Anti-cholangiocarcinoma and toxicity evaluation of ...ethesisarchive.library.tu.ac.th/thesis/2016/TU_2016...6.1 Preparation and qual ity control of the plant extract and CMC oral pharmaceutical

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ANTI-CHOLANGIOCARCINOMA AND TOXICITY

EVALUATION OF Atractylodes lancea (Thunb.) DC. AND

Kaempferia galanga Linn. HERBAL EXTRACT

PREPARATIONS FOR DRUG DEVELOPMENT

BY

MR. ASMARE AMUAMUTA LIMAENEH

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR

PHILOSOPHY (BIOCLINICAL SCIENCES)

CHULABHORN INTERNATIONAL COLLEGE OF MEDICINE

THAMMASAT UNIVERSITY

ACADEMIC YEAR 2016

COPYRIGHT OF THAMMASAT UNIVERSITY

Ref. code: 25595729320217IWP

ANTI-CHOLANGIOCARCINOMA AND TOXICITY

EVALUATION OF Atractylodes lancea (Thunb.) DC. AND

Kaempferia galanga Linn. HERBAL EXTRACT

PREPARATIONS FOR DRUG DEVELOPMENT

BY

MR. ASMARE AMUAMUTA LIMAENEH

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR

OF PHILOSOPHY (BIOCLINICAL SCIENCES)

CHULABHORN INTERNATIONAL COLLEGE OF MEDICINE

THAMMASAT UNIVERSITY

ACADEMIC YEAR 2016

COPYRIGHT OF THAMMASAT UNIVERSITY

Ref. code: 25595729320217IWP

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Dissertation Title

ANTI-CHOLANGIOCARCINOMA AND

TOXICITY EVALUATION OF Atractylodes

lancea ( Thunb. ) DC. AND Kaempferia galanga

Linn. HERBAL EXTRACT PREPARATIONS

FOR DRUG DEVELOPMENT

Author

Mr.Asmare Amuamuta Limaeneh

Degree

Doctor of Philosophy (Bioclinical Sciences)

Major Field/Faculty/University

Chulabhorn International College of Medicine

Thammasat University

Dissertation Advisor

Professor Kesara Na-Bangchang, PhD

Academic Year

2016

ASTRACT

Cholangiocarcinoma (CCA) is an important public health problem in

many tropical and subtropical parts of the world particularly Southeast Asian

countries. Chemotherapy against CCA is largely ineffective and discovery and

development of effective chemotherapeutics is urgently needed. The aim of the

research dissertation was to evaluate the toxicity and anti-CCA activity of the prepared

CMC (chemistry manufacturing and control) oral formulation of the crude ethanolic

extract of Atractylodes lancea (Thunb.) DC. rhizomes (AL) for further first-in-human

clinical evaluation. In addition, the toxicity and anti-CCA activity of the crude

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ethanolic extract of Kaempferia galanga Linn. rhizomes (KG) was also investigated.

Quality control of the CMC oral pharmaceutical formulation of AL rhizome extract

was performed using HPLC based on the identification of the marker compound

atracylodin (with content of 1.14 + 0.09 mg/g or 0.114% w/w). Quality control of the

crude ethanolic extract of KG rhizome was performed using HPLC based on the

identification of the marker compound ethyl-p- methoxycinnamate (with peak area of

94.09% of total content of the extract preparation).

Based on the results of acute and subchronic (90 days repeated oral

dosing) toxicity evaluation in Wistar rats, the maximum tolerated dose (MTD) of the

CMC oral pharmaceutical formulation of the AL crude ethanolic extract was at least

5,000 mg/kg body weight. This formulation can be used in the next step of product

development for further first-in-human study. Nevertheless, further studies including

chronic toxicity and genotoxicity of the extract formulation are necessary to ensure

tolerability of the product in human uses for treatment of CCA. The KG rhizome

extract exhibited moderate to high cytotoxic activity against human CCA CL-6 cell

line. Results of the toxicity study in mice (acute and subacute toxicity testing)

suggested the MTD of the KG extract of 1,000 mg/kg body weight. In CCA (CL-6)-

xenografted nude mouse model, the KG rhizome extract exhibited significant anti-

CCA activity at the maximum dose level of 1,000 mg/kg body weight as shown by

TGI of 58.41%, prolongation of survival time until 62 (53.2-71.8) days, and inhibition

of lung metastasis by 33% compared with the untreated control mice. Preparation of

the CMC oral pharmaceutical formulation of the KG rhizome extract is needed before

further studies in humans. Further studies including subchronic toxicity and other

pharmacological activities with the extract should be further investigated for

determining the oral safety dose in an effort for developing an effective drug option

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against CCA from this potential herbal agent.

Keywords: Cholangiocarcinoma, Atractylodes lancea (Thunb.) DC., Kaempferia

galanga Linn., cytotoxicity, CL-6, toxicity.

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ACKNOWLEDGEMENTS

First of all, I would like to express my special thanks and appreciation to

my advisor, Prof. Dr. Kesara Na-Bangchang for her generosity, valuable guidance and

encouragement since the beginning to the end of my study and ultimately made what I

thought impossible to be possible.

I also want to express my sincere gratitude for my co-advisor Asst. Prof.

Dr. Tullayakorn Plengsuriyakarn for his enormous guidance, understanding and

encouragement throughout my study not only as a mentor but also as a friend.

I would also like to thank my advisor committee members, Prof. Dr. Juntra

Karbwang, Asst. Prof. Dr. Wanna Chaijaroenkul, and Dr. Mayuri Tarasuk for their

valuable guidances.

My deep appreciation also goes to Dr. Thunyatorn Yimsoo, Dr. Werayut

Yingmema, Mr. Noppadon Suttirak, and Mr. Noppanan Kotsaouppara and all other

staff members there for their professional guidance and compassionate help during my

long time work and study at the Laboratory Animal Center, Thammasat University.

I wish to express my sincere gratitude also to Thammasat University (TU)

particularly Chulabhorn International College of Medicine (CICM) and Center of

Excellence in Pharmacology and Molecular Biology of Malaria and

Cholangiocarcinoma, National Research University Project of Thailand (NRU) and

Office of Higher Education Commission of Thailand for funding me the scholarship

grants and support for the completion of this study.

I would also like to thank my home University (Bahir Dar University,

College of Medicine and Heath Sciences) for allowing me to pursue my Ph.D. study

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leave and all the favorable supports and encouragement I received from it throughout.

CICM staff members, my classmates, TU community and all Thai

individuals I met also deserve my appreciation and heartfelt thanks for every kind of

support and lessons I gained from them during my study and stay in Thailand.

I would like to owe a special thanks to my dear father Mr. Amuamuta

Limeneh and my dear mother Mrs. Tangut Birhanae and also my gratitude to my

entire family (brothers, sisters and relatives), who have made valuable support, love,

and prayers throughout my entire life that makes me constantly feel alive. Helen

Endale, my love partner, also deserve my special thanks for her best wishes,

encouragement and trust on me.

Finally, it is my wish to forward my appreciation and gratitude to all of

my friends and anyone involved and contributed for the success of this work and to

my life.

Mr. Asmare Amuamuta Limeneh

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TABLE OF CONTENTS

Page

ABSTRACT (1)

ACKNOWLEDGEMENTS (4)

TABLE OF CONTENTS (6)

LIST OF TABLES (11)

LIST OF FIGURES (14)

LIST OF ABBREVIATIONS (18)

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 REVIEW OF LITERATURE 5

2.1 Cholangiocarcinoma (CCA) and treatment approaches 5

2.2 Screening methods for development of anticancer and anti-CCA

drugs and/or phytochemicals

11

2.2.1 In vitro methods for screening anticancer compounds 11

2.2.2 In situ models 12

2.2.3 In vivo methods for screening anticancer compounds (screening

using animal models)

12

2.3 Medicinal plants with anti-CCA activities 19

2.4 Description of the study plants 20

2.4.1 Atractylodes lancea (Thunb.) DC. 20

2.4.1.1 Plant description and medicinal properties 20

2.4.1.2 Phytochemistry 23

2.4.1.3 Safety and toxicity 23

2.4.2 Kaempferia galanga Linn. 25

2.4.2.1 Plant description and medicinal properties 25

2.4.2.2 Phytochemistry 29

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2.4.2.3 Safety and toxicity 30

CHAPTER 3 OBJECTIVES 35

3.1 General objective 35

3.2 Specific objectives 35

CHAPTER 4 RESEARCH METHODOLOGY 37

4.1. Reagents and cell lines 37

4.2 Preparation of test materials 37

4.2.1 Chemistry, manufacturing and control (CMC) oral

pharmaceutical formulation of Atractylodes lancea (Thunb.) DC.

37

4.2.2 Kaempferia galanga Linn. 38

4.3 Identification of marker compounds 41

4.3.1 CMC oral pharmaceutical formulation of Atractylodes

lancea (Thunb.) DC.

