Evaluation of Three Plant Extracts Activity to the Stopping of …عاليات... · 2020-05-13 · Chapter One: Introduction and Literature review 0-0 Introduction 0 ... Chapter

Republic of Iraq

Ministry of Higher Education &

Scientific Research

University of Baghdad

College of Science

Evaluation of Three Plant Extracts Activity

to the Stopping of Bleeding in Albino Mice

A Thesis

Submitted to the Biology Department

College of Science


In Partial Fulfillment of the Requirements for the Degree of

Master of Science in Biology

By

Zahraa Abdul Elah M.A. Al-Naqqash

B.Sc. in Biology, 2002

Supervised

By

Dr. Abdul-Latif M. Jawad Dr. Ayyad W. Raof

Professor Assist. Professor

3102 A. D. 0121 A. H.

يقاف نزيفإثالث مستخلصات نباتية في فعاليةم يتقي

الفئران البيض الدم في

جامعة بغداد –رسالة مقدمة إلى كلية العلوم

وهي جزء من متطلبات نيل درجة ماجستير علوم الحياة

من قبل

زهراء عبداالله محمدعلي النقاش

4228ة، الحيا بكالوريوس علوم

بإشراف

االستاذ المساعد االستاذ

د. أياد وجيه رؤوف اللطيف محمد جواد د. عبد

هـ 3656

م 4235

جمهورية العراق

وزارة التعليم العالي والبحث العلمي

جامعة بغداد / كلية العلوم

قسم علوم الحياة

الهذيه آمنوا منكم والهذيه أوتوا العلم يزفع للاه

بما تعملون خبيز ج درجات وللاه

صدق هللا العلي العظيم { ((00االية )) -سورة المجادلة }

اإلهــــــــــــــداء

الى ساقي عطاشى كربالء رمز

التضحية والفداء

االمام العباس

السالم عليه

Acknowledgements

First of all dear GOD thanks for all the blessings which lightened my way in

life and encouraged me to accomplish this study.

I would like to show my appreciation and thanks to the dedicated

researcher, honorable person who bare a remarkable passion to science, my

supervisor Professor Dr. Abdul-Latif M. Jawad, Department of Biology,

College of Science, University of Baghdad for his enormous work helping

me editing my research for which he literally read this thesis word by word

and follow my work day by day with the same momentum till the end of it.

Also I would like to show my gratitude and respect to my second supervisor

Assistant Professor Dr. Ayyad W. Raof, Department of Biology, College of

Science, University of Baghdad for his great scientific help and kindness.

For all that I wish them all the happiness in their life.

I would like to show my appreciation and gratitude to Dr. Salah Mahdi

Muhsen, Biotechnology Research Center, Al-Nahrain University for his

magnificent help and guidance during the period of my study, also I would

like to thank Dr. Farah Ali Husain FICMS/ Patho-hematology, Baghdad

medical city for her great help with the platelets count test and special

thanks to Dr. Hind H. Obaid Department of Biology, College of Science,

University of Baghdad for her moral support. I would like to show great

gratitude to my friend Roaa Mohamad Hassan for helping me with every

possible aid can offer and also my dear friend Safa Hisham Al-Ibrahimi for

her scientific and moral support.

Finally I would like to thank my father´s soul for inspiring me all the time,

my dear mother for her great blessings, my dear brothers for their moral

and financial support and no words can express my gratitude to my beloved

supportive husband Estabraq and lastly thanks to my little angel Ruqaia for

just being in my life.

Zahraa

SUPERVISORS CERTIFICATION

We certify that thesis entitled (Evaluation of Three Plant Extracts Activity

to the Stopping of Bleeding in Albino Mice ) was prepared by (Zahraa

Abdul Elah M.A. Al-Naqqash) under our supervision at the College of

Science, University of Baghdad, as a partial fulfillment of requirements for

the degree of master in Science of Biology/ Botany (plant physiology).

Supervisors


Professor/ Phycology Assistant Professor/ Plant Physiology

Department of biology Department of biology

College of Science College of Science

University of Baghdad University of Baghdad

In view of the available recommendation, we forward this thesis for debate

by the examining committee.

Dr. Sabah N. Alwachi

Professor

Head of Biology Department

College of Science


Committee´s Certification

We, the examining committee, certify that we have read this thesis and

have examined the student Zahraa Abdul Elah M.A. Alnaqqash in its

contents and that in our opinion it is adequate with good standing as a thesis

for the Master degree in Biology (Botany).

Dr. Sabah N. Alwachi

Professor

Chairman

32 /3/3102

Dr. Abdul-Kareem Jasim Dr. Salah Mahdi Muhsen

Assist. Professor

Member Member

32/ 3/3102 32/3 /3102


Professor Assist. Professor

Adviser Adviser

32 /3 /3102 32/3 /3102

Approved for the College of the committee of graduate studies.

Prof. Dr. Saleh M. Ali

Dean

College of Science

Baghdad University

/ / 3102

DECLARATION

This is to certify that the dissertation / thesis entitled:

"Evaluation of Three Plant Extracts Activity to the Stopping of

Bleeding in Albino Mice"

Submitted by: ZAHRAA ABDULELAH M.A. AL-NAQQASH

Department: BIOLOGY

College: SCIENCE

Has been linguistically corrected and its language in its present form is

acceptable.

Name: DR. NAZAR AZIZ AUDA

Address: Department of Biology, College of Science, University of

Baghdad.

Signature

List of contents Subject Page No. Chapter One: Introduction and Literature review

0-0 Introduction 0

0-3 Literature review 1

0-3-0 Active Components in Medical Plants 1

0-3-0-0 Alkaloids 1

0-3-0-3 Phenols 5

0-3-0-2 Terpens 3

0-3-3 Zingiber officinale Roscoe 3

0-3-3-0 Plant Description 3

0-3-3-3 Plant Common Names 8

0-3-3-2 Plant Distribution over the World 8

0-3-3-1 Plant Chemical Composition 8

0-3-3-5 Plant Medicinal Uses 01

0-3-2 Thymus vulgaris L. 02



0-3-2-2 Plant Distribution Over the World 05



0-3-1 Acacia arabica L. 02



0-3-1-2 Plant Distribution Over the World 08



0-3-5 Haemostasis (the stopping of bleeding) 30

Chapter Two: Materials and Methods

3-0 Chemicals and Apparatus 35

3-0-0 Chemicals 35

3-0-3 Apparatus 33

3-3 Collection of Plant Samples 32

3-2 Preparation of Plant Extracts 38

3-1 Preparation of different concentrations of plant extracts 21

3-5 Compounds' Detection 21

3-3 Haematological Tests 23

3-3-0 Haematological Test in vivo 23

3-3-3 Bleeding Time Measurement in vivo 23

3-3-2 Clotting Time Measurement in vitro 23

3-3-1 Platelets Count 22

3-2 Statistical Analysis 22

Chapter Three: Results

2-0 Plants Extract Preparation 23

2-3 Haematological Tests: Bleeding time, Clotting time &

platelets count

22

2-3-0 Effect of Alkaloids Extract in Bleeding Time, Clotting

Time and Platelets Count

22

2-3-3 Effect of phenols Extract in Bleeding Time, Clotting Time

and Platelets Count

28

2-3-2 Effect of Terpens Extract in Bleeding Time, Clotting Time

and Platelets Count

13

Chapter Four: Discussion

1-0 Plant extracts yield and their chemical constituents 13

1-3 Haematological Tests: Bleeding time, Clotting time &

platelets count

18

1-3-0 Effect of Alkaloids Extract in Bleeding time, Clotting time

and platelets count

18

1-3-3 Effect of Phenols Extract in Bleeding time, Clotting time

and platelets count

50

1-3-2 Effect of terpens Extract in Bleeding time, Clotting time

and platelets count

52

Conclusions and Recommendations

Conclusions 53

Recommendations 52

References 58

List of Tables

Table No. Title Page

No.

(3-0) The apparatus used in this study. 32

(3-3) The chemicals used in this study. 31

(2-0) Compounds yielded from plant parts expressed as % 23

(2-3) Zingiber officinale crude alkaloids extract effect on

the tested blood characteristics by using different

concentrations

22

(2-2) Thymus vulgaris crude alkaloids extract effect on the

tested blood characteristics by using different

concentrations

25

(2-1) Acacia arabica crude alkaloids extract effect on the


concentrations

23

(2-5) Zingiber officinale crude phenols extract effect on the


concentrations

28

(2-3) Thymus vulgaris crude phenols extract effect on the


concentrations

21

(2-2) Acacia arabica crude phenols extract effect on the


concentrations

10

(2-8) Zingiber officinale crude terpens extract effect on the


concentrations

13

(2-1) Thymus vulgaris crude terpens extract effect on the


concentrations

11

List of Figures Figure & plate

No.

Title Page

No.

Figure (0) Zingiber officinale Roscoe plant 2

Figure (3) zingiberene (A), gingerols (B) and shogaols (C)

structures

1

Figure (2) Thymus vulgaris: (A) plant in bloom, (B) leaf seen

from under surface, magnified 1 diam., (C) flower

seen from the side, magnified 5 diam.

01

Figure (1) principal component of Thymus vulgaris 05

Figure (5) Acacia arabica plant 08

Figure (3) Zingiber officinale Roscoe dry rhizomes 35

Figure (2) Thymus vulgaris dry leaves 35

Figure (8) Acacia arabica dry gum 33

Figure (1) The effect of Zingiber officinale crude alkaloids

extract of three concentrations on bleeding time

and clotting time.

21

Figure (01) The effect of Zingiber officinale crude alkaloids

extract of three concentrations on platelets count.

21

Figure (00) The effect of Thymus vulgaris crude alkaloids


and clotting time.

25

Figure (03) The effect of Thymus vulgaris crude alkaloids


23

Figure (02) The effect of Acacia arabica crude alkaloids


and clotting time.

22

Figure (01) The effect of Acacia arabica crude alkaloids


22

Figure (05) The effect of Zingiber officinale crude phenols


and clotting time.

28

Figure (03) The effect of Zingiber officinale crude phenols


21

Figure (02) The effect of Thymus vulgaris crude phenols


and clotting time.

11

Figure (08) The effect of Thymus vulgaris crude phenols


11

Figure (01) The effect of Acacia arabica crude phenols extract

of three concentrations on bleeding time and

clotting time.

10

Figure (31) The effect of Acacia arabica crude phenols extract

of three concentrations on platelets count.

13

Figure (30) The effect of Zingiber officinale crude terpens


and clotting time.

12

Figure (33) The effect of Zingiber officinale crude terpens


12

Figure (32) The effect of Thymus vulgaris crude terpens


and clotting time.

