has been seen. This interest is due to the fact that

conventional cancer treatment procedures (such as surgery, radiotherapy and

chemotherapy) are not effective in all cases and result in serious side effects, in addition

to the drug resistance associated with chemotherapeutic drugs. Furthermore, the limited

availability and the expensiveness of most chemotherapy drugs, also the trust that

natural products are safer, all have contributed to the popularity of traditional medicine.

This study was originated to fulfill the aim of discovering new anticancer agents from

natural materials.

Cultured cancerous cell lines with comparison to normal healthy cell lines are

commonly used to assess the anticancer activities of isolated phytochemicals and

extracts of medicinal plants. Some herbs may be harmful to the human body if they are

used improperly, and some herbs may cause serious toxicity when taken excessively or

under inappropriate circumstances. The successful treating material is that which

exhibits its therapeutic effect without causing serious side effects or exerting toxicity to

the body cells and organs. Therefore, it is not enough to investigate the therapeutic

activities of a given material without assessing its cytotoxicity against normal cells. If

this cytotoxicity was not excluded or at least reduced, then this material is not

considered a successful therapeutic drug.

The present study objectives were: assessing the antileukemic effects of the

Origanum syriacum, Hibiscus sabdariffa, Thymus vulgaris, and Pelargonium

graveolens extracts on THP-1 cells, also investigation of their inhibitory effect against

normal PBMCs to assess their selectivity. In addition, the detection of cytotoxicity of

the extracts on leukemic cells by LDH assay was a third objective.

Anticancer materials which induce cancer cell death and apoptosis are called

cytotoxic and those which do not kill cancer cells but instead stop the proliferation are

called cytostatic. Whether a drug seems cytostatic or cytotoxic may be drug independent

and instead depend on the dose used, the schedule of administration, the phase of the

51

cell cycle in which the drug acts and in which the cell resides, and the cellular context

(Rixe & Fojo, 2007).

MTT cell proliferation assessment kit was used to evaluate the growth inhibition

activity of the four plants extracts on both leukemic and normal cells. MTT system

developed by Mosmann (1983) is a means of measuring the activity of living cells via

mitochondrial dehydrogenases. The cleavage of the tetrazolium salt MTT (3- (4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into a blue colored product

(formazan) by mitochondrial dehydrogenases is potentially very useful for assaying cell

survival and proliferation. The conversion takes place only in living cells and the

amount of intracellular formazan produced, which can be measured

spectrophotometrically after dissolving it, is proportional to the number of viable cells

present.

Because both cultures with died cells and those with cells that have stopped

proliferation will result in a decreased MTT absorbance values, there was a need for

another assay that discriminates between cytotoxic and cytostatic effects of the extracts.

Lactate dehydrogenase (LDH) assay was used for this purpose. The LDH assay

(Korzeniewski & Callewaert, 1983) is a means of measuring membrane integrity as a

function of the amount of cytoplasmic LDH released into the medium. The assay is

based on the reduction of NAD by LDH. The resulting reduced NAD (NADH) is

utilized in the reduction of a tetrazolium dye to colored formazan product that is

measured spectrophotometrically. When cell death takes place in culture either by

necrosis or by apoptosis, the end of died cells is lysis and therefore, the intracellular

materials including LDH enzyme are released in to the extracellular medium. Cell

cytotoxicity can be estimated by assaying the cell free culture medium after exposing

cells to the test material for a period of time.

In this study, the only extract that resulted in both reduced viability and

increased cytotoxicity is the O. syriacum extract that is most likely to have apoptotic or

necrotic activity. On the other hand, based on above discussion, the P. graveolens and

T. vulgaris extracts have only exhibited cytostatic and not cytotoxic activities at the

applied concentrations and exposure times. These extracts had likely halted the cells

proliferation at one level or the other and did not kill the cells. H. sabdariffa extract was

not investigated by LDH assay in this study.

52

The extract of H. sabdariffa has showed a dose dependent inhibitory effect on

THP-1 leukemic cells with an IC50 value of 15.47 mg/mL, but its inhibitory effect on

normal cells was roughly five-fold stronger (IC50= 3.514 mg/mL). This finding means

that H. sabdariffa extract at certain concentrations was significantly more potent to

normal cells than leukemic cells and at the concentrations that did not inhibit PBMCs

(figure 4.8), it had not any inhibitory effect on the THP-1 leukemic cells.

Concurred with these results, one previous study showed that H. sabdariffa

crude aqueous extract did not exhibit significant inhibitory effect on some cancer cell

lines as Hep G2/C3A, Hep 3B, and SK-HEP-1 hepatoma cells (Lin et al., 2002). On the

other hand, most previous studies showed that H. sabdariffa has anticancer and/or

antileukemic activities with little cytotoxicity toward normal cells. For example,

Hibiscus anthocyanins (HAs) extracted from the roselle calyx as well as Hibiscus

polyphenol-rich extracts (HPE) were found to have a concentration-dependent

inhibitory effect on the growth of the cell lines: NIH3T3, Hep G2, MCF-7, KB, Caco-2,

Hep 3B, HL-60, and AGS cells (Chang et al., 2005; Hou et al., 2005; Lin et al., 2005).

The least cytotoxicity of both HAs and HPE was toward the normal mouse fibroblast

cell line. Another study by Akim et al. (2011) concluded that H. sabdariffa juice

exhibits anticancer activities on human breast cancer, human cervical cancer and human

ovarian cancer cell lines without investigating the cytotoxic effect against normal cells.

However, the apparent contrary of the above studies with the present study may

has resulted from the fact that in all of these studies, if the normal cells were used to

assess the cytotoxicity of the H. sabdariffa, the selected cells were the normal mouse

fibroblast cells (NIH3T3), and none has used the peripheral blood mononuclear cells

(PBMCs) as a normal cells model as was done in this study. It is known that NIH3T3

cell line grows better in culture conditions than the primary PBMCs cells and this may

lower their sensitivity to the extract in comparison to the PBMCs sensitivity. In a study

of H. sabdariffa extract effective protection of cultured PBMCs from the cellular death

induced by H2O2, the PBMCs were exposed to incremental doses of H. sabdariffa

extract (up to 2000 µg/mL) for 24 h and the viability of PBMCs was more than 95% but

when the extract concentration exceeded this limit (2 mg/mL), it exhibited a

cytotoxicity toward PBMCs (Beltrán-Debón et al., 2010). This finding is more or less

similar to the present study results where IC50 of H. sabdariffa extract on PBMCs was

3.514 mg/mL.

53

The effect of O. syriacum extract was similar to that of H. sabdariffa extract but

the IC50 value on THP-1 cells was 2.126 mg/mL, and its inhibitory effect on normal

cells (PBMCs) was extremely stronger (IC50= 0.4247 mg/mL). Not only the inhibition

of O. syriacum extract was not selective to leukemic cells, but also it does no longer

exist toward THP-1 leukemic cells at the non cytotoxic concentrations (figure 4.9).

With LDH assay, the effect of O. syriacum extract against THP-1 cells was

distinguished to be cytotoxic or apoptotic rather than cytostatic and LC50 value was

9.646 mg/mL. This would be a much more valuable finding if the effect of O. syriacum

extract was selective to the leukemic cells only.

The present study is the first to investigate the anticancer effect of O. syriacum

against leukemic cells. No previous studies were found in the literature that have

assessed the antileukemic effect of O. syriacum, but there are few studies which

investigated its anticancer activity against cancer cell lines other than leukemia cells. El-

Desouky et al. (2009) and Al-Kalaldeh et al. (2010) found that the ethanol crude

extracts of O. syriacum showed antiproliferative activity to adenocarcinoma of breast

cell line (MCF7) and human cervical adenocarcinoma (HeLa) cells with IC50 values of

6.40 μg/mL and 474.2 μg/mL, respectively. However, none of these studies investigated

the cytotoxicity of the O. syriacum extracts against PBMCs. There is only one study

(Al-Ali et al., 2013) found in the literature that deals with the O. syriacum cytotoxicity

against normal cells. And however the normal cells used by that study researchers were

the diploid human embryonic fibroblasts (MRC-5 cell line) and not PBMCs, their

results are consistent with the present study results where the crude methanolic extract

of O. syriacum has showed cytotoxic activity against the normal MRC-5 cells and IC50

was >64 µg/mL.

Not as other extracts, T. vulgaris extract has showed different results. It has

exhibited an inhibitory effect toward THP-1 leukemic cells (IC50= 0.1569 mg/mL) in a

dose dependent manner and this inhibition was twofold more potent than the extract

inhibition on normal mononuclear cells (IC50= 0.3345 mg/mL). This finding suggests

that T. vulgaris extract inhibition is more selective to leukemic cells than normal cells,

and at certain concentrations, T. vulgaris may be used to inhibit leukemia without

causing significant cytotoxicity toward normal body cells.

54

Consistent with these results, Amir and Karimi (2001) in their study found that

the ethanolic extract of T. vulgaris showed a dose dependent inhibition of proliferation

of human breast cancer (SK-Br-3, MDA-MB-435) and leukemia (U937 and K562) cell

lines as well as cervical epithelial carcinoma (HeLa) cell line which was the least

sensitive cell line to the extract. The most sensitive cells were U937 and MDA (50%

inhibition at 10 µg/mL). Also they found that the effect of T. vulgaris on normal Vero

cell line and PBMCs was stimulatory at low concentrations (10-100 µg/mL) and

inhibitory when extract concentration increased (at 400 µg/mL, the inhibition of Vero

cells was 27.6%). In contrary, Berrington and Lall (2012) in another study have

different results and found that acetone extract of T. vulgaris inhibits the normal Vero

cell line (IC50= 138.4 μg/mL) more than the HeLa cells (IC50> 200 μg/mL).

Furthermore the essential oil of T. vulgaris was tested on head and neck squamous cell

carcinoma (HNSCC) by Sertel et al. (2011) and also was found to have a relatively low

toxicity with an IC50 value of 369 μg/mL.

The IC50 value of T. vulgaris extract on THP-1 cells was low in comparison to

other extracts values. However, the effect of this extract on THP-1 cells was only

cytostatic, and at the concentrations used in this study with LDH assay there was no

detected cytotoxic or apoptotic effect of this extract. Whether the T. vulgaris extract

effect was cytotoxic or cytostatic, it is a hopeful finding that this plant contains cancer

therapeutic agents that are more selective to leukemic cells than normal cells at

relatively low concentrations.

The carvacrol and thymol may be the most constituents of T. vulgaris extract

which are responsible for its anticancer activities. Many studies have reported the

anticancer effect of these essential oils. Carvacrol and thymol significantly reduced the

level of DNA damage induced in K-562 cells by the strong oxidant H2O2 (Horvathova

et al., 2007). Furthermore, carvacrol has an important in vitro antitumor effect against

tumor cell lines like Hep-2 (Stammati et al., 1999), B16 (He et al., 1997) and A-549

(Tansu Koparal & Zeytinoglu, 2003; Zeytinoglu et al., 2003). The effect of carvacrol

and thymol on cell cycle progression in K-562 tumor cells was examined by Jaafari et

al. (2012) with flow cytometry after DNA staining in order to investigate the molecular

mechanism of their cytotoxic activity. The results revealed that carvacrol stopped the

cell cycle progression in S phase; however, thymol stopped it in G0/G1 phase.

55

P. graveolens extract has not exerted any effect, neither cytotoxic nor growth

inhibitory, against the THP-1 leukemic cells at the studied concentrations. However, it

has exhibited inhibitory activity against PBMCs (figure 4.7). There are no studies found

in the literature that have assessed the P. graveolens anticancer activities using the plant

crude extracts while many studies have detected the plant's essential oils (citronellol,

geraniol, and transgeraniol) anticancer and antileukemic behaviors (subsection 2.2.4).

The contrary of this study finding with those studies' results may be due to the use of the

plant crude extract in the present study where the concentrations of the essential oils

with detected anticancer activities are low in comparison to the separated and purified

essential oils used in those studies.