41

4.3.2 Standardization of the crude ethanolic extract of Kaempferia

galanga Linn. rhizomes

42

4.4 Animal stocks, handling and ethical approval 42

4.5 Toxicity evaluation of the CMC oral pharmaceutical formulation

of the crude ethanolic extract of Atractylodes lancea (Thunb.) DC.

rhizomes

45

4.5.1 Acute toxicity evaluation 45

4.5.2 Subchronic toxicity evaluation 45

4.6 Cytotoxicity, toxicity testing and anti-CCA activity evaluation of

the crude ethanolic extract of K. galanga Linn. rhizomes

47

4.6.1 Cytotoxic activity evaluation 47

4.6.2 Toxicity testing of K. galanga Linn. Extract 48

4.6.3 Anti-CCA activity of K. galanga Linn. extract in CCA-

xenografted BALB/c nude mice

50

4.6.3.1Tumor growth inhibition 53

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4.6.3.2 Survival time and metastasis rate 53

4.7. Data management and analysis 54

CHAPTER 5 RESULTS 55

5.1 Preparation of the test materials and identification of marker

compounds

55

5.1.1 CMC oral pharmaceutical formulation of Atractylodes

lancea (Thunb.) DC. extract

55



55


of the crude ethanolic extract of Atractylodes lancea (Thunb.) DC.

rhizomes

60

5.2.1 Acute toxicity test 60

5.2.1.1 Clinical signs, mortality, and body weight changes 60

5.2.1.2 Autopsy and histopathological findings 60

5.2.2 Subchronic toxicity test 62

5.2.2.1 Clinical signs, mortality, and body weight changes 62

5.2.2.2 Autopsy and histopathology findings 62

5.2.2.3 Hematology and serum biochemical evaluation 63

5.3 Cytotoxicity, toxicity testing and anti-CCA activity evaluation for


69

5.3.1 Cytotoxic activity the ethanolic extract of K. galanga Linn.

rhizomes against CL-6

69

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5.3.2 Toxicity evaluation of the ethanolic extract of K. galanga Linn.

rhizomes

70

5.3.2.1 Acute toxicity test 70

5.3.2.2 Subacute toxicity test 76

5.3.3 Anti-CCA activity of the crude ethanolic extract of K. galanga

Linn. in CCA-xenografted BALB/c nude mice

91

5.3.3.1 Tumor growth inhibition 91

5.3.3.2 Survival time and tumor metastasis 91

CHAPTER 6 DISCUSSION 100

6.1 Preparation and quality control of the plant extract and CMC oral

pharmaceutical formulation

100

6.1.1 CMC oral pharmaceutical formulation of the crude ethanolic

extract of A. lancea (Thunb.) DC. rhizomes

100

6.1.2 Crude ethanolic extract of K. galanga Linn. 100


of the crude ethanolic extract of A. lancea (Thunb.) DC. rhizomes

101



102

6.3.1 Cytotoxic activity evaluation 102

6.3.2 Toxicity testing of K. galanga Linn. rhizome extract 103

6.3.3 Anti-CCA activity of the crude ethanolic extract of K. galanga

Linn. rhizome in CCA-xenografted BALB/c nude mice

105

CHAPTER 7 CONCLUSIONS AND RECOMMENDATIONS 107

7.1 Toxicity evaluation of the CMC oral pharmaceutical formulation 107

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of the crude ethanolic extract of A. lancea (Thunb.) DC. rhizomes



107

REFERENCES 110

APPENDICES 120

Appendix A 120

Appendix B 121

Appendix C 122

Appendix D 122

Appendix E 123

Appendix F 123

Appendix G 124

Appendix H 125

Appendix I 126

Appendix J 131

Appendix K 133

Appendix L 140

Appendix M 149

Appendix N 152

Appendix O 154

Appendix P 164

BIOGRAPHY 168

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LIST OF TABLES

Tables Page

1. Animal models in current use for screening and development of

cancer chemo preventive agents

17

2. Phytochemical test results detected in different extracts of K.


31

3. Volatile oil components, retention time and peak area (%) of K.

galanga Linn. oil

33

4. Summary of possible mechanisms of action associated with the

bioactive constituents of K. galanga Linn. for the specific

pharmacological activities

34

5. Experimental design of the anti-CCA activity evaluation of the

KG extract in comparison with control groups (vehicle control

and 5-FU) in CCA-xenografted BALB/c nude mice

52

6. Percent yield (w/w) of the crude KG rhizome extract using

different extraction methods

58

7. Haematological parameters of male and female rats in the

subchronic toxicity evaluation of the AL extract formulation in

comparison with control (fed with 20% tween-80)

65

8. Haematological parameters of female rats in the subchronic

toxicity evaluation of the AL extract formulation in comparison

with control (fed with 20% tween-80)

66

9. Serum biochemistry parameters of male rats in the subchronic



67

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Tables Page

10. Serum biochemistry parameters of female rats in the subchronic



68

11. Cytotoxic activities expressed as the IC50 values of the crude

ethanolic extract of KG rhizomes and 5-FU in CL-6 and OUMS

cell lines at in vito

71

12. Vital organ weights of male and female mice following treatment

with 5,000 mg/kg body weight of the KG extract and control

group in the acute toxicity evaluation

74

13. Survival of mice receiving the oral dose administration of KG

extract & 20% tween-80 (control group) in the acute and subacute

toxicity evaluation

79

14. The body weights (in grams) of male and female ICR mice in

subacute toxicity test of the KG extract at the three dose levels,

i.e.,5,000, 3,000, and 1,000 mg/kg body weight

81

15. Median (95% CI) organ weights (in grams) as median (95% CI)

of mice in subacute toxicity test of the KG extract at 5,000, 3,000,

and 1,000 mg/kg body weight dose levels

82

16. Haematological parameters of male mice receiving the KG extract

at high (5,000 mg/kg body weight), medium (3,000 mg/kg body

weight), and low (1,000 mg/kg body weight) and 20% tween-80

(control) in the subacute toxicity test

87

17. Haematological parameters of female mice receiving the KG

extract at high (5,000 mg/kg body weight), medium (3,000 mg/kg

body weight), and low (1,000 mg/kg body weight) and 20%

tween-80 (control) in the subacute toxicity test

88

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Tables Page

18. Serum biochemistry parameters of male mice receiving the KG




89

19. Serum biochemistry parameters of female mice receiving the KG




90

20. Representative tumors of the CCA (CL-6)-xenografted nude mice

following treatment with KG extract at low (100 mg/kg body

weight), medium (500 mg/kg body weight), and high (1,000

mg/kg body weight) dose levels, 5-FU (reference control: 40

mg/kg body beight) and 20% tween-80 (untreated control)

95

21. Representative tumor metastases at autopsy of the CCA (CL-6)-

xenografted nude mice following treatment with KG extract at

low (1,000 mg/kg body weight), medium (3,000 mg/kg body

weight), and high (5,000 mg/kg body weight) dose levels, 5-FU

(reference control: 40 mg/kg body weight) and 20% tween-80

(untreated control)

98

22. Summary of the comparison of toxicity and anti-CCA activity

findings for AL rhizome extract and its CMC oral pharmaceutical

formulation

109

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LIST OF FIGURES

Figures Page

1. Worldwide annual incidence (per 100,000) of liver cancer in

males (1995)

6

2. Age-standardized incidence rates of liver cancers world wide per

100,000 in males

8

3. Representative some liver fluke (O. viverrini) recovered

specimens

9

4. Life cycle of O. viverrini (A), and the freshwater fish species,

Puntius orphoides (B)

10

5. General scheme for uptake and pharmacokinetics of dietary

phytochemicals

13

6. Contemporary preclinical drug development cascade 14

7. Screening procedure using patient derived tumors for the

establishment of in vitro and in vivo models

18

8. Atracylodes lancea (Thunb.) DC. leaves (a) and its rhizomes (b) 22

9. The chemical structures of major components of A. lancea

(Thunb.) DC. rhizome extract

24

10. (A) Flower, (B) fresh rhizomes, (C) dried rhizomes, and (D)

sprouting plant from rhizomes of K. galanga Linn .

28

11. Chemical structures of some important phytoconstituents

isolated from K. galanga Linn. extract

32

12. A diagram showing extract preparation using 7 day maceration of

the KG rhizomes

40

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Figures

13. Overall schematic study design of the toxicity testing and anti-

CCA evaluation of the crude ethanolic extract of K. galanga Linn.

rhizomes (KG) and CMC oral pharmaceutical formulation of the

crude ethanolic extract of A. lancea (Thunb.) DC. rhizomes (AL).