11

Figure (31) The effect of Thymus vulgaris crude terpens


15

List of Abbreviations

Abbreviation Key

ABS Ankaferd Blood Stopper

ADP Adenosine Diphosphate

BT Bleeding Time

CT Clotting Time

GA Gallic acid

SCA Spontaneous contractile activity

Abstract This study included the identification of extracted crude alkaloids,

phenols and terpenes compounds from dry rhizomes of Zingiber officinale

Roscoe, dry leaves of Thymus vulgaris L. and dry gum of Acacia arabica

L. Evaluation of their activity as a coagulant factor through observing the

changes occurred on bleeding time, clotting time and platelets count in mice

under study were determined.

Results showed that alkaloids yielded from Z. officinale dry rhizomes

extract were the highest weight percentage; while terpens extracted from T.

vulgaris dry leaves were the highest yield percentage. However phenols

extracted from A. arabica dry gum were the highest.

In this study seventy five male lab mice from balb-c breed with average

body weight of (2±82) gm were divided randomly into twenty five groups

treated with three concentrations (0mg/ml, 5mg/ml, 01 mg/ml) for each

plant extract with a single dose (1.1ml) once a day for seven days

successively. Then the mice undergone bleeding time, clotting time and

platelets count tests to observe the changes made by the given extracts.

In general the results showed significant differences at (P<.0.5) in the

parameters of the test; bleeding time and clotting time were decreased

significantly compared with the control while platelets count increased

significantly compared with control. Crude alkaloids extract of Z. officinale

was most effective at (01 mg/ml) concentration comparing with other

compounds.

While crude terpens extract of T. vulgaris dry leaves was the most

effective at (01 mg/ml) with a slight difference followed by crude terpenes

extract of Z. officinale dry rhizomes at (01 mg/ml). However crude phenols

extract of A. arabica dry gum was the most effective at (0 mg/ml) on the

tested parameters compared with the control and also in comparison with

plants under study because results showed higher yield percentage and the

most effective concentration was 0mg\ml which make crude phenols extract

of A. arabica the best plant product pharmaceutically and commercially.

Chapter one

Introduction

&

Literature Review

3-3 Introduction

One of God's graces on human kind is the creation of plants to be a

source of food and medicine. Based on that fact, mankind directed his

thinking to get full benefit from what God provided him. So human

invented many ways to use these plants as a source of medicine since

ancient times and managed to create the first pharmacy that hold various

remedies for different kind of illnesses. Those primitive storage places

(what now a day called pharmacies) hold roots, leaves, seeds, weeds and

other plants products as medicine (Akeil, 2..6).

Medicinal plants contain some organic compounds which produce

definite physiological action on the human body and these bioactive

substances include tannins, alkaloids, carbohydrates, terpenoids, steroids

and flavonoids (Edeoga et al., 2..5). Many of these natural products have

vital roles as mediators of ecological interactions; that is, they have

functions in ensuring a continued survival of particular organisms in often

hostile environments where there is competition with other organisms

(Mann, 879±). Such roles include being attractant to pollinators, allelo-

pathic agents or defense against predators and pathogens. For example,

ipsdienol, a major constituent of the floral fragrance of several orchid

species and azadichtin, present in Azadiracta indica, have roles as

attractant to bees and defense mechanism against insects respectively (Hill,

87±5; Swaminathan and Kochhar, 87±7).

Medicinal plants are of great importance to the health of individuals

and communities (Edeoga et al., 2..5). Many of these indigenous

medicinal plants were used as spices and food plants. They were

sometimes added to foods meant for pregnant and nursing mothers for

medicinal purposes (Okwu, 8777; Okwu, 2..8). Medicinal plants are

generally used in traditional medicine for the treatment of many ailments

(Ogukwe et al., 2..4; Njoku and Ezeibe, 2..9). From the plants that have

been used medicinally for a long time ago till now Zingiber officinale

Roscoe, Thymus vulgaris L. and Acacia Arabica L.

Zingiber officinale Roscoe which belongs to the family zingiberacea is

used worldwide for different purposes such as a cooking spice, condiment

and herbal remedy. The Chinese have used ginger for at least 25.. years as

a digestive aid, antinausea remedy, treat bleeding disorders and

rheumatism; it was also used to treat baldness, toothache, snakebite, and

respiratory conditions (Duke and Ayensu 87±5). Thymus vulgaris L. which

belongs to the family lamiaceae is also one of the plants that have many

uses. It adds a distinctive aromatic flavoring to sauces, stews, stuffing,

meats, and poultry; it possesses antispasmodic, antiseptic, expectorant,

carminative and anti-oxidative properties (Omidbaigi and Nejad 2...;

Dapkevicius et al., 2..2). Acacia arabica L. belongs to the family fabaceae;

Acacia seeds were often used for food and a variety of other products. It

was used as an effective medicine for diarrhea (Spicer et al., 2..9).

Because the three above mentioned plants also share their usage as

hemostatic drugs (coagulant agent) as they proved their activity in the

control of bleeding due to gastrointestinal cancers, mediastinal bleeding,

hemorrhage after retro pubic radical prostatectomy and post-tonsillectomy

bleeding (Baykul et al., 2.8.; Ercetin et al.,2.8.; Tuncer et al., 2.8.).

Aim of the study:

This study was conducted to:

8- Detect the presence of major chemical compounds (alkaloids,

phenols and terpens) in Zingiber officinale Roscoe, Thymus vulgaris L.

and Acacia arabica L. by extracting them specifically.

2- Determine the yield percentage of each active group in the plants

under the study.

3- Check if there are any coagulant properties in the extracts and which

plant has higher coagulant effect.

3-4 Literature Review

3-4-3 Active Components in Medical Plants

Plants produce high diversity of secondary metabolites with a

prominent function of protecting plants against predators and microbial

pathogens due to their biocidal properties against microbes or repellence

to herbivores. Some metabolites were also involved in defense mechanisms

against a biotic stress (e.g., UV-B exposure) and are important in the

interaction of plants with other organisms (e.g., attraction of pollinators)

(Rosenthal, 8778; Schafer and Wink, 2..7). It was believed that most of the

8..,... known secondary metabolites were involved in plant chemical

defense systems, they seemed to be appeared as a response of plants to

the interactions with predators throughout the millions of years of co-

evolution (Wink, 8777). Secondary metabolite compounds were divided

into three main groups which are: Alkaloids, Phenols and Terpenes

(Goodwin and Mercer, 87±3).

3-4-3-3 Alkaloids

Alkaloids rank among the most efficient and therapeutically significant

plant substances (Okwu, 2..5). Five thousands and five hundreds of

alkaloids were known and they comprise the largest single class of

secondary plant substances which contain one or more nitrogen atoms,

usually in combination as part of a cyclic structure (Harborne, 8793). They

are usually organic bases and form salts with acids and when soluble gives

alkaline solutions. Examples include nicotine, cocaine, morphine and

codeine (Papaver sominferum), quinine (Cinchona succirubra), reserpine

(Rauwolfia vomitoria), which has a large demand worldwide. Alkaloid

production is a characteristic of all plant organs. They exhibit marked

physiological activity when administered to animals (Okwu and Okwu,

2..4). Furthermore, alkaloids are often toxic to man and many have

dramatic physiological activities, hence their wide use in medicine was for

the development of drugs (Harborne, 8793; Okwu, 2..5). Alkaloids are

usually colorless, but often optically active substances. Most are crystalline

but a few are liquid at room temperature. Alkaloids have bitter tastes. The

alkaloid quinine for example is one of the bitterest tasting substances

known and is already significantly bitter at a molar concentration of 8x8.-5

(Harborne, 8793). Pure, isolated plant alkaloids and their synthetic

derivatives are used as basic medicinal agents for their analgesic, anti

spasmodiac and bactericidal effects (Stary, 877±). Quinine with a molecular

formula of C2.H24N2O2 is an anti-malarial drug extracted from the bark of a

cinchona tree (C. succirubra). Quinine is highly valued in the treatment of

unusually resistant strains of malaria.

3-4-3-4 Phenols

Phenols, sometimes called phenolics, are a class of aromatic organic

compounds consisting of one or more hydroxyl groups attached to an

aromatic hydrocarbon group (Scott, 2..9). Phenol is a benzene derivative

and is the simplest member of the phenolic chemical. Its chemical formula

is C6H5OH and its structure is a hydroxyl group (-OH) bonded to a phenyl

ring.

The presence of phenols is considered to be potentially toxic to the

growth and development of pathogens (Okwu and Okwu, 2..4). The

structural classes of phenolic compounds include the polyphenolic

(hydrolysable and condensed tannins) and monomers such as ferulic and

catechol (Okwu, 2..5). Polyphenols might interfere in several of the steps

that lead to the development of malignant tumors, may play a role in

inactivating carcinogens and inhibiting the expression of mutagens

(Urquiaga and Leighton, 2...). The types of phenols were flavonoids,

tannins and coumarins.

3-4-3-5 Terpenes (Terpenoids)

The terpenes are a structurally diverse and widely distributed family of

natural products containing well over 3.,... defined compounds identified

from all kingdoms of life (Buckingham, 877±). The majority of terpenes

have been isolated from plants where they serve a broad range of roles in

primary metabolism (including several plant hormones and the most

abundant plant terpenoid, phytol, the side chain of the photosynthetic

pigment chlorophyll) and in ecological interactions (as chemical defenses

against herbivores and pathogens, pollinator attractants, allelopathic

agents, etc.). Many terpenes are of economic importance, including the

essential oils, carotenoid pigments and natural rubber (Davis and Croteau,

2...). Terpenoids are synthesized from acetate units, and as such they

share their origins with fatty acids. They differ from fatty acids is that they

contain extensive branching and cyclized (Cowan, 8777). Terpenes were

classified into four groups: Saponins, Glycosides, Resins and Volatile

oils.

3-4-4 Zingiber officinale Roscoe

3-4-4-3-Plant description:

Zingiber belongs to the group of Monocotyledon plants which belongs

to the zingiberacea family and for its importance it calls zingiber family;

Linnaeus was the first scientist who described this plant. In 8±.9, the

English botanist William Roscoe (8953-8±38) gave the plant the name

Zingiber officinale Roscoe (Foster, 2...).

