A Study on the Effect of Some Plant Extracts on Certain...

Islamic University – Gaza Dean of Higher Education Faculty of Science Master of Biological Sciences

A Study on the Effect of Some Plant Extracts on Certain Malignant Cell Lines in Vitro

Prepared by

Saib H. Al Owini

Supervisors

Dr. Abboud EL Kichaoui Dr. Basim Ayesh

Submitted in Partial Fulfillment of the Requirements for the degree

of Master of Biological Sciences/Zoology

Department of Biology, Faculty of science

2006

ii

سم اهللا الرمحن الرحيمب

والتني والزيتون(

وطور سينني *

وهذا البلد األمني *

*

لقد خلقنا اإلنسان في أحسن تقويم

).التني()

فنبذناه بالعراء وهو سقيم (

وأنبتنا عليه شجرة من يقطني *

(

) الصافات (

صدق اهللا العظيم

iii

إن خياطا دعا رسول ا : عن أنس بن مالك رضي ا عنه قالفذهبت مع رسول : صلى ا عليه و سلم لطعام صنعه، قال أنس

عليه و سلم فرأيته يتتبع الدباء من حوايل القصعة، ا صلى ا.فلم أزل أحب الدباء من يومئذ: قال

ھو القرع الیقطینيلدباءا

iv

DEDICATION

Dedicated to my father and my mother

To my wife

To my son Ahmad

To my daughters Shahd, Tasneem and Malak.

To all my family members

v

DECLARATION

I hereby declare that this submission is my own work and that, to

the best of my knowledge and belief, it contains no material

previously published or written by another person nor material

which to a substantial extent has been accepted for the award of

any other degree of the university or other institutes, except where

due acknowledgment has been made in the text. Signature

Saib H. Al Owini

Copyright

All rights reserved: No part of this work can be copied, translated or stored in

retrieval system, without prior permission of the author.

vi

ABSTRACT

In Palestine like in other countries of the world, cancer is one of the most

serious health problems that affect the duration and quality of the individuals’

life. Enormous efforts are invested to cope with this problem, but unfortunately

limited success has ever been achieved with most of the therapeutic

strategies. These efforts are usually complicated with the need for well

experienced surgeons, lack of specificity and high cost, as well as being

usually accompanied with a wide range of side effects.

As the conventional therapeutic strategies fail to fulfill the major requirements

for a successful cancer therapy, the use of naturally developed anticancer

agents has evolved as an alternative safe, low-cost and convenient one.

Therefore, the use of plant extracts with potential anticancer therapeutic

effects might be particularly significant, especially in Palestine, which is rich in

thousands of plant species known for their medical uses. Moreover, the lack

of expertise, the scares economical resources and the complicated political

situation in Palestine don’t allow the application of sophisticated surgical,

chemo- and radio-therapies to cure cancer.

Therefore, the current study, investigates the effect of crude water extracts

from Bottle gourd (Lagenaria siceraria), Fig (Ficus carica) and Nettle (Urtica

pilulifera) on cell lines derived from different human tissue origins (Hep3b:

Hepatocellular carcinoma; Hela: cervical epithelial cancer; and PC-3: prostate

cancer).

The results showed a concentration-dependent reduction in the final number

of cancer cells in consequence to treatment with the aforementioned crude

extracts. Two kinds of anticancer effects were evaluated and found to

contribute to this reduction: the antiproliferation effect (decreased number of

metabolically active cells) and cytotoxicity (decreased number of live cells).

The three plants examined possess both of the effects with various degrees.

Urtica pilulifera possess the strongest and most profound effects on the three

cell lines, mainly by induction of cell death. On the other hand Lagenaria

siceraria probably affects the three cell lines by a combination of cytotoxicity

vii

and antiproliferation almost to a similar degree. Ficus carica most probably

reduces the final number of metabolically active cells mainly by its

antiproliferative effect.

Both Ficus carica and Lagenaria siceraria are edible plants that were chosen

on the bases of being mentioned in the holy Quran. Therefore, although their

effect is lower than that of Urtica pilulifera their amount in the diet or as a

treatment can be safely scaled up when ingested in their native form. On the

other hand, despite its possible toxicity Urtica pilulifera is frequently orally

used as a medication in many conditions by traditional medicine.

Further studies are needed to assess the active ingredients of Ficus carica,

Lagenaria siceraria and Urtica pilulifera, involved in the antiproliferative or

cytotoxic effects of these plants. These studies must involve the

establishment of in vivo animal models and the application of more efficient

extraction and fractionation techniques.

viii

دراسة حول تأثیر بعض المستخلصات النباتیة على عدد من الخالیا السرطانیة

مستخلصال

لى مدى فع، في عصرنا الحدیثالصحیة التي تواجھ العالم من أخطر التحدیات السرطان مرضیعتبر

و على الرغم من التقدم ."السبب الثاني للوفاة" المختلفة أنواعھ ھذا المرض بالعقدین السابقین شكل

ھذا باإلضافة لألعراض الجانبیة، كانت محدودةنجاح النسبة أن إال العالجالعلمي الكبیر في أسالیب

في فلسطین وكثیر من دول وصاص العالجات وكذلك ارتفاع ثمنھا ونقص الخبرات خالناجمة من ھذه

لعودة إلى استعمال المواد المستخلصة طبیعیا من الكائنات الحیة ھذه األسباب دعت الباحثین ل .العالم

الحیوي النباتي فكان من الضروري أن التنوع فلسطین تشتھر بوحیث أن .وخاصة النباتات والطحالب

. بعض ھذه النباتات على مرض السرطانریأثتدراسة یتم

لقد تم و. والیقطین، والقریص، ھي التینو ثالث نباتات معروفة في فلسطین تم اختیارفي ھذا اإلطار

فإنھ من أشھر كنباتات مباركة أما القریص األول والثاني ذكرا في القرآن الكریم أناالختیار بناءا على

وماتیزم، الحصى وأمراض الر( النباتات الطبیة والتي تستعمل شعبیا لعالج العدید من األمراض

).الكلى

من النباتات الثالث كل مستخلصات مائیة تم تحضیر السرطانیة الخالیا النباتات على ولدراسة أثر ھذه

ضمن )بروستاتا، عنق الرحم، كبد( السرطانیة الخالیاثالث أنواع من إضافتھا على كما تم ، على حده

ونسبة ، أثر ھذه المستخلصات على معدل انقسام الخالیاوقد تم قیاس .الوسط الغذائي لھذه الخالیا

. وقد تم تأكید ھذه النتائج بدراسة تغیراتھا الشكلیة،) السمیة (موتھا

أو بزیادة نسبة االنقسام سواء بخفض معدل ، األشد أثراولقد دلت النتائج على أن نبات القریص ھو

أثرا فقد أظھرالتین والیقطینأما بالنسبة ل . الخالیاللھ ھو قتمما یدل على أن األثر األرجح ، السمیة

سبة معینة نوب،ن سمیة التین كانت األقل أضف إلى ذلك أ انقسام الخالیا جیدا من حیث خفض معدل

. فقط أثرھما على الخالیا لم یكن بقتل الخالیا أندعمیالیقطین مما

یا أو على حیوانات التجارب ضروریة لتأكید أثر مزیدا من الدراسات حول أثر ھذه النباتات سوءا معمل

. ھذه النباتات على السرطان

جید للحمایة والوقایة من بشكل للتركیز على تناول التین والیقطین قوي ھذه النتائج دافع تمثل

.السرطان

" حافظا وھو أرحم الراحمینخیر واهللا"

ix

Acknowledgment

Praise be to Allah, the lord of the worlds, and peace and

blessings of Allah be upon the noblest of the Prophets and Messengers,

our Prophet Muhammad.

I wish to express my great thanks to Dr. Abboud El-kichaoui,

Associated Professor of Mycology and Director of Biological Science

Master program, the Supervisor of this work, for his kind assistance,

continuous and valuable advices.

I would like also to express my deepest gratitude and

appreciation to Dr. Basim Ayesh Associated Professor of Molecular

Biochemistry, the Supervisor of this work, for initiating and planning this

study, enlightening supervision, useful assistance during the course of

the study.

I delighted to acknowledge for Dr. Maged M. Yassin Associated

Professor of physiology for his kindly efforts and valuable suggestion

during the thesis review.

I would like also to express my sincere thanks and gratitude to my

uncle Shehada M.El-Ebweini Master of science in mother and child

health, for valuable advices and continuous support during the course of

this study.

Finally I would like to thank all my friends and colleagues for their

contributed in some way or another to the present work.

x

CONTENTS

DECLARATION-------------------------------------------------------------------------------- i

COPYRIGHT------------------------------------------------------------------------------------ i

ABSTRACT------------------------------------------------------------------------------------- ii

ARABIC ABSTRACT------------------------------------------------------------------------- iv

ACKNOWLEDGMENT--------------------------------------------------------------- v

CONTENTS----------------------------------------------------------------------------- vi

LIST OF FIGURES AND TABLES------------------------------------------------ ix

LIST of ABBREVIATIONS xi

INTRODUCTION 1

I. Overview------------------------------------------------------------------------------- 1

II. Objectives----------------------------------------------------------------------------- 4

LITERATURE REVIEW 5

I. Conventional cancer therapy----------------------------------------------------- 5

II. Plants with anticancer effects---------------------------------------------------- 5

A. Plants mentioned in Islamic literature with therapeutic activity--------------- 6

B. Plants studied in this work---------------------------------------------------- 10

1. Bottle gourde( Lagenaria siceraria ) ---------------------------------- 10

2. Fig (Ficus carica)----------------------------------------------------------- 13

3. Romman nettle (Urtica pilulifera)--------------------------------------- 14

III. Cancer cell lines ------------------------------------------------------------------- 16

A. Hepatocellular carcinoma (Hep3b) cell line------------------------------ 17

B. Cervical epithelial cell line (Hela) ------------------------------------------ 17

C. Prostate cell line (PC-3)------------------------------------------------------ 18

xi

MATERIAL AND METHODS 20

I. Materials ------------------------------------------------------------------------------ 20

II. Methods ------------------------------------------------------------------------------ 21

A. Plant collection------------------------------------------------------------------- 21

1. Romman nettle (Urtica pilulifera)---------------------------------------- 21

2. Bottle gourde( Lagenaria siceraria )----------------------------------- 22

3. Fig (Ficus carica)----------------------------------------------------------- 22

B. Preparation of the crude extract -------------------------------------------- 23

C. Cell culture ----------------------------------------------------------------------- 24

D. Experimental design ---------------------------------------------------------- 25

E. Determination of growth characteristics: -------------------------------- 25

F. Determination of plant extract working concentrations --------------- 25

G. Cell proliferation assay ------------------------------------------------------- 26

H. Trypan blue exclusion viability assay-------------------------------------- 27

I. Determination of morphological changes of the cells in culture ---- 27

J. Statistical analysis -------------------------------------------------------------- 28

RESULTS 29

I. Determination of the cell lines growth characteristics---------------------- 29

II. Determination of the different plant extract working concentrations--- 30

III. Effects of plants extracts on cancer cells proliferation activity in

culture -----------------------------------------------------------------------------------

32




IV. Effect of plant extracts on cancer cell lines viability in culture--------- 39

xii




V. Determination of morphological changes of cell lines in response to

treatment of different plant extracts .----------------------------------------------

45




DISCUSSION--------------------------------------------------------------------------- 51

CONCLUSION ------------------------------------------------------------------------- 63

RECOMMENDATIONS -------------------------------------------------------------- 64

REFERENCES ------------------------------------------------------------------------- 65

xiii

LIST OF FIGURES AND TABLES

Figure 1 Taxonomy of Urtica pilulifera. 21

Figure 2 Taxonomy of Lagenaria siceraria. 22

Figure 3 Taxonomy of Ficus carica. 23

Figure 4 Laboratory instruments used for extract preparations. 24

Figure 5 Growth curves of Hep3b, Hela and PC-3 cell lines.. 29

Figure 6 A preliminary experiment of the effect of Urtica pilulifera, Lagenaria

siceraria and Ficus carica on the viability of Hepatocellular carcinoma

Hep3b cells.

31

Figure 7 Reduction of proliferation activity of Hep3b cells in response to plant

extracts from Urtica pilulifera, Lagenaria siceraria and Ficus carica.

34

Figure 8 Reduction of proliferation activity of Hela cells in response to plant


36

Figure 9

Reduction of proliferation activity of PC-3 cells in response to plant


38

Figure10 Reduction of Hep3b cells viability in response to extracts from Urtica

pilulifera, Lagenaria siceraria and Ficus carica.

40

Figure 11 Reduction of Hela cells viability in response to extracts from Urtica


42

Figure12 Reduction of PC-3 cells viability in response to extracts from Urtica


44

Figure 13 Morphology of Hep3b cells in culture with or without extract. 46

Figure 14 Morphology of Hela cells in culture with or without extract. 48

Figure 15 Morphology of PC-3 cells in culture with or without extract. 50

Figure16 Reduction of viability and proliferation activity of Hep3b (a), Hela (b)

and PC3 (c) cells in response to Treatment with Urtica pilulifera

extract.

55


and PC3 (c) cells in response to Treatment with Lagenaria siceraria

extract.

56

xiv


and PC3 (c) cells in response to Treatment with Ficus carica extract

57

Table 1 Literature summary of studies that examined the therapeutic

potential of plant extracts.

7

Table 2 Literature summary of studies that examined the therapeutic

potential of Islamic plant extracts.

8

Table 3 Summary of calculated concentrations of the three plant extracts

Urtica pilulifera, Lagenaria siceraria and Ficus carica that give 50%

(IC50) reduction in proliferation activity of Hep3b, Hela and PC-3

cancer cell lines. The R2 value for each curve equation is illustrated.

54

Table 4 Summary of calculated concentrations of the three plant extracts

Urtica pilulifera, Lagenaria siceraria and Ficus carica that give 50%

(IC50) reduction in the % viability of Hep3b, Hela and PC-3 cancer

cell lines. The R2 value for each curve equation is illustrated.

54

xv

LIST OF ABBREVIATIONS

ATCC American Type Culture Collection

ADA Adenosine deaminase activity

PBS Phosphate Buffer Saline.

BPH Benign Prostate Hyperplasia

DMEM Dulbecco's Modified Eagle's Medium.

EDTA Ethylene-Diamine Tetra-acetic Acid.

ELISA Enzyme-linked Immunosorbent Assay

EGF Epidermal Growth Factor

HCC Hepatocellular Carcinoma.

HPV Human Papilloma Virus.

Hep3b Human Hepatocellular carcinoma cell line.

Hela Human cervical epithelial cell line. .

HL-60 Human, peripheral blood, leukemia, acute myeloid cell line

HepG2 Human hepatocellular liver carcinoma cell line

HepA-H Liver cancer cells.

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Buffer

hpcps Prostate stromal compartment

IC50 50% Inhibitory concentration.

Lncap Human, prostate, carcinoma cell line

PC-3 Human prostate cancer cells.

R Coefficient of correlation

R2 Related coefficient of determination

SE Standard Error of the mean

SHBG Sex hormone binding gluobin

XTT Cell proliferation assays, A colorimetric method based on

the tetrazolium salt

1

INTRODUCTION

I. Overview Cancer is one of the most serious health problems worldwide, affecting

individuals from different sexes, ages, and races. It is a group of diseases,

characterized by uncontrolled cellular growth with frequent cancer cells invasion

to different body parts and spreading to other organs, a process referred to as

Metastasis. Metastasis is the major cause of cancer related mortality (1). In

2005, cancer was the second leading cause of death among both men and

women and accounted for 13% of the total 58 million deaths worldwide (1). In

2006, about 10.9 million new cancer cases are expected to be diagnosed

worldwide and more than 7.8 million cancer patients may die (1).

According to the latest report of cancer registry unit in Gaza strip, 5500 cases

have been reported over the period from January, 1995 to December, 2003 (2).

In addition, 1026 cancer patients died in 2004 in the Palestinian territories with a

mortality rate of 28.2 per 100.000 (2).

Cancer is also a problem of economical dimensions with a very high level of

expenses associated to it. For example the National Institute of Health, USA

estimates that an overall of $209.9 billion were invested worldwide in 2005, for

the sake of cancer research and management (3).

Cancer is a heterogeneous illness which can originate from many different

organs of the human body. However, the most frequent cancer types in the

world are lung, prostate, stomach, colorectal, and esophagus in men; and breast,

lung, stomach, colorectal and cervical in women (1).

Prostate cancer is the most frequently diagnosed and the second leading cause

of cancer death among men, with 234460 new cases estimated to occur in USA

during 2006, and 27350 American men will die as a result of this disease(3). In

Palestine, the mortality rate of prostate cancer was 1.4 per 100000 during the

period from January, 1995 to December, 2002 (2). Despite the fact there are

2

several cell types in the prostate, nearly all of the prostate cancers are

adenocarcinoma, originating in the gland cells (3).

Liver cancer ranks as the sixth most common type of cancer world wide (3).

According to the Palestinian ministry of health liver cancer mortality rate was 1.6

per 100000 over the period from January, 1995 to December, 2003 (2). Many

different liver related tumors are identified depending on the type of cells where

they originate, from these types about 83% are hepatocellular carcinoma (HCC)

that begin in the hepatocytes, the main type of liver cells.

Cervical cancer is the most common cause of cancer death among women in

developing countries and the second most common caner in women worldwide

(1). It is caused by a change in the epithelial cells, which line the wall of the

cervix, and the most common risk factor for this type of cancer is the human

Papillomavirus (HPV) (1).

In the last decades there were great advances in the diagnosis of cancer as well

as in the field of molecular oncology. However, the cure rate of most cancers

remains low. Several strategies have been used to cure cancer among which the

most common are surgery, chemotherapy, radiotherapy, and immunotherapy.

Other modern approaches such as hormonal and gene therapy were proposed

by researchers to replace conventional cancer therapy, with variable degrees of

success (3, 4). All of these therapies have undesired side effects, they are

usually not available all the time and they are expensive. For instance, in surgery

the immune system is compromised due to the large amount of cortisole

released subsequent to the surgery, which increase the probability of cancer

relapse (5). Moreover, the current use of chemotherapy is accompanied with

difficult side effects. It inhibits bone marrow stem cells proliferation leading to

immune suppression (5). Radiotherapy which is widely used in the world is also

accompanied by a great deal of side effects. Lymphocytes are most readily

affected by radiation resulting in prolonged T-cell suppression (5). Other side

effects such as, bone necrosis, lung fibrosis, skin devascularization, ulceration,

3

nausea, vomiting, and renal damage are also associated with all types of

conventional therapies.

As the conventional cancer therapies failed to completely fulfill the criteria for a

successful cancer therapy, the use of naturally developed anticancer agents has

evolved as an alternative safe, low-cost and convenient one. Nontoxic

chemoprevention agents from natural resources were proposed by researchers

for this purpose.

Historically, plants with known therapeutic potential have long been used to cure

a wide range of diseases. An example for these drugs is Morphine, which is a

plant product discovered in 1861 as an analgesic agent. Later, Quinine the

active component of Cinchona bark was isolated in 1820 as an effective anti-

malaria drug (5, 6). Our Arabic tradition is particularly rich in medical plants that

have been used by pioneer Arabic physicians to establish the basis for modern

therapies. These were also recommended by our Profit Mohammad ( م وسلصلى اهللا عليه )

and the Holly Quran. Nigella, Garlic, Onion and Fenugreek are famous examples

for these plants that were recently proven to have therapeutic effects on several

illnesses.

The use of potentially curative plants might be particularly significant in the

Palestinian territories where the plain and mountains are covered with more than

2600 plant species of which more than 700 are known for their uses as medicinal

herbs or as botanical pesticides (7).

In this thesis we studied the therapeutic potential of three Arabic and Islamic

traditional plants as anticancer agents. These were Fig (Ficus carica) and Bottle

gourd (Lagenaria siceraria) that were mentioned in the holy Quran in more than

one occasion as paradise and favorite plants and were selected based on that.

Nettle (urtica pilulifera) was also tested as a popular traditional plant used for

healing and diuretic purposes (8).

4

II. Objectives

The aim of the current study is to investigate the effect of Bottle gourd (Lagenaria

siceraria), Fig (Ficus carica) and Nettle (Urtica pilulifera), extracts on Hep3b from

human Hepatocellular carcinoma; Hela: human cervical epithelial cancer; and

PC-3- human prostate cancer cells in vitro on the basis of Arabic and Islamic

traditional medicine.

Specifically this study aims at:

1- Determination of the proliferation activities of each cell line in response to

each plant extract treatment.

2- Determination of percent viability of each cell line in response to each

plant extracts treatment.

3- Determination of any morphological changes of each cell line in each time

performing viability testing assay in parallel.

5

LITERATURE REVIEW

I. Conventional cancer therapy Despite the latest advances in medical sciences, and progress in strategies of

cancer treatment, cancer currently remains a tragic disease and is one of the

major causes of death worldwide. The principal methods of cancer treatment

include chemotherapy radiotherapy and surgery.

Chemotherapy is a systemic treatment, to which the whole body is exposed.

Among the most successful chemotherapeutic agent are Cisplatin, Mitomycin

and Docetaxel. All of these agents enhance serious side effects or long term

complication (9, 10 and 11). These side effects include kidney damage, hearing

loose, lower blood count, liver damage, nerve damage, and blood vessel damage

(12, 13).

External beams of Radiotherapy are associated with unacceptably high levels of

local-regional toxicity (13). Particularly, it affects the rapidly dividing cells of

mucosa, causing irritative urinary and blood loss (13). Later toxic effects result

from damage to the more slowly proliferating cells such as fibroblasts,

endothelial, or paranchymal stem cells causing chronic fibrosis and vascular

damage (13).

Other undesired side effects, such as, immune suppression, bone necrosis, lung

fibrosis and skin devascularization are seen with all types of conventional

therapies (11, 14).

II. Plants with anticancer effects Plants are the chief source of natural products that are used in medicine. Even

Aspirin, the world best known and most universally used medication, has its

natural origins from the glycoside salicin which is found in many species of the

plant genera Salix and Populus (15).

6

The scientific literature is rich in epidemiological studies that support significant

differences in the occurrence of cancers between oriental and occidental

populations (16). Generally, populations that consume a high level of natural

herbal products have a reduced incidence of cancer. An example is the low

incidence of colon cancer in Asian countries with high consumption of soybean

products (16). Soy beans are the major dietary source of saponins, which have

been suggested as possible anticancer agents (17).

There is lately a great interest in screening for plants to be used in cancer

prevention and treatment. For this reason, extracts from different plants have

been extensively studied. Table (1) summarizes some of these plants with

confirmed anticancer activity, while the following sections review some of the

studies relevant to this work.

A. Plants mentioned in Islamic literature with therapeutic activity The use of many types of medicinal plants has been practiced by Arabic and

Moslem physicians long before they were shown to possess any therapeutic

value by modern research. For example, members of the Ginger family

(Zingiberaceae) were mentioned in the Holly Quran in more than occasion and

recently shown to protect mice against experimentally-induced mutagenesis and

tumorigenesis (18). It also induces apoptosis in various immortalized or

malignant cell lines (18). Pomegranate (Punica granatum), is also among the

plants mentioned in Quran as a Paradise plant, and many studies highlighted its

chemo-preventive potential (19, 20). In addition, Nigella (Nigella sativa),

Fenugreek (Trigonella foenum graecum), and Henna (Lawsonia inermis) were

also recommended by the Profit Mohammed (P.B.U.H) as medicinal plants.

Extracts from these plants were recently shown to possess an anticarcinogenic

effect (21). Refer to Table (2) for a summary of these and other studies.

7

Table (1): Literature summary of studies that examined the therapeutic potential of plant extracts

Common name

Latin name Type of

extraction Proposed active

material Subject Effect Ref.

Neem flowers Azadirachta Indica

(Meliaceae ) Methanol Crude Rat liver Chemopreventive 22

Turmeric Curcuma longa

(Zingiberaceae) Aqueous Gingerol and paradol HL-60 Antiporliferative 23

Green tea Camellia sinesis

(Theaceae) Methanol

Epiallocatechin-3-gallat

( EGCG) T-cell leukemia ATL

Inducing apoptotic

cell death 24

Green tea Camellia sinesis

(Theaceae) Aqueous

Epiallocatechin-3-gallat

( EGCG)

Human stomach cancer

cells Antiproliferative 25

Soybean Glycine max Aqueous Crude Adenocarcinoma form

colon cancer(HT-29) Antiproliferative 26

Cranberry Vacciniumm acrocarpon Organic Polyphenole 9 cancer cell lines Colon,

oral, prostate Antiproliferative 27

Bamboo Bamboo Methanol Dihydroxypurpurin Colo320, leukemia CMK-7 Inducing apoptotic

cell death 28

Salomn's seal Polygonum tinctorium Organic Ethyl acetate -and

tryptanthrin crude F344 rats Chemopreventive 29

Radix Coptidis rhizome Aqueous Crude F344 Chemopreventive 30

Lemon grass Cymbopogon citratus stapf 80% ethanol Crude F344 rats Antimutagenic 31

8

Table (2): Literature summary of studies that examined the therapeutic potential of Islamic plant extracts

Common name

Latin name Type of

extraction Proposed active

material Subject Effect Ref.

Pomegranate Punica granatum

(Punicaceae )

- Aqueous,

and organic

pericarp

Polyphenols Human breast cancer

cell line

Inhibition of

proliferation 32


(Punicaceae ) Seed oil Polyphenols Rat skin tumor Chemopreventive 19


(Punicaceae ) Seed oil Polyphenols

Mouse mammary organ

culture Chemopreventive 20


(Punicaceae ) Seed oil Linolenic acid Rat colon cancer

Suppress

Azoxymethane

induced cancer

33


(Punicaceae )

Aqueous or

oily

compartment

Ellagi, caffeic,

luteolin and punic

acid

Prostat cancer cell line Antiporliferative 34

Garlic Allium sativum

( Liliaceae ) Oil- soluble

Organosulfur

compounds Liver cancer Chemopreventive 35

Henna Lawsonia inermis

( lythraceae) Chloroform Tocopherol Liver carcinoma cell line Antioxidant 36

9

Henna Lawsonia inermis

( lythraceae) Aqueous Crude Rat Hepatocarcinoma

Reduction of

severity 37

Grape Vitis vinifera

(Vitaceae) Aqueous Flavanol derivatives

Hepatorenal carcinoma

rat Chemopreventive 38

Nigella Nigella sativa

( Ranunculaceae ) Aqueous Crude

Natural Killer cells

activity Augmentation 21

10

B. Plants studied in this work

1. Bottle gourd (Lagenaria siceraria)

Bottle gourd (Lagenaria siceraria) is one of the cucurbitaceous crops present in

Palestine and other parts of the world. It was mentioned in the Holly Quran as

mercy and therapy for the Profit Yonis ( عليه الصالة والسالم) . It is classified under the

Lagenaria genus of the cucurbitaceae family. The medicinal benefits of

Lagenaria siceraria are rarely dealt with in the scientific debate. In one study the

Lagenin fraction which was extracted from seeds of the Bottle gourd, was

regarded as a novel ribosome–inactivating protein with a ribonucleolytic activity

(39).

The Cucurbitaceae family is composed of thirty four genus members and ninety

three taxa overall. Many members of these genera have been reported as

medicinal plants, and some of these members were considered as anticancer

plants. The Cucurbita L, Ecballium A, Lagenaria Ser, Siraitia, Luffa P, Momordica

L. and Trichosanthes L, are examples for these genera.

The Momordica genus is composed of four species: Momordica charantia,

Momordica balsamina, Momordica dioica and Momordica cochinchinesis. The

Momordica charantia species (Bitter gourd) contains an array of novel and

biologically active phytochemicals, including (triterpenes), proteins and steroids.

Triterpenes in (bitter melon) have been clinically demonstrated to possess the

ability to inhibit the guanylate cyclase enzyme (40). This enzyme is thought to be

linked to the mutagenic signaling of leukemia, and solid tumors (40). Other

phytochemicals that have been documented with cytotoxic activity are a group of

ribosome-inactivating proteins named alpha- and beta - momorcharin, momordin,

and cucurbitacin B. A chemical analog of these proteins was developed (MAP-

30) and reported to be able to inhibit prostate tumor growth (41). The

phytochemical momordin has a clinically demonstrated cytotoxic activity against

Hodgkin’s lymphoma in vivo (42). Several other in vivo studies have

demonstrated the cytotoxic and antitumor activity of the entire plant of bitter

11

melon (43). In one study, a water extract blocked the growth of rat prostate

carcinoma (44). Another study reported that a hot water extract of the entire

plant inhibited the development of mammary tumors in mice (45). Numerous in

vitro studies also demonstrated the anti-cancer activity of bitter melon against

numerous cell lines including liver cancer, human leukemia, melanoma and solid

sarcomas (46, 47).

Momordica cochinchinensis, also known as (Gac fruit) is a bright-red fruit rich in

carotene and lycopene. Similar to Momordica charantia, Momordica

cochinchinensis is postulated to contain some antioxidant components in the fruit

extract. Recently, Gac fruit was found to contain a higher lycopene concentration

than other fruits (48). According to a clinical trial study at the Medical School of

Hanoi University, Vietnam, an oil extract of Gac fruit was found to be effective in

the treatment of liver cancer (49).

In a recent study the ability of Gac fruit water extract to suppress colon cancer

cell line growth in vivo and in vitro was studied. It was found that this extract

inhibited the growth of the colon 26-20 adenocarcinoma cell line, transplanted in

Balb/c mice, reducing the tumor wet weight by 23.6% (49). Moreover, it was

found to inhibit cell proliferation in the (colon 26-20) and (hepG2) cells in a

concentration dependent manner (49).

Cucurbita L. genus is one of the most famous members in cucurbitaceae family,

it is composed of nine species of which are, Cucurbita pepo L (pumpkin),

Cucurbita moschata (crookneck squash ), Cucurbita maxima (winter squash )

and others.

Cucurbita pepo, (pumpkin) is one of the popular plants in Palestine. Its fruits

consist of up to 50% fatty acid, carotenoides, proteins, tocopherol and

phytosterol (50). Pumpkin seeds have been used for a long time in the traditional

medicine in North America and Mexico as an antihelmintic agent and as a

supportive treatment of the bladder disorders and urination difficulties (50). The

childhood enuresis nocturna and irritable bladder have also been treated

12

successfully with pumpkin seeds (50). Pumpkin has also been used to eradicate

tapeworm (50). Furthermore, Pumpkin seeds are considered an alternative

treatment for stage I and II benign prostatic hyperplasia and for irritable bladder

(51). Muscatine is a novel ribosome–inactivating protein that has been recently

purified from pumpkin seeds, and shown to have a strong inhibitory activity to

protein synthesis (52). It has been used for construction of an immunotoxin that

can efficiently and selectively kill cultured human melanoma cells (52).

A novel immunotoxin Muscatine (Ng76) was prepared successfully form pumpkin

extracts and found to efficiently inhibit the growth of targeted (M21) melanoma

cells (53). These results implied that Muscatine could be used as a new potential

anticancer agent. Moreover, the pumpkin seeds extract can modulate the

immunopathological pathways induced by interferon-γ (50). This finding suggests

an immunoregulatory potential of compounds contained in pumpkin seeds.

The anticancer and antiinflamatory activities of squash (Cucurbita maxima) are

reported by a study carried out on breast, lung, and central nervous system

cancer cell lines (54).

Siraita grosvenorii (Momordica grosvenorii) is the only species in the genus

Siraitia form cucurbitaceae family; its fruits have been used for the treatment of

pharyngitis, and antitussive medicine in Japan (55). Extracts from this fruit also

where shown to possess an anticancer effect. For example they exhibit a

significant inhibitory effect on the two stage mouse-skin carcinogenesis in vivo,

induced by 7,12-dimethyle benz[a]anthracene (DMBA) and 12-0-

tetradecanoylphorbol –13-acetat (TPA) (56, 57).

Trichosanthes Kirilowii (Maximowii) a species of the Trichosanthes genus from

the cucurbitaceae is a popular herbal medicine in China. It has been used to

induce midterm abortion and to treat ectopic pregnancies, hydatidiform and

trophobalstic moles (58). Maximowii has also been found to posses various

pharmacological properties including immunomodulatory, anti- human

immunodeficiency virus, and anti tumor activities (58).

13

Trichosantin is a ribosome inactivating protein produced from the root tubers of

Trichosanthes Kirilowii. This protein has been regarded as an effective anti-tumor

agent that is highly specific to choriocarcinoma cells from trophoplast (59, 60).

The growth inhibitory effect of Maximowii extract on the liver cancer cells (HepA-

H) and the cervical cancer (Hela) cells was investigated in vitro. The data

showed that the Maximowii extract has a strong cytotoxicity and induction of

apoptosis of Hela cells (61, 62 and 63).

Ecballium elaterium L. Rich, Squirting cucumber is one of the most distributed

cucurbitaceous herb in Palestine, locally known as" Qitha al Hamir". In an

ethnobotanical study carried out in Palestine it was reported that "Qitha al Hamir"

is used as a diuretic, and to treat urine’s retention, piles, swollen testicles, and

yellow fever (64). Cucrbitacin B a protein separated from this plant was reported

as an anti-inflammatory and antitumor agent (65).

2. Fig (Ficus carica)

Ficus carica (the common edible fig) is a member of Ficus L. genus which

belongs to the Moraceae family. Many species of this genus such as, Ficus

racemosa, Ficus bengalensis, Ficus awkeotsang Makino, Ficus microcarpa,

Ficus pumila and Ficus carica are of a medicinal value.

For example ethyl acetate extract of Ficus racemosa resulted in significant tumor

growth inhibition by 76% in in-vivo study (66). An extract from Ficus racemosa

was used to contra diet the carcinogenic effect of Ferric nitrilotriacetate a well

known renal carcinogen (67). When rats were orally treated with this extract, they

showed a significant decrease in lipid peroxidation, xanthin oxidation, H2O2

generation, blood urea nitrogen, DNA synthesis and incidence of tumors (67).

These data suggest that Ficus racemosa extract is a potent cancer preventive

agent.

An aqueous extract of Ficus pumila species was reported as an active preventive

agent against cervical cancers in vivo (68). Pectin esterase, an inhibitor of a

14

group of cationic polypeptides, was extracted from Jelly fig (Ficus awkeotsang

Makino). This polypeptide displayed a strong growth inhibition of human

leukemia cell lines via induction of apoptosis in a dose and time dependent

manner (69).

According to an ethnopharmacological study carried out in the United States of

America, Ficus bengalensis has also been reported as a medicinal plant used for

reduce tumor growth on bone (70). In a different study that aimed to demonstrate

the possible antioxidant role of Ficus bengalensis against a cholesterol rich diet,

it was found that treatment of rabbits with a water extract decreased the serum

cholesterol, and triacylglycerol levels (71). In addition, treatment with the same

extracts led to decrease in lipid peroxidation (71).

In an ethnopharmacological survey carried out in Palestine, edible fig was

reported as one of the most popular plants that are used to cure Warts, kidney

stones, respiratory system, asthma and cholesterol (7). A similar study carried

out in Pakistan indicated that edible fig fruits are useful in the case of the head

blood leprosy, nose bleeding, while the root is tonic and useful in leucoderma

and ringworm (72).

An in vitro assessment of the antihilmintic effect of Papaya, Fig and Kiwi against

gastrointestinal nematodes showed that fig and papaya caused marked damage

of the cuticle of Heligmosomoides polygyrus (73).

The antidiabetic effect of Ficus carica leave extracts has been reported. In one

study, the parameters related to oxidative stress in rats were measured and the

results confirmed that the antioxidant status is affected in diabetes, and that

Ficus carica extract tends to normalize it (74).

3. Roman nettle (Urtica pilulifera)

Urtica pilulifera, a facultative wetland plant, is common in many parts of the

world. It is a member of the urticaceae family which is considered as an invasive

plant. Members of the genus Urtica, including Urtica massaica, Urtica parviflora,

15

Urtica pilulifera, Urtica urens, and Urtica dioica are known for the stinging

sensation caused by the injection of histamine, acetylcholine, and Serotonin into

the skin by fine, needle-like projections found on the leaves and stems of the

plant (8, 75). Despite this deterrent, many of these nettle species have been

used for generations in the preparation of herbal medications (76, 77).

Many plants of the family Urticaceae are commonly used in the world for their

anti-inflammatory properties, particularly Urtica leptuphylla. However, Urtica

dioica is by far the most widely used plant of the family, both culturally and

medicinally (78, 79).

Urtica dioica is a popular anti-diabetic drug, acting against hyperglycemia (80).

To determine this role a water soluble extract of Urtica dioica was administered

to rats under oral glucose tolerance test. The results showed that rats receiving

the extract had 33% lower glucose levels than the control (80). Recently, a

number of studies have been conducted to investigate the effect of Urtica dioica

in the treatment of rheumatoid arthritis as well as the mechanisms through which

it acts (81). Urtica dioica is also believed to have an antioxidant role against

oxidative damage (81, 82). Antioxidants halt the free radical chain reaction,

thereby playing a preventive role in cancer, mutations, and accelerated aging.

Oxidative damage is usually caused due to the cellular and tissue destructive

effects of some chemicals usually formed as byproducts of normal metabolism.

These include, superoxide, hydrogen peroxide, hydroxyl radicals, or singled

oxygen all of which are common byproducts of normal metabolism, attack cells

and tissues. An imbalance between active oxygen and antioxidants may cause

stimulation of peroxisomes and polymorphonuclear leukocytes and

macrophages, which can result in diseases such as arthritis and accelerated

aging (81).

In a study on the antioxidant activity of non cultivated plants, it has been shown

that the Urtica dioica extract inhibits lipid peroxidation by more than 50% (82). In

a similar study, it was reported that an aqueous extract of Urtica dioica is the

16

most effective antioxidant among series of some other medicinal plants (83, 84

and 85). Urtica dioica also showed a potential antioxidant effect on ischemic

muscle tissue, generated by a turniquet in rats (86). In another study, antioxidant

activity of Urtica dioica extract was determined to be iron-promoted oxidation of

phospholipids, linoleic acid, and deoxyribose (87). Many of the phytochemicals

shown to exhibit antioxidant activity are polyphenols, such as caffeic malic acid,

common in Urtica extract (88).

Extracts of Urtica dioica were also shown to have an Antiproliferative effects in

vitro. Such effects were determined by testing methanolic aqueous extracts from

deride milled stinging nettles root (89). Lymph node carcinoma of the prostate

cell (Lncap), and human primary culture of the prostate stromal compartment

(hpcps) were cultivated without extract (control) or treated with various extract

concentrations at different time intervals. The cell proliferation was determined

colorimetrically, and the cytotoxicity was tested by trypan-blue dye exclusion test.

The results demonstrated that the methanolic-aqueous extract treated Lncap

cells are reduced in proliferation in comparison to the untreated controls, while

hpcps remained unchanged.

In an ethno-botanical survey carried out in the west bank, Palestine to evaluate

the most popular traditional plants, Urtica pilulifera was reported as a popular

traditional plant used as aphrodisiac, diuretic, and fresh young leaf are eaten to

treat kidney stone Rheumatism and bleeding (64).

Another survey conducted in the west bank and Negev desert, of Palestine

concluded that Urtica pilulifera is a traditional plant widely used in the treatment

of cancer (7). It was suggested that a standard decoction be prepared from 50 g

plant leaves in one liter and taken orally, 150 ml, 3-4 times/day until the condition

improves (7) .

III. Cancer cell lines A cell line refers to cells derived from a single cell type that have been adapted to

grow continuously in the laboratory and are used in research (90).

17

Prostatic cancer cell line PC-3, cervical epithelial cancer cells (Hela) and

Hepatocellular carcinoma cell line (Hep3b) were used in the study.

A. Hepatocellular carcinoma (Hep3B) cell line

The Hep3b cell line is a hepatocellular carcinoma derived from 8 years human

male juvenile (91). Analysis of the cell culture fluid form Hep3b revealed that 17

of the major human plasma proteins are synthesized and secreted by this cell

line. Also, Hep3b produces the two major polypeptides of Hepatitis B virus

surface antigen. This cell line was used to generate an animal model for

metastatic hepatocellular carcinoma by injection into athymic mouse (101).

The Hep3b cell line was used to determine the cytotoxic effects of 6-xanthone

compounds from the peel of fruits of Garcinia mangostana on hepatocellular

carcinomas using the MTT proliferation assay (102).

A Similar study examined the effect of Ginko biloba extract on cell proliferation

and cytotoxicity in human hepatocellular carcinoma cells using this cell line in

addition to HepG2 cell line (103). In this study cell proliferation and cytotoxicity

were determine by the (MTS) assay.

B. Cervical epithelial cell line (Hela) The Hela cell line was derived from a cervical epithelial cancer from a 31

years old female, and it has been reported to contain Human Papillomavirus18

(HPV-18) (91). Hela cells have been used in many studies which aimed to

investigate the anticancer role of artificial and natural products.

The antiproliferative effect of Chlorophytum comosum root extract was tested in

vitro against Hela, HL-60, and U937 cells (97). The extract was found to have

antiporliferation effect, and to induce apoptosis in these human cell lines (97).

The cytotoxic effect of the anticancer drug Rhodamine and Herbal extracts of

Glcyrhizan Radix, Rhei Rhizoma and Zingibers Rhizoma on Hela cells was also

suggested (98). Hela cells were used to indicate that the combination of

18

anticancer drugs with some herbal extracts contributes to the enhancement of

clinical outcomes in cancer thereby (98).

In a cytotoxicological study of Iranian conifers on Hela cells, a colorimetric assay

(MTT) was used, and the findings showed that Junperus Sabina extract posses

an inhibitory effect against Hela cells by reducing the cell viability to less than

50%(99). Another similar study investigated the suppressant effects of Vandellia

cordifolia on human cervical cancer Hela cell line. The result indicated that this

plant extract suppressed Hela cell line proliferation by cell cycle progression

inhibition at the G1 to S phase transition (100).

C. Prostate cell line (PC-3)

The Prostate cell line (PC-3) was initiated from a grade-IV bone metastasis

Prostate adenocarcinoma from a 62 years-old Caucasian male (91). The

establishment, characterization, and tumorigenicity of (PC-3) cell line were

reported (92). The cultured cells showed anchorage-independent growth in both

monolayers and in soft agar suspension and can produce subcutaneous tumors

in nude mice.

Electron microscopic studies revealed many features common to neoplastic cells

of epithelial origin including numerous microvilli, junctional complexes, abnormal

nuclei and nucleoli, abnormal mitochondria, annulate lamellae, and lipoidal

bodies. In general, the functional and morphological characteristics of PC-3 are

those of a poorly-differentiated adenocarcinoma (92).

The PC-3 cell line has been used in vivo and in vitro in many studies in the

screening for new anticancer agents. PC-3 cells were used in a recent study to

test the cytotoxic and antiproliferative effects of the crude extract of Thai

medicinal plants for cancer treatment (93). A similar study evaluated the effects

of plant hormones on PC-3 cell line in vitro reported that PC-3 cells exhibited

differential sensitivities to three hormones, and PC-3 proliferation was

significantly inhibited (94). The human prostate carcinoma PC-3 was also used to

determine the chemosensetivity of prostate cancer to genistein isoflavon

19

(metabolites of soy) and ß-lapachone (plant product) in vitro using trypan-blue

and MTS bioassay (95). The type of cell death, apoptosis or necrosis in response

to treatment of PC-3 cells was determined using different assays such as

annexin V-FITC and PI/TUNEL apoptosis assays (96).

20

MATERIALS AND METHODS

I. Materials

Reagents and disposables Biological Industries

Biet Haemic, Israel • DMEM powdered medium

• 0.05% trypsin-EDTA solution

• Cell Proliferation kit (XXT kit)

Sartorius.

UK • Sartolab RF/BT Vacuum Filtration Units

(0.2µm filter )

• Minisart 0.2µm syringe filter.

Greiner bio-one. Germany

• Cell culture flasks.

• PS Plate six-well.

• PS Plate-96-well.

Buffers and solutions DMEM medium • 10% fetal calf serum (inactivated at 55°C for

30 min)

• 25mM HEPES (pH 7.4),

• Penicillin (180 units/ml)

• Streptomycin (100μg/ml)

• Amphotericin-B (0.2 µg/ml)

HEPES (Buffer) • 280 mM NaCl, 1.5 mM Na2HPo4 and 25mM

HEPES

21

II. Methods A. Plant collection

Three plants were studied in this thesis: Urtica pilulifera, Lagenaria siceraria and

Ficus carica. They were collected at different times, localities and conditions.

1. Roman nettle (Urtica pilulifera)

Urtica pilulifera locally named “Qurrais” (قریص), or “Huraiq” ( ریقح ) belong to the

family Urticaceae which is a member of the Urticales order (Figure1). It is an

annual plant that grows to 1 meter high by 0.5 meter wide and lives in many

types of soils, especially light ones, with a PH ranging from acid to alkaline and a

full sun with moderate moisture. This plant withstands frost and is distributed in a

weed of cultivated lands, waste places, hedges and on damp roadsides or near

dwelling. From all of these sites in the middle districts of Gaza, nettle was

collected during March and April, 2005 (the end of winter and the beginning of

spring in Palestine). All of the plant parts including roots, leaves, and seeds were

harvested by drawing the stem from the soil. All plant parts were washed under

tap water and then derided in the shadow for three to five days. Dried leaves and

seeds were grounded by hand and stored in dry and clean bottles until the time

of experiments.

Figure1. Photograph of Urtica pilulifera in its habitat, shot by the

researcher, April 2005.

22

2. Bottle gourd (Lagenaria siceraria)

Figure 2 shows the vigorous annual herb Bottle gourd, locally named “Yaktein”

which is a member of the Cucurbitaceae family. The stems prostrate or ,(یقطین)

climb up to 5m long, and the leaves are simple, shortly and softly hairy, and non-

aromatic when crushed. Lagenaria siceraria is growing mainly on alluvial sandy

soil and red loam, on flat areas and moderate slopes, on rocky ridges, on river

banks and in dry riverbeds.

Lagenaria siceraria was collected during May and June (beginning of the

summer). Also all parts of the plant (roots, leaves, and fruits) were harvested by

drawing the plant stem. The seeds were isolated form the fruits, then seeds and

leaves were washed under tap water and dried in shadow places for seventh to

ten days. The dried plant parts were grounded by hand and stored in dry and

clean bottles until the time of experiments.

Figure 2. Photograph of Lagenaria siceraria in its habitat, shot by

the researcher, may 2005.

3. Edible fig (Ficus carica)

The edible fig belongs to the family Moraceae (Figure3), centers around the

Mediterranean region, and is commonly cultivated in mild–temperate climates.

Ficus carica is a tree of small dimensions; (3-9m) high with numerous branches

and a trunk rarely more than (17.5cm) in diameter, and contains copious milky

latex. Lateral spread of roots is extensive and, in certain soils, the roots are quit

deep. Plant leaves were harvested in summer 2005, washed under tap water and

23

shadow-derided for a week at least. Deride leaves were grounded and stored in

a dry and clean bottles until the time of experiments.

Figure 3. Photograph of Ficus carica in its habitat, shot by the

researcher, June 2005.

B. Preparation of the crude plant extracts

Twenty grams of grounded dry parts of the upper-indicated parts of each plant

were soaked in 80 ml distilled water (20% dry wt/v). The extraction was carried

out by using a reflux condenser (Figure 4a) at boiling temperature for 30 min.

The condenser returns the extract vapor to the boiling flask. The extract was

cooled at room temperature and filtered using a Buchenr funnel (Figure 4b) with

0.4µm cellulose filter paper. Finally the extract was sterilized-filtered using

vacuum filter with 0.2µm cellulose filter paper (Sartorius, UK). About 80% of the

extract volume was collected after filtration, and stored in sterilized bottles at 4˚C

(Stock extract). The different working dilutions were prepared in the cell culture

media as indicated (104, 105, 106, 107 and108).

24

a b

Figure 4. Laboratory instruments used for extract preparations.

(a) Reflux condenser and (b) Buchenr funnel.

C. Cell culture

The human cervical carcinoma cell line (Hela), hepatocellular carcinoma cell line

(Hep3b) and Prostate cancer cell line PC-3 were obtained from the Hebrew

university of Jerusalem. They were chosen based on there high proliferation

rates and availability. The cell lines were routinely maintained as a monolayer in

Dulbecco's modified Eagle's medium (DMEM) Containing 10% fetal calf serum

(inactivated at 55°C for 30 min), 25mM HEPES (pH 7.4), penicillin (180 units/ml),

streptomycin (100μg/ml) and amphotericin-B (0.2 µg/ml)(4,109). The cells were

grown to confluency in a humidified incubator with 5% CO2 in polystyrene culture

flasks. They were subcultured by removing the medium and adding 4-6 ml of

0.05% trypsin-EDTA solution. The cells were allowed to detach at (37˚C) for 5-10

min. About 1/6 of the trypsinized Hep3b or 1/4 of the other cells was passed

twice a week to new flasks containing fresh-medium.

25

D. Experimental design

- In the first (preparatory) experiments, the extracts were prepared from the

desired plant parts. The working extract concentrations were then

determined by testing an array of extract dilutions on one cell type.

- The working extract concentrations were tested for each plant against

each of the three cell lines in terms of cellular proliferation.

- The effects of the same extracts working concentration were further

analyzed by viability assay to determine the type of cellular effect.

- Observation of the morphological changes was carried out in parallel to

the viability assays.

- The data was statistically analyzed and comparison of the results from

different methods was done and reported.

E. Determination of growth characteristics Cells from Hela, Hep3b and PC-3 cell lines were seeded in 6-well plats, at a

density of 100000 cells/well, as indicated earlier. Three wells from each cell line

were trypsinized and harvested after 24, 48 and 72 hours and the medium was

changed for the cells continuing to grow at each time point. The harvested cells

were counted on hemocytometer and the average number of 3 wells was used

for the growth curve.

F. Determination of plant extract working concentrations

Plant extract-DMEM preparations were prepared by incorporation of sterile stock

extracts into the DMEM, media preparation (the plant extract volume was

included in final volume calculation). The highest extract-DMEM plant

concentration achieved this way was 16% dry wt/v (Stock extract-DMEM), and

the desired extract-DMEM concentrations were prepared by dilution with the

proper volume of complete DMEM medium.

26

To determine the concentration with a 100% cytotoxic effect, 200000 cells were

seeded onto 25cm2 flasks containing DMEM media for 24 hours. The medium

was then replaced by 8ml of 16, 8, 4 or 0.0% plant extract-DMEM. The flasks

were prepared in triplicates, and the cells were incubated in extract presence for

24 hours. The viability of cells was determined as described in the following

section.

G. Cell Proliferation Assay

A commercially purchased colorimetric kit was used to determin the proliferation

activity of cells (Biological Industries, Biet Haemic, Israel). The method is based

on the ability of metabolically active cells to reduce the tetrazolium salt XTT to

orange-colored compound of formazon. The formed dye is water soluble and the

dye optical intensity can be determined at 490 nm. The intensity of the dye is

proportional to the number of metabolically active cells. The test procedure

includes cultivation of cells in 96-well plates, addition of the XTT reagent and

incubation for 2-24 hours, during which an orange color is formed. The greater

the number of metabolically active cells in the well, the greater the activity of

mitochondrial enzymes and the higher the concentration of the dye formed,

which can then be measured and quantified (117).

The Hela and PC-3 cells were seeded in 96-well plats at a density of 7000

cells/well, whereas the Hep3b cells were seeded at density 5000 cells/well. The

cells were maintained at (37˚C) for 24 hours in the presence of 100 µl DMEM.

The medium was replaced with 8, 4, 2, 1, 0.5 or 0.25% plant extract-DMEM

concentration in triplicates. Twenty four hours later, 50 µl of the XTT reaction

solution were added to each well and the plates were incubated at 37˚C for 3

hours. The absorbance was measured with ELISA reader at a wave length of

490 nm. The reference absorbance (non specific background reading) was

measured at 630nm. Negative control cells were incubated with no extract in the

medium .

27

The cells proliferative activity was estimated by calculating the ratio of remaining

viable cells in each well in comparison to the control and expressed as (% of

control). Each assay was repeated for tow additional times and the mean and

standard error was calculated for each extract concentration (113, 114, 115 and

116).

H. Trypan-blue dye exclusion Viability assay

The trypan-blue dye exclusion assay was used to determine the plant extract-

mediated cell death (89,110, 111 and 112). 200000 cells/well were seeded in 6-

well plates and grown as previously described. After 24 hours the medium was

replaced with different plant extract-DMEM concentration (8, 4, 2, 1, 0.5 and

0.25%) in triplicates. After 48 hours incubation the medium was discarded and

the cells were harvested by trypsinization and washed twice with (PBS). A

volume of 0.4% trypan-blue stain that is equal to the residual PBS was then

added. After 5 min incubation, the cells were counted with a hemocytometer by

compound light microscope. The unstained (viable) cells and the blue-stained

(dead) cells were counted separately.

Negative control cells were incubated with DMEM media without any extract and

treated the same way. Positive control cells were incubated for 10 min with

0.5mM H2O2 before being harvested and counted as described.

The % cell viability was calculated using the following equation:

% cell viability = total viable cells (unstained) X 100 / total cells (stained + unstained)

Each experiment was carried out in triplicate and repeated for one more time and

the average of 6 wells was considered for each extract concentration.

I. Determination of morphological changes of the cells in culture

The different cell lines normally grown in 6-well plates or incubated with the

desired extract concentration for the purpose of viability testing were monitored

by an inverted microscope in 24 hours intervals. Any morphological changes in

28

the cells shape, level of adhesion and any other alterations were observed and

documented by photography before end of experiment (104, 114, 117 and 118).

J. Statistical analysis

All values expressed as mean ± standard error of the mean by Microsoft Excel.

The data were statistically analyzed by SPSS by performing the correlation,

regression and one-way ANOVA tests.

For each experiment the data obtained was blotted against extract concentration

and the obtained curve equation and R2 value were calculated by the Microsoft

Excel software. The extract concentration that gives 50% or 100% reduction in

the number of metabolically active cells or in viability of cells (IC50 and IC100

respectively), was determined by substitution in the obtained equation (89.119).

29

0

100

200

300

400

500

600

700

800

900

1000

1100

0 24 48 72 96Hours

Cel

l num

ber×

1000

Hep3bHelaPC3

RESULTS

I. Determination of the cell lines growth characteristics Figure 5 shows the growth curve of each of the hepatocellular carcinoma Hep3B,

cervical epithelial Hela and Prostate PC-3 cell lines in normal DMEM culture

media. The three cell lines maintained exponential growth characteristics until the

end of experiments (96 hours). The Hep3b cells grew faster than the other two

cell lines. However, no cell line growth curve reached a plateau at the end of the

experiments (four days). It should be emphasized that the time of all of the

following experiments did not exceeded this time.

Figure 5. Growth curves of Hep3b, Hela and PC-3 cell lines.

Hepatocellular carcinoma Hep3b, Cervical epithelial Hela and

Prostate PC-3 Cells were seeded at a density of 100000 cells/well.

The wells were prepared in triplicates for each time point and were

incubated at 37˚C in 5% CO2. Three wells from each cell line were

harvested and counted at each time point and the rest of wells

allowed continuing growing. The numbers of cells were blotted

against the growth duration.

30

II. Determination of the different plant extracts working

concentrations Plant extracts-media were prepared by introducing sterile plant extracts into the

DMEM culture media (the plant extract volume was included in final volume

calculation). The plant extract sterilization by a 0.2µl filter was difficult to perform

as the filter was blocked by the fine pieces of extract. Thus the highest plant

concentration achieved (stock concentration) was 20% (wt/v). The examined

extract concentrations were 0, 4, 8 and 16% for each plant type. The effect of

such concentrations from Urtica pilulifera, Lagenaria siceraria and Ficus carica

on the viability of Hep3b cells is illustrated in Figure 6. Urtica pilulifera

concentrations less than 4 % (wt/v) showed a profound effect on the viability of

cells, while 4% and higher concentrations gave maximal effect of 100% cell

mortality. According to these result the gap between 0% and 4% is critical and a

wider range of concentrations in this interval are necessary. Lagenaria siceraria

gave weak reduction of Hep3b cells viability and the testing concentrations did

not give more than 70% effect. Ficus carica had lower but considerable effect on

the viability of Hep3b cells, and the highest concentrations did not reach the 50%

inhibition.

From Figure 6 it’s noticed that concentrations lower than 4% are needed to be

introduced into the experiment to assess cell death before reaching maximal

values. Moreover, it seems futile to examine concentrations higher than 8% as all

of the extract effects reach plateaus at those concentrations. Therefore the

concentrations (0.25, 0.5, 1, 2, 4 and 8%) of each plant extract were used to be

examined in the rest of the study. Any effect within this range of concentrations

would be amplified theoretically if we increased the concentrations.

31

0

10

20

30

40

50

60

70

80

90

100

110

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Extracts concentrations.(dry wt / v)

Per

cent

via

bilit

yUrtica piluliferaLagenaria sicerariaFicus carica

Figure 6. A preliminary experiment of the effect of Urtica pilulifera,

Lagenaria siceraria and Ficus carica on the viability of

Hepatocellular carcinoma Hep3b cells. The percent viability of cells

was calculated as stated previously and plotted against the plants

concentrations.

32

III. Effect of plant extracts on cancer cells proliferation activity in

culture A. Hepatocellular carcinoma cell line Hep3b

The Human hepatocellular carcinoma cell line Hep3b was maintained in the

presence of increasing concentrations of each of the three plant extracts as

indicated in each experiment. The following sections describe the effect of each

plant extract on this cell line in terms of proliferation activity (Figure 7).

1. Effect of Urtica pilulifera

Extracts from the plant Urtica Pilulifera were able to inhibit the proliferation of the

Hep3b cells in a dose response manner. The proliferation activity of the cells was

found to be significantly, inversely related to increasing the extract concentration

in the medium (P =0.013, R =-0.86). This inhibitory effect was initiated at low

extract levels and reached a 50% proliferation inhibition (IC50) when the cells

were grown in media with 1.9% extract concentration (R2 =0.83). The highest

proliferation inhibition level was about 85% of the control group, however was

obtained at the maximum extract concentration tested (4%).

2. Effect of Lagenaria siceraria

A dose response effect was seen when the Hep3B cells were incubated with

increasing concentrations of the Lagenaria Siceraria extracts in the culture

medium. The degree of proliferation is also inversely related to the increase in

medium extract content. This relationship is significant with P-values of less than

or equal to 0.01and R values equal to -0.98. However, the extract inhibitory effect

was initiated at higher concentrations than in the case of Urtica Pilulifera. Higher

concentrations were thus needed to reach a 50% proliferation inhibition

(IC50=3.4%, R2= 0.97). The maximum extract concentration tested (8%) was able

to reach proliferation inhibition levels as high as in the case of Urtica pilulifera

(>85% of the control group).

33

3. Effect of Ficus carica

The results indicate that the proliferation of the Hep3B cells is inversely related to

the increased levels of Ficus carica extracts in there culture media. This behavior

is significant with P-values of ≤0.005 and R =-0.908. The extract concentrations

needed to elicit a considerable inhibitory effect were higher than the Urtica

pilulifera and Lagenaria Siceraria extracts (IC50=5.7%, R2=0.87). The maximal

inhibition observed (60% of the control group) was seen in media with 8% extract

content.

34

0102030405060708090

100110120

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5Extracts concentrations (dry wt / v )

Pro

lifer

atio

n ac

tivity

( %

of c

ontro

l )Lagenaria siceraria

Urtica pilulifera

Ficus carica

Figure 7. Reduction of proliferation activity of Hep3b cells in

response to plant extracts from Urtica pilulifera, Lagenaria siceraria

and Ficus carica.

5000 Hep3b cells/well were seeded in 96-well plates. The cells

were grown in DMEM culture media containing 8, 4, 2, 1, 0.5 or

0.25% of the indicated plant extract in triplicates. The cells’

proliferation activity was determined by the tetrazolium salt XTT

assay as described in the material and methods. Each experiment

was repeated for additional two times and the average of a total of

9 wells was calculated for each concentration. The results were

calculated as percent of the control group with no plant extract and

blotted against the respective extract concentration.

35

B. Cervical epithelial cell- line Hela

The cervical epithelial cell line Hela was grown in the presence of increasing

concentrations of plant extracts from Urtica pilulifera, Lagenaria siceraria and

Ficus carica. The following sections depict the effects of each plant extract on

Hela cells proliferation activity (Figure 8).


Urtica pilulifera extract showed a steep inhibitory effect on the proliferation of

Hela cells (figure 8). The proliferation activity of the cells was found to be inversly

related to the increase in extract concentration. A dose rsponse effect was

initiated at the lowest extract level used in the experiments (0.25%), and a 50%

inhibition of the cells proliferation activity was achevied at extract level of

(0.132%).

2. Effect of Lagenaria siceraria The proliferation activity of Hela cells was inhibited in a dose response manner

by Lagenaria siceraria. The proliferation activity of cells was found to be

significantly, inversely related to the increase in extract concentrations (P<0.01,

R=-0.938). The inhibitory effect of this extract was also started at the lowest

extract concentration used (0.25%) however higher concentrations were needed

(3.1%) to reach a 50% inhibition, than in the case of urtica pliulifera. Higher

concentration also were needed to reach the maximum levels of inhibition than in

the case of urtica pilulifera.

3. Effect of Ficus carica The results indicate that Ficus carica extract is also capable of reducing the Hela

cells proliferation in a concentration dependent manner. The proliferation activity

is also inversely related to the increase in extract concentration. This relationship

is significant with P ≤ 0.05 and R= to -0.87. Although a considerable inhibitory

effect was induced by lower concentrations, IC50 was reached at 3.1% extract

concentration. The effect of Ficus carica is slightly different from that of

Lagenaria siceraria, but with minor differences.

36

0

1020

30

40

50

6070

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Extracts concentrations ( dry wt /v)

Pro

lifer

atio

n ac

tivity

( %

of c

ontro

l )

Lagenaria siceraria

Ficus carica

Urtica piluifera

Figure 8. Reduction of proliferation activity of Hela cells in response

to plant extracts from Urtica pilulifera, Lagenaria siceraria and Ficus

carica.

7000 Hela cells/well were seeded in 96-well plates. The experiment

conditions were as in figure 7.

37

C. Prostate cell line PC-3

The prostate cells PC-3 were incubated in the presence of plant extracts from

Urtica pilulifera, Lagenaria siceraria of different concentrations. The inhibition of

proliferation activity of PC-3 is illustrated in the following sections (Figure 9).


Urtica pilulifera extract showed a strong inhibition of cell proliferation in a dose

response manner. The proliferation activity is inversely related to the increase in

extract concentration. This relationship is significant with P=0.004 and R= -0.916.

The 50% proliferation inhibition (IC50) was achieved at 2.3% extract content in the

medium. 4% extract concentration was able to reduce the proliferation activity to

reach about 10% only.


The results indicate that the proliferation activity of PC-3 cells is inversely related

to the increased levels of Lagenaria siceraria extracts concentration. Although

this proliferation inhibition was weaker than Urtica pilulifera effect, a significant

dose response manner was shown (P=0.00, R=-0.995). A relatively high extract

concentration 7.9% was necessary to reach 50% proliferation inhibition.

3. Effect of Ficus carica Ficus carica extract showed a significant inhibition of PC-3 proliferation activity

P= 0.001, R= -0.954. The lowest concentrations of this extract were able to

induce inhibitory effects more than the other extracts; despite of this noticeable

effect, IC50 was 5.5% extract concentration.

38

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Extratcs concentrations ( dry wt /v )

Pro

lifer

atio

n ac

tivity

( %

of c

ontro

l )

lagenaria siceraria Urtica pilulifera Ficus carica

Figure 9. Reduction of proliferation activity of PC-3 cells in

response to plant extracts from Urtica pilulifera, Lagenaria siceraria

and Ficus carica.

7000 PC-3 cells/well were seeded in 96-well plates. The

experiment conditions were as in figure 7.

39

IV. Effect of plant extracts on cancer cell lines viability in culture A. Hepatocellular carcinoma cell line Hep3B

Hep3b cells viability was determined in the presence of increasing concentrations

of plant extracts from Urtica pilulifera, Lagenaria siceraria and Ficus carica.

Figure 10, depicts the percent viability of Hep3b cells after incubation for 48

hours with different extracts at different concentrations. Hep3b cells which were

maintained in DMEM without any plant extract have a percent viability ranging

from 92 to 94.5% according to the three experiments.


The percent viability of Hep3b cells was found to be significantly, inversely

related to the increase in Urtica pilulifera extract concentration (P=0.03, R=-

0.787). The lowest concentration 0.25% of this extract was capable to reduce the

percent viability of the cells by slightly less than 30%. The 50% viability reduction

was obtained by 2.1% plant extract concentration. At 4% extract concentration

100% reduction in cell viability was already obtained.


The percent viability of Hep3b cells was reduced in a significant, dose response

manner due to treatment with Lagenaria siceraria (P=0.002, R=-0.937). Although

there is no noticeable inhibitory effect at the lowest concentration, the highest

concentration examined (8%) was able to elicit viability reduction by 60%. In

contrast with Urtica pilulifera behavior Lagenaria siceraria effect is weaker, and

IC50 was obtained at 7.1% extract concentration.


The results show that increasing the levels of Ficus carica in the DMEM media is

inversely related to the percent viability of Hep3b cells. This behavior is

significant with P= 0.001 and R=-0.94. Despite of that both Ficus carica and

Lagenaria siceraria behave similarly at most of the concentrations examined,

higher Ficus extract concentration (18.1%) are estimated to be needed for

40

reaching the 50% reduction in cell viability, than Lagenaria siceraria extract

(6.7%). The difference between both extracts arrows at high concentration (8%).

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5Extracts concentrations(dry wt /v)

Per

cent

via

bilit

y (%

of c

ontro

l)

Urtica pilulifera

Lagenaria siceraria

Ficus carica

Figure 10. Reduction of Hep3b cells viability in response to extracts

from Urtica pilulifera, Lagenaria siceraria and Ficus carica.

20000 Hep3b cells/well were seeded in 6-well plates. The cells

were grown in DMEM culture media containing 8, 4, 2, 1, 0.5 or

0.25% of the indicated plant extract in triplicates. The percent

viability was determined by the Trypan-blue test as described in the

materials and methods. Each experiment was repeated for one

additional time and the average ± Standard error of the mean, of a

total of 6-wells was calculated for each concentration. The results

were calculated as percent of the control group with no plant extract

and blotted against the respective extract concentration.

41

B. Cervical epithelial cells Hela

The percent viability of Hela cells was determined in the presence of increasing

concentrations of Urtica pilulifera, Lagenaria siceraria and Ficus carica plant

extracts. Figure11 shows the effect of each plant extract on this cell line in terms

of percent viability. In normal conditions without any extract treatment the percent

viability ranges from 90 to 95% according to the results of the three experiments.


Urtica pilulifera extract shows a steep and strong reduction of cell viability in a

dose response manner. This effect is significant with P=0.004, R=-0.911. This

effect was noticeable at 1% extract concentration, whereas to reach 50% viability

reduction, 2.8% extract concentration was required. A complete elimination of

viable cells was obtained at 4% extract concentration, and maintained at higher

concentrations.


The results illustrated in Figure11 indicate that Lagenaria siceraria extract causes

a significant viability reduction with P=0.00 and R=-0.982. This inhibitory effect is

weaker than the effect of Urtica pilulifera with IC50 equals to 14.1% Lagenaria

siceraria extract concentration.


Ficus carica behaved similar to Lagenaria siceraria extract in causing some

reduction in percent viability of Hela cells. A dose response effect was observed

and significant with P= 0.003, R=-0.921. The fifty percent inhibition of cell viability

was at 15.7%. No cell viability reduction was achieved as much as in the case of

Urtica pilulifera even with the concentration 8% of both Lagenaria siceraria and

Ficus carica.

42

0

10

20

30

40

50

60

70

80

90

100

110

120

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5Extarcts concentrations (dry wt /v)

Per

cent

via

bilit

y (%

of c

ontro

l)Urtica piluliferaLagenaria sicerariaFicus carica

Figure11. Reduction of Hela cells viability in response to extracts

from Urtica pilulifera, Lagenaria siceraria and Ficus carica.

The experiments’ condition as in figure 10.

43

C. Prostate cell line PC-3

Figure 12 shows the percent viability of PC-3 cells grown in the presence of

increasing concentrations of plant extracts from Urtica pilulifera, Lagenaria

siceraria and Ficus carica. The following sections describe the effect of each

plant extract on this cell line in terms of percent viability. In normal conditions

without any extract addition, PC-3 cells showed a lower percent viability than

Hep3b and Hela cells. According to the three different experiments the percent

viability of PC-3 at normal DMEM was ranging between 85.5 and 90.5%.


Percent viability of PC-3 affected by Urtica pilulifera extract was significantly

inhibited in a dose response manner (P= 0.00 and R= -0.992). A smooth and a

steep relationship was initiated at the lowest extract concentration. The 50%

viability was obtained by 3.6% extract concentration, while 8% extract

concentration resulted in 100% elimination of any viable cells.


The percent cell viability was found to be significantly, inversely related to the

increase in extract concentrations (P= 0.001, R= -0.955). Despite of that this

effect is weaker than the previous extract effect, IC50 of Lagenaria siceraria was

achieved at 9.4% extract concentration.


A dose response effect was also seen when PC-3 was grown in increasing

concentrations of Ficus carica. Percent viability was significantly, inversely

related to the increasing concentrations of this extract (P=0.00, R =-0.974).

Higher concentrations were needed to reach a 50% viability inhibition (IC50

=13.8% extract concentration).

44

0

10

20

30

40

50

60

70

80

90

100

110

120

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5Extracts concentrations (dry wt /v)

Perc

ent v

iabi

lity

(% o

f con

trol)

Urtica piluliferaLagenaria sicerariaFicus carica

Figure 12. Reduction of PC-3 cells viability in response to extracts

from Urtica pilulifera, Lagenaria siceraria and Ficus carica

The experiment conditions where as in figure 10.

45

V. Determination of morphological changes of cell lines in

response to treatment with different plant extracts The morphological characteristics of the Hep3b, Hela and PC-3 cell lines were

evaluated in parallel to each time performing viability testing assay. In each

viability testing experiment the 6-well plates were examined for any

morphological changes in response to treatment with the three plant extracts

(Urtica pilulifera, Lagenaria siceraria and Ficus carica) compared to the normally

maintained cells (no treatment). Representative wells were pictured and shown in

this study (Figures 13, 14 and 15). A few cell culture flasks were frequently

monitored at short time intervals.

A. Hepatocellular carcinoma cell line Hep3B The normally maintained Hep3b cells were proliferating with high rate, and

formed a monolayer growth with no less than 90% confluency within 48 hours

(Figure 1a).


Urtica pilulifera showed noticeable morphological alterations with weaker

adhesion level at different concentrations as evident by the cells floating. After

48 hours, the 2% extract concentration showed a potent activity. It induced

Hep3b cells to round and form a grape-shaped cluster. At 24 hours after

treatment of hep3b with 4 and 8% extract most of cells were detached and

aggregated in clusters (Figure 16b)

2. Effect of Lagenaria siceraria Hep3b cells treated with Lagenaria siceraria 4 and 8% concentrations for 24

hours suffered from noticeable alterations such as shrinkage, sparse, and spindle

shape formation (Figure16c). At the highest concentration (8%) the level of

adhesion was reduced and many cells were detached.

46


Ficus carica treated cells showed a high proliferation rat at low extract

concentrations (0.25 and 0.5%). While at 4% extract concentration their growth

was inhibited and slight cell shrinkage was observed (figure 16d). On the other

hand 8% Ficus carica extract concentration induced obvious alterations to the

cells morphology such as, the rounding up and detachment of many cells.

a b

c d

Figure 13. Morphology of Hep3b cells in culture with or without

different plant extracts.

Hep3b cells were grown in DMEM culture medium without any

extract (a) or with 4% Urtica pilulifera extract for 24 hours (b), 8%

Lagenaria siceraria extract for 48 hours (c) and 4% Ficus carica for

48 hours (d). Cells in a, c and d were visualized by inverted

microscope at 10X and in b at 20X.

47

B. Cervical epithelial cells Hela

The proliferation rate of Hela cells maintained in normal media was lower than

Hep3b, as about 80% confluency was obtained after 48 hours of growth (Figure

17a). Apparent alterations in cell morphology and detachment of cells from the

culture surface were observed after 24 and 48 hour of treatment with different

extract concentrations.


Five to ten hours after treatment of Hela cells with Urtica pilulifera (2, 4 and 8%

concentrations) the cells began to round up and the level of adhesion was

influenced. Many cells detached easily from the surface of the plastic flasks and

moved into medium. After 24 hours of treatment with 2% Urtica pilulifera, most of

cells were rounded and formed sparse clusters (Figure 17b). Higher

concentrations (4 and 8%) had more pronounced effects and nearly all cells were

rounded up and detached.


When Hela cells were treated with 2% Lagenaria siceraria extract for 24 hours a

number of cells were dead as evident by cellular rounding up, floating and

fragmentations. The time and concentration dependent extract effect was

observed in the degree of confluency. Figure 7c illustrates the effect of 8%

extract concentration after 48 hours of incubation.


Treatment of Hela cells with Ficus carica 4 and 8% for 48 hours resulted in

reduced density of the monolayer, and some of the cells were round and

detached (Figure 17d).

48

a

dc

b

Figure 14. Morphology of Hela cells in culture with or without


Hela cells were grown in DMEM culture medium without any extract

(a) or with 2% Urtica pilulifera extract for 24 hours (b), 8%


48 hours (d). Cells in all were visualized by inverted microscope at

20X.

49

C. Prostate cell line PC-3:

Figure 18a shows PC-3 cells which are characterized by a considerably lower

proliferation rate than both Hela and Hep3b cell lines in normal conditions.

1. Effect of Urtica pilulifera After 24 hours of PC-3 treatment with 4% Urtica pilulifera the cells were rounded

up, and detached from the monolayer (Figure 8b). There were relatively large

amounts of cellular fragments and cytoplasm condensation that appeared at 4%

Urtica pilulifera concentration.

2. Effect of Lagenaria siceraria PC-3 cells treated with 4 and 8% Lagenaria siceraria showed a noticeable

decrease in confluency after 48 hours of treatment, but no evident morphological

changes (Figure 18c).


The reduction of confluency degree was visible in PC-3 cells when incubated for

48 hours in the highest extract concentration used, but also with no noticeable

morphological changes (Figure 18d).

50

a b

dc

Figure 15. Morphology of PC-3 cells in culture with or without


PC-3 cells were grown in DMEM culture medium without any

extract (a) or with 4% Urtica pilulifera extract for 24 hours (b), 8%


48 hours (d). Cells in all were visualized by inverted microscope at

20X.

51

DISCUSSION

Cancer is a term describing conditions characterized by unscheduled and

uncontrolled cellular proliferation (120). It is a very common disease, and its

incidence is increasing at an average annual rate of 1.2% (120). Lately, there has

been improvement in the treatment strategies of cancer, which has resulted in

prolonged survival of patients with chronic cancer disease. However there is a

growing need for additional means of cancer therapy, in the form of both

palliative and curative treatments. The strategies available today, are

sophisticated, and are only able to affect 50 to 60% of cancer patients, while the

others will eventually die from the disease (121, 122 and 123).

Chemotherapy has been used for cancer treatment for more than 50 years;

sometimes in combination with or parallel to surgery and radiotherapy. After

surgical ablation of progressive cancer, metastasized tumor cells continue to

progress and this is one of the faultiest associated with surgery. One the other

hand, radioactive rays and most anticancer chemotherapeutic agents damage

DNA or suppress DNA duplication to kill the rapidly growing tumor cells. At the

same time, they also affect normal cells causing serious adverse effects, such as

bone marrow function inhibition, bone necrosis, lung fibrosis, skin

devascularization, ulceration, nausea, vomiting, renal damage and alopecia

(124). Thus it is evident that a wide array of selective and potent components is

needed to match the growing problems associated with cancer.

Plants and natural products play an important role in medicine and provide

important prototypes for the development of novel drugs (125). They offer a

valuable source of compounds with a wide variety of biological activities and

chemical structures. Many anti cancer agents have been derived from natural

sources; directly as pure native compounds, or as semi-synthetic analogues

(126, 127 and 128).

52

Arabic and Islamic tradition is particularly rich in medical plants that have been

used by pioneer Arabic physicians to establish the basis for modern therapies.

But a few of these plants have been examined scientifically. In this extent we

studied the potential effect of three Arabic and Islamic traditional plants as

anticancer agents. These were Fig (Ficus carica) and Bottle gourd (Lagenaria

siceraria) that were mentioned in the holy Quran in more than one occasion and

Roman nettle (Urtica pilulifera) which is traditionally used as a popular

medication (8). To fulfill this aim, the proliferation, viability and morphological

characteristics of three cell lines (Hep3b, Hela and PC-3) were studied in

response to treatment with extracts from the aforementioned plants.

The cellular proliferation activity was tested by a colorimetric method which is

based on the ability of metabolically active cells to reduce the tetrazolium salt

XTT to orange-colored compounds of formazon. The intensity of the formed

water soluble dye is proportional to the number of metabolically active cells. The

proliferation activity of the treated cells was normalized to that of normally

growing cells from the same type with no treatment. This normalized value was

expressed as percent of the control group. Theoretically, any reduction in the

number of metabolically active proliferating cells might mean that the proliferation

pathway itself was halted (cell cycle arrest), or that a fraction of the cells went

through a death pathway. Therefore, a viability test was necessary to determine

which of these two options may play a role in this study. Therefore, the trypan-

blue dye exclusion test was used to determine the plant extract-mediated cell

death. The unstained cells (viable) and the blue stained (dead) were counted

separately, and the percent cell viability was calculated as previously described.

The viability assay was performed with the same cell-extract combinations as in

the proliferation assay. The percent viability of normally grown cells from the

same types was determined for comparison with extract-treated cells. If we

consider the results of the proliferation and viability assays in parallel, then we

would be able to say whether reduction of the number of the metabolically active

cell has resulted from death of part of them or due to any other reason. Any

conclusion drawn from such comparison may be confirmed morphologically

53

following up the treated cells in comparison with normally maintained control

cells. In this study a combination of the three different assays was performed in

parallel for each plant extract and each cell line.

In order to assess the degree of proliferation inhibition and viability reduction

obtained by each extract in comparison to the others, the IC50 values were

summarized in tables 3 and 4. The findings of this study indicated that the

proliferation activity of Hep3b cells was inhibited by all of the three plant extracts

(Urtica pilulifera, Lagenaria siceraria and Ficus carica). A dose response effect

was obtained with all of these extracts, but the degree of this effect varied from

one extract to the other. The results indicated that Urtica pilulifera is the most

potent extract, even at low concentrations with IC50 of (1.9%) followed by

Lagenaria siceraria with IC50 of (3.4%) and Ficus carica with of IC50 of (5.7%). At

the highest extract concentration used in the study (8%) both Urtica pilulifera and

Lagenaria siceraria reached a similar proliferation inhibition level of about (90%).

However this degree of inhibition was reached at lower concentration of Urtica

pilulifera extract than of Lagenaria siceraria extract (Figure7). Ficus carica on the

other hand did not reach the same degrees of proliferation inhibition obtained by

both Urtica pilulifera and Lagenaria siceraria even at the highest concentration.

When considering the results of Heb3b cells viability assay, the three plant

extracts showed various degrees of Heb3b cells viability reduction. Urtica

pilulifera showed a steep reduction of the cells viability with IC50 of 2.1% extract

concentration, which equals the IC50 of the same extract in cell proliferation

assay. These compatible results (Figure 16) suggest that reduction in the

metabolically active Hep3b cells in consequence to Urtica pilulifera extract

treatment is due to cell death. However such compatibility was not evident in the

case of Lagenaria siceraria (Figure 17) and Ficus carica (Figure 18). Lagenaria

siceraria has a less potent cytotoxic effect against Hep3b cells (IC50 = 7.1%).

This might indicate that this extract counteracts the proliferation activity of Hep3b

cells mostly by mechanisms other than induction of their death. This deviation

between the cytotoxic effect and proliferation effect was more profound in the

54

case of Ficus carica with IC50 of 18.1% in case of cytotoxicity compared to 5.7%

in the case of proliferation.

Table 3. Summary of calculated concentrations of the three plant

extracts Urtica pilulifera, Lagenaria siceraria and Ficus carica that give

50% (IC50) reduction in proliferation activity of Hep3b, Hela and PC-3

cancer cell lines. The R2 value for each curve equation is illustrated.

Plant Hep3b cells Hela cells PC-3 cells

IC50 1.98 0.132 2.31 Urtica pilulifera

R2 0.837 0.6022 0.8563

IC50 3.4 3.1 7.9 Lagenaria siceraria

R2 0.97 0.9761 99.82

IC50 5.7 3.1 5.5 Ficus carica

R2 0.8675 0.9059 0.9583

Table 4. Summary of calculated concentrations of the three plant

extracts Urtica pilulifera, Lagenaria siceraria and Ficus carica that give

50% (IC50) reduction in the % viability of Hep3b, Hela and PC-3 cancer

cell lines. The R2 value for each curve equation is illustrated.

Plant Hep3b cells Hela cells PC-3 cells

IC50 2.1 2.8 3.6 Urtica pilulifera

R2 0.7056 0.8305 0.9843

IC50 7.16 14.18 9.4 Lagenaria siceraria

R2 0.8496 0.9585 0.9367

IC50 18.12 15.7 13.8 Ficus carica

R2 0.8886 0.852 0.9442

55

R 2 = 0 .837

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

E xtrac t concentrations (dry wt /v)

Per

cent

of

effe

ct

p ro liferationof viab ili ty

R2 = 0.6022

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Extract concentrations (dry wt /v)

Perc

ent o

f effe

ct

proliferationviability

R 2 = 0.85 63

-10

0

10

20

30

40

50

60

70

80

90

100

110

0 0 .5 1 1.5 2 2.5 3 3 .5 4 4.5 5 5.5 6 6 .5 7 7.5 8 8 .5

Extrac t c onc en trations(dry wt /v)

Per

cent

of e

ffect

P roli fe ra tionViabili ty

Figure16 : Reduction of viability and proliferation

activity of Hep3b (a), Hela (b) and PC3 (c) cells in

response to Treatment with Urtica pilulifera extract.

The best-fit trend line are shown. The equation for each

trend line was used to estim ate the IC50, IC100 and R2

values by substitution. R2 values Are indic ated next to

the respective lines.

a b

c

56

R 2 = 0.8496

R 2 = 0.97

0

10

20

30

40

50

60

70

80

90

100

110

120

0 0.5 1 1.5 2 2 .5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8 .5

Extrac t conc entrations (dry wt /v)

Per

cent

of e

ffect

viability pro life ration

R 2 = 0 .9585

R 2 = 0.9761

0

10

20

30

40

50

60

70

80

90

100

110

0 0 .5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Extrac t c oncentrations(dry wt /v)

Per

cent

of e

ffect

P roli feration viabili ty

R2 = 0.9367

R2 = 0.9982

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5Extract concentrations (dry wt /v)

Perc

ent o

f effe

ct

proliferationViability



response to Treatment with Lagenaria siceraria

extract.


trend line was used to estimate the IC50, IC 100 and R2

values by substitution. R 2 values Are indicated next to

the respective lines.

a b

c

57

R2 = 0.9442

R2 = 0.9583

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5Extract concentrations (dry wt / v )

Perc

ent o

f effe

ct

ProliferationViability

R2 = 0.852

R2 = 0.9059

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5

Extract concentrations (dry wt /v)

perc

ent o

f effe

ct

Proliferationviabiltity

R2 = 0.8886

R2 = 0.8675

0

10

20

30

40

50

60

70

80

90

100

110

0 0.5

1 1.5

2 2.5

3 3.5

4 4.5

5 5.5

6 6.5

7 7.5

8 8.5

Extract concentrations(dry wt / v)

pece

nt o

f effe

ct

viability proliferation



response to Treatment with Ficus carica extract.


trend line was used to estimate the IC50, IC100 and R2

values by substitution. R2 values Are indicated next to the

respective lines.

a b

c

58

These data were confirmed by the inspection of morphological alterations of

Hep3b cells due to treatment with the three plant extracts (Figure13). As shown

in figure 13b Urtica pilulifera had the most potent effect compared to the control

(Figure 13a). Most of cells were rounded up and formed a grape shaped cluster,

which is one of the prominent morphological features of cellular death in culture.

Such features were also noticeable to lesser extent when the cells were

incubated with Lagenaria siceraria, and could hardly be detected in the case of

Ficus carica. Moreover, the confluency of Heb3b cells was reduced in response

to treatment with the three plant extracts and in accordance to the previously

discussed results of proliferation inhibition ( 104,114, 118).

Similarly Hela cells proliferative activity was inhibited with various levels due to

their treatment with the three plant extracts. As shown in Figure 8, Urtica

pilulifera has a strong inhibitory effect on the proliferation activity in extract

concentration dependent manner with IC50 of 0.13% extract concentration. Both

Ficus carica and Lagenaria siceraria had a similar but weaker effect on

proliferation of Hela cells, and the IC50 for both extracts was 3.1%. The percent

viability of Hela cells was also reduced in a dose response manner by all of these

plant extracts. Urtica pilulifera extract was the most effective with IC50 of 2.8%,

while the Lagenaria siceraria and Ficus carica effect on cells viability were

similarly less potent with IC50 of 15.7% for Ficus carica and 14.1% for Lagenaria

siceraria. Being the most potent plant, Urtica pilulifera mostly exerts its effects by

inducing cell death although other effect can be present (IC50 for

proliferation=0.13% and for cytotoxicity =2.8%). The difference between the IC50

of both Ficus carica and Lagenaria siceraria in proliferation inhibition and the IC50

of cell viability reduction indicates that these extracts exert their effect by cell

cycle arrest with low cytotoxicity. The morphological observations of Hela cells

maintained in increasing concentrations of the three plant extracts show that

Urtica pilulifera had the most rapid and potent effect of these extracts. Figure 14b

shows that most of the cells were rounded up and formed sparse clusters due to

treatment with 2% Urtica pilulifera. These results suggest that most of Hela cells

were dead in the case of Urtica pilulifera. A reduction of Hela cells confluency

59

was the most prominent observation when these cells were incubated with Ficus

carica or Lagenaria siceraria, but cell death indications were almost not seen.

These observations indicate an antiproliferative effect of both Ficus carica and

Lagenaria siceraria but less likely cytotoxicity.

The proliferation activity of the prostate cell line PC-3 was inversely related to

increasing the levels of the three plant extracts. The degree of proliferation

reduction varied from one plant extract to the other. As shown in figure 9, Urtica

pilulifera was again the most potent extract with IC50 of 2.3%, whereas Ficus

carica comes second with IC50 of 5.5%, and Lagenaria siceraria comes last with

IC50 of 7.9%. When comparing these results with results from PC-3 cells viability

assay, we find that Urtica pilulifera also has the strongest effect with IC50 of 3.6%.

These results are supported by the morphological changes shown in figure15b

including cells rounding up and detachment from the monolayer indicating that

cell death might has occurred.

Lagenaria siceraria induced a reduction of PC-3 cell viability with IC50 of 9.4%

extract concentration similar to IC50 from the same extract in the proliferation

assay. This fact suggests that this extract also caused PC-3 cell death. The

reduction of the cells confluency due to Lagenaria siceraria treatment observed

in figure 14c gives evidence of the effect of this extract. A Higher concentration of

Ficus carica was needed to reach a 50% viability inhibition (IC50 =13.8%) than

that needed to reach 50% inhibition in cell proliferation (IC50=5.5%). This again

suggests that the effect of this extract did not occur only through cell death.

Based on the previous results, Urtica pilulifera have a potent antiproliferative

effect on the three tested cancer cell lines. While there is no previous studies

about the role of Urtica pilulifera extract as anticancer agent, many studies

investigated the role of its genus member Urtica dioica. Urtica pilulifera and

Urtica dioica are similar in many chemical and morphological aspects (75, 8). Our

results on Urtica pilulifera are in agreement with previous studies on Urtica

dioica. For example an aqueous extract of Urtica dioica roots was shown to

60

directly inhibit the proliferation of Hela cells and block its binding by epidermal

growth factor (EGF) (129). In the same study a polysaccharide mixture from an

aqueous root extract was shown to exert an anti-inflammatory activity in a rat

Paw oedema test. Investigation of the effect of a 20% methanol extract of Urtica

dioica roots on prostate cell line (LNCap) resulted in a selective and significant

concentration-dependent proliferation reducing effect on prostate cells (89). In

the same study the cell proliferation activity was determined by a colorimetric

assay and the cytotoxicity was examined by Trypan-blue test. Our results are

also comparable with data obtained from an in vitro study that aimed to

investigate the effect of Urtica dioica leaves aqueous extract on the enzyme

activity of prostate cancer tissue (130). The results of the study indicated that the

extract caused a significant inhibition of adenosine deaminase activity (ADA) of

these tissues in a dose dependent manner. These data might be of importance

because ADA is a key enzyme in the nucleotide metabolism and DNA turn over.

Extracts of Urtica dioica were also used in the treatment of adult mouse with

bingeing prostate hyperplasia (131). Five differently prepared root extracts were

tested on these rats. The 20% methanol extract was the most effective with

51.4% inhibition of induced growth.

Despite all of these data the mechanism(s) of action of Urtica dioica extract as an

antiproliferative agent has yet to be established. Different modes of actions are

proposed in this regard. For example it has been observed that some sterols and

hydroxyl-fatty acids, given their low concentrations in Urtica dioica, can inhibit

aromatase, which is a key enzyme in steroid hormone metabolism mediating the

conversion of androgens into estrogens (132). Another mechanism involves a

dose dependent inhibition of the binding of sex hormone binding gluobin (SHBG)

to its receptor in response to Urtica dioica extract (133). Some lignans present in

Urtica dioica were shown to interfere with the binding of androgens to SHBG,

thereby reducing the transport capacity of androgens (132). Based on the

similarity between Urtica pilulifera and Urtica dioica we would suggest that Urtica

pilulifera might exerts its antiproliferative effect via a similar fashion.

61

The results of this study strongly indicate a possible cytotoxic effect of Urtica

pilulifera against cancer cell lines. This was evident from the results of Trypan-

blue viability assay as well as from the inspection of cells morphology in culture.

The cytotoxicity of fixed and volatile oils extracted from Urtica pilulifera leaves

and seeds were tested on Swiss albino mice (134). The results indicated that

both oils of Urtica pilulifera are completely nontoxic even at doses reaching 12.8

ml/kg. However this study involved only the oil fractions of Urtica pilulifera

extract. Other components of the extract may play a role in the observed toxicity

in our study. Moreover, the aforementioned study was performed in vivo on mice

and the only cytotoxicity parameter considered was the mice death. No other

mice toxicity indicators were analyzed such as, histopathological alterations of

the different mice tissues and organs.

According to results discussed earlier the inhibitory role of Ficus carica on the

proliferation of the three cell lines was more profound than its cytotoxic effect on

the same cell lines. This conclusion is in accordance with the results of previous

studies. For instance, the proliferation of different cell lines was inhibited by

components of Ficus carica (135). Such proliferation inhibition was also obtained

when cow teat papillomatosis skin surface benign tumors, were treated by Ficus

carica latex (136). Furthermore, Ficus carica was found to have an in vivo

antioxidant effect after being consumed by human (137). Accordingly, the dried

fruits of this plant should be eaten more frequently as they are rich in phenol

antioxidants and fibers. Compared with vitamins C and E, the well known

antioxidants, the dried fruits of Ficus carica were found to be a superior one.

Lagenaria siceraria was found to posses both antiproliferative effects as well as a

cytotoxic effect to a considerable degree. Unfortunately no literature was found

regarding the anticancer activities of this plant. However, members of its family

(Cucurbitaceae), were shown to have a class of biologically active compounds

(Cucurbitacins) with well established anticancer cytotoxic activity (138,139,140).

For example, Cucurbitacins were shown to have strong cytotoxic effect on KB

cell line, derived from human nasopharyngeal carcinoma and on Hela cells by

62

different proposed mechanisms (140, 141). Moreover, (Cucurbitacins) were

shown to have a proliferative inhibitory activity in vivo against several carcinoma,

sarcoma, and leukemia models (142, 143 and 144).

The level of these compounds and/or any of their derivatives in Lagenaria

siceraria is unknown. However the results of this thesis indicate both

antiproliferative and cytotoxic activity of Lagenaria siceraria. Therefore we expect

Lagenaria siceraria extracts to behave in a similar manner like other members of

its family via (Cucurbitacins) involvement.

63

CONCLUSION

In conclusion, all of the three plants examined in this study possess varying

levels of anticancer activity in vitro. This is evident by the concentration

dependent manner reduction in the final number of cancer cells as a

consequence to treatment. Two kinds of anticancer effects were examined and

found to take part in this study. The first is anti-cell proliferation effect (decreased

number of metabolically active cells) and the second is cytotoxicity (decreased

number of live cells). The three plants examined possess both of the effects with

various degrees. Urtica pilulifera possess the strongest and most profound

effects on the three cell lines, probably by cytotoxicity mainly. On the other hand

Lagenaria siceraria probably affects the three cell lines by combination of

cytotoxicity and antiproliferation almost to a similar degree. Ficus carica most

probably reduces the final number of metabolically active cells mainly by its

antiproliferative effect, although cytotoxicity likely contributes to viability reduction

at high concentrations.

Both Ficus carica and Lagenaria siceraria are edible plants that were chosen on

the bases of being mentioned in the holy Quran. Therefore, although their effect

is lower than that of Urtica pilulifera their amount in the diet or as a treatment can

be safely scaled up when ingested in their native form. On the other hand,

despite its possible toxicity Urtica pilulifera is frequently orally used as a

medication in many conditions by traditional medicine.

64

RECOMMENDATIONS

1. Further studies are needed to assess the active ingredients of Ficus

carica, Lagenaria siceraria and Urtica pilulifera, involved in the

antiproliferative or cytotoxic effects of these plants. These studies must

involve the establishment of in vivo cancer models and their treatment by

these plants in their native crude extracts or by their purified active

components. More efficient extraction techniques and instruments have to

be used in order to obtain the crude extracts or their derivatives in

convenient quantities and concentrations.

2. The type(s) of effects exerted by the three plants component(s) involved in

their activity have to be further studied by molecular or other relevant

techniques.

3. Both Ficus carica and Lagenaria siceraria may be ingested more

frequently in different forms, if not to treat cancers, then as a prophylactic

against them. Urtica pilulifera can be administered for treating relevant

conditions including cancer with reasonable pre-studied amounts.

4. Many other plants are waiting to be examined for any possible therapeutic

effects and if present they may represent a convenient and cheap means

to cope with many types of cancer.

65

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