Page

44

14. HPLC chromatograms of (A) A. lancea (Thunb.) DC. ethanolic

extract and (B) atractylodin standard

56

15. Characteristic appearance of the KG ethanolic extract after

concentration

57

16. HPLC chromatograms of (A) standard ethyl-p-

methoxycinnamate (EPMC) and (B) the ethanolic extract of KG

rhizomes

59

17. Median body weight (g) in male and female rats (n=10 for each

group) following a single oral dose of the CMC oral formulation

of AL rhizome extract at the highest dose level of 5,000 mg/kg

body weight

61

18. Median body weight changes (in grams) of (A) male and (B)

female rats following repeated 90 days oral dosing of the CMC

formulation of the AL rhizome extract at 5,000, 3,000, 1,000

mg/kg body weight and 20% tween-80 (control group)

64

19. Changes in body weights (g) of male and female mice (n=10 for

each group) following a single oral dose of 5,000 mg/kg body

weight of the KG rhizome extract and 20% tween-80 (control

group) for acute toxicity evaluation

20. The net body weight gains (in grams) of male (A) and female (B)

mice on the 15th day of treatment with KG at the highest dose of

5,000mg/kg body weight and 20% tween-80 (control group) in

the acute toxicity evaluation

72

73

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Figures Page

21. Representative haematoxylin-eosin staining of various vital

organs (liver, spleen, heart, lungs, and kidneys) collected at

autopsy from mice (males and females) treated with the KG

extract at the high dose level (5,000 mg/kg body weight) and 20%

tween-80 (untreated control mice) in the acute toxicity evaluation

75

22. Time to death (days) and survival curves for male and female

mice during subacute toxicity evaluation of the KG extract

80

23. Representative haematoxylin-eosin staining of various vital organs

and tissues collected at autopsy from male mice in the KG-treated

mice at high (5,000 mg/kg body weight), medium (3,000 mg/kg

body weight), and low (1,000 mg/kg body weight) doses and 20%

tween-80 (control) in the subacute toxicity testing

84

24. Representative haematoxylin-eosin staining of various vital

organs and tissues collected at autopsy from female mice in the

KG-treated mice at high (5,000 mg/kg body weight), medium

(3,000 mg/kg body weight), and low (1,000 mg/kg body weight)

doses and 20% tween-80 (control) in the subacute toxicity testing

86

25. Anti-CCA activity and tumor volume progression (mm3) of the

CCA (CL6)-xenografted nude mice following treatment with the

KG extract at low (100 mg/kg body weight), medium (500 mg/kg

body weight), and high (1,000 mg/kg body weight) dose levels, 5-

FU (reference control: 40 mg/kg body weight) and 20% tween-80

(untreated control) during the investigation period

93

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Figures Page

26. Tumor volumes (mm3) of the CCA (CL-6)-xenografted nude mice

at the end of treatment (on day 31st) with KG extract at low (100

mg/kg body weight), medium (500 mg/kg body weight), and

high (1,000 mg/kg body weight) dose levels, 5-FU (reference

control: 40 mg/kg body weight) and 20% tween-80 (untreated

control)

94

27. Median survival time (days) of the CCA (CL-6)-xenografted nude

mice following treatment with the KG extract at low (1,000

mg/kg body weight), medium (3,000 mg/kg body weight), and

high (5,000 mg/kg body weight) dose levels, 5-FU (reference

control: 40 mg/kg body weight) and 20% tween-80 (untreated

control) during the investigation period

96

28. Gross appearance of the representative primary CCA tumor at

autopsy in a CCA (CL-6)-xenografted nude mouse

97

29. Representative primary CCA tumors and lung metastases at

autopsy of the CCA (CL-6)-xenografted nude mice following

treatment with KG extract at low (1,000 mg/kg body weight),

medium (3,000 mg/kg body weight), and high (5,000 mg/kg body

weight) dose levels, 5-FU(reference control: 40 mg/kg body

weight) and 20% tween-80 (untreated control)

99

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LIST OF ABBREVIATIONS

Symbols/Abbreviations Terms

AL Atractylodes lancea (Thunb.) D.C.

ALP

ALT

AST

AVMA

Alkaline phosphatase

Alanine aminotransferase

Aspartate aminotransferase

American Veterinary Medical Association

BUN

BW

Blood urea nitrogen

Body weight

oC

CA19-9

CAM

Degree celsius

Carbohydrate antigen 19-9

Complementary and alternative medicine

CCA Cholangiocarcinoma

CEA

CI

CICM

Carcinoembryonic antigen

Confidence interval

Chulabhorn International College of

Medicine

CL-6 Cholangiocarcinoma cell line

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CNS

CO2

COX

CYP450

Central nervous system

Carbon dioxide

Cyclooxygenase

Cytochrome P450

dH2O Distilled water

DMEM

DMN

DMSO

EC

EDTA

EPMC

Dulbecco's Modified Eagle's Medium

Dimethyl nitrosamine

Dimethyl sulfoxide

Ethyl cinnamate

Ethylene diamine tetraacetic acid

Ethyl-p-methoxycinnamate

FBS Fetal bovine serum

FDA Food and Drug Administration

5-FU

GAE

g/dl

H

Hb

5-Fluorouracil

Galic acid equivalent

Gram per deciliter

Hour

Hemoglobin

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HCC

HCF

HCT

H & E

HDL

HepG2

Hepatocellular carcinoma

Human cardiac fibroblast

Hematocrit

Hematoxylin and eosin

High density lipoproteins

Hepatocarcinoma cell line

HPLC

HTS

High performance liquid chromatography

High through-put assays

IC50

ICR

Inhibitory concentration by 50%

Imprinting control region

ID50 Inhibitory dilution by 50%

KG Kaempferia galanga Linn.

LD50

LDL

Lethal dose concentration by 50%

Low density lipoproteins

µg

µl

MCV

MCH

MCHC

Microgram

Microliter

Mean corpuscular volume

Mean corpuscular hemoglobin

Mean corpuscular hemoglobin

concentration

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mEq/L Milliequivalent per liter

mm Millimeter

MPV

MST

Mean platelet volume

Mean survival time

MTD Maximum tolerated dose

MTT

NBF

Nm

Methyl thiazoldiphenyl tetrazolium

Neutral buffered formalin

Nanometer

NOAEL

NSS

No observed adverse effect level

Normal saline solution

OD Optical density

OECD

Organization for Economic Co-operation

and Development

% Percent

PBS

PCT

PDW

Pg

PK

PD

Phosphate buffered saline

Plateletcrit

Platelet distribution width

Picogram

Pharmacokinetics

Pharmacodynamics

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RBC

RDW

Red blood cell (s)

Red cell distribution width

Rpm Revolutions per minute

RPMI

SC

SD

SE

Roswell Park Memorial Institute

Subcutaneous

Standard deviation

Standard error

TGI

TM

TNF-α

TPC

TU

Tumor growth inhibition

Traditional medicine

Tumor necrosis factor-α

Total phenolic content

Thammasat University

TV

U/l

Tumor volume

International unites per liter

v/v

WBC

w/w

WHO

Volume by volume

White blood cell (s)

Weight by weight

World Health Organization

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CHAPTER 1

INTRODUCTION

Cholangiocarcinoma (CCA) is an important public health problem in

several parts of the world and in particular in Asia including Thailand. CCA or

adenocarcinoma of the bile ducts arises from the epithelial cells of bile ducts

anywhere along the intrahepatic and extra hepatic biliary tree excluding the papilla of

Vater and the gall bladder (Mosconia et al., 2009). The highest prevalence of CCA in

Northeast of Thailand is associated to the consumption of improperly cooked and

fermented or preserved cyprinid fish species food which contains the fluke,

Opisthorchis viverrini (Dholwani et al., 2008; Haswell-Elkins et al., 1992;

Khuhaprema and Srivatanakul, 2008; Singh and Facciuto, 2012). In other Asian

countries like China, the parasite known as Clonorchis sinensis is the main cause of

CCA (WHO, 2003).

The major challenge for CCA control and treatment is the lack of early

diagnosis and multidrug or radio-resistant nature of CCA tumor (Na-Bangchang and

Karbwang, 2014; Namwat et al., 2008). This requires an ongoing and urgent need for

the discovery and development of effective alternative diagnostic tools and

chemotherapeutics. Even though the clinical response rate is low and the recurrence

rate is extremely high, surgical resection of detectable tumors and combination

therapy with standard chemotherapeutic agents including 5-fluorouracil (5-FU) leads

to an improvement in the 5-year survival rate in the present therapeutic approaches

(Lee et al., 2004; Prabhleen and Todd, 2005). The promising therapeutic options in

many types of cancers including CCA is the use of combination therapies of standard

treatments and in conjunction with alternative therapy with dietary phytochemicals

(Vapiwala et al., 2006). There is a pressing need of drug discovery research for new

alternative drugs against cancers in general and CCA in particular.

Plants have formed the basis of traditional medicine (TM) systems in

many parts of the world which have been used for thousands of years and the use of

plant-based therapeutic systems continues to play an essential role in health care

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2

system (Zia-Ul-Haq et al., 2013). The use of plant-derived material or preparation

with therapeutic or other human health benefits which contains either raw or

processed ingredients of one or more plants and in some conditions, materials of

inorganic or animal origin may also be added is referred as herbal medicine (WHO,

1993). Approximately 80 % of the population in developing countries rely on TM for

their primary health care (Hostesttmann and Marston, 2002; Zia-Ul-Haq et al., 2013).

In China, the use of traditional herbal preparations account for 30 to 50% of the

medicines (WHO, 2003). In developed countries, therapeutic approaches of TM also

known as complementary and alternative medicine (CAM), also plays an important

role in the health care system of 20% of the population. In addition to such therapeutic

options, TM has also afforded a rich source of remedies or drugs with diverse

chemical structures and bioactivities against several health disorders including cancer

and several modern anticancer drugs approved by FDA (including vinblastine,

vincristine, etoposide, teniposide, paclitaxel, vinorelbine, docetaxel, topotecan, and

irinotecan) have been developed from plant sources (Dholwani et al.,2008; Na-

Bangchang and Karbwang, 2014; Schwikkard and van Heerden, 2002; Sudhanshu et

al., 2003).