As shown in (Fig. 8) it is a perennial herb with a subterranean digitately

branched rhizome producing stems up to 805.m in height with linear

lanceolate sheathing leaves (5–3.cm long and ±–2. mm wide) that are

alternate, smooth and pale green. Flower stems shorter than leaf stems

and bearing a few flowers, each surrounded by a thin bract and situated in

axils of large, greenish yellow obtuse bracts, which are closely arranged at

end of flower stem forming collectively an ovate-oblong spike. Each flower

shows a superior tubular calyx, split part way down one side; an orange

yellow corolla composed of a tube divided above into 3 linear oblong, blunt

lobes; 6 staminodes in 2 rows, the outer row of 3 inserted at mouth of

corolla; the posterior 2, small, horn-like; the anterior petaloid, purple and

spotted and divided into 3 rounded lobes; an inferior, 3-celled ovary with

tufted stigma. Fruit is a capsule with small arillate seeds (Youngken, 875.;

Keys, 8796).

Figure (3) Zingiber officinale Roscoe plant

http://www.en.wikipedia.org

3-4-4-4 Plant Common Names

The name of the genus, Zingiber, derives from a Sanskrit word denoting

"horn-shaped," in reference to the protrusions on the rhizome (Foster,

2... ( . Zingiber has different names over the world and the most popular

one is ginger. It called Sheng Jiang in China, in Spain gengiber, in France

http://www.en.wikipedia.org/

gingermber, in Poland imbir lekarski, in Denmark and Norway ingefaer; in

Indonesia jahe; in Greece ziggiber (Leonard, 2...) and in Iraq it called

zangabel or erk har (Al-zubaidy et al., 8776).

3-4-4-5 Plant Distribution over the World

The plant grows in moist fertile soils under abundant shadows in the

tropics (Evans, 87±7). Native to southern Asia now cultivated extensively in

almost all tropical and subtropical countries, especially China, India,

Nigeria, Australia, Jamaica, and Haiti (Grieve, 8797; Reineccius, 8774;

Bruneton, 8775; Budavari, 8776; Leung and Foster, 8776). In Iraq it was

imported from the tropics and sold in the herbarium markets and shops all

over the country (Townsend and Guest, 87±5).

3-4-4-6 Plant Chemical Composition

Zingiber officinale contains a number of antioxidants such as beta-

carotene, ascorbic acid, terpenoids, alkaloids, and polyphenols such as

flavonoids, flavones glycosides, rutin, etc. (Aruoma et al., 8779).

The active ingredients in ginger are thought to reside in its volatile oils,

which comprise approximately 8-33 of its weight (Newall et al., 8776). The

major active ingredients in ginger oil are the sesquiterpenes: bisapolene,

zingiberene, and zingiberol (Connell and Sutherland, 8767; Yoshikawa et

al.,8773); it also contains other types of sesquiterpenes such as

sesquiphellandrene and curcurmene and phenolic compounds such as

shogaols and gingerols , the shogaols are formed from gingerols when

ginger is dried or cooked. Zingerone is also produced from gingerols during

this process, and it is less pungent and has a sweet aroma (Malu et al.,

2..±). Other compounds were found in zingiber such as 6-

dehydrogingerdione, galanolactone, gingesulfonic acid, zingerone, geraniol,

neral, monoacyldigalactosylglycerols and gingerglycolipids (Kemper, 8777).

The compounds 6-gingerol and 6-shogaol have been shown to have a

number of pharmacological activities, including antipyretic, analgesic,

antitussive, and hypotensive effects (Suekawa et al., 87±4). Representative

structures of zingiberene, gingerols and shogaols are presented in the Fig

(2) below.

(A)

(B)

(C)

Figure (4) zingiberene (A), gingerols (B) and shogaols (C) structures

3-4-4-7 Plant Medicinal Uses

Ginger has been used as a medicine since ancient times, recorded in

early Sanskrit and Chinese texts and ancient Greek, Roman, and Arabic

medical literature (Bone, 8779). In Ayurveda, it has been recommended for

use as carminative, diaphoretic, antispasmodic, expectorant, peripheral

circulatory stimulant, astringent, appetite stimulant, anti-inflammatory

agent, diuretic and digestive aid (Johri and Zutshi, 8772).

The anti-inflammatory properties of ginger have been known and

valued for centuries. The original discovery of ginger's inhibitory effects on

prostaglandin biosynthesis in the early 879.s has been repeatedly

confirmed. This discovery identified ginger as an herbal medicinal product

that shares pharmacological properties with non-steroidal anti-

inflammatory drugs. Ginger is a strong anti-oxidant substance and may

either mitigate or prevent generation of free radicals. It is considered a safe

herbal medicine with only few and insignificant adverse/side effects

(Bhargava et al., 2.82).

In Malaysia, it has been used as a food and medicinal plant for over

2... years for treating diabetes, high blood pressure, cancer and many

other illnesses (Ghasemzadeh et al., 2.8.). In recent studies, ginger

varieties have been reported as a good potential source for anti-cancer,

anti-microbial and anti-inflammation (Mohd Habib et al., 2..±); and the

aromatic, spasmolytic, carminative, and absorbent properties of ginger

suggest it has direct effects on the gastrointestinal tract (Tyler, 87±6).

Ginger has long been used as a remedy to decrease nausea and

vomiting associated with several conditions (Lien et al., 2..3); also it is

used to ameliorate symptoms of nausea in pregnancy (Blumenthal, 2..3).

Studies indicate that ginger is definitely an anti-hyperlipidemic agent

(Bhandari et al., 877±) and is used as a stimulant, carminative, and was

used frequently for drypepsia and colic; also ginger has a sialagogue action,

which stimulates the production of saliva, and could be used to disguise the

taste of medicines. The gingerols could make ginger available for treatment

of stomach acidity and may have analgesic and sedative properties (O’Hara

et al., 877±).

Ginger has been found effective by multiple studies for treating nausea

caused by seasickness, morning sickness and chemotherapy (Ernst and

Pittle, 2...). Ginger has been reported to be effective for the treatment of

inflammation, rheumatism, cold, heat cramps, and diabetes (Al-Amin,

2..6; Afshari, 2..9) and it was also reported that sesquiterpenoids are the

main component of ginger which attributes its antibacterial activity (Malu

et al., 2..±). Ginger promotes the release of bile from the gallbladder

(Opdyke, 8794; Kato et al., 8773; O’Hara et al., 877±). Ginger may also

decrease joint pain from arthritis, may have blood thinning and cholesterol

lowering properties and may be useful for the treatment of heart diseases

and lungs diseases (Opdyke, 8794; Kato et al., 8773; O’Hara et al., 877±;

Kuschener and Stark, 2..3).

Zingiber has been shown to have an antipyretic, antitussive,

hypotensive

(Mamoru et al., 87±4) ,cardiotonic (Kobayashi et al., 87±9), antiplatelet

(Guh et al., 8775), antiangiogenic (Kim et al., 2..5), anti-inflammatory and

analgesic (Young et al., 2..5), cytotoxic, apoptotic (Wei et al., 2..5),

antitumor (Surh et al., 8777 ), anticancer (Dorai and Aggarwal, 2..4),

antioxidant (Masuda et al., 2..4 ), antihepatotoxic (Hikino et al., 87±5),

antifungal (Ficker et al., 2..3), vanilloid receptor agonistic (Dedov et al.,

2..2 ), cholagogic (Yamahara et al., 87±5) and antiemetic (Kawai et al.,

8774) activities.

Ginger has been noted to treat migraine headaches without side-effects

(Mustafa and Srivastava, 877.); it has also been used to relieve symptoms

of colds and arthritis due to its anti-inflammatory properties (Kapil et al.,

877.). Previous studies on the antioxidant properties of various ginger

species had been confined only to the rhizomes (Ying et al., 2..2; Yingming

et al., 2..4) which have been reported to have tyrosinase inhibiting

properties (Lee et al., 8779). Recently, skin-lightening cosmeceutical

products have been developed from the rhizomes of gingers (Rehman et

al., 2..3). Gallic acid (GA) which is one of the ginger chemical constituents

was reported as a free radical scavenger and as an inducer of

differentiation and apoptosis in leukemia, lung cancer, and colon

adenocarcinoma cell lines, as well as in normal lymphocyte cells (Inoue et

al., 8774; Sohi et al., 2..3). It has been postulated that GA plays an

important role in the prevention of malignant transformation and cancer

development same as quercetin. Hence, the results of a recent research

showed that flavonoids are important components of this plant, and some

of its pharmacological effects could be attributed to the presence of these

valuable constituents (Ghasemzadeh et al., 2.8.).

In traditional medicine ginger has been used as antipyretic and in the

treatment of pain, rheumatism and bronchitis (Afzal et al., 2..8); it is also

used for the treatment of gastrointestinal disorders including gastric

ulcerogenesis (Agrawal et al., 2...; Mohsen et al., 2..6). At a molecular

level, it reduces retinoid binding protein (RBP) mRNA expression levels in

the liver and visceral fat resulting in improved lipid metabolism (Matsuda et

al., 2..7). Zingiber reduces the stickiness of blood platelets, hence may

help reduce the risk of artherosclerosis and heart attacks (Verma et al.,

2..4). In a laboratory test, aqueous ginger extract reduced platelet

thromboxane and also inhibited platelet aggregation (Srivastava, 87±6),

and in a research study it was demonstrated that the aqueous extract of

fresh ginger lowers blood pressure via endothelium-dependent

(cholinergic) and endothelium-independent vasodilator pathways (Ghayur

et al., 2..5). The rhizomes of ginger contain potent inhibitors against

prostaglandin biosynthesizing enzyme (PG synthetase) which are directly

associated with anti-inflammatory and anti-platelet aggregation activities

(Couch et al., 8772).

3-4-5 Thymus vulgaris L.

3-4-5-3 Plant description:

Thymus belongs to the group of angiosperm plants which belongs to

the lamiaceae family; Linnaeus (8953) was the first scientist who described

it.

An aromatic perennial sub-shrub, 2.–3. cm in height, with ascending,

quadrangular, greyish brown to purplish brown lignified and twisted stems

bearing oblong-lanceolate to ovate-lanceolate greyish green leaves that are

pubescent on the lower surface. The flowers have a pubescent calyx and a

bilobate, pinkish or whitish, corolla and are borne in verticillasters (Fig. 3).

The fruit consists of 4 brown ovoid nutlets (Youngken, 875.; Mossa et al.,

87±9 and Bruneton, 8775). Leaf is 4–82 mm long and up to 3mm wide; it is

sessile or has a very short petiole. The lamina is tough, entire, lanceolate to

ovate, covered on both surfaces by a grey to greenish grey indumentum;

the edges are markedly rolled up towards the abaxial surface. The midrib is

depressed on the adaxial surface and is very prominent on the abaxial

surface. The calyx is green, often with violet spots, and is tubular; at the

end are 2 lips of which the upper is bent back and has 3 lobes on its end;

the lower is longer and has 2 hairy teeth. After flowering, the calyx tube is

closed by a crown of long, stiff hairs. The corolla, about twice as long as the

calyx, is usually brownish in the dry state and is slightly bilabiate (European

pharmacopoeia, 8775).

Figure (5) Thymus vulgaris: (A) plant in bloom, (B) leaf seen from under

surface, magnified 6 diam., (C) flower seen from the side, magnified 7

diam.

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Thymus Vulgaris L. known as Thyme or Common thyme, farigola,

garden thyme, herba timi, herba thymi, mother of thyme, red thyme,

rubbed thyme, ten, thick leaf thyme, Thymian, time, timi, tomillo (British

herbal phamacopoeia ,8797; European pharmacopoeia, 8775 and

Farnsworth, 8775) and it is also locally known as “ za'ater ” (Behnia et al.,

2..±).

3-4-5-5 Plant Distribution over the world

Thymus vulgaris is indigenous to the Mediterranean region and is

grown in Southern and Southeastern Europe in countries that include

France, Portugal and Spain. Thyme can also be cultivated commercially in

other parts of the world with a temperate climate (Van Wyk and Wink,

2..4).


Thymus contains about 2053 but not less than 80.3 of volatile oil. The

composition of the volatile oil fluctuates depending on the chemo type

under consideration. The principal components of Thymus vulgaris are

thymol [8] and carvacrol [2] (up to 643 of oil) as shown in (Fig. 4), along

with linalool, p-cymol, cymene, thymene, α-pinene, apigenin, luteolin, and

6-hydroxyluteolin glycosides, as well as di-, tri- and tetramethoxylated

flavones, all substituted in the 6- position (for example 5,4'-dihydroxy-6,9-

dimethoxyflavone, 5,4'-dihydroxy- 6,9,3'-trimethoxyflavone and its ±-

methoxylated derivative 5,6,4'-trihydroxy- 9,±,3'-trimethoxyflavone)

(British herbal pharmacopoeia, 8797; Mossa et al.,87±9; Ghazanfar, 8774

and European pharmacopoeia, 8775).

The main constituents of the oils are the phenols, thymol and carvacrol.

Other leaf constituents responsible for activity include tannins, triterpenes

and polysaccharides (Blumenthal et al., 2...; VanWyk and Wink, 2..4).

Figure (6) principal component of Thymus vulgaris


Thymus vulgaris is cultivated for culinary, cosmetic and medicinal

purposes. The most commonly used parts of the plant are the leaves and

the flowering tops. The leaves can be used fresh, dried or powdered.

Thymus vulgaris is commonly used in the household as a flavoring agent in

most meat, fish and poultry dishes. It is also used in herbal butters and

cheeses and the dried leaf could be used to prepare herbal tea (Blumenthal

et al., 2...; VanWyk and Wink, 2..4). Its oil is used in the manufacturing

of products that include mouthwashes, toothpastes, detergents and

perfumes. The essential oil of thyme, constituting thymol, is widely used for

antiseptic effects, fumigants and antioxidant; it is also active against

Salmonella.

Externally the oil may be used as a lotion for the treatment of wounds.

It has been included in the formulation of mouth washes and for the

sterilization of bandages. It could be gargled for the relief of laryngitis and

tonsillitis. Thyme tea and tinctures have also been used for the treatment

of respiratory infections, coughs, bronchitis, ease chest congestion, and

stimulate production of saliva and other digestive infections. Other

biological properties include anti-inflammatory, antibacterial and

antispasmodic, carminative, astringent, anti-flatulent and anti-worm

(Baytop, 87±4; Leung & Foster, 8776; Blumenthal et al., 2...; Jellin et al.,

2...; Barnes et al., 2..2; Baranauskiene et al., 2..3 and VanWyk & Wink,

2..4 ). It has anti-fungal activity against number of species including

Cryptococcus neoformans Aspergillus, Sapralegnia and Zygorohynchus sp.

(WHO, 8777 and Zambonelli et al., 2..4). Thymol shows spontaneous

contractile activity (SCA) of smooth muscle strips from the stomach and

vena portae of guinea pigs (Beer et al., 2..9).

3-4-6 Acacia arabica L.

3-4-6-3 Plant Description:

Acacia described first in Africa by the Swedish Botanist Carl Linnaeus

in 8993. It belongs to the group of angiosperm plants which belongs to the

fabaceae family.

Acacia arabica also known as Acacia nilotica (Fig. 5) which is small tree,

usually 405-6 to 82 m., twigs glabrous to subtomentose grey or brown to

purplish-black, sometimes shining, cortex striate but not cracking. Stipules

spinescent, .05-± cm., divergent. Pinnae in 2-88 pairs, the leaf rhachis often

with glands between at least some pairs; leaflets in 9-25 pairs, glabrous or

pubescent, only the midrib visible below, the lateral veins quite obsolete, 3-

9 x .05-805 mm. (Townsend and Guest 8794). Flowers in globular ±-82 mm.

diameter heads on axillary peduncles of 82-85 mm. , involucel in lower half

of peduncle. Calyx tubular-campanulate, 5-dentate, 805 mm. corolla 3 mm.,

lobes 8 m.,yellowish-green. Filaments 5m., bright yellow.pod very variable

in form, indehiscent, straight or curved, glabrous or downy, thick, the

sutures straight or constricted between the seeds. Seeds suborbicular,

blackish-brown, compressed, 9mm., areole large (Townsend and Guest

8794). Acacia seeds can be difficult to germinate; research has found that

immersing the seeds in vario s temperat res ( s ally aro nd ±. C) and

manual seed coat chipping can improve yields to approximately ±. percent

(Clemens et al., 8799). The small flowers have five very small petals, almost

hidden by the long stamens, and are arranged in dense globular or

cylindrical clusters; they are yellow or cream-colored in most species,

whitish in some, even purple (Acacia purpureapetala) or red (Acacia leprosa

Scarlet Blaze) (Singh, 2..4).

The crown is low, or rounded umbrella shape (in free standing

specimens). The bark is very spreading and almost symmetrical, and could

be flattened dark brown to black with deep regular vertical grooves in older

specimens (Burkill, 8775).

Figure (7) Acacia arabica tree

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Common names of Acacia are Egyptian mimosa, Egyptian thorn, red

thorn also known as babul and kikar in India. Burkill gives at least 827

different names for this plant as a whole or for the fruit and seeds (Burkill,

8775). Acacia also known as shok al-hind, sunt and qaradh (Townsend and

Guest, 8794).

3-4-6-5 Plant Distribution over the world

Very rare found once in the desert region of Iraq (Guest 8684).

According to the collector note: “A single thorny tree abo t 2.ft. high (6-9

m.) growing on the edge of the desert near a date garden; apparently self-

sown and s b spontaneo s” ; also more recently c ltivated at the

horticultural Station near Baghdad (Townsend and Guest, 8794).

Acacia is widely spread in subtropical and tropical Africa from Egypt to

Mauritania southwards to South Africa, and in Asia eastwards to Pakistan

and India. It has been introduced in China, the Northern Territory and

Queensland in Australia (where it is considered to be a pest plant of

national importance), in the Caribbean, Indian Ocean Islands, Mauritius,

United States, Central America, South America and the Galápagos Islands).

It has naturalized in several countries where it has been introduced as a

medicinal, forage and fuel wood plant (Ellery and Ellery, 3991).


It contains Gum arabic, tannins, mucilage, catechin, arabic acid, malic

acid, gallic acid, megnesium, potassium, calcium and flavonoid compounds

(Cragg and Newman, 2..5)


Leaves, fruits and seeds of Acacia arabica have been used in traditional

medicine as appetizer, mild laxative, diuretic and anti-fungal medication

and people in some Asian and African countries have used it for many years

(Akhtar and Ajmal, 87±8; Ezmirly et al., 87±8; Al Sadhan and Almas, 8777;

Darout et al., 2..2; Al-Otaibi et al., 2..3; Almas et al., 2..5; Darmani et

al., 2..6).

The gum of this plant is described in the British pharmacopoeia as a

source of useful medicaments. It is believed to be value for treating

gingivitis and for reducing plaque (Gazi, 3115). It is also used as astringent,

emollient, liver tonic, antipyretic and antiasthmatic (Baravkar et al., 3118).

Acacia arabica used as astringent, demulcent, anti dysentric,

aphrodisiac, expectorant, antacid, nutritive and tonic (Cole, 0111).

Decoction of the bark is used in vaginitis, leucorrhea and gonorrhea

(Erdogrul, 3113). Gum in the form of mucilage is used for the treatment of

diarrhea, dysentery and diabetes. Powdered gum is used to treat

hemorrhages (Cos et al., 3113). Gum is useful nutritive tonic and

aphrodisiac in sexual debility (Mahmood et al., 3111). Pods are used in

cough. Pods are expectorant (Gupta et al., 3115). The decoction made from

the leaves is useful in spongy gums sore throat and as wash in hemorrhagic

ulcers and wounds (Gohl, 0125).

Tender growing tops rubbed into paste with sugar and water act as

demulcent in coughs (Rojas et al., 3113), watery extract is given to allay

irritation in acute gonorrhea, especially in cases complicated with leprosy

and leucorrhea (Qureshi et al., 3112). Plant is considered to be antacid,

antidysentric and antispasmodic (Diallo et al., 0111). The decoction of the

bark is largely used as a local astringent douche or enema in vaginitis,

leucorrhoea, cystitis, gonorrhea, piles, prolapsus ani and prolapsus uteri

(Jeevam et al., 3111; Kloucek et al., 3115). It represents a new class of plant

stress metabolites capable of activating stress adaptation and suppressing

proinflammatory components of the immune system in human cells by redox

regulation (Bessong and Obi, 3113). This plant has antibacterial activity

(Sandhu and Heinrich, 3115; Banso, 3111).

Acacia inhibits tumor cell growth and induces apoptosis, in part, by

perturbing mitochondrial function (Das et al., 0182). It is selectively toxic to

tumor cells at very low doses. It has been shown to have potent cytotoxic

activity (apoptosis) against human T-cell leukemia (Agrawal and Agarwal,

0111). Antiplatelet aggregator activity of the extract of Acacia nilotica is

mainly due to the blockade of Ca3+

channels (Shah et al., 0112; Gilani et al.,

0111). Evidence also suggests the involvement of protein kinase C (Hussein

et al., 0111; Hussein et al., 3111).

Acacia nilotica has a wealth of medicinal uses. It is used for stomach

upset and pain, the bark is chewed to protect against scurvy, an infusion is

taken for dysentery and diarrhea. It has also been used to eliminate

stomach worms, as an antiseptic for open wounds and as an expectorant

for treating coughs (Spicer et al., 2..9). In the form of a solution or

mucilage it is an agreeable lenitive for irritated and inflamed membranes,

and for this purpose is frequently used in medicinal preparations for

coughs, colds, hoarseness, pharyngitis, gastric irritation and inflammation

(Felter, 2..8).

Acacia nilotica leaf used as anticancer, astringent, anti-inflammatory

and Alzheimer’s diseases (Kalaivani and Mathew, 3101; Shittu, 3101 and

Kalaivani et al., 3100). Studies have confirmed anti-diabetic activities.

However, pods and tender leaves are considered very beneficial in folk

medicine to treat diabetes mellitus (Gilani et al., 0111). In folk medicine

Acacia was used as Antitussive, decongestant, demulcent and

neurostimulant (Duke, 0182). It was found that it is safe to be used as

amebicide, antibacterial, hypoglycemic, hypotensive, molluscicide,

Protisticide, Taenicide, hepatotonic and vermifuge (Duke et al., 3113).

Recently this ayurvedic herb was found to be useful as dentifrice and anti-

hemorrhagic agent (Pradeep et al., 3101).

1-2-5 Haemostasis (The Stopping of Bleeding)

Haemostasis is a protective physiological mechanism that has several

important functions:

0- To maintain blood in a fluid state while it remain circulating within

the vascular system.

3- To arrest bleeding at the site of injury or blood loss by formation of a

haemostatic plug.

2- To ensure the eventual removal of the plug when healing is complete.

Normal physiology thus constitutes a delicate balance between these

conflicting tendencies and a deficiency or exaggeration of any one

may lead to either thrombosis or haemorrhage.

There are at least five different components involved in coagulation

process: blood vessels, platelets, plasma coagulation factors, their inhibitors

and fibrinolytic system (Lewis and Bain, 3113; Baklaja et al., 3118);

disorders of this system are the leading immediate cause of mortality and

morbidity in the modern society. The most prominent of them is thrombosis;

the intravascular formation of clots that obstruct blood flow in the vessels

(Davies, 3111).

During the last twenty years, the haemostasis system was a subject of

intense interest in this field; reviews are available that describe these

theoretical studies of blood coagulation and platelet-dependent haemostasis

and thrombosis (Ataullakhanov and Panteleev, 3115; Panteleev et al., 3112;

Xu et al., 3100 and Xu et al., 3103).

Platelets are small fragments of cytoplasm derived from

megakaryocytes. On average 0.5- 2.5µm in diameter but may be larger in

some disease states. They do not contain a nucleus and are bounded by a

typical lipid bilayer membrane. Beneath the outer membrane lies the

marginal band of microtubules, which maintain the shape of the platelet and

depolymerize when aggregation begins (Ruggeri, 0112; Nurden, 0111).

Platelets have at least three roles in haemostasis (George, 3111):

0- Adhesion and aggregation forming the primary haemostatic plug.

3- Release of platelet activating and procoagulant molecules.

2- Provision of a procoagulant surface for the reactions of the

coagulation system.

Chapter Two

Materials

&

Methods

4- Materials and Methods

4-3 Chemicals and Apparatus

4-3-3 Chemicals

Chemicals used in this study are listed in Table (2-8).

Table (4-3): Chemicals used in this study.

Origin Chemicals

BDH / England Ammonium hydroxide

BDH / England Anhydride acetic acid

Scharlau / Spain Chloroform

Scharlau / Spain Ethanol absolute

Thomas Baker / India Ethyl acetate

BDH / England FeCl3

BDH / England HgCl2

BDH / England Hydrochloric acid

BDH / England KI

BDH / England Lead acetate

Fluka / Switzerland Methanol

Anaylt / U.K. N-Propanol

BDH / England Potassium ferric cyanide

BDH / England Sodium chloride

Merk / Germany Sulphuric acid

BDH / England Tannic acid

4-3-4 Apparatus

The apparatus used in this study are listed in Table (2-2).

Table (4-4): The apparatus used in this study.

Company The apparatus

DMD- DISPO /Syria Anticoagulant tubes

Cell-DYN Emerald / Abotte

USA

Automated platelet count device

Hettich / Germany Centrifuge

Adam / England Electric balance

Moulinex / France Electric Blender

El-arabi / Egypt Electric Mill

Baird & Tatlock London

Limited / England

Electric shaker

Whatman / England Filter paper

Vitrex / Denmark Non heparinized capillaries

Memmert / Germany Oven

Milwaukee / USA pH-meter

Ishtar / Iraq Refrigerator

Heidolph / Germany Rotary evaporator

Electrothemol / England Soxhlet extractor

Gallenkamp / England Water bath

4-4 Collection of plant samples

Plant samples included Zingiber officinale dry rhizomes (Fig. 6), dry

leaves of Thymus vulgaris (Fig.9) and dry gum of Acacia arabica (Fig. ±)

were obtained from local herbarium market in Baghdad city. Each sample

was grinded down to powder form in an electric mill to facilitate dealing

with them in the extraction steps. These powdery samples were stored in

dry and clean conditions until use.

Figure (8) Zingiber officinale Roscoe dry rhizomes

Figure (1) Thymus vulgaris dry leaves

Figure (8) Acacia arabica dry gum

4-5 Preparation of Plant Extracts

a- Alkaloids

The extract was prepared according to the method of Harborne (87±4).

A quantity of 8..g of plant powder was homogenized in electrical blender

with 35. ml of (4:8) ethanol: D.W. the sample was filtered through muslin

and then through a filter paper in Bouknner funnel, then acidified by drops

of (23 sulphuric acid) until the pH level dropped between 8 and 2. The

solution was re-extracted with chloroform 3 times in the separating funnel

until we got two layers; the upper one was neglected and the lower one

was used. Drops of concentrated ammonium hydroxide were added to this

layer until the pH became between 7 and 8.. Then the solution was again

extracted in the separation funnel with (8:3) chloroform: methanol twice

and once with chloroform alone. After that the solution was separated into

two layers; the upper layer (solvent) was neglected and the lower layer was

evaporated in a rotary evaporator at 4.4c for (8-2) hours then oven dried

until it turned into powder and the powder was kept in refrigerator until

use.

b- Phenols

Phenols were extracted according to Ribereau-Gayon (8792) and

Harborne (87±4). 2.. g of plant powder were divided into 2 equal portions,

one was mixed with 3.. ml of D.W. and the other one was mixed with 3..

ml of 83 Hydrochloric acid. Then samples were homogenized in electric

shaker for 5 minutes. The two mixtures were transferred to boiled water

bath for 3.-4. minutes, then cooled and filtered through muslin cloth and

centrifuged with speed of 3... rpm for 8. minutes. The two supernatants

were mixed. Equal quantity of N-propanol was added to the mixture prior

to sodium chloride was added until the solution was separated into two

layers. The lower layer extracted in separating funnel with Ethyl acetate,

and concentrated by using rotary evaporator at 4.°C for (8-2) hours. The

upper layer was dried by rotary evaporator at 4.°C for (8-2) hours. The

dried material of both layers were mixed and dissolved in 5ml of 763

ethanol, then left in oven until it turned into powder and kept in

refrigerator until use.

c- Terpens

Terpens were extracted according to the method of Harborne (87±4).

85g of plant powder was successively extracted in a soxhlet extractor for 24

hours with 2.. ml chloroform. The solvent was removed by rotary

evaporator at 4.4C. Then the extract dried in the oven at 4.4C until it

turned into powder and kept in the refrigerator until use.

4-6 Preparation of different concentrations of plant extracts:

Alkaloid, phenol and terpenoid extracts were prepared by dissolving

certain weight of each plant extract powder according to the needed

concentration in D.W. Different concentrations (8, 5 and 8.) mg / ml of

plant extracts were prepared according to the following equation:

Concentration mg/ml ═ Weight ×3222

Volume

4-7 Compounds' Detection

A- Alkaloid Detectors

3- Mayer reagent

This reagent (Jones and Kinghorn, 2..6) was used for the detection of

alkaloids. The stock solution (8) was prepared by dissolving 8305g HgCl2 in

6. ml H2O, stock solution (2) was prepared by dissolving 5g KI in 8. ml

water. Then (8) and (2) were combined and completed with8.. ml D.W.,

then 8-2 ml of Mayer reagent was added to 5 ml of alcohol extract. A

creamy or white precipitate indicates the presence of alkaloids.

4- Tannic acid reagent

This acid was used to precipitate alkaloids (Al-Salami, 877±). 83 tannic

acid was prepared, and then 8-2ml of reagent was added to 5ml of the

extract. The white turbidity was appeared indicating the presence of

alkaloids.

B- Phenolic Detectors

3- Lead acetate reagent

It is aqueous or alcohol solution of lead acetate 83. Amount of reagent

was added to equal amount of alcohol extract then white precipitate

appeared indicating the presence of phenols (Al-Salami, 877±).

4-Ferric Chloride and potassium ferric cyanide reagent

It was used to detect the general phenols. This reagent was prepared by

two equal amounts of aqueous solution of ferric chloride 83 and potassium

ferric cyanide 83. Blue-green color appeared indicating the presence of

phenols (Harborne, 87±4).

C- Terpenoid Detectors

3- HgCl4 reagent

This reagent (Al-Salami, 877±) was used for saponine detection by

adding 8-2 ml of 83 HgCl2 to 5ml of the extract, appearance of white

precipitate indicated the presence of terpenes.

4- Acetic anhydride reagent

According to Al-Bid (87±5), 8-2 drops of chloroform, 8 drop of anhydride

acetic acid and 8 drop of concentrated H2SO4 were added to 8 ml of the

extract. The appearance of brown color indicates the presence of terpens,

and the appearance after a period of time of black-blue indicated the

presence of steroids.

4-8 Haematological Tests:

4-8-3 Haematological Test in vivo

In this study seventy five male Albino mice from Balb-c. breed were

used; their average weight was (2±82) gm; they were divided randomly into

twenty five groups kept in clean lab cages (three mice in each cage) in the

animal house of the Biotechnology Research Center in Al- Nahrain

University under optimal conditions of light and ventilation; fed on

standard food and adequate amount of water. The groups were labeled

with the plant name, its active group i.e. (alkaloids, phenols and terpens)

and the degraded concentrations (8, 5, and 8.) mg\ml of these three active

groups extracts.

Intra gastric dose of (.04ml) of the replicates for each concentration of

the three active groups of the plants under study were given to the mice

once daily for seven days continuously )Spehaat, 2..±).

2-6-2 Bleeding Time Measurement in vivo

Mouse tail was cut with a scalpel 3-4 cm proximal from the end and

bleeding time was calculated from the time of starting of bleeding till

bleeding stopped. Spots were made with the bleeding tail on a blotting

paper every 37 seconds till bleeding stopped and bleeding time was

calculated accordingly or the time taken between the appearances of blood

to the cessation of bleeding is taken as the bleeding time expressed in

minutes (Shrivastava and Das, 3981; Dacie et al., 3997).

4-8-5 Clotting Time Measurement in vitro

Blood was drawn into a capillary tube. The time of appearance of the

blood drop on the cut tail was noted. The capillary glass tube is then kept

between the palms of both hands for 52 second to keep it at body

temperature. After 52 second the tube was taken out and small portion of

the capillary tube was broken at regular intervals of 32 seconds, until a

thread of clotted blood appears between the two pieces of capillary glass

tube. The time interval between the appearance of the drop of the blood

and the thread of the blood clot was the clotting time of the blood sample

of the mouse expressed in minutes (Shrivastava and Das, 3981; Dacie et al.,

3997).

2-6-4 Platelets Count

Platelets count was measured by putting the blood samples of the

tested mice in anticoagulant tubes and measured them in automated

platelet count device. This method was recommended because it is more

accurate than the classic manual methods (Harrison et al., 4227).

2-9 Statistical Analysis

The Statistical Analysis System- SAS (4226) was used to study the effect

of different factors in the study parameters. The significant differences

between the means in this study were determined by using Least

Significant Difference (LSD) test.

Chapter Three

Results

5- Results

5-3 Plant extracts preparation

The dry rhizomes of Zingiber officinale Roscoe, dry leaves of Thymus

vulgaris L. and dry exudates (gum) of Acacia arabica L. were extracted for

detection of alkaloids, phenols and terpens. The yield of major compounds

in each extracts was determined (table 3-8).

Table (5-3) Compounds quantity yielded from plant parts expressed as %

Plants Name

Type of extract

Yield (%)

Zingiber officinale Roscoe

Dry rhizomes

Alkaloids

50±6

Phenols

204

Terpens

302

Thymus vulgaris L.

Dry leaves

Alkaloids

8052

Phenols

805±

Terpens 405

Acacia arabica L.

Dry gum

Alkaloids

8032

Phenols

52039

Terpens

-

(-): not detected.

5-4 Haematological Tests

Bleeding Time, Clotting Time & Platelets Count

This section describes the results of various experiments which include

bleeding time, clotting time and platelets count which are related to blood

coagulatory processes.

5-4-3 Effect of Alkaloids Extract in Bleeding Time, Clotting Time

and Platelets Count

The effect of crude alkaloid extract of Zingiber officinale dry rhizomes

which described in table (3-2) and Fig (7, 8.) showed that (8.mg/ml) was

the most effective conc. in bleeding time, clotting time and also platelets

count than other two concentrations compared with the control group.

Table (5-4) Zingiber officinale crude alkaloids extract effect on the tested blood characteristics

using different concentrations

Blood

Characters

Conc. of Zingiber officinale crude alkaloid extract LSD

Value Control 8 mg/ ml 5 mg/ ml 8. mg/ ml

Bleeding

time(Min.) 80±± ± .028 808± ± .0.3 808± ± .0.4 .074 ± .087 .0492 *

Clotting

time(Min.) 2052 ± .0.4 208. ± .0.3 203. ± .0.± 8023 ± .0.7 .02.6 *

Platelet

count (x *

8.7/ L) 357069882044 837303387±034 92±069884.052 85630..8862082

3±5039 *

* (P<.0.5).

Figure (9) the effect of Zingiber officinale crude alkaloids extract of three concentrations on

bleeding time and clotting time.

0

0.5

1

1.5

2

2.5

3

Bleeding Time Clotting Time

Min

.

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (32) the effect of Zingiber officinale crude alkaloids extract of three concentrations on

platelets count.

The effect of crude alkaloid extract of Thymus vulgaris dry leaves which

described in table (3-3) and Fig. (88,82) showed that (5mg/ml) was the

most effective conc. in bleeding time, clotting time and also platelets count

than other two concentrations compared with the control group.

0

200

400

600

800

1000

1200

1400

1600

Platelets Count

359

1393

728

1563

Control

1 mg/ml

5 mg/ml

10 mg/ml

Table (5-5) Thymus vulgaris crude alkaloids extract effect on the tested blood characteristics


Blood

Characters

Conc. of Thymus vulgaris crude alkaloid extract LSD


Bleeding

time(Min.) 80±± ± .028 8065 ± .089 80.2 ± .023 802± ± .0.8 ..5±6 *

Clotting

time(Min.) 2052 ± .0.4 2082 ± .0.8 8029 ± .0.4 806± ± .086 .0299 *

Platelet

count (x *

8.7/ L) 357069882044 66±06982.053 8276069855023 ±360338825097 229042 *

* (P<.0.5).

Figure (33) the effect of Thymus vulgaris crude alkaloids extract of three concentrations on


0

0.5

1

1.5

2

2.5

3


Min

.

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (34) the effect of Thymus vulgaris crude alkaloids extract of three concentrations on

platelets count.

The effect of crude alkaloid extract of Acacia arabica described in table

(3-4) and Fig. (83,84) showed that (8.mg/ml) was the most effective conc.

in bleeding time, clotting time and also platelets count than other two

concentrations compared with the control group.

0

200

400

600

800

1000

1200

1400

Platelets Count

359

668

1296

836

Control

1 mg/ml

5 mg/ml

10 mg/ml

Table (5-6) Acacia arabica crude alkaloids extract effect on the tested blood characteristics


Blood

Characters

Conc. of Acacia arabica crude alkaloid extract LSD Value

Control 8 mg/ ml 5 mg/ ml 8. mg/ ml

Bleeding

time (Min.) 80±± 8.028 8034 ± .0.5 8029 ± .0.9 802. ± .0.2 .0392 *

Clotting time

(Min.) 2052 8.0.4 2024 ± .0.3 20.4 ± .0.2 8037 ± .0.5 .0889 *

Platelet

count (x *

8.7/ L)

357069882044 ±6±0..8822064 7±803388540.. 8876033822209

2 4±5083 *

* (P<.0.5).

Figure (35) the effect of Acacia arabica crude alkaloids extract of three concentrations on


0

0.5

1

1.5

2

2.5

3


1.88

2.52

1.34

2.24

1.27

2.04

1.2 1.39

Min

.

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (36) the effect of Acacia arabica crude alkaloids extract of three concentrations on

platelets count.

5-4-4 Effect of phenols Extract in Bleeding Time, Clotting Time

and Platelets Count

The effect of crude phenol extract from Zingiber officinale which

described in table (3-5) and Fig. (85,86) showed that (8.mg/ml) was the

most effective conc. in bleeding time, clotting time and also platelets count

than other two concentrations compared with the control group.

0

200

400

600

800

1000

1200

Platelets Count

359

868

981

1196

Control

1 mg/ml

5 mg/ml

10 mg/ml

Table (5-7) Zingiber officinale crude phenols extract effect on the tested blood characteristics


Blood

Characters

conc. of Zingiber officinale Roscoe crude phenol extract LSD


Bleeding

time(Min.) 80±± ± .028 8038 ± .0.7 802. ± .08. 808± ± .0.6

.0428

*

Clotting

time(Min.) 2052 ± .0.4 2026 ± .082 8069 ± .022 8027 ± .084 .0499*

Platelet

count (x *

8.7/ L) 357069882044 8.2±069832604± 822±0..8285022 84930..83.40±8

±.±092

*

* (P<.0.5).

Figure (37) the effect of Zingiber officinale crude phenols extract of three concentrations on


0

0.5

1

1.5

2

2.5

3


Min

.

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (38) the effect of Zingiber officinale crude phenols extract of three concentrations on

platelets count.

The effect of crude phenol extract of Thymus vulgaris which described

in table (3-6) and Fig. (89,8±) showed that (8.mg/ml) was the most

effective conc. in bleeding time, clotting time and also platelets count than

other two concentrations compared with the control group.

0

200

400

600

800

1000

1200

1400

1600

Platelets Count

359

1028

1228

1473

Control

1 mg/ml

5 mg/ml

10 mg/ml

Table (5-8) Thymus vulgaris crude phenols extract effect on the tested blood characteristics


Blood

Characters

Conc. of Thymus vulgaris crude phenol extract LSD Value


Bleeding

time(Min.) 80±± ± .028 8043 ± .0.5 803. ± .0.3 8089 ± .0.4 .0362 *

Clotting

time(Min.) 2052 ± .0.4 2085 ± .0.3 20.2 ± .0.8 8035 ± .0.9 .0847 *

Platelet

count (x *

8.7/ L)

3570698

82044 58803388605± 9920..8866069 ±860..88.±0.± 32506± *

* (P<.0.5).

Figure (31) the effect of Thymus vulgaris crude phenols extract of three concentrations on


0

0.5

1

1.5

2

2.5

3


Min

.

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (38) the effect of Thymus vulgaris crude phenols extract of three concentrations on

platelets count.

The effect of crude phenol extract of Acacia arabica which described in

table (3-9) and Fig. (87,2.) showed that (8mg/ml) was the most effective

conc. in bleeding time, clotting time and also platelets count than other two

concentrations compared with the control group.

0

100

200

300

400

500

600

700

800

900

Platelets Count

359

511

772 816

Control

1 mg/ml

5 mg/ml

10 mg/ml

Table (5-1) Acacia arabica crude phenols extract effect on the tested blood characteristics


Blood

Characters

conc. of Acacia arabica crude phenol extract LSD


Bleeding

time (Min.) 80±± ± .028 .0±± ± .086 8023 ± .0.9 8056 ± .024 .06.2 *

Clotting time

(Min.) 2052 ± .0.4 8065 ± .089 2022 ± .0.6 203. ± .085 .0374 *

Platelet

count (x *

8.7/ L)

357069882044 783069828204

6

6±703388460.

5

6.60.. ±

89±043

588066

*

* (P<.0.5).

Figure (39) the effect of Acacia arabica crude phenols extract of three concentrations on


0

0.5

1

1.5

2

2.5

3


Min

.

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (42) the effect of Acacia arabica crude phenols extract of three concentrations on

platelets count.

5-4-5 Effect of Terpens Extract in Bleeding Time, Clotting Time

and Platelets Count

The effect of crude terpen extract of Zingiber officinale which described

in table (3-±) and Fig. (28,22) showed that (8.mg/ml) was the most

effective conc. in bleeding time, clotting time and also platelets count than

other two concentrations compared with the control group.

0

100

200

300

400

500

600

700

800

900

1000

Platelets Count

359

913

689

606 Control

1 mg/ml

5 mg/ml

10 mg/ml

Table (5-8) Zingiber officinale crude terpens extract effect on the tested blood characteristics


Blood

Characters

conc. of Zingiber officinale crude terpen extract LSD


Bleeding

time(Min.) 80±± ± .028 8085 ± .0.3 8089 ± .0.9 80.7 ± .0.4 .0367 *

Clotting

time(Min.) 2052 ± .0.4 2022 ± .0.6 2023 ± .082 803± ± .0.9 .0259 *

Platelet

count (x *

8.7/ L)

357069882044 962033895069 6830..8390.9 8.56033868083 89.079 *

* (P<.0.5).

Figure (43) the effect of Zingiber officinale crude terpens extract of three concentrations on


0

0.5

1

1.5

2

2.5

3


Min

.

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (44) the effect of Zingiber officinale crude terpens extract of three concentrations on

platelets count.

The effect of crude terpen extract of Thymus vulgaris which described in

table (3-7) and Fig. (23,24) showed that (8.mg/ml) was the most effective

conc. in bleeding time, clotting time and also platelet count than the other

two concentrations compared with the control group.

0

200

400

600

800

1000

1200

Platelets Count

359

762

613

1056

Control

1 mg/ml

5 mg/ml

10 mg/ml

Table (5-9) Thymus vulgaris crude terpens extract effect on the tested blood characteristics


Blood

Characters

Conc. of Thymus vulgaris crude terpen extract LSD Value


Bleeding

time(Min.) 80±± ± .028 8083 ± .0.4 80±. ± .029 .069 ± .089 .0625 *

Clotting

time(Min.) 2052 ± .0.4 8067 ± .028 2085 ± .0.3 8038 ± .0.2 .0356 *

Platelet

count (x *

8.7/ L)

3570698

82044 9960..88±099 636069868034 8.±4033886907. 273097 *

* (P<.0.5).

Figure (45) the effect of Thymus vulgaris crude terpens extract of three concentrations on


0

0.5

1

1.5

2

2.5

3


Min

,

Control

1 mg/ml

5 mg/ml

10 mg/ml

Figure (46) the effect of Thymus vulgaris crude terpens extract of three concentrations on

platelets count.

0

200

400

600

800

1000

1200

Platelets Count

359

776

636

1084

Control

1 mg/ml

5 mg/ml

10 mg/ml

Chapter Four Discussion

6- Discussion

6-3 Plant extracts yield and their chemical constituents

The relative proportion between the amounts of plant used for

extraction and the crude products was variable depending on several

factors such as methods of extraction, solvent used in extraction process,

and the plant parts or species (Henning et al., 2..3).

Results in table (3-8) showed that the yield of alkaloids obtained from

Zingiber officinale dry rhizomes was the highest (50±6 %) than alkaloids

yielded from Thymus vulgaris dry leaves and Acacia arabica dry gum which

were (80523),(8032%) respectively.

Phenols obtained from A. arabica dry gum was the highest (52039%),

followed by Z. officinale dry rhizomes (204%) and T. vulgaris dry leaves

(805±3).

The yield of terpens obtained from T. vulgaris (4053) was higher than

that obtained from Z. officinale (3023) while A. arabica showed no terpens

detected.

Results showed that alkaloids extract yielded form Z. officinale was

higher than that obtained by Akintobi et al. (2.83) who mentioned that

alkaloids percentage was (80463) while Spehaat (2..±) reported that no

alkaloids in the crude extraction of Z. officinale were detected. These

differences could be due to place of sampling (i.e. different herbarium

markets in which they differ in the means of preserving the samples).

Phenol extract yielded from Z. officinale was similar to the results

obtained by Kaushik and Goyal (2.88) and Setty et al. (2.88) who

mentioned positive result for the presence of phenols without reporting

the amount of phenols yielded from this plant.

In this study the amount of terpens yielded from Z. officinale was (302

%); however Anosike et al. (2..7) mentioned that terpenes yielded from

crude extract of Z. officinale were present in a very high concentration

while Motawi et al. (2.88) showed that terpene presented in moderately

high concentration.

These authors didn´t mentioned the percentage of terpens yielded

from this plant.

Terpenoid yield of T. vulgaris as shown in table (3-8) was higher than

both alkaloid and phenol extracts. This result is similar to what obtained by

Yarnell (2..9) and also agreed with what Oyewole et al. (2.8.) found as

they mentioned positive result for terpenoid presence without reporting

the amount of terpens yielded from this plant.

Sharafzadeh et al. (2.8.) indicated that thymol was 56073 while in this

study the percentage of phenols was (805± %). This is probably due to

nutrients affect growth, essential oil and total phenolic content of thyme

(Sharafzadeh, 2.88); Shapiro and Guggenheim (8775) mentioned that

Thymol and carvacrol are the main phenolic compounds responsible for

most of the therapeutic properties. Alkaloids showed the lowest

percentage among phenols and terpenoids.

As results shown A. arabica phenols yield was too high compared with

other plants in this study and similar to reported results by Sharma (2.8.)

which was (24-42 %); Panchawat et al. (2.8.) also reported high positive

results for phenols while Banso (2..7) recorded negative results which

authenticate the absence of these phytochemicals. Saini et al. (2..±) and

Jigam et al. (2.8.) both mentioned the presence of alkaloids in leaves while

Raghavendra et al. (2..6) reported negative results of alkaloids.

Terpens of A. arabica dry gum showed negative result while Malviya et

al. (2.88) showed positive result for terpens without reporting the amount

or percentage in the plant.

In this study as far as in our knowledge, there was no one mentioned

the weight percentage of any compound extracted from the plants used in

this study.

6-4 Haematological Tests:

Bleeding Time, Clotting Time and Platelets Count.

Haemostasis which is the arrest of blood loss from severed blood

vessels and the maintenance of the blood fluidity involves coagulation and

fibrinolysis. A wound or cut on blood vessels causes vasoconstriction and

thrombin activation which is then accompanied adhesion and platelet

activation, fibrin formation from circulating fibrinogen and coagulation

inactivation mechanism

(Rang et al., 8777). The present study was carried out to determine the

potentials of Zingiber officinale dry rhizomes, Thymus vulgaris dry leaves

and Acacia arabica dry gum on the hemostatic mechanism, with primary

interest on how it affects bleeding time, clotting time and platelets count.

Bleeding time evaluates the vascular and platelet responses with

hemostasis (Dapper et al., 2..9; Weremfo et al., 2.88), whereas the

clotting time measures the intrinsic clotting factors (I, II, V, VIII, IX, X, XI and

XII). Clotting time test is a qualitative measurements of factors involved in

the intrinsic pathway (Ochei and Kolhatkar, 2...). Therefor the deficiency

in these factors will affect the results.

6-4-3 Effect of Alkaloids Extract in Bleeding Time, Clotting Time and

Platelets Count

After the intra gastric administration of the mice under the test with

three concentrations (8, 5, 8. mg/ml) of Z. officinale crude alkaloid extract

for 9 days successively.

Results of the bleeding time (BT), clotting time (CT) and platelets count

tests showed variations compared with the control group as the extract

decreases the bleeding time and clotting time while increases the platelets

count at significant difference of (P<.0.5). This agreed with De Caterina et

al. (8774) who mentioned that there is an inverse relationship between

bleeding time and platelets count.

Bleeding time is affected by many factors including vasoconstrictive

effect of blood vessels, the formation of hemostatic plug and platelet

activity. In general, anticoagulants and aspirin have been reported to

increase BT in animals and humans while coagulants have opposite effect

(Hadi et al., 2.8.).

In table (3-2) the results showed that both concentrations (8mg/ml)

and (5mg\ml) of crude alkaloid extract decreased the BT to (808± min.)

compared with control (80±± min.) and also decreased the CT to (208. min.)

and (203. min.) respectively compared with control (2052 min.) while the

third concentration of the extract (8. mg/ml) was the most effective

because it decreased the BT to (..74 min.) and the CT to (8023 min. )

compared with the control value mentioned above. However Prasad et al.

(2.82) who used crude extract of fresh rhizomes juice of Z. officinale to

investigate BT and CT on Albino Wister rats reported an increase in BT

compared to control and mentioned that CT didn´t changed due to the

extract; these differences might be due to plant part used and also the type

of lab animals.

Cessation of bleeding indicates the formation of hemostatic plugs,

which are in turn dependent on an adequate number of platelets and on

the ability of the platelets to adhere to the sub endothelium and to form

aggregates (Rodgers and Levin, 877.). The platelets count also significantly

changed but adversely with BT and CT, the normal value (control) was

(357*8.7/L) while the platelets count of the concentrations (8mg/ml;

5mg/ml; 8.mg/ml) was (8373*8.7/L) (92±*8.7/L) (8563*8.7/L)

respectively. As mentioned above (8 mg/ml) concentration was more

efficient than (5 mg/ml) this might happened due to negative response to

the extract that has been given to the mice under investigation which might

increases the blood fluidity rather than decreasing it (Zokian, 2..5). In

general for the three parameters (8. mg/ml) of crude alkaloid extract of Z.

officinale was the most effective concentration.

Results in table (3-3) crude alkaloids extract of T. vulgaris dry leaves

also revealed significant differences at (p> .0.5). The concentration

(8mg/ml) and (8. mg/ml) decreased the BT to (8065) and (802±)

respectively compared with the control (80±±) and also decreased the CT to

(2082 min.) and (806± min.) respectively compared with control (2052 min. )

while the BT of the (5 mg/ml) concentration was (80.2 min. ) and CT was

(8029 min.), The results recorded in this study are in consonance with the

reports of Okoli et al., (2..9) on the haemostatic activities of the leaf

extract of Aspilia africana which arrested bleeding from fresh wounds by

reducing both bleeding and clotting times. While the platelets count of the

experiment concentrations (8mg/ml) and (8. mg/ml) increased significantly

to (66±*8.7/L) and (±36*8.7/L) respectively compared with control.

However the platelets count of the concentration (5 mg/ml) was higher

than the other concentrations which was (8276*8.7/L) compared with

control. In general for the three parameters (5 mg/ml) of crude alkaloid

extract of T. vulgaris was the most effective concentration.

Thymus vulgaris generally showed decrease in both BT and CT while

platelets count increased significantly; but it has not been studied

separately before just as a component of mixture of different plants

extracts for hemostatic activity evaluation for example the Ankaferd Blood

Stopper (ABS) in which T. vulgaris represents one of its component; ABS

was found to be effective in shortening the duration of bleeding and

decreasing the amount of bleeding (Cipil et al., 2..7; Kurt et al., 2.8.).

In table (3-4) results for crude alkaloid extract of A. arabica showed

significant differences at (p> .0.5). The concentrations (8 mg/ml), (5

mg/ml) and (8. mg/ml) decreased the BT compared with control to (8034

min.), (8029 min.) and (802. min.) respectively; as well as decreased the CT

compared with control to (2024 min.), (20.4 min.) and (8037 min.)

respectively. These results obtained in this work reflecting that there was

an increase in one or more of the clotting factors involved in the intrinsic

pathway. Plasma fibrinogen which was not measured in this study has been

known to facilitate the rate of fibrin polymer formation which ultimately

leads to more effective clot formation (Guyton and Hall, 2...) while Hadi

et al. (2.8.) showed marked effect of gum arabic on the coagulation

system of rats that it prolongs the BT and CT.

In this study platelets count increased significantly compared with the

control to (±6±*8.7/L), (7±8*8.7/L) and (8876*8.7/L) respectively. In

general for the three parameters (8. mg/ml) of crude alkaloid extract of A.

arabica was the most effective concentration.

Crude alkaloids activity of the three plants under study revealed that

crude alkaloids of Z. officinale dry rhizomes was the most effective as the

yield percentage was higher than crude alkaloids of T. vulgaris dry leaves

and Acacia arabica dry gum respectively as well as it was efficient at the

concentration 8mg/ml while the efficient concentration for T. vulgaris was

5 mg/ml and for A. arabica was 8. mg/ml which confirms the results

obtained in this study as alkaloids yield percentage of each plant under the

study was compatible with their most effective concentrations. This makes

crude alkaloids extract of Z. officinale dry rhizomes is the best plant product

therapeutically and commercially.

6-4-4 Effect of Phenols Extract in Bleeding Time, Clotting Time and

Platelets Count

The results of the crude phenols extract of Z. officinale in table (3-5)

revealed significant differences at (p> .0.5). The concentrations (8 mg/ml),

(5 mg/ml) and (8. mg/ml) decreased the BT compared with control to (8038

min.), (802. min.) and (808± min.) respectively. Miller (877±) mentioned

that some cautious physicians have advised that zingiber may alter bleeding

time. These concentrations also decreased the CT compared with control to

(2026 min.), (8069 min.) and (8027 min.) respectively; while platelets count

increased significantly compared with control to (8.2±*8.7/L),

(822±*8.7/L) and (8493*8.7/L) respectively. Crude phenols extract may

have positive effect on haemostasis possibly by acting on the integrity of

the blood vessel or involvement of platelets forming the haemostatic plug

or both. Another possible explanation may be the inhibition of the

formation of prostaglandin by the vessel walls during injury.

Prostaglandins released during injury are responsible for vessel

relaxation, which leads to increase in bleeding of blood (Bunting et al.,

8796). In general for the three parameters (8. mg/ml) of crude phenols

extract of Z. officinale was the most effective concentration.

In table (3-6) results of the crude phenols extract of T. vulgaris showed


mg/ml) and (8. mg/ml) decreased the BT compared with control to (8043

min.), (803. min.) and (8.89 min.) respectively; as well as decreased the CT

compared with control to (2085 min.), (2..2 min.) and (8.35 min.)

respectively; while platelets count increased significantly compared with

control to (588*8.7/L), (992*8.7/L) and (±86*8.7/L) respectively which

agreed with Turhan et al. (2.88) who showed histopathological results of

coagulative necrosis which revealed platelets aggregation due to increase

in platelets count and also Upton (2..8) who mentioned that plant phenols

of the same family T. vulgaris belonging to (e.g. Vitex agnus-castus L. )

showed increase in platelets count due to the effect on one of the

coagulation factors While Okazaki et al. (2..2) mentioned that thymol,

isolated from the leaves of thyme, was found to inhibit platelet aggregation

induced by collagen, ADP, arachidonic acid and thrombin. In general for the

three parameters (8. mg/ml) of crude phenols extract of T. vulgaris was

the most effective concentration.

Results for crude phenols extract of A. arabica in table (3-9) showed


mg/ml) and (8. mg/ml) decreased the BT compared with control to (.0±±

min.), (8023 min.) and (8056 min.) respectively; also decreased the CT

compared with control to (8065 min.), (2022 min.) and (203. min.)


control to (783*8.7/L), (6±7*8.7/L) and (6.6*8.7/L) respectively, which

agreed with Bamidele et al. (2.8.) who mentioned that the methanolic leaf

extract of Ageratum conyzoides exhibited haemostatic activities by

decreasing bleeding, prothrombin and clotting times due to the presence of

Tannins which have been implicated in the haemostatic activity of plants

where they arrest bleeding from damaged or injured vessels by

precipitating proteins to form vascular plugs (Okoli et al., 2..9). Therefore,

the haemostatic mechanism of A. arabica also may be related to the

presence of these phytochemicals. In general for the three parameters (8

mg/ml) of crude phenols extract of A. arabica was the most effective

concentration.

Crude phenols activity of the three plants under the study revealed that

crude phenols of A. arabica dry gum was the most effective as the yield

percentage was higher than crude phenols of Z. officinale dry rhizomes and

T. vulgaris dry leaves respectively as well as it was efficient at the

concentration 8mg/ml while the efficient concentration for Z. officinale and

T. vulgaris was 8. mg/ml which confirms the results obtained in this study

as phenols yield percentage of each plant under the study was compatible

with their most effective concentrations. This makes crude phenols extract

of A. arabica dry gum is the best plant product therapeutically and

commercially.

6-4-5 Effect of Terpens Extract in Bleeding Time, Clotting Time and

Platelets Count

The results of the crude terpenes extract of Z. officinale in table (3-±)

revealed significant differences at (p> .0.5). The concentrations (8 mg/ml),

(5 mg/ml) and (8. mg/ml) decreased the BT compared with control to (8085

min.), (8089 min.) and (80.7 min.) respectively; as well as decreased the CT

compared with control to (2022 min.), (2023 min.) and (803± min.)


control to (962*8.7/L), (683*8.7/L) and (8.56*8.7/L) respectively, which

doesn´t comply with Spehaat (2..±) who mentioned that the crude

extraction of Z. officinale showed increase in BT, CT but agreed with

platelets count results compared with control. As results showed that (8

mg/ml) was more efficient than (5 mg/ml) this might happened due to

negative response to the extract that has been given to the mice under the

test which might increases the blood fluidity rather than decreasing it

(Zokian, 2..5). In general for the three parameters (8. mg/ml) of crude

terpenes extract of Z. officinale was the most effective concentration.

In table (3-7) results of the crude terpenes extract of T. vulgaris showed


mg/ml) and (8. mg/ml) decreased the BT compared with control to (8.83

min.), (8.±. min.) and (..69 min.) respectively, while Chan et al. (2..9)

reported increased BT in Gingko biloba leaves extract due to the presence

of terpenes in high concentration. In this study terpens extract of T.

vulgaris also decreased the CT compared with control to (8067 min.), (2.85

min.) and (8.38 min.) respectively; while platelets count increased

significantly compared with control to (996*8.7/L), (636*8.7/L) and

(8.±4*8.7/L) respectively. As mentioned above (8 mg/ml) concentration

was more efficient than (5 mg/ml) as mentioned previously this might

happened due to negative response to the extract that has been given to

the mice under the test which might increases the blood fluidity rather than

decreasing it (Zokian, 2..5). As mentioned before T. vulgaris was studied as

a part of mixture of plant extracts called Ankaferd blood stopper which as

reported provides hemostasis independently from coagulation factors and

the standard coagulation cascade. As the mechanism of action, it forms a

structural net by interacting with proteins, especially with fibrinogen, in

blood and hence, provides vital aggregation of erythrocytes (Goker et al.,

2..±; Aydin, 2..7; Cipil et al., 2..7). In general for the three parameters

(8. mg/ml) of crude terpenes extract of T. vulgaris was the most effective

concentration.

Crude terpenes activity of the plants under the study revealed that

crude phenols of T. vulgaris dry leaves was the most effective as the yield

percentage was higher than crude terpenes of Z. officinale dry rhizomes as

well as it was effective at the concentration 8. mg/ml and the efficient

concentration for Z. officinale was also 8. mg/ml which revealed slight

difference in their activity as as both of them were effective at the same

concentration despite the differences in the yield percentage of terpenes of

the other two plants. This makes crude terpenes extract of T. vulgaris dry

leaves is the best plant product therapeutically and commercially.

Conclusions

& Recommendations

Conclusions

In this study the following findings were concluded:

8- Extraction of crude alkaloids, phenols and terpenes of Zingiber

officinale dry rhizomes, Thymus vulgaris dry leaves and Acacia

arabica dry gum revealed the following:

a- Higher yield percentage of alkaloids was obtained from Z. officinale

dry rhizomes.

b- Higher yield percentage of phenols was obtained from A. arabica dry

gum.

c- Higher yield percentage of terpenes was obtained from T. vulgaris

dry leaves.

2- Plants extracts showed effect on coagulation parameters (bleeding

time, clotting time and platelets count) causing decrease in both

bleeding time and clotting time, and increase in platelets count.

3- Crude alkaloids extract of Z. officinale dry rhizomes was the most

effective followed by phenols extract and terpenes extract

respectively.

4- Crude phenols extract of A. arabica dry gum was the most effective

followed by alkaloids extract among the three plants.

5- Crude terpenes extract of T. vulgaris dry leaves was the most

effective followed by alkaloids extract and phenols extract

respectively.

6- In a comprehensive comparison between the plants under study,

crude phenols extract of A. arabica dry gum was the best

therapeutically and commercially because it gave very high yield

percentage and it was effective in the lower concentration used in

the study.

Recommendations

From all obtained and analyzed results the following points can be

recommended:

8- Further study of chemical separation and purification of the active

components in Zingiber officinale dry rhizomes, Thymus vulgaris dry

leaves and Acacia arabica dry gum.

2- Using plant extracts as spray or ointment instead of intra gastric

administration recommended to be studied.

3- Study the histopathological effects of Z. officinale dry rhizomes, T.

vulgaris dry leaves and A. arabica dry gum extracts.

4- Study the biochemical and enzymatic activity, toxicological,

therapeutic and immunological effects of the active groups tested in

Z. officinale dry rhizomes, T. vulgaris dry leaves and A. arabica dry

gum extracts.

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