STUDIES ON THE UTER0TONIC ALKALOIDS

OF

FRUCTUS EVODIAE

BY

KING CHEUNG-LAM

B. SC. (HON.), C.U.H.K.

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENT FOR THE DEGREE OF

MASTER OF PHILOSOPHY

IN

BIOCHEMISTRY

May, 1979

DEPARTMENT OF BIOCHEMISTRY

THE CHINESE UNIVERSITY OF HONG KONG

1

TO ALL EXAMINERS AND GRADUATE SCHOOL OFFICE

Evodia rutaecarpa is a plant under investigation

in a World Health Organization sponsored research contract.

We are not allowed to disclose any original results without

prior World Health Organization clearance.

Any citation or public disclosure of these

results may jeopardize our patent position and forfeit

further World Health organization support.

ACKNOWLEDGEMENT

I wish to express my sincere gratitude to Dr. Y.C.

Kong for his valuable guidance throughout the entire peroid

of this investigation.

I would also like to thank Prof. U. Sankawa for

his helpful discussions and a generous gift of synephrine

and N,N-dimethvl-5-methoxvtrvptamine.

I am indebted to Dr. C.Y. Lee, Dr. H.W. Yeung and

Prof. H.H.S. Fong for supervision and many helpful discussions.

My grateful thanks are due to Mr. T.T. Yip, Mr. K.H

Ng, Mr. Y.T. Chun, Miss S.K. Wong, Miss K.L. Lau and Miss Y.L.

Yau for their technical assistance and valuable discussions.

This work is partly supported by World Health

Organization, Special Programme for Research in Human

Reproduction.

TABLE OF CONTENTS

Page No.

1. INTRODUCTION 1

1.1 ANATOMY AND PHYSIOLOGY OF THE UTERUS 2

1.1.1 Actions of ovarian hormones on

the myometrium 4

1.1.2 Actions of ovarian hormones on

the endometrium 5

1.2 THE CONTRACTILE MECHANISM OF MYOMETRIUM 7

1.3 NATURAL UTERINE STIMULANTS 10

1.4 USE OF HERBAL MEDICINES FOR

ANTIFERTILITY CONTROL 13

1.5 EVODIA RUTAECARPA 19

1.5.1 History 20

1.5.2 Phytochemistry 21

1.5.3 Pharmacology 21

2. EXPERIMENTAL 26

2.1 PURIFICATION AND STRUCTURE DETERMINATION

OF EVODIA RUTAECARPA ALKALOIDS 26

2.1.1 Isolation and identification of

rutaecarpine 26

2.1.2 Isolation of dehydroevodiamine

hydrochloride( DHE) 31

2.1.3 Isolation of N,N-dimethyl-5-methoxy-

tryptamine and synephrine 31

2.1.4 Detection of alkaloids 31

2.1.4.1 Mayer reagent 31

2.1.4.2 Dragendorff reagent 34

2.2 SPECTROSCOPIC ANALYSIS 35

2.2.1 UV-visible spectroscopy 35

2.2.2 IR spectroscopy 35

2.2.3 NMR spectroscopy 35

6

Page No.

2.3 PHARMACOLOGICAL STUDIES 35

2.3.1 The animal model 35

2.3.1.1 In vivo test 35

2.3.1.2 In vitro test 36

2.4 ENZYME ASSAY 36

2.4.1 Bovine erythrocyt

acetylcholinesterase 36

2.4.2 Rat uterus acetylcholinesterase 37

2.5 STATISTICAL ANALYSIS 37

3. RESULTS 38

3.1 PURIFICATION OF EVODIA RUTAECARPA

ALKALOIDS 38

3.1.1 Purification of rutaecarpine 38

3.1.2 Purification and identification

of DHE 40

3.2 PHARMACOLOGICAL OBSERVATION 42

3.2.1 Uterotonic effect of DHE 42

3.2.2 Potentiation effect of DHE on

uterine stimulants on isolated

rat uterus 47

3.2.3 Uterotonic effect of rutaecarpine 48

3.2.4 Uterotonic effect of

N,N-dimethyl-5-methoxytryptamine 48

3.2.5 Relaxation effect of synephrine

on contracting uterus 48

3.3 EFFECT OF DHE ON ACETYLCHOLINESTERASE

ACTIVITY 63

4. DISCUSSIONS 67

4.1 SUITABILITY OF THE ANIMAL MODEL 67

4.2 UTEROTONIC EFFECT OF EVODIA RUTAECARPA

ALKALOIDS 69

5

Page No.

4.3 POTENTIATION EFFECT OF DHE ON UTERINE

CONTRACTION 71

4.4 PHARMACOLOGICAL EFFECT OF EVODIA RUTAECARPA

IN ETHNOMEDICAL PREPARATION 72

4.5 PROSPECT OF USING DHE AS ANTIFERTILITY

AGENT 74

5. SUMMARY 79

6. REFERENCES 81

1. INTRODUCTION

The uterus is composed of myometrium and endometrium

It provides the site of the attachment for the fetus, an

environment for the fetal growth during gestation and a

mechanism for its expulsion at term. Since the uterus is an

organ of reproduction, it will undergo histological and

biochemical changes during the reproductive cycles under

the influences of reproductive hormones. In general, the

myometrial activity is enhanced by estrogen and depressed

by progesterone. The endometrium is specially sensitive to

the changes of the ovarian hormones.

Theoretically, a uterine stimulant is a potential

contraceptive agent. Since a quiescent uterus is essential

for the implantation process and for the maintenance of

pregnancy, uterine stimulants could be intensified as

postcoital or once-a-month contraceptives. Uterine stimulants

can also be used for the treatment of postpartum hemorrhage,

dystocia as well as amenorrhea.

According to Chinese medical literature, there

are hundreds of plants used in gynecology and obstetrics.

They are mainly used as emmenagogue, abortifacient and ecbolic.

These plants were thought to contain uterine stimulants and

some of them had been isolated and identified( Kong et a l.,

1976a,b Ghosal, Singh and Battacharya, 1971 Sizov, 1969).

Of these natural uterine stimulants, sparteine and ergonovine

2

are most frequently used in obstetrics nowadays. They arE

alkaloids isolated from serveral Leguminosae species and

ergots respectively.

The rapid population growth puts a constant

demand on new and effective contraceptive agents. The

present study represents an attempt to select potential

antifertility agents from Chinese medicinal herbs. In

this investigation uterotonic assay is used to moniter

the drug activity.