Thailand is a country which is rich in a wide range of tropical habitats,

remarkable biodiversity, and uses traditional medicines for treatment of various

infectious illnesses and chronic diseases including cancer (Subchareon, 1998;

Thiengsusuk et al., 2013). Ethnopharmacological studies of many candidate medicinal

plants or herbal formulations commonly used in Thai TM practice for treatment of

various diseases have been screened for their anti-CCA activities (Mahavorasirikul et

al., 2010; Na-Bangchang and Karbwang, 2014; Plengsuriyakarn et al., 2012).The

extracts from seven plant species (Atractylodes lancea (Thunb.) DC., Kaempferia

galanga Linn., Zingiber officinale Roscoe., Piper chaba Linn., Mesua ferrea Linn.,

Ligusticum sinense Oliv. cv. Chuanxiong, Mimusops elengi Linn.) and one folklore

recipe (Pra-Sa-Prao-Yhai) exhibited promising activity against CL-6 cell line with

IC50 (concentration that inhibits cell growth by 50%) values reported less than

50μg/ml (Mahavorasirikul et al., 2010). Among the above extract and recipe

preparations (including Atractylodes lancea (Thunb.) DC., Kaempferia galanga Linn.,

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Zingiber officinale Roscoe., Piper chaba Linn., Mesua ferrea Linn., and Pra-Sa-Prao-

Yhai recipe), potent cytotoxic activity were found with mean IC50 values of 24.09,

37.36, 34.26, 40.74, 48.23 and 44.12 μg/ml, respectively. The crude ethanol extract

from A. lancea (Thunb.) DC. showed a potent activity against CL-6 cell lines (IC50 =

24.09 + 3.40 μg/ml and selectivity index = 8.6) and HepG2 cell line (IC50 = 76.68 ±

15.94 and SI = 2.7) at in vitro (Mahavorasirikul et al., 2010). In a similar protocol

(Mahavorasirikul et al., 2010), K. galanga Linn. extract showed cytotoxic activity

against CL-6 cell lines (mean + SD IC50 = 37.36 ± 3.98μg/ml and selectivity index =

2.9) and against HepG2 cell line (mean + SD IC50 =115.47 ± 26.23 and SI= 0.9).

Moreover, the anti-CCA activity and safety of the crude ethanolic extract of A. lancea

(Thunb.) DC. rhizomes was also demonstrated CCA-xenografted nude mice at

different dose levels (1,000, 3,000, and 5,000 mg/kg body weight) (Plengsuriyakarn et

al., 2012) that showed significant reduction in tumor size, prolongation of survival

time, and inhibition of lung metastasis as compared with the standard drug (5-FU) and

the untreated control.

Upon reviewing the current literature, there has been little evidence or

report found especially on the in vivo toxicity and anti-CCA activity of K. galanga

Linn.. On the other hand, previous in vitro and in vivo studies conducted on the crude

ethanol extract of A. lancea (Thunb.) DC. rhizomes indicated its potential role for

treatment of CCA and its safety as described above (Mahavorasirikul et al., 2010;

Plengsuriyakarn et al., 2012). In hamster model, the ethanolic extract of A. lancea

(Thunb.) DC. showed satisfactory safety and a significant anti-CCA activity

particularly at the highest dose level of 5,000 mg/kg body weight compared with the

standard drug 5-FU and untreated control animal groups (Plengsuriyakarn et al.,

2015). Moreover, the in vivo anti-CCA study (Plengsuriyakarn et al., 2015) conducted

on CCA-induced mice using PET-CT imaging following treatment with high dose β-

eudesmol (100 mg/kg body weight for 30 days), the bioactive compound isolated

from A. lancea (Thumb.) DC. rhizomes showed significant reduction in tumor size

and lung metastasis. The survival time of mice was prolonged by 64.4% compared

with the untreated control. Based on such preliminary screening results (in vitro and

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in vivo) of various plants used in traditional medicine against CCA in Asia, including

Thailand, there is a need to select and further study the toxicity (safety) and efficacy

of some promising candidate herbal agent preparations including A. lancea (Thunb.)

DC. formulation and K. galanga Linn. extract in an effort for new drug discovery and

development option.

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CHAPTER 2

REVIEW OF LITERATURE

2.1. Cholangiocarcinoma (CCA) and treatment approaches

Hepatocellular carcinoma (HCC) is the most frequent and important of

liver cancers which poses a significant disease burden in many parts of the world,

particularly in Africa and Asia (WHO, 2003). In high incidence regions or countries

of HCC, chronic infection with hepatitis B virus (HBV) is its principal underlying

cause and in countries like Japan, it’s associated with high prevalence of hepatitis C

infection. On the other hand, liver cirrhosis associated to chronic alcohol abuse is a

major cause of HCC in the Western or developed countries. Unlike HCC,

cholangiocarcinoma (CCA) has a different geographical distribution, with peak

incidences in Asia especially in the Northeast of Thailand (WHO, 2003).

CCA is classified into three major groups, i.e., intrahepatic, perihilar, and

distal extra hepatic and generally CCA accounts for approximately 15% of liver

cancers worldwide (Nakanuma et al., 2003). Intrahepatic type of CCA arises from any

portion of the intrahepatic bile duct epithelium or intrahepatic small bile ducts, as well

as from the right and left hepatic ducts at or near their junction called hilar CCA

which is considered an extrahepatic lesion (Nakanuma et al., 2003). Intrahepatic CCA

is the most common case of CCA in Thailand and the disease in the area has been

definitely related to chronic infestation with Opisthorchis viverrini (O. viverrini)

(Sripa et al., 2011; Watanapa and Watanapa, 2002). According to the World Health

Organization reports in 1994 (WHO, 1994), the age standardized incidence of

intrahepatic CCA in Khonkaen region of Thailand was 88 per 100,000 in males and

37 per 100,000 in females (Figure 2). Approximately 90% of the confirmed cases of

liver cancers were intrahepatic CCA, and almost all the CCA cases were found to be

related to chronic O. viverrini infection in the area of this study.

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Figure 1 Worldwide annual incidence (per 100,000) of liver cancer in males (1995).

Numbers on the map indicate regional average values (WHO, 2003).

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O. viverrini (OV) infections is pathologically associated to a number of

hepatobiliary diseases including cholangitis, obstructive jaundice, hepatomegaly,

cholecystitis, and cholelithiasis are associated with (Harinasuta et al., 1984). Both

experimental and epidemiological evidences strongly indicated that chronic infection

with OV liver fluke as the etiology of CCA (Elkins et al., 1996; Haswell-Elkins et al.,

1992). Moreover, the pathological consequences of OV infection (i.e., epithelial

desquamation, inflammation, epithelial hyperplasia, goblet cell metaplasia,

adenomatous hyperplasia, and periductal fibrosis) appear to be similar in both human

and animals (Riganti et al., 1989).

In addition to limitations of therapeutic options, early detection of CCA is

also a major problem for controlling CCA. Among the serum tumor markers,

carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are used as

candidate biomarkers for assessment and monitoring during and after the treatment of

several gastrointestinal cancers. At present, surgical resection of detectable tumors

leads to an improvement in the 5-year survival rate and additional chemotherapy with

standard drugs including 5- fluorouracil (5-FU) has been indicated to improve local

control and prolong survival time of patients (Prabhleen and Todd, 2005). However,

the recurrence rate is extremely high, with a 5-year survival rate of less than 40%

even with operable tumors (Sirica, 2005; Thongprasert, 2005). Other reports also

indicated that chemotherapeutic treatment of CCA is largely ineffective; and

treatment with 5- FU always produces low clinical response rates (Lee et al., 2004;

Patt et al., 2001; Thongprasert, 2005). Moreover, in severe stages of CCA, metastatic

tumors in the lungs develop by spreading from the liver origin through the

bloodstream or lymphatic system, which is one of the main determinants of therapy

and patients with such metastatic disease (inoperable CCA) should be considered for

systemic chemotherapy with gemcitabine and cisplatin (Namwat et al., 2008).

Generally, CCA is considered to be a multidrug and radio-resistant tumor that still

requires new approaches of treatments.

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Figure 2 Age-standardized incidence rates of liver cancers worldwide per 100,000 in

males (WHO, 1994).

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Figure 3 Representative liver fluke (Opisthorchis viverrini) recovered specimens.

(A) An adult specimen of O. viverrini recovered from a patient after chemotherapy

and purgation, (B) A metacercaria of O. viverrini detected in a freshwater fish Puntius

orphoides, and (C) A young adult of O. viverrini recovered after an experimentally

infected hamster with the metacercariae in Cambodia (Sohn et al., 2012).

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(A)

(B)

Figure 4 Life cycle of (A) O. viverrini, and (B) the freshwater fish species Puntius

orphoides (Sohn et al., 2012).

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The growing trend and promising activity to improve survival of cancer

patients including CCA, is to combine conventional therapy such as advanced surgical

techniques in combination with chemotherapy and alternative chemotherapeutic

options from dietary phytochemicals (Vapiwala et al., 2006). In general,

chemotherapy with plant-derived compounds or dietary phytochemicals has emerged

as an accessible and alternative promising approach to cancer control and

management in many countries (Surh, 2003). In such efforts to develop new and

alternative drugs against CCA and other cancers from natural products, an ongoing

preclinical research is required and should be conducted to evaluate the safety,

efficacy and essential pharmacokinetic aspects of the test substances.

2. 2. Screening methods for development of anticancer and anti-CCA drugs

and/or phytochemicals

To combat the problem of resistance, newer drugs are urgently required

and an unprecedented number of anticancer discovery and development projects are

now underway throughout the world. Its recognized that most of the methods for

anticancer screening and investigating the principal mechanisms of liberation,

absorption, distribution, metabolism and elimination (LADME) have originated from

the pharmaceutical research/new drug development sector, but the same procedure

may be applied and optimized for phytochemicals as well (Birgit and Gary, 2004).