It is also expected that the cytotoxicity of the used extracts in this study against

the normal PBMCs was relatively high due to the application of the crude extract and

not purified compounds. The crude extract contains many unpurified and unspecified

materials that may exert non specific inhibition against the already difficult to grow

PBMCs and not necessarily that the compounds which posses anticancer or

antileukemic activities are the same compounds that exhibit non specific inhibition

against the normal cells. This thesis is a screening study and many subsequent more

detailed studies can be performed to study the antiproliferative and cytotoxic effect of

the purified compounds of the extracts which have showed antileukemic activities in

this preliminary study.

Furthermore, it is expected that the studied extracts if investigated in vivo, they

will exhibit less cytotoxicity against normal cells than that exhibited when the

investigation occurs in vitro. The in vitro cytotoxic effect exerted by the extracts against

the normal cells may be due to the fact that the primary blood mononuclear cells

behavior outside the body is different from that inside the body because the culture

conditions even when are optimal are not as the physiological environment in vivo. This

difference may affect normal cells more than leukemic cancerous cells that have gained

some growth advantages during their multistage progress towards cancer.

There is a study by Lakis et al. (2012) in which the T. vulgaris and O. syriacum

essential oils cytotoxic effect on normal cells was investigated in vivo. The

hematological and biochemical routine parameters of Wistar rats were assayed in order

to determine a possible secondary effect after the oral administration of T. vulgaris and

56

O. syriacum essential oils. The oral dose used in the study corresponded to 0.03g

carvacrol/Kg bw/day in the case of T. vulgaris essential oil and 0.015g thymol/Kg body

weight/day in case of O. syriacum essential oil. No significant variations of the

hematological or biochemical parameters were noticed after the oral treatment with T.

vulgaris essential oil (29.74% thymol) and O. syriacum essential oil (66.64% carvacrol)

compared to the control group treated with physiological solution.

57

Chapter 6 Conclusion and Recommendations

6.1 Conclusion

In this study the in vitro antiproliferative and cytotoxic effects of Origanum

syriacum, Hibiscus sabdariffa, Thymus vulgaris, and Pelargonium graveolens extracts

on human leukemic THP-1 cells were assessed. Also the inhibitory effects of these

extracts on normal peripheral blood mononuclear cells were evaluated.

It was found that both extracts Origanum syriacum and Hibiscus sabdariffa

exert concentration dependent antiproliferative effect on leukemia as well as on normal

cells. Origanum syriacum extract also exhibited cytotoxic activity against THP-1

leukemic cells which was a significant finding in this study. These extracts need to be

more selective toward leukemic cells than normal cells before they are considered

successful antileukemic agents.

Pelargonium graveolens extract did not exhibit any inhibitory effect neither

antiproliferative nor cytotoxic toward leukemic cells at the applied concentrations and

exposure time. In addition, it has exerted antiproliferative effect against normal cells. As

a result, it is unpredicted that this extract may possess significant therapeutic

characteristics for leukemia.

The fourth extract, Thymus vulgaris, has had more selective antiproliferative

effect against leukemic cells and its effect was only cytostatic and not cytotoxic at the

applied concentrations and exposure time. At relatively low concentrations, this extract

was able to inhibit THP-1 cells proliferation more than normal cells proliferation and

this inhibition was in a dose dependent manner. This important finding gives a hope that

Thymus vulgaris active materials can be useful in leukemia treatment development.

6.2 Recommendations

It is recommended that further research should be undertaken in the following areas:

• Investigation of the Origanum syriacum, Hibiscus sabdariffa, and Thymus

vulgaris extracts toxicity in vivo at the effective antileukemic concentrations.

58

• Purification of Origanum syriacum, Hibiscus sabdariffa, and Thymus vulgaris

extracts active materials and assessment of the antiproliferative and cytotoxic

effects of these purified materials against leukemic cells as well as normal cells

both in vitro and in vivo.

• Following the mode of action of these extracts against leukemia cells at the

molecular level to understand their antileukemic mechanisms.

• Assessment of these extracts anticancer effect against malignant cells of cancer

types other than leukemia.

• Assessment of other medicinal plants extracts on both leukemia cells and other

cancer cells.

59

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