1.1 ANATOMY AND PHYSIOLOGY OF THE UTERUS

Human uterus is a pyriform body 9.0 cm in

length, 6.5 cm in width and 3.5 cm in thickness. It is

divided anatomically and functionally into body and cervix.

The uterine wall consists of three layers, an outer serosal

layer( peritoneum), a firm, thick, intermediate coat

of smooth muscle( myometrium) and an inner mucosal lining

( endometrium).

The myometrial cell is a contractile system

surrounded by an excitable membrane( Csapo, 1962 ).The

cells are small, spindle-shaped with a length of 20-60 um

and a maximum diameter of 2-10 um. But their dimensions

are altered by hormonal and physical factors. The myometrial

cells are exposed to structural and functional changes from

puberty till menopause, during the menstrual cycles, pregnancy,

3

and postpartum involution. The largest cells are observed

in pregnant animals at term. The individual cells form

fibre bundles of about 100 um in diameter and are embedded

in a connective tissue network. Three layers can be

distinguished. The outer layer bundles are arranged parallel

to the long axis of the uterus body contraction of this

layer tends to shorten the uterus cephalo-caudally. Its

main function is to expel the fetus at term. In the second

layer, the muscle fibres are interlaced with the blood

vessels in the connective tissues. Contraction of this layer

produces a hemostatic effect, although its expulsive action

during labour is no less important. The inner muscle layer

consists of circular fibres, contraction in this layer

serves to constrict the uterus lumen.

The endometrium of the uterus is about 3-4 mm

thick and it consists of a surface epithelium, uterine

glands and stroma. The cuboidal epithelial cells are ciliated

in some animals such as human and rabbit( Fleming, Tweedle

and Roddick, 1968 )they play a major role in the secretion

of endometrial fluids which are important for the survival

and transport of the gametes. The uterine glands of endometrium

are simple tubules during post-menstrual phase but become

corkscrew-shaped near menstruation. The stroma intervenes

between the surface epithelium and the myometrium, it

consists of spindle-shaped cells, blood vessels and lymphatics.

4

1.1.1 Actions of ovarian hormones on the myometrium

Uterine atrophy due to deficiency in estrogen was

already noted many centuries ago( Nilsson, 1958a). The

contractile proteins and high energy phosphates were decreased

after ovariectomy. Substitution therapy in estrogen-deficient

animals induces mark effects on the uterus. It restores

the contractile system and its working capacity.

The suppression of the uterine motility during

pregnancy has been called' inactivation' by Reynolds( 1965)

or' progesterone block' by Csapo( 1956a, b). In rabbit,

the inhibitory effect of progesterone on the myometrial

response to oxytocin( Csapo, 1961a, b) was observed.

Schofield( 1957) showed that the uterus was refractory

to oxytocin action throughout pregnancy. Allen and

Reynolds( 1953) revealed the inhibitory effect of progesterone

on rabbit uterus by the method of intrauterine pressure

recording. Csapo and Takeda( 1965) pointed out that

progesterone reduced the amplitude but not the frequency of

the contractions of the rabbit uterus in vivo. Ichikawa

and Bortoff( 1970) observed an increase in intercellular

resistance in the progesterone-dominanted myometrium. They

speculated that this factor may be important in preventing

the propagation of action-potential and thus inhibit the

uterus activity.

5

1.1.2 Actions of ovarian hormones on endometrium

There are morphological and biochemical changes

during the estrus and menstrual cycles under the influence

of estrogen and progestins.

Administration of estrogen causes a rapid uptake

of water in rodent uterus resulted in both intercellular

oedema and fluid accumulation in uterine lumen( Carroll,

1945). The cellular response in the endometrium is most

pronounced in the epithelium. The cells of both the luminal

and glandular epithelia increase in size, especially in

height(. Nilsson, 1958a, b, c). The effect is initially

caused by water infiltration into the cell and followed by

true cell growth. The amount or the size of many subcellular

organelles such as ribosome, endoplasmic reticulum, mitochondria,

golgi apparatus and nuclei were also increased( Schultz

et al., 1969 Wilson, 1963 Elftman, 1963 Nilsson, 1958a).

Estrogen stimulates the growth of the mucosal lining of

the endometrium. It also primes the endometrium for the

action of progesterone.

The straight glands become conspicuously

tortuous during the luteal phase by the action of progesterone

In human and some primates, gland proliferation is initiated

by estrogen and the glands are then converted to a secretory

condition by progesterone( Engle and Smith, 1938 Kohorn

and Tchao, 1969 ).Similar cases were observed in rat, mouse

6

and guinea pig.

The normal cyclic changes in endometrium are closely

correlated to those in the ovary. The proliferative phase

and secretory phase of endometrium correspond to the

follicular phase and luteal phase of the ovarian cycle

respectively. In human, the 28-day menstrual cycle can

be divided into three stages, i.e., menstruation, proliferative

phase and secretory phase.

The first stage, where menses occur during 3-5

days, is characterized by hemorrhage in the endometrial

stroma and breakdown of the glandular epithelium.

Days 5-14 is proliferative phase, under the

influence of estrogen, there is rapid growth and proliferation

of the epithelial cells. During this time, the epithelial

cells increase in size and become columnar. The glands

increase in length and tortuosity. The stromal cells show

increasing complexity during this phase. Cell proliferation

and hypertrophy continue until before ovulation.

The secretory phase begins on the fifteenth day

and persists until the onset of menstruation. The endometrium

is influenced by the combination of estrogen and progesterone.

It is characterizied by the appearance of the subnuclear

vacuoles in the glandular epithelium. The fluid in these

subnuclear vacuoles consists of glycogen and mucin, the

7

function of which is presumably nutritive for the fertilized

ovum if it is implanted. The content of the vacuoles are

discharged into the glandular lumen by day 19. Oedema occurs

in the stromal cells in early secretory phase and reaches

its peak by day 22. After that, no further growth is

observed in the normal cycle of the non-pregnant uterus.

Around day 24, glandular epithelial involution begins. The

stromal cell is infiltrated by leukocytes and later by

erythrocytes.

1.2 THE CONTRACTILE MECHANISM OF MYOMETRIUM

Analytical study of the mechanism of myometrial

function began with the isolation of the protein complex

actomyosin, the high energy phosphate compound ATP, and the

in vitro examination of their interaction( Csapo, 1955

1962). Actin-free myosin has been prepared from uterine

actomyosin( Csapo, 1959) while myosin-free actin has also been

obtained from the uterus in 1967( Needham and Shoenberg,

1967),

The proportion of actin to myosin in smooth muscle

actomyosin is 1: 4, comparable to that of skeletal muscle

actomyosin( Needham and Williams, 1963b but the total

amount of actomyosin present is 6-10 mg/g wet wt which is

considerably lower than that found in skeletal muscle

( 70 mg/g wet wt)( Needham and Williams, 1963a).