Several methods including in vitro, in situ and in vivo screening bioassay methods

described below are routinely used for anticancer drug development evaluations

(Birgit and Gary, 2004).

2.2.1 In vitro methods for screening anticancer candidates

Researchers use cell-free and cell-based screenings (e.g. artificial

membranes, cells, tissues, and organ cultures) to study a broad range of endogenous

processes outside the body under controlled and standard conditions (Birgit and Gary,

2004). In the majority of cases, either cellular or target-based high-throughput assays

(HTS) are performed before in vivo evaluation of potential anticancer drugs/test

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compounds. Permanent human tumor cell lines (their immortal nature and

reproducible growth behavior) makes cell-based screening assays in cancer research

are suitable test systems (Birgit and Gary, 2004). The cell-based assay formats can be

performed in 96- to 384-well plates but the detection method (which could depend on

the cell number used and desired sensitivity) is of critical importance and the earliest

broadly used detection method (growth inhibition assay) is the methyl thiazoldiphenyl

tetrazolium (MTT) assay (Mosmann, 1983).

2.2.2 In situ models

In situ models such as continuous, single-pass perfusion in animals, and

humans, provide a powerful research tool for the investigation of intestinal transport

and metabolism of test compounds (Birgit and Gary, 2004).

2.2.3 In vivo methods for screening anticancer compounds (screening

using animal models)

It is difficult to study complex interactions in simplistic models like in

vitro and only in vivo models provide the opportunity to integrate the dynamic

components of the mesenteric blood circulation, the mucous layer, and all other

factors that can influence ADME (absorption, distribution, metabolism, and excretion)

and the subsequent pharmacodynamic effects (Birgit and Gary, 2004) (Figure 5).

In general, compounds effective in in vitro screening tests are taken up for

in vivo evaluation with the appropriate animal models/organisms (Figure 6). Despite

the fact that the in vivo methods are expensive and time consuming, rodent xenograft

models are the current traditional preclinical test-bed and form an excellent model for

initial basic pharmacologic research and safety investigations (Malaney et al, 2014;

Peldschus and Ittrich, 2014) and currently even for clinical therapeutic response

studies (Burger and Fiebig, 2014).

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Figure 5 General scheme for uptake and pharmacokinetics of dietary phytochemicals


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Figure 6 Contemporary preclinical drug development cascade (Burger and Fiebig,

2014; Rudek et al., 2014).

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Animal based (in vivo) bioassays afford a strategic framework for

evaluating evidence of agent efficacy, and also serve to generate valuable dose-

response, toxicity, and pharmacokinetic data which are required prior to phase I

clinical safety testing (Steele et al., 2005). It has been indicated that some key

elements are necessary in choosing the ideal animal model for cancer chemotherapy

or chemoprevention testing; (1) the animal model should bear relevance to human

cancers (including in terms of specific organ sites and in producing cancerous lesions

of similar pathology); (2) the animal model should have relevant intermediate lesions

that simulate the human cancer process both histologically and molecularly; (3) the

genetic abnormalities of these lesions should resemble those of humans; (4) the model

should be capable of producing a consistent tumor burden of greater than 80% lesions

within a reasonable period of time (less than 6 months); and (5) the carcinogen or

genetic defect used to produce cancer should bear relevance to that encountered by

humans (Steele et al., 2005).

Research and development of better animal models is ongoing, although

no current animal model is considered ideal or best (Steele et al., 2005) and a review

of some of the currently used animal models for cancer chemoprevention efficacy

testing is presented in Table 1.

The Freiburg Experience: The Freiburg xenograft panel is derived

directly from patient explants instead of established from permanent human tumor

cell line material (Figure 7) (Fiebig et al., 2004). In this panel, comparison of the

efficacy of a standard drug or drug combinations in patients and their tumors in nude

mice, a total of 21 patients reached a remission and the same result was observed in

19 tumors growing as xenografts (Fiebig et al., 2004). Moreover, 59 mice did not

respond to treatment and the same result was found in 57 mice. Overall, xenografts

provided a correct prediction for resistance in 97 % (57/59) and for tumor

responsiveness in 90 % (19/21).

For ethical and other pragmatic reasons, the most frequently used animal

models for in vivo profiling is the rat (Kararli, 1995). However, interspecies

differences between humans and animals would mean that the endpoints of such

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studies may not reflect the human situation. Several factors attributed to differences in

anatomy and physiology which include gut length and motility, amount and

composition of gastrointestinal microflora, enterohepatic circulations, as well as

intestinal transit time may all have profound effects on pharmacokinetic and

pharmacodynamic activities of a test compound in different species (Birgit and Gary,

2004).

In general, at dietary levels, food constituents including phytochemicals

are recognized as safe. As a result, human studies of dietary phytochemicals at dietary

doses should always be considered as the method of choice, which provides a great

advantage over drug studies. The disadvantage and challenge is the complexity of the

food matrix that needs to be studied to simulate dietary conditions, the low

concentrations of most phytochemicals in food, and the complexity of the human

organism. Considering such aspects, it is necessary to address the gaps in knowledge

on the processes of liberation, absorption, distribution, metabolism, and elimination

(LADME) and complement human in vivo data with appropriate in vitro data obtained

by studies on human tissues, cells, or subcellular fractions and animal in vivo data


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Table 1 Animal models in current use for screening and development of cancer

chemo preventive agents (Steele et al., 2005).

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Figure 7 Screening procedure using patient derived tumors for the establishment of in

vitro and in vivo models (Fiebig et al., 2004).

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2.3. Medicinal plants with anti-CCA activities

Treatment of malignancies by using herbal medicine in Asian countries

has been used since early times (Efferth et al., 2007) and TM is also commonly used

as an alternative treatment for cancers by Thai peoples (Subchareon, 1998). It is

recognized that considerable researches have been performed on these plants to

treatment of cancers, and some plant products have been marketed as anticancer drugs

based on the traditional uses and scientific evidences (Mahavorasirikul et al., 2010;

Na-Bangchang and Karbwang, 2014).

Numerous cancer research for chemotherapeutic potential of medicinal

plants have been carried out in an effort to discover new therapeutic agents that lack

the toxic effects associated with current cancer therapeutic agents and it is known that

several conventional anticancer drugs approved by the FDA and being used in

patients with different types of cancers are derived from plants (Dholwani et al.,

2008). These include vinblastine, vincristine, etoposide, teniposide, paclitaxel,

vinorelbine, docetaxel, topotecan, and irinotecan. Several Thai traditional folklores

have been shown to possess anticancer activities in various human cancerous cell

lines with some promising candidates (Itharat et al., 2004; Mahavorasirikul et al.,

2010; Prayong et al., 2008).

Specific ethnobotanical and in vitro investigations of the candidate

medicinal plants or herbal formulations which are commonly used in Thai traditional

medicine for their anti-CCA activity screening (Mahavorasirikul et al., 2010). The

extracts from seven plant species, i.e., Atractylodes lancea (Thunb.) DC., Kaempferia

galangal Linn., Zingiber officinale, Piper chaba, Mesua ferrea, Ligusticum sinense,

Mimusops elengi, and one folklore recipe Pra-Sa-Prao-Yhai were shown to exhibit

promising activities against the human CCA cell line (CL-6) with IC50 (concentration

that inhibits cell growth by 50%) values of less than 50 μg/ml (Mahavorasirikul et al.,

2010). The corresponding mean IC50 values for these test agents were 24.09, 37.36,

34.26, 40.74, 48.23 and 44.12 μg/ml, respectively. The crude ethanol extract from

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Atractylodes lancea (Thunb.) showed the most potent activity with selectivity index

(SI) of 8.6. In addition, its activity against the cancer cell HepG2 was also potent with

mean IC50 of 76.68 μg/ml and SI of 2.7 (Mahavorasirikul et al., 2010). Kaempferia

galanga Linn. extract also showed cytotoxic activity against CL-6 cell line with mean

IC50 of 37.36 μg/ml and SI of 2.9; its mean IC50 and SI values for the HepG2 cell were

115.47 μg/ml and 0.9, respectively.

Another ethnopharmacological study conducted in the United States also

confirmed the anticancer activity of A. lancea (Thunb.) DC. in murine neuroblastoma

cells originally derived from a spontaneous malignant tumor and its activity was

considered moderate to strong with LC50 (50% lethal concentration) of 0.70 mg/ml

(Mazzio and Soliman, 2010). Moreover, the anti-CCA activity of A. lancea (Thunb.)

DC. extract and its active compound known as β–eudesmol were also demonstrated at

in vivo in CCA-xenografted nude mice (Plengsuriyakarn et al., 2012) and in hamster

model (Plengsuriyakarn et al.,2015). Such results attract researchers’ attentions to

further investigate the safety anticancer property of both K. galanga Linn. extract and

A. lancea (Thunb.) DC. extract preparations.

2.4 Description of the study plants

2.4.1 Atractylodes lancea (Thunb.) DC.

2.4.1.1 Plant description and medicinal properties

Atractylodes lancea (Thunb.) DC., belonging to the Compositae

Family, also commonly known as Khoad- Kha- Mao (Thailand) and So- Jitsu (Japan), is

widely distributed in Asia. It is known as “Cang Zhu” in traditional Chinese medicine

(Florae et al., 2005). It has long been used as crude drugs to treat a lot of diseases

such as rheumatic diseases, digestive disorders, night blindness, and influenza (Xiao,

2002).

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A. lancea (Thunb.) DC. has been used as diuretic and gastro-intestinal

(against stomachic damage and dyspepsia) drugs in Japanese (Kitajima et al., 2003)

and Korean (Tatsuta and Iishi, 1993) traditional medicines. In Thai traditional

medicine, the dried rhizome of A. lancea (Thunb.) DC. has been used to treat fever

and the common cold, and to relieve gastro-intestinal symptoms such as dyspepsia,

flatulence, nausea, and noninfectious diarrhea (Qian et al., 2006).

Ethnopharmacological studies also suggest as the A. lancea (Thunb.)

DC. extract exhibits anti-inflammatory and antimicrobial activities as well as activities

on the nervous, cardiovascular, and gastro-intestinal systems (Koonrungsesomboon et

al., 2014). Results of cytotoxic testing in the human CCA cell line CL-6 demonstrated

potent activity of A. lancea (Thunb.) DC. crude extract (IC50 = 24.09 μg/ml) and most

selective against CCA (SI= 8.6) (Mahavorasirikul et al., 2010). The crude ethanolic

extract of A.lancea (Thunb.) DC. and its bioactive compound (β-eudesmol)

demonstrated significant reduction of tumor size, tumor metastasis inhibition, and

increased survival time when compared with standard drug 5-FU and untreated

control in OV/DMN-induced CCA hamster model (Plengsuriyakarn et al., 2015) and

CCA-xenografted mice (Plengsuriyakarn et al., 2012). The in vivo anti-CCA study

conducted in CCA-xenografted nude mice indicated significant reduction in tumor

size and lung metastasis by 91.6% (of baseline) and 95% (of total lung mass),

respectively following treatment with high dose β-eudesmol (100 mg/kg body weight

for 30 days), one of the bioactive compounds isolated from A. lancea (Thunb.) DC.,

by using PET-CT imaging study (Plengsuriyakarn et al., 2015). Moreover, the

survival time of mice was prolonged by 64.4% compared with the untreated control.

The pharmacokinetics of the radiolabeled β-eudesmol was also evaluated in healthy

and CCA-xenografted nude mice showing the systemic clearance of the compound

was rapid (particularly during the first 60 min) and the compound was distributed to

the vital organs following oral and intravenous administrations (Plengsuriyakarn et

al., 2015).

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(A)

(B)

Figure 8 (A) Atracylodes lancea (Thunb.) DC. leaves and (B) its rhizomes (Subhuti,

2003).

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2.4.1.2 Phytochemistry

The major essential oil constituents and sesquiterpenoid glycosides of

A. lancea (Thunb.) DC. rhizomes (Figure 9) which exhibit several pharmacological

functions include β-eudesmol, hinesol, atractylone, and atractylodin. β-eudesmol

accounts for about 6% of the total yield, while atractylodin and atractylone account

for about 14 and 9% of the total yield, respectively, following activity guided

fractionation (Ji et al., 2001; Ouyang et al., 2012; Zhou et al., 2012). The essential

oils of A. lancea (Thunb.) DC. rhizomes possess anti-inflammatory and anti-ulcer

properties, scavenging activity on trichloromethyl (CCl3) radical, inhibitory activity

on lipid peroxidation and xanthine oxides, inhibitory activity on tert-butyl

hydroperoxide-induced cytotoxicity and lipid peroxidation in primary culture of rat

hepatocytes, and inhibitory activity on the growth of esophageal carcinoma cells and

tumor cells (Yang et al., 2002).

2.4.1.3 Safety and toxicity

The maximum tolerated dose of A. lancea (Thunb.) DC. rhizomes

extract was 5,000 mg/kg body weight in three species (hamsters and mice) and no

significant toxicity except stomach irritation and general CNS depressant signs

(reduced alertness and locomotion and diminished response to touch and balance)

were observed following acute and subacute toxicity studies (Plengsuriyakarn et al.,

2012; Plengsuriyakarn et al., 2015).

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β-Eudesmol Atractylodin

Atractylone Hinesol

Figure 9 The chemical structures of major components of A. lancea (Thunb.) DC.

rhizome extract (Ji et al., 2001).

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2.4.2 Kaempferia galanga Linn.

2.4.2.1 Plant description and medicinal properties

K. galanga Linn. is one of the plants in Zingiberaceae family and a

genus of rhizomatous herbs distributed in the tropics and sub tropics of Asia and

Africa, being cultivated for its aromatic rhizomes (Geetha et al., 1997). Since the

rhizomes of this plant contain volatile oils and other important compounds of

enormous medicinal values, they are very demanding to the traditional health care

practitioner and is used as traditional medicinal plant in many countries (Mohammad,

2008). It is well known for its action as remedy for inflammation and various ethno

botanical studies indicated that its rhizome extract is used to relieve pain and various

disorders around Southeast Asia (Kanjanapothi et al., 2004; Mohammad, 2008).

(1) Anti-inflammatory and analgesic activity

Aqueous extracts of K. galanga Linn. (leaves and rhizomes)

administered at different doses (30, 100, and 300 mg/kg body weight) to rats

produced significant anti-inflammatory and analgesic effects in hot plate and formalin

tests (Sulaiman et al., 2008). An oral dose of 200 mg/kg body weight of KG rhizome

extract produced anti-nociceptive effect more potent than aspirin at the dose of 100

mg/kg body weight, but lesser than morphine at the dose of 5mg/kg body weight, in

another study (Ridtitid et al., 2008). The capacity of the extracts to block abdominal

constriction demonstrated in the hot plate and formalin tests (Sulaiman et al., 2008)

indicates that analgesic activity of A. lancea (Thunb.) DC. rhizomes involves both

central (opioid receptors), and peripheral (cyclooxygenase, COX) mechanisms.

A study conducted using n-hexane rhizome extract of K. galanga

Linn. in acetic induced writhing test in mice demonstrated the highest analgesic effect

(62.31% inhibition) and the chloroform extract showed moderate analgesic activity

(38.19% inhibition) as compared with the activity observed by the positive control

diclofenac sodium (63.82% inhibition) (Chowdhur et al., 2014).

(2) Antimicribioal activity

K. galanga Linn. extract has also been demonstrated to exhibit

antimicrobial activity against a number of organisms including Staphylococcus

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aureus, Streptococcus pyogenes, Candida albicans, Escheriachia coli, Klebsiella

pneumonia, Salmonella typhi, Seratia marcescens, Vibrios cholera, Vibrio

sparahaemolyticus, Enterococcus faecalis, and Pseudomonas aeruginosa with

minimum inhibitory concentration (MIC) of 0.81, 3.25, 25, >6.5, > 6.5, >6.5, >6.5,

>6.5, >6.5,1.625 and >6.5 μg/ml, respectively (Mekseepralard et al., 2010). Moderate

antibacterial activity (including gram positive and gram negative bacteria) has also

been demonstrated by another study (Dash et al., 2014) with different K. galanga

Linn. extracts as compared with the standard drug ciprofloxacin (5 μg/disc).

Significant antibacterial and antifungal properties were also

demonstrated by the rhizome extract of K. galanga Linn. by using different solvents

(ethanol, methanol, and aqueous). The ethanolic extract showed potent activity

against Staphylococcus aureus, Aspergillus niger, A. flavus, A. fumigatus and

Candida albicans with mean + SD inhibition zone of 21.3±0.08, 16.4±0.45,

15.3±0.36, 14.0±0.48, 12.2±0.45 mm, respectively (Kochuthressia et al., 2012). On

the other hand, the methanolic rhizome extract showed potent activity against

Aspergillus niger (inhibition zone of 14.2±0.26 mm).

(3)Anti-oxidant activity

Anti-oxidant activity of K. galanga Linn. extract was also observed

in various studies (Chan et al., 2008; Mekseepralard et al., 2010). The total phenolic

content (TPC) of ethanolic extract was found to be 146 mg galic acid equivalent

(GAE)/100 g from the leaves of K. galanga Linn. and 57 mg GAE/100g from the

rhizomes of K.galanga Linn. (Chan et al., 2008). The antioxidant activities of the

leave and rhizome extracts were 77 mg ascorbic acid/100 g and 17 mg ascorbic

acid/100 g, respectively, and this anti-oxidant activity was further reduced by drying

which was in turn prevented if the plant was subjected to freeze-drying (Chan et al.,

2008). However, it was reported in another study that the volatile oil extract from K.

galanga Linn. was inactive for antioxidant activity with IC50 >100 µg/ml (Tewtrakul

et al., 2005).

(4) Anti-hyperglycemic effects

Different solvent extracts of K. galanga Linn. were shown to exhibit

antihyperglycemic activity in in vivo models. The crude methanolic extract and

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chloroform soluble fractions of K. galanga Linn. extract (200 mg/kg body weight)

showed remarkable blood glucose level reduction (with 61.2 and 89.63 % reduction,

respectively), which were more prominent than that of standard drug glibenclamide

(34.78% reduction compared to control) after 30 minutes of glucose loading

(Chowdhur et al., 2014). The percentage of maximal inhibition of glucose load for

that of the n-hexane extract was 91.63%, which was also higher than the value

observed for that of the standard drug glibenclamide (Chowdhur et al., 2014).

(5) Antineoplastic and apoptotic activity

K. galanga Linn. extract has also been reported to possess

antineoplastic activity with inhibitory effect being observed on the tumor-promoting

stage of neoplasia (Koh, 2009; Vimala et al., 1999). A study conducted with the

methanolic extract of K.galanga Linn. showed inhibitory activity in 12-O

tetradecanoyl-phorbol-13-acetate (TPA)-induced activation of Epstein barr virus early

antigen in Raji cells and this led to the inhibitory activity on tumor promoting stage

even though the inhibition of this tumor promoting stage was partial (Vimala et al.,

1999). At the dose of 320 μg/ml, the inhibition observed was 80% and the inhibition

was even increased up to a maximum of 90% at 640 μg/ml in this study. In a recent

study (Mahavorasirikul et al., 2010), K. galanga Linn. ethanolic extract showed

cytotoxic activity against CL-6 and HepG2 cell lines with mean (+SD) IC50 values of

37.36+3.98 and 115.47+26.23 µg/ml, respectively. Moreover, the methanolic extracts

of K. galanga Linn. has been shown to exert inhibitory effect on human cardiac

fibroblast (HCF-7) and human T-cell leukemia (HT-29) cell lines but only at doses

higher than 250 μg/ml (Kirana et al., 2003).

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(A) (B)

(C) (D)

Figure 10 (A) Flower, (B) fresh rhizomes, (C) dried rhizomes, and (D) sprouting

plant from rhizomes of K. galanga Linn. (Katzer, 2011).

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2.4.2.2 Phytochemistry

Preliminary phytochemical group tests revealed that different K.

kalanga Linn. extracts contain carbohydrates, tannins, flavonoids, proteins, steroids,

alkaloids, and resins (Dash et al., 2014) as summarized in Table 2.

The major constituents of the volatile oil extracts of K.galanga

rhizome include cineol, borneol, 3-carene, camphene, kaempferol, kaempferide,

cinnamaldehyde, p-methoxycinnaamic acid, ethyl cinnamate, and ethyl p-

methoxycinnamate (Figure 11) (Sutthanont et al., 2010). Ethyl–para-

methoxycinnamate (EPMC) and ethyl-cinnamate (EC) are found to be the most

dominant and vital constituents in the methanol and water extracts (Huang et al.,

2008; Sirisangtragul and Sripanidkulchai, 2011; Tewtrakul et al., 2005). Among the

volatile oil content obtained from the rhizomes of K. galanga Linn., the highest

content (peak chromatogram area 31.77%) was EPMC (Tewtrakul et al., 2005)

(Table 3). A much higher EPMC content (80 mg %) was reported from the

dichloromethane extract of K. galanga Linn. rhizomes (Sirisangtragul and

Sripanidkulchai, 2011). About 98.98% of essential oil constituents from K. galanga

Linn. have been isolated and identified; the remaining 1.11% constituents are still

unknown (Sutthanont et al., 2010).

It has been reported that EPMC of K. galanga Linn. is involved in

many biological activities including anticancer (Liu et al., 2010; Zheng et al., 1993),

antimicrobial (Kanjanapothi et al., 2004), and anti-monoamine oxidase activities

(Noro et al., 1983). This chemical constituent has been also found to inhibit

proliferation of HepG2 cell line in a dose-dependent manner (Liu et al., 2010).

Mechanistic-based studies using annexin-fluorescein isothiocyanate and propidium

iodide staining showed an increased early apoptotic population in human

hepatocellular carcinoma cells. It was proposed that EPMC induced translocation of

phosphatidyl serine of HepG2 cells to cell surface, resulting in an increase in sub-G

cell population (Liu et al., 2010). A significant anti-inflammatory potential has also

been demonstrated by suppressing interleukin-1, tumor necrosis factor-α (TNF-α),

and angiogenesis by blocking endothelial functions (Umar et al., 2014). EC, EPMC

and p-methoxycinnamic acid from the methanolic extract of K. galanga Linn. also

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showed larvicidal activity against the second stage larva of dog roundworm, Toxocara

canis (Kiuchi et al., 1988). The pharmacological activities of various chemical

constituents identified from K. galanga Linn. including their possible mechanisms of

action are summarized in Table 4.

2.4.2.3 Safety and toxicity

Administration of K. galanga Linn. ethanol extract to rats up to the

dose level of 5,000 mg/kg body weight (acute toxicity test) neither resulted in

mortality nor any significant difference in body and organ weights between control

and test animals (Kanjanapothi et al., 2004). However, in the subacute toxicity testing

at the dose levels of 25, 50, and 100 mg/kg body weight for 28 days, a slight decrease

in the lymphocyte count was observed in male rats.

In a study conducted with the dichloromethane extract of K. galanga

Linn. (100 mg/kg body weight) and EPMC (120 and 160 mg/kg body weight) in mice

for 28 consecutive days of subacute toxicity testing, a significant effect on death,

body or organ weights of mice were not observed in both treated and control groups

(Sirisangtragul and Sripanidkulchai, 2011). Oral administration with the K. galanga

Linn. extract and EPMC for evaluation of its effect on hepatic microsomal

cytochrome P450 (CYP450) enzyme activities demonstrated a reduction in

microsomal P450 content, whereas CYP1A1 and CYP2B activities were increased but

no effect was observed on CYP1A2 and CYP3A4 activity (Sirisangtragul and

Sripanidkulchai, 2011). It has been indicated that more than 50% of conventional

drugs are metabolized via these CYP isoforms (CYP1A1 and CYP2B) (Anzenbacher

and Anzenbacherova, 2001) and the results observed by such study (Sirisangtragul

and Sripanidkulchai, 2011) might suggest certain safety of this plant with regard to

herbal-drug interaction and human risk assessment. Moreover, the slight reduction of

CYP2E1 activity was observed (Sirisangtragul and Sripanidkulchai, 2011) which

could suggest slight anti-carcinogenic activity since CYP2E1 is involved in metabolic

activation of many low molecular weight toxins and carcinogens (such as carbon

tetrachloride, chloroform, methylchloride, and vinylchloride) (Anzenbacher and

Anzenbacherova, 2001).

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Table 2 Phytochemical test results detected in different solvent extracts of K. galanga

Linn. rhizomes (Dash et al., 2014).

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Ethyl-cinnamate (EC) Ethyl-p-methoxycinnamate (EPMC)

Kaempferol Kaempferide

Figure 11 Chemical structures of some important phytoconstituents isolated from K.

galanga Linn. extract (Sutthanont et al., 2010).

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Table 3 Volatile oil components, retention time and peak area (%) of K. galanga

Linn. oil (Tewtrakul et al., 2005).

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Table 4 Summary of possible mechanisms of action associated with the bioactive

constituents of K. galanga Linn. for the specific pharmacological activities.

Pharmacological

activity

Responsible active

Constituent

Possible mechanism of

action

Mosquito

repellant,

larvicidal and

antimalarial

activity

Ethyl p-methoxycinnamate,

ethyl-cinnamate, 3 carene, 2-

propionic acid (Kim et al.,

2008; Sutthanont et al., 2010)

Destruction of ionic

regulation in the anal gills

(Insun et al., 1999)

Analgesic and

anti-inflammatory

…….. Central mechanism involving

opioid receptors and

peripheral mechanism

involving COX

pathway(Ridtitid et al., 2008;

Sulaiman et al., 2008)

Antimicrobial

activity

Ethyl-p-methoxycinnamate

(Kanjanapothi et al., 2004)

……..

Anti-oxidant

activity

Total phenolic content and

flavovoids including luteolin

and apigenin (Mustafa et al.,

2010)

……..

Antihyperglycemic

activity

…… May probably possess an insulin-like effect or stimulate

the pancreatic ß-

cells(Chowdhur et al., 2014)

Antineoplastic

activity

Ethyl-p-methoxycinnamate (Liu

et al., 2010)

Translocation of

phosphatidylserine of Hep G2

cells to cell surface, resulting

in an increase in sub-G cell

population (Liu et al., 2010)

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CHAPTER 3

OBJECTIVES

3.1. General objective

The overall objective of the research dissertation was to evaluate the

safety/toxicity of the two candidate plants for further development as alternative

chemotherapeutics for cholangiocarcinoma (CCA). These included the CMC

(chemistry, manufacturing and control) oral pharmaceutical formulation of the crude

ethanolic extract of A. lancea (Thunb.) DC. (AL) rhizomes prepared for first-in-human

clinical study. The safety profile and anti-CCA activity of the crude ethanolic extract

of this plant has previously been shown in a series of in vitro and in vivo model

(Mahavorasirikul et al., 2010; Plengsuriyakarn et al., 2012). In addition, anti-CCA

activity of the crude ethanolic extract of K. galanga Linn. (KG) leaves was initially

evaluated in vitro models (Mahavorasirikul et al., 2010).

3.2 Specific objectives

3.2.1 To evaluate potential of toxicity of the CMC oral pharmaceutical

formulation of the crude ethanolic extract of AL rhizomes:

3.2.1.1 To evaluate the acute toxicity of the CMC oral pharmaceutical

formulation of AL in rats;

3.2.1.2 To evaluate the subchronic (90 days repeated dosing) toxicity

of the CMC oral pharmaceutical formulation of AL in rats;

3.2.2 To evaluate potential cytotoxic activity against CCA, toxicity, and

anti-CCA activity of the crude ethanolic extract of KG rhizomes:

3.2.2.1 To standardize the extraction procedure for preparation of the

crude ethanolic extract of KG rhizomes;

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3.2.2.2 To evaluate cytotoxic activity of the KG rhizome extract in

CCA cell line (CL-6);

3.2.2.3 To evaluate potential of toxicity (acute and subacute toxicity)

of the crude ethanolic extract of KG rhizomes in mice; and

3.2.2.4 To evaluate the anti-CCA activity of the KG rhizome extract in

CCA-xenografted BALB/c nude mouse model.

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CHAPTER 4

RESEARCH METHODOLOGY

4.1. Reagents and cell lines

The solvent used for plant extraction (95% ethanol) was commercial

grade which was obtained from Labscan Asia Co. Ltd. (Bangkok, Thailand). HPLC

grade methanol and distilled water were obtained from Fisher Scientific

(Leicestershire, UK). The cell culture reagents including RPMI 1640, DMEM, fetal

bovine serum (FBS), phosphate buffered saline (PBS), penicillin and streptomycin

antibiotics were purchased from Gibco Life Technologies (NY, USA). The CCA cell

line (CL-6) used for both in vitro and in vivo study was kindly provided by Associate

Professor Adisak Wongkajornsilp, Department of Pharmacology, Faculty of

Medicine, Siriraj Hospital, Bangkok, Thailand. The normal fibroblast cell line used

(OUMS) was obtained from JCRB cell bank (Osaka, Japan). Methyl thiazoldiphenyl

tetrazolium (MTT) reagent was obtained from Life Technologies (CA, USA) and

dimethyl sulfoxide (DMSO) was obtained from MP Biomedicals (CA, USA).

Atractylodin (standard compound) and 5-FU were purchased from Wako Pure

Chemical Industries Ltd. (Osaka, Japan) and tween-80 was purchased from Sigma-

Aldrich (MO, USA). Neutral buffered formalin (NBF) used for organs/tissue fixation

was purchased from Bio-Optica (Milano, Italy). Isoflurane used for euthanasia was

purchased from Minrad Inc. (PA, USA). All other remaining chemicals and reagents

were purchased from commercial suppliers and were of high purity grade. The

standard ethyl-p-methoxycinnamate (EPMC) was kindly supplied by Dr. Sumet

Kongkiatpaibo, Drug Discovery and Development Center, Thammasat University,

Thailand.

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4.2 Preparation of test materials

4.2.1 Chemical, manufacturing and control (CMC) oral

pharmaceutical formulation of Atractylodes lancea (Thunb.) DC.

The CMC oral pharmaceutical formulation of the crude ethanolic extract

of AL rhizomes was prepared by Ms. Thananchanok Rattanathada, the Center of

Excellence in Pharmacology and Toxicology and Molecular Biology of Malaria and

Cholangiocarcinoma. The AL powder formulation was prepared as AL crude extract

(from AL rhizomes) mixed with lactose (25% w/w) and the methods used for

extraction and preparation of the AL formulation included maceration, rotary

evaporation, and freeze-drying.

4.2.2 Kaempferia galanga Linn.

The dried and powdered rhizomes of the plant K. galanga Linn. (KG) were

obtained from Thailand, Nakhon Pathom Province. Authentication of the plant

material/rhizome was carried out at the herbarium of the Department of Forestry,

Bangkok, Thailand, where the herbarium voucher has been kept (Mahavorasirikul et

al., 2010).

The plant rhizome powder was extracted by sonication, heating under

reflux, and maceration (1, 3, and 7 days) using 95 % ethanol to obtain the optimized

method for preparation of the extraction with high and consistent yield. For the

sonication method, a sample powder of about 10 g was mixed with 95% ethanol (300

ml) and sonicated with the ultrasonicator (Meditop, Bangkok, Thailand) for 75

minutes. The plant rhizomes of about 10 g in 300 ml of 95% ethanol was also used for

the heating under reflux method in a water bath (MemmertTM, Schwabach, Germany)

for about 50 minutes according to the previously described methods with modification

(Khoddami et al., 2013; Sasidharan et al., 2011).

For the maceration method, the powdered KG rhizomes (10 g) was mixed

with 300 ml of 95% ethanol in a small glass flask and stored at 25 oC for 1, 3, and 7

days. Later on, maceration method for 7 days was optimized and about 1,000 g

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powdered rhizomes was added in each three large clean flasks containing 5 liters of

95% ethanol for 7 days at 25-30 °C with occasional shaking and stirring. The extracts

were separated first (filtered) with gauze and then through Whatman no. 1 filter paper

(GE HealthcareTM, Maidstone, England). The residues were remacerated for

additional 24 h and then filtered as indicated above. The extract solvent from was

evaporated using a rotary evaporator (HeidolphTM, Schwabach, Germany) and further

concentrated by heating at 60 oC in a water bath (Builders et al., 2011). The crude

extracts obtained using different methods were weighed and the median (range)

percent yields were calculated. The extracts were stored in small glass bottles

(covered with aluminum foil) at -20 °C until used (Figure 12) to ensure the apparent

stability and activity (Builders et al., 2011; Sasidharan et al., 2011).

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K. galanga Linn. (KG) rhizomes

Maceration in 95% of ethanol for 7 days, followed by remaceration for 24 hours

Filtration through gauze and Whatman no. 1 filter paper

Concentration with rotary evaporator (at 60oC) and then in a water bath

Storage in small glass bottles (covered with aluminum foil) at -20 oC until use

Figure 12 A diagram showing extract preparation using 7 day maceration of the KG rhizomes.

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4.3 Identification of marker compounds

4.3.1 CMC oral pharmaceutical formulation of Atractylodes lancea

(Thunb.) DC.

Atracylodin was used as a marker compound for quality control of the

preparation process for preparation of the AL extract and CMC oral pharmaceutical

formulation (Ji et al., 2001; Ouyang et al., 2012). The stock solution was prepared by

accurately weighing and dissolving the compound in methanol to obtain the final

concentration of 1,000 µg/ml. Working solutions at desired concentrations were

prepared by diluting the stock standard solution with methanol. The AL rhizome

extract CMC oral pharmaceutical formulation sample solution was also prepared by

accurately weighing 100 mg of each sample and dissolved in methanol (5 ml). To

enable complete dissolution, each sample was sonicated for 60 min (triplicate each).

Prior to injection onto the HPLC system, each solution was filtered through a 0.22 µm

nylon membrane filter. Semi-quantitative determination of atractylodin content of the

extract CMC oral formulation was performed by using high performance liquid

chromatography (HPLC) according to the previously described procedures

(Kongkiatpaiboon et al., 2015). The HPLC system consisted of an Agilent 1260 series

equipped with a 1260 quaternary pump, 1260 ALS autosampler, 1260 TCC column

thermostat, and 1260 DAD detector (Agilent Technologies, CA, USA). Sample

separation was achieved on a Hypersil BDS C18 column (4.6 x 100 mm i.d., 3.5 µm)

with a C18 guard column (Thermoschientific, MA, USA) and the mobile phase

consisting of (A) water and (B) methanol using gradient elution: 50% B in A to 100%

B for 15 min; 100% B for 10 min according to the previously described procedures

with modification (Kongkiatpaiboon et al., 2015. The column was equilibrated with

50% B in A for 10 min prior to each analysis and the flow rate was set at 1.0 ml/min

with controlled temperature at 25C. Sample injection volume was 5 µl and UV

detection was set at the wavelength of 360 nm for the analysis.

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Ethyl-p-methoxycinnamate (EPMC) which is the major bioactive

chemical constituent of the KG extract (Sirisangtragul and Sripanidkulchai, 2011;

Tewtrakul et al., 2005) was used as a marker compound for quality control of the

preparation process of the KG rhizome extract. Semi-quantitative determination of

EPMC content of the extract was performed using HPLC according to the previously

described method with modification (Sirisangtragul and Sripanidkulchai, 2011). The

extract and the standard EMPC were prepared at the concentrations of 20 and 10

µg/ml, respectively. The HPLC system used consisted of HPLC instrument 1200

series (Agilent Technologies, CA, USA), and photo diode array detector (PDA)

detector (Thermoschientific, MA, USA); and sample separation was performed using

the ThermoscientificTM Hypersil Gold Column (250 x 4.6mm i.d., 5 µm particle size,

reversed phase C18) with an isocratic mobile phase consisting of a mixture of

methanol and distilled water (54:46% v/v) at a flow rate of 1 ml/min. Sample

injection volume was 10 μl , the column temperature was maintained at 250C and UV-

detection was set at 270 nm for this analysis.

4.4 Animal stocks, handling and ethical approval

The toxicity (acute and 90-days subchronic toxicity) testing of the CMC

oral pharmaceutical formulation of the crude ethanolic extract of AL rhizomes was

evaluated in Wistar albino (strain Crl:W1 or Han) rats of both genders (6-9 weeks of

age, and weighing 160-400g). The toxicity testing (acute and subacute toxicity) of the

crude ethanolic extract of KG rhizomes was evaluated in ICR (Imprinting Control

Region) male and female mice, 6-7 weeks of age, weighing 26-40 g. The anti-CCA

activity of the KG rhizome extract was evaluated in BALB/c nude mice of both

genders (7-8 weeks of age, and weighing 15-24 g). All the animal stocks were

obtained from the National Laboratory Animal Center, Thailand. Animals were

housed under standard conditions and acclimatized for abou

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