3

Actin is present in the thin filaments, 30-80 R in diameter,

lying largely parallel to the longitudinal axis of the cell.

There are no differences observed among smooth muscle actin,

skeletal muscle actin and cardiac muscle actin of various

species(Carsten and Katz, 1964). The amino acid

compositions are also similar for these actins( Grosslin-

Rey et al., 1969 Bray and Thomas, 1975).

Since myosin forms thick filaments in all other

types of muscle and since they are essential for the classical

sliding filament theory of contraction, it was expected

that thick filaments must also be present in smooth muscles.

However, there are some debates about the presence of myosin

filament. Hanson and Lowy( 1964) identified only actin

filaments but not myosin filaments in guinea pig taenia coli.

Elliot( 1967) could only demonstrate actin pattern in X-ray

diffraction in most smooth muscles.

During the 1960's, thick filaments were seen in

some smooth muscle cells in a number of laboratories,

but they were observed only in muscles that had received

unphysiological pretreatments( See review by Shoenberg

and Needham, 1976). Recently, many workers observed the

thick filaments in different types of smooth muscles.

The thick filaments of vertebrate smooth muscles differ

strikingly from those of other muscle and it is not yet

known how much of the myosin in these muscles is actually

in filament form in vitro. Furthermore, the smooth muscle

9

myosin is so labile during fixation that the electron

microscopic observations thus obtained cannot be counted and

exact structure of the myosin in smooth muscle remains a

problem.

Tropomyosin has also been found in smooth

muscle( Sheng and Tsao, 1955 Needham and Williams, 1963a

and is present in higher proportion to actomyosin than

in skeletal muscles, i.e., 1: 2-3 in smooth muscle

( Needham and Williams, 1963b) compared to 1: 5-6 in

skeletal muscle( Perry and Corsi, 1955). Recently,

both troponin and a-actin have been isolated from smooth

muscles( Ebashi, 1969).

Bagdy et al.( 19 71) amd Small( 1974) have

studied the contraction in isolated smooth muscle cell and

concluded that the contractile elements are grouped in

fibrils which lie obliquely to the cell axis. In Hanson

and Huxley's( 1955) classical sliding filament theory

of contraction, both actin and myosin are in filament form and

it has generally been assumed that this is a prerequisite

if the system is to operate. In vertebrate smooth muscle

there is no doubt that the actin is in filament form. But

it has not yet been shown conclusively that all the

myosin is also aggregated into filaments. However, there

were no significant differences found between striated and

smooth muscle in biochemical and mechanical characteristics.

Nor does the fine structure of smooth muscle exclude the

10

operational principle of a sliding mechanism. Therefore,

one may consider that a similar contraction mechanism

is operated in smooth muscle.

1.3 NATURAL UTERINE STIMULANTS

There are many compounds having oxytocic effect,

such as amines, peptides, alkaloids and fatty acids.

Although there are a great number of uterine stimulants, only

a few of them are clinically useful. Many others, when

administered in adequate doses to increase uterine activity,

evoke significant side effects. The clinical oxytocics

found their greatest use in the management of postpartum

hemorrhage, which is today the chief cause of puerperal

maternal death. They may also be used in the treatment of

incomplete abortion, missed abortion, uterine hypotonicity

and certain menstruation disorders.

Oxytocin is a hormone secreted from the posterior

pituitary gland. It is a nanopeptide in which a ring

structure is formed by the closure of an S-S bond between the

cysteine molecules. Clinically, oxytocin is used to induce

labour. The exact mechanism of the stimulatory action

is not clear. It is well established that oxytocin

increases the influx of extracellular calcium ion together

with the mobilization of intracellular calcium pools.

Calcium ion triggers the contraction process by an unknown

11

pathway. Dousa( 1977) reported that calcium ion stimulates

the generation of cGMP. He speculated that cGMP may be

the primary factor for uterine contraction.

Prostaglandins( PG's) are fatty acid derivatives.

PGE and PGF stimulate the pregnant or non-pregnant human

uterus. Prostaglandin is very useful for midtrimester

abortion but they bear many side effects such as actions

on the cardiovascular system and gastrointestinal tract.

Intra-uterine or intra-amniotic administration is more

effective and the side effects are decreased.

There are many classes of alkaloids that have

uterotonic effect, (Table 1.) only the most commom ones

are mentioned here.

Ergot alkaloids are indole derivatives. Generally

speaking, the ergot alkaloids form a pair of isomers and

only the levorotatory forms are pharmacologically active.

Of these natural ergot alkaloids, ergonovine( ergometrine)

is the most important clinically. The pregnant human

uterus is highly sensitive to ergonovine at term. The

action on the uterus is prompt and powerful which may

persist for several hours. This ergot alkaloid is always

used for the managemant of-postpartum hemorrhage. Post-

partum uterus is extremely sensitive to ergonovine and

effects were observed with doses' less than 0.05 mg

intravenously. Undesirable side effects are rare following

12

the administration of therapeutic doses.

The only clinically useful abortifacient plant

product known at present is sparteine( pachycarpine)

( Zhvaniya, 1970 Manske, 1955). It is an alkaloid derived

from serveral Leguminosae species. Sparteine was first

introduced into clinical practice as an oxytocic in 1939

and thousands of patients have been treated successfully

with it. Numerous reports confirm its oxytocic action both

in vitro and in vivo. Undesirable side effects in human

are almost always due to overstimulation of the myometrium

thus, the dose of this alkaloid must be carefully monitored.

'Quinine is often used with castor oil to induce

labour in human. Quinine has been reported to have uterotonic

effect in vivo and in vitro( Acton, 1921). Evidences

suggest that the motility of the human puerperal uterus

is enhanced by small doses and depressed by large doses

of quinine.

Leonurine. (4-guanidino-n-butyl syringate) was

isolated from the leaves of the Chinese motherwort Leonurus

artemisia. Kong et al.( 1976b) reported that leonurine

stimulated the isolated rat uterus in vitro at a dose

of 0.2 ug/ml. Linear responses were observed between 0.2-

1.0 pg/m1.

Hydrastine, which is isolated from Hydrastis

canadensis L.( golden-seal), is structurally related to

13

narcotise. Hydrastine stimulates the guinea pig uterus

both in vivo and in vitro( Reynolds, 1940). Reynolds

observed marked contractions at a dose of 20pg/ml of

hydrastine. The isolated rabbit uterus responses differently

at different doses. It is stimulated by low dose( 5 pg/ml)

and suppressed by high dose( 83 pg/ml)( Welch and

Henderson, 1934). Hydrastine hydrochloride has been

employed in uterine hemorrhage, the therapeutic dose is

10 mg.

1.4 USE OF HERBAL MEDICINES FOR ANTIFERTILITY CONTROL

The world population explosion necessitates

continuous efforts to develop new and effective contraceptive

agents with a minimum of side effects. Current contraceptive

methods such as steroidal pills, intrauterine devices( IUP)

and prostaglandins are effective but carry some definite

defects( See review of Josimovich, 1973). They have

limitations with respect to safety, efficacy, acceptability

and simplicity of use. The greatest concern to women using

steroidal contraceptive pills are their association with

thromboembolic disease, heart disease and tumors. Many

other minor side effects such as nausea, menstrual irregularities

and chloasma, though not lethal, are unacceptable to some

users. Intrauterine devices also cause some unpleasant

side effects such as bleeding and abdominal pain, they are

14

Table 1. Alkaloids with uterotonic effect

Ergonovine

HOCH2CHNHOC

N

CH3

NH

Sparteine

Quinine

H2C CH

H

NHO

N

CH3O

N

N

15

Leonurine

CH30

HO

CH3O

COOCH2CH2CH2CH2NHC

NH

NH2

Hydrastine

N

CH3

CH3

OCH3

16

troublesome enough to undermine its acceptability for some

women.

In most developing countries, most modern

contraceptice devices are either unavailable or too

expensive for chronic use. Moreover, medical check-up

twice,a year, as it was recommended for pill or IUD

users,requires a dedicated medical superstructure which

is not possible for most countries. Thus, some inexpensive

safe and long-term active contraceptives are urgently

required.

Practically all major research effort involved

with the search for new oral contraceptives has been of the

synthetic types. Recently, attention has been directed to

the plant kingdom( World Health, 1978). There are three

approaches to select the potential antifertility plants,

i.e., a) plants with folkloric reputation suggesting that

they are actually used as oral contraceptives, b) plants

containing constituents that could theoretically affect

the female reproductive system to produce antifertility

effects,( estrogenic sterols, coumesterols and isoflavones)

c) plants that are uterotonic.

The first step for the selection of potential

antifertility plants for laboratory studies is ethnomedical

information collection. In China, abundant and systematic

knowledge in medicinal plants are recorded in a special

17

format' Peng Ts'ao'. From here there are about 200 plants

frequently used as abortifacients or contraceptives in

traditional Chinese medicine(Farnsworth et al., 1975

Kong et al., 1976a). The second step is the examination

of the literatures about the chemical compositions of the

plants. Certain plants containing compounds that can

theoretically affect the reproductive system. For example,

stigma sterol in Glycine max(Soy bean), 13-sitosterol

in cereal grains, and diosgenin in Dioscorea spp.( yams).

These compounds are structurally similar to steroid hormones

and used for the synthesis of steroids( Breger, 1960).

The third step concerns the plant.sources. The plants

should be collected in sufficient amounts for phytochemical

and pharmacological investigations. Detailed ethnobotanical

and taxonomic studies is the last and the most important

step prior to laboratory investigation. Different pharmacological

effects and chemical constituents were obtained from

different species. It should also be known that geographic

variations result in various chemical composition for the

same species.

Some well known Chinese herbal drugs affecting

the human reproductive functions are listed in Table 2.

in most developing countries, the use of indigenous

medicinal herbs is still very mupha part of their health

care system. For instance, Chinese medicinal herbs have

18

Table 2. Plants affecting human reproductive functions

Plant Function

AbortifacientPrunus persica

Crocus sativus Abortifacient

Achranthes bidentata Abortifacient

AbortifacientSophora japonica

Pistacia lantisus Ecobolic

Ricinus communis Ecobolic

EcobolicVaccaria pyrarnidata

Cyperus rotundus Emmenagogue

Angelica sinensis Emmenagogue

EmmenagogueRuta graveolens

Polygonum hydropiper For menorrhagia

Capsella bursa-pastoris Uterine contraction

Evodia rutaecarpa Uterine contraction

Leonurus artemisiaExpelling placenta

or dead fetus

17

been used for more than two millennia, the therapeutical

values and other pharmacological effects are constantly

experienced by millions of people today. The universal

acceptability for herbal medicine among ethnic Chinese

transcends the economic status and education background.

The discovery of effective antifertility agents from

indigenous medicinal plants will eventually be proved

economically sound and_ acceptable in developing countries

It can also provide an alternative for people in the

developed countries who,for one reason or another, are

incompatible with steroidal pills.

1.5 EVODIA RUTAECARPA

Evodia rutaecarpa( Juss) Benth.( Rutaceae),

an evergreen shrub or small tree, usually 2.5-5.0 m tall.

Leaves opposite, imparipinnate leaflets subsessile,

elliptic, accuminate, entire, surface woolly pubescent,

5-15 cm long and 2-6 cm wide, 2-4 pairs. Inflorescence,

a paniculate cyme, flowers small, unisexual, dioecious,

light yellow. Fruit,a capsule with 5 loculi. Mainly

distributed in the provinces of Szech'uan and Kwangsi

in southwest China. It usually grows at roadside or

under tall trees. The unripe fruits collected in autumn

were used medicinally.

20

1.5.1 History

Evodia rutaecarpa( Wu Zhu Yu) has been used

for a long time in Chinese medicine. It is classified as

a' hot' drug. The earliest written record of Evodia

rutaecarpa appears in Sheng Nung Peng-ts'ao Ching( 100-

180 A.D.). It is a drug of the medium category, indicating

that it is not a tonic and should be prescribed only to

cure disease. In Peng-ts'ao Kang Mu( Li, 1593) it

was recorded that the tree was formerly planted at the

side of a well, so that the leaves might fall into the

water. Drinking the water achieved the prophylactic

effect against contagious diseases. The fruits are used

as stimulant, carminative, stomachic, deobstruent, astringent

and anthelmintic remedies. The root and bark are used

as astringent and anthelmintic remedies and in the treatment

of rheumatism.

In. traditional medicine, Evodia rutaecarpa

is incorporated in standard formulae for the treatment of

many diseases. For example, it can be used in combination

with Coptis chinensis in the treatment of some gastro-

intestinal diseases( Tso Chin Wan, Zhu, 1350). 左 金 丸 )(Zhu,1350)

Anther well known prescription is the Chi Kung Wan ( 啟 客 丸 )

Which consits of Evodia rutaecarpa,Cnidium monniei,

Polygala tenuifolia,zingiber officinale and moschus moschiferus

for the treatment of menstruation disorders.

1.5.2 Phytochemistry

Rutaceae family includes many important medicinal plants, such as Citrus aurantium ( ) , Evodia

rutaecarpa, Murraya paniculata

and Phe1lodendron amurense. Up to now, over300 alkaloids were isolated from 182 species (Mester,1973; 1977 ). Some of them have significant biological activities. For example, acronycine from Acronychia bauari

has anti-cancer properties ( Lam, 1977a ); and berberine from Evodia meiiaefolia has uterotonic effect ( Imaseki, Kitabatake and Taguchi; 1961 ).

More than 20 alkaloids were isolated from Evodia spp ( Mester, 1973; 1977 ). At least three of them show significant uterotonic activity. They are skimmianine, dictamine and berberine ( Panashchenko, 1967; Kovalenko, 1946;Imaseki, Kitabatake and Taguchi, 1961 ).

Chemical Studies on the fruits of Evodia rutaecarpa

were reported by serveral investigators. The compounds identified were listed in Table 3.

1.5.3. Pharmacology

Parallel to the folkloric used, serveral pharmacological effects of Evodip rutaecarpa have been observed in this century. These include anthelmintic

21

22

Table 3. Compounds isolated from the fruits

of Evodia rutaecarpa

Compound Physical data References

Evodiamine C19H17N30 Li and Huang, 1966

mp 278°C Asahina and Kashiwaki,

1915

Rut aecarp me C18H13N30 Li and Huang, 1966

mp 258°C Asahina and Kashiwaki,

1915

Wuchuyine C13H1302N Chen and Chen, 1933

mp 237.5°C

Hydroxyevodiamine C19H17N302 Yamazaki and Kawana

mp 192'C 1967( Rhetsinine)

Evocarpine C22H33NO Tschesche and Werner.

1967

Hirose et al., 1969

23

Table 3.( Cont.)

Compound Physical data References

Evodene C10H1 Asahina and Kashiwaki

bp 67-68°C 1915( Ocinene)

Evodin C26H3008 Asahina and Ishio,

mp 285°C( Limonin) 1915

Fujita and Akatsuka

1949

Rutaevin C26H3009 Hirose, 1971

mp 3000C

Evodinone C26H3209 Chu, 1951

mp 295-297'C

Evogin C24H2808 Chu, 1951

mp 279-281°C

Evodol C26H2809 Hirose, 1963

mp 281-282°C

Goshuyuic acid C14H2402 Kurona et al., 1973

24

Table 3.( Cont.)

Compound References

1-methyl-2-pentadecyl- Kamikado et al., 1976

4 (1H)- quino lone

1-methyl-2-tridecyl- Kamikado et al., 1976

4(1H)-quinolone

( Dihydroevocarpine)

1-methyl-2-undecyl- Kamikado et al., 1976

4(1H) -quino1one

25

effect( Wu, Xu and Xiao, 1948), diuretic effect( Anon,

1961) and antibacterial and antiviral effect( Cheng, 1952

Tsao, 1957). The hypertensive effect on the anesthetized

dog was also reported( Anon, 1959). Methanol extract

of Evodia rutaecarpa was reported to have analgesic effect

( The encyclopedia of Chinese medicine, 1977). Although

Evodia rutaecarpa is used to cure dysmenorrhea, there is no

report about any effect of Evodia rutaecarpa on reproductive

functions.

26

2. EXPERIMENTAL

2.1 PURIFICATION AND STRUCTURAL DETERMINATION OF

EVODIA RUTAECARPA ALKALOIDS( FIG. 1)

Dry fruits of Evodia rutaecarpa were purchased

from the herbal stores. According to the dealer, these

fruits were originated from Szech'uan province in China.

A voucher sample was saved for future reference.

2.1.1 Isolation and identification of rutaecarpine

5.3 Kg of the fruits were grounded and defatted

with petroleum ether( 60-80°C), followed by exhaustive

extraction with methanol. The methanolic extract was

concentrated to a thick syrup in vacuo. 5% Hydrochloric

acid was added and the residual methanol was subsequently

removed in vacuo. The aqueous solution was made alkaline

with 287 ammonia which was extracted with chloroform for

6 times. The chloroform solution was dehydrated with anhydrous

sodium-sulfate and concentrated. The chloroform extract

obtained was subjected to column chromatography on silica

gel 60( 70-230 mesh). The column was developed with

benzene and 16 ml fractions were collected. The elution

profile and the yield were tabulated in Table 4. Fraction I

was allowed to crystallize. The crystal obtained was named

Er II and exhibited mp 262-264°C. The physical data

Rutaecarpine.HC1

N

H

N

N

H

C1

DHE

Evodiamine.Hcl

N

H

H3

Evadiamine.HC1

CI

N

N

H

H3CN

N

0

0

0

C1

H

Fig. 1 Alkaloids isolated from the fruits of

Evodia rutaecarpa

28

Synephrine

HO

OH

H

N CH3

N,N-dimethy1-5-methoxytryptamie

CH30

NN

H3C CH3

Fig. 1( Cont.)

29

Table 4. Elution profile and the yield of fractions

of the column chromatography

Fraction No. Tube No. Solvent Dry wt.( g)

A 1-70 benzene 0.12

B 71-109 benzene 0.23

C 110-120 benzene 1.00

D 121-170 benzene 0.7

E 171-239 benzene 0.4

240-370CHC13 245*

0.3

C 371-400 0.15

K 401-430 CHCl3 0.5

I 431-820 CHC13 1.9

J 821-870 CHC13 0.15

K 871-1240 CHC13 0.7

1241L 1241-1279 3% MeOH/CHC13 0.95

M 1280-1560 3% MeOH/ CHC13 3.55

N 1561-1750 3% MeOH/CHC13 0.95

0 1751-1849 3% MeOH/CHC13 0.3

P 1850-1939 3% Me0H/ CHC13 0.2

20211940-2340 5% MeOH/ CHC 13 0.7Q

2350R 2341-2430 10% MeOH/CHC13 0.2

S 2431-2449107 MeOH/CHC1

0.3

F

CHC13

30

Table 4.( Cont.)

Fraction No. SolventTube No. Dry wt.(g)

T 2450-2649 0.95107 MeOH/CHC1 3

U 2650-2740 10% MeOH/CHC 13 0.3

2887V 2741-2900 0.2

20% MeOH/CHC1 3

W 2901-3150 20% MeOH/CHC13 0.3

3153X 50% McOH/CHC13'3151-3260 0.3

Y 50% MeOH/CHC13 1.03261-3420

Z 3421-3840 100% McOH 0.6

It denotes the tube No. at which the solvent

was changed

31

( UV, IR, TLC, 1H-NMR )were in good agreement with the

reference sample of rutaecarpine. The procedure of

purification was summarized in Fig. 2.

2.1.2 Isolation of dehydroevodiamine hydrochloride( DHE)

The chloroform extract obtained as above

cotaining mainly tertiary alkaloids was converted to the

hydrochloride salt by the use of IN HCl and named fraction F.

When fraction F was repeatedly recrystallized from ethanol,

a yellow crystalline compound was obtained. TLC investigation

revealed that it is the main constituent in fraction F.

2.1.3 Isolation of N,N-dimethyl-5-methoxytryptamine

and synephrine

N,N-dimethyl-5-methoxytryptamine and synephrine

were supplied through the courtesy of Prof. U. Sankawa.

These compounds were isolated from the fruits of Evodia

rutaecarpa of Japanese origin. The purification procedure

was summarized in Fig. 3.

2.1.4 Detection of alkaloids

2.1.4.1 Mayer reagent

The sample were dissolved in 1N HC1 and Mayer

reagent was added dropwise formation of white precipitates

show the presence of alkaloids.

32

Evodia rutaecarpa

dry fruits

extract with petroleum etner

Petroleum ether extractPlant'material

extract with methanol

Methanol' extractPlant material

concentrate in vacuo

add 5% HCl to the thick syrup

( 5: 1 v/v)

remove the residual methanol

Acid solution

alkalinized with 28% ammonia

up to pH 11.

partition with CHC13 for 6 times

CHC1 phaseAqueous phase

dehydrated with Anh

Na2SO4

concentrate in vacuo

CHC1 extract

Column Chromatographyadd iN HCi

Fraction IFraction F

DHE Er II( rutaecarpine)

Fig.% Purification of DHE and rutaecarpine

33

Evodia rutaecarpa

dry fruits

extract with hot water

Aqueous extract

IRC-50 (H+) column chromatography

Eluate Absorbed

eluted with 0.5N ammonia

Eluate

concentrated

Basic fraction

IRA-410 (0H) column

chromatography

Absorbed Eluate

eluted with

N,N-dimethyl-5-methoxy-1N HC1

tryptamineEluate

neutralized with

IR-45 (OH--)

Prep. TLC

Synephrine

Fig. 3 Purification of synephrine and N,N-dimethyl-5-methoxy

tryp tamine.

34

Preparation of Mayer reagent:

Stock solution 1.: 13.55 g mercury chloride in 20 ml

of water

Stock solution 2.: 49.8 g potassium iodide in 20 ml

of water

Working solution 10 ml of stock solution 1 mixed with

10 ml of sotck solution 2. The mixture

was diluted to 500 ml with water and

50 ml of 17% HC1 was added.

2.1.4.2 Dragendorff reagent

The samples were applied to a filter paper

and sprayed with Dragendorff reagent. Ai orange-yellow

color indicates the presence of alkaloids.

Preparation of Dragendorff reagent

Stock solution 1, 0.85 g of bismuth subnitrate in 10 ml

of glacial acetic acid and 40 ml of

water.

Stock solution 2 8 g of potassium iodide was dissolved

in 20 ml of water.

Working solution 5 ml of stock solution 1 and solution 2

mixed with 20 ml glacial acetic acid

and 10 ml of water.

35

2.2 SPECTROSCOPIC ANALYSIS

2.2.1 UV-visible spectroscopy

Samples were dissolved in methanol and the

absorption spectra were obtained by scanning from 550 nm

to 220 nm in 1 cm thick quartz cuvettes with a Beckman

Model 25 double beam spectrophotometer-recorder.

2.2.2 IR spectroscopy

IR spectroscopy was recorded with Beckman

IR 10 spectrometer in KBr disc.

2.2.3 MAR spectroscopy

Samples were dissolved in deuteromethanol

NMR spectra were obtained with Jeol C-60HL NMR spectrometer

2.3 PHARMACOLOGICAL STUDIES

2.3.1 The animal model

Uterine contraction in vivo was measured by

recording intrauterine pressure( IUP)( Bengtsson, 1968).

Uterine contraction in vitro was measured by isometric

contraction of the isolated uterine strips.

2.3.1.1 In vivo test

Estrus Sprague Dawley rats( vaginal smear)

36

were anaesthetized with sodium pentobarbital( 40 mg/Kg,

intraperitoneally). The lower mid section of abdomen

was opened. The lower portion of the right uterine horn

was lifted up by a pair of hemostat. A small incision

( 3-5 mm) was made at a position 0.8-1.0 cm from the

cervix. A catheter with a sponge tip filled with physiological

saline was inserted into the uterine cavity. The other end

of the catheter was attached to a Statham P 23 BB transducer

coupled to a Beckman 9872 strain gauge coupler in a Beckman

R 5llA dynograph.

2.3.1.2 In vitro test

Uteri were isolated from estrus Sprague Dawley

rats. The middle third segment of the uterus was mounted

in a 10 ml organ bath. It was maintained at 32°C and constantly

gassed with 95% 02: 5% CO2. The physiological medium

was modified Van Dyke and Hasting solution( Munsick, 1973).

Isometric contraction was recorded on a Beckman R 511A

dynograph through a Statham UC 3 tension transducer and

Beckman 9853A coupler.

2 ENZYME ASSAY

2.4.1 Bovine erythrocyte-acetylcholinesterase

Enzyme and all reagents were dissolved in 0.1 M

phosphate buffer, pH 7.6. Acetylthiocholine iodide and

37

bovine erythrocyte acetylcholinesterase were obtained from

Sigma Co. The enzyme has a specific activity of 3. 3 U/mg.

The assay procedure was modified after Ellman

et al.( 1961). 10 p1 of 2 mg/ml acetylcholinesterase

were added to 3 ml of 0.423 mM 5,5'-dithiobisnitrobenzoic

acid( DTNB, Sigma). After incubating at 370C for 15

minutes, 20 pl of 0.75 M acetylthiocholine iodide was

added and the absorbance was measured at 412 nm with

Beckman Model 25 double beam spectrophotometer-recorder.

Enzyme activity was presented in iimole/min.

2.4.2 Rat uterus acetylcholinesterase

Rat uteri were excised, weighed and suspended

in 17 Triton-X 100 in a ratio of 1: 5( w/v). The

preparation was then homogenized with a polytron( Type

PFA-10-35) homogenizer. The homogenate was centrifuged

at 15,000 x g for 10 minutes in a Sorvall RC2-B centrifuge

50 it of the supernate was used in each assay.

2.5 STATISTICAL ANALYSIS

All data were represented as mean+ S.E.M.,

with(n) denoting the frequency( number of animals/

samples tested). Student's t test was applied to evaluate

the differences between sets of data.

38

3. RESULTS

3.1 PURIFICATION OF EVODIA RUTAECARPA ALKALOIDS

Four alkaloids were obtained from the fruits

of Evodia rutaecarpa( from China and Japan) for this

study. The yield of the alkaloids was.. listed as follows

Alkaloid Yield

DHE 0.03%

Rutaecarpine 0.00026%( 2.6 ppm)

Synephrine* 0.18%

N,N-dimethyl-5-methoxy

Not determinedtryptamine%*

Isolated from Evodia rutaecarpa of Japanese origin

3.1.1 Purification of rutaecarpine

22.7 g of chloroform extract was applied to

column chromatography on silica gel 60( 70-230 mesh).

13.9 mg of Er II was crystallized from 1.9 g of Fraction I.

Er II and rutaecarpine have the same Rf value on silica gel

TLC in several solvent systems( Fig. 4).

The absorption maximun of Er II was identical to

rutaecar ine. For Er II AMeOH nm log p max( 278( 3.73),

290( 3.78), 332( 4.40), 345( 4.54) and 364(4.44).

For rutaecarpine, 278( 3.83), 290( 3.88), 332( 4.49),

39

1. 2. 3 1. 2. 3

Solvent: CHC13 Solvent: BuOH: HOAc: H2O

( 4: 1: 2 v/v)

Fig. 4 TLC of 1. Fraction I

2. Er II

3. rutaecarpine

Detection: 1. UV light

2. Dragendorff spray

40

345( 4.54) and 3 64( 4.44)

Both Er II and rutaecarpine showed the same

-1KBr

IR absorption characteristics cm 3345. 1650.max

1601, 1540, 1385, 1350, 1310, 1210, 1130, 750 and 720.

Er II and rutaecarpine showed identical spectra.

CD3OD3.2( 2H, J=7 Hz), 4.5( 2H, J=7 Hz), 7.2-7.6

TMS

( 5H, m), 8.2( 1H, J=8 Hz) and 9.3( 1H, m)

3.1.2 Purification and identification of DHE

10.5 Kg of powdered fruit was processed. The

yield of different fractions was tabulated as follows..

Fraction Weight Yield

Starting material 10.5 Kg 100

Chloroform fraction 0.5962.0 g

Fraction F 0.1314.2 g

DHE 0.033.5 g

TLC of DHE on silica-,gel _GF 254 in BuOH: HOAc

H2O( 4 1: 5, v/v, upper phase) revealed that there

were three spots detected under UV, but only the major

peak at Rf 0.35 has a positive reaction to Dragendorff

reagent( Fig. 5)

The UV spectrum of DHE differed from that

MeOHof rutaecarp ine. For DHE nm ( log)max

41

1. 2. 3

Solvent: BuOH: HOAc: H2O

( 4: 1: 5 v/v, upper phase)

Fig. 5 TLC of 1. Fraction F

2. DHE

3. DHE from rhetsinine

Detection: 1. UV light

2. Dragendorff spray,

42

230( 3.75), 250( 3.69) and 3.72( 3.90)( Fig. 6)

The IR spectrum of DHE is shown in Fig. 7.

-1KBrv cm 3400, 1700, 1608, 1544, 1333, 1210, 1101 and 756

max

The NMR spectrum of DHE is very similar to that

of evodiamine( Asahina and Ohta, 1916) and indicated the

presence of eight aromatic protons, two methylene located

in the adjacent position and N-methyl group.

NMR 6CD3OD: 3 .38( 2H, J=7 Hz), 4.44( 3H, J=7 Hz),TMq

4.56( 2H, t) J=7 Hz), 7.2-7.8( 7H, m), 8.32( 1H) dd,

J=8 Hz and J=1.5 Hz)( Fig. 8).

Elemental analysis,: Found: C, 66.61 H, 4.75

N, 11.97. Calculated for C19H16N30C1.4 H2O C, 66.67

H, 4.85 N. 12.27.

3.2 PHARMACOLOGICAL OBSERVATION

3.2.1 Uterotonic effect of DHE

Intravenous injection of DHE caused a dose-

dependent increase in intrauterine pressure( IUP) in vivo

The minimum effective dose was 80 Llg/Kg body wt. The

response was linear between 80 jig/Kg and 2400 jig/ Kg. The

contraction persisted for more than 20 minutes and up to

90 minutes. The results are presented in Fig. 9 and

Fig. 10.

43

3.9

3.8

3.7

3.6

3.5

3.4

230 250 270 290 310 330 350 370 390 410

WAVE LENGTH nm

Fig. 6 The UV-visible spectrum of DHE

44

?000 1800 1600 1400 1200 1000 800 600

WAVE NUMBER cm-1

Fig. 7 The fingerprint region of the IR spectrum of DHE

45

s 9 8 7 6 5 4 3 2 1 O

46

Fig. 8 The proton NMR spectrum of DHE

47

The minimum effective dose in vitro was 0.6 pc,/ml

to 6.6 pg/ml( 1.8 x 10-6M to 2.0 xl0-5M). The results

are presented in Fig. 11 and Fig. 12. As shown in

Fig. 11, maximum contraction was achieved at 3.6 ug/ml,

but total contraction would further increase due to the

increase in frequency of contractions at higher doses.

The inhibitory effect of methysergide on DHE is also

observed.

3.2.2 Potentiation effect of DHE on uterine stimulants

on isolated rat uterus

The uterotonic effects of acetylcholine were

compared in the absence and presence of 0.33 ig/ml of

DHE. Acetylcholine was added 5 minutes after addition of

DHE. Maximum peak contractions were recorded and compared.

The results are presented in Fig. 13. Significant

difference( P 0.001) was observed between the control

group and the test group.

The potentiation effect of DHE on serotonin

( 5-hydroxytryptamine) was demonstrated in Fig. 14.

Oxytocin stimulated the estrus rat uterus both

in vivo and in vitro. The potentiation effect of DHE

on oxytocin in vitro is shown in Fig. 15. The dose range

of oxytocin is from 0.02 mU/ml to 0.32 mU/ml. Significant

differences were observed at the doses of 0.08 mU/ml,

48

0.16 mU/ml and 0.32 mU/ml

Barium chloride( BaCl2) stimulates the isolated

rat uterus in vitro. Barium chloride was tested between

the doses of 4.1 x 10-5M and 1.23 x 10-3M. The uterotonic

effect of barium chloride was potentiated by DHE as shown

in Fig. 16. Significant difference was also observed

between the control group and the potentiated group.

3.2.3 Uterotonic effect of rutaecarpine

Rutaecarpine hydrochloride was used in the

uterotonic assay. Exact concentration is not known (til jig/ml)

since it is only slightly soluble in water. The relative

potency of rutaecarpine hydrochloride is only about 20%

with respect to DHE.

3.2.4 Uterotonic effect of N,N-dimethyl-5-methoxytryptamine

Dose response curve was constructed between

0.1 ug/ml and 10.0 pg/ml( 4.6 x 10-7M and 4.6 x 10-5M).

The uterotonic effect of N,N-dimethyl-5-methoxytryptamine

was inhibited by 1 x 10-9M of methysergide. The results are

presented in Fig. 17.

3.2.5 Relaxation effect of synephrine on contracting

uterus

49

DHE1 min

80 ug/kg

10 mmH9

DHE

240 ug/kg

DHE

800 ug/Mg

DHE

2400 ug/kg

Fig. 9 Effect of DHE on rat uterus in intact rat.

Arrow indicated the administration of DHE.

iv

iv

iv

iv

50

Intrauterine

pressure(mmHg)

30

20

10

0

(3)

(3)

(3)

(3)

80 240800 2400 ug/Kg body wt

DHE

Fig.10 Uterotonic effect of DHE. Maximum peak contractions

were measured as response. Three rats were used

in this experiment.

51

OHE

D H E+ MS

3.0

2.5

2.0

1.5

1.0

0.5

0

(4)

(21)

(7) (23)

(12)

(12)

(4)(4)(4)(4)(4)

0.33 0.6 1.1 2.0 3.6 6.6 g/ml

DHE

Fig. 11 Dose response curve of DHE. The maximum peak

tension within 20 minutes after drug addition

was measured as the response. Its effect was

totally suppressed by 3x10-9M methysergide (MS)

52

(15)OHE

DHE+ MS

12.0

10.0

(3)

8.0

(23)

6.0

4.0

(12)

2.0

(12)

(4)(4) (4)(4) (400

ug/ml0.33

DHE

Fig. 12 Dose response curve of DHE. Total contractions

( sum of contraction forces) within 20 minutes

after drug addition was measured. The effect

of DHE was totally suppressed by 3xl0-9M methysergide

CONTRACTION(9-force)

(7)

0.6 1.1 2.0 3.6 6.6

53

CONTROL

D H E

4.0

3.0

2.0

1.0

0

1 3 10 30

(8)

(7)

(6)

(8)

Fig. 13 Potentiation effect of DHE on the stimulatory

effect of acetylcholine( ACh) on isolated

rat uterus.. The maximum peak contraction was

recorded as response. For the potentiation group,

0.33 Ug/ml DHE was added with each dose of ACh.

P<0.001 P<0.05

CONTRACTION

(g-force)

10-6M

ACh

54

CONTROL

OHE

(4)

(4)(4)

(4)4

3

2

1

0

1 3 10 30 10- M

SER

Fig. 14 Potentiation effect of DHE on the stimulatory

effect of serotonin( SER) on isolated rat

uterus. The maximum peak contraction was recorded

as response. For the potentiation group, 0.33 ig/ml

DHE was added with each dose of SER.

P<0.001 P<0.01

CONTRACTION

(g-force)

55

CONTRACTION

(g-force)

3.0

2.0

1.0

CONTROL

DHE

(4)

(4)

(4)

(4)

(4)

0.020.04

0.08 0.160.32 mU/ml OT

56

Fig. 15 Potentiation effect of DHE on the stimulatory

effect of oxytocin( OT) on isolated rat uterus.

The maximum peak contraction was recorded as response.

For the potentiation group, 0.33 jig/ml DHE was

added with each dose of OT.

*P0.000* P0.05

57

CONTROL

D H E

(4)

(4)

3.0

(4)2.0

(4)1.0

0

4.1 10-5 m

BaCI2

Fig. 16 Potentiation effect of DHE on the stimulatory

effect of barium chloride( BaCl2) on rat

uterus in vitro.The maximum peak contraction

was measured as response. For the potentiation

group, 0.33 ug/ml DHE was added with each dose

of BaC1. *P0.001** P<0.01

12.3 41123

CONTRACTION

(g-force)

58

DM

DM MS

(4)1.0

(4)0.8

(4)

0.6 (4)

(4)

0.4

0.2

0

0.1 0.3 1.0 3.0 10.0 ug/ml

DM

Fig. 17 Effect of N,N-dimethyl-5-methoxytryptamine( DM)

on isolated rat uterus. The inhibitory effect

of methysergide( MS) on DM is also presented.

The maximum peak contraction was measured as

response.

CONTRACTION(g-force)

59

(4)SYN

100SYN+PRO

(4)

80

60

(4)

(4)40

20

ug/ml SYN0 0.6 2.0 6.0 20

Fig. 18 Relaxation effect of synephrine tartrate( SYN)

on isolated rat uterus. The inhibitory effect

of propranolol( PRO) on SYN is also shown.

The maximum peak contraction was recorded as

response. The spontaneous contraction was regarded

as 100%

%

ACTIVITY

P<0.001P<0.05

60

SYN +1 xlO-5M ACh

EPI +3 x1O-5M ACh

(4)

100

(4)80

(4)

60

(4)

40

20

0

0 1.2 4.13 1 2 41.3 10 M

SYN/ EPI

%

Activity

61

Fig. 19 Relaxation effect of synephrine tartrate( SYN)

and epinephrine( EPI) on the stimulatory

effect of acetylcholine on isolated rat uterus.

In the test group, acetylcholine was added

2 minutes after addition of SYN or EPI. The

maximum peak contraction within 5 minutes was

measured as response. The maximum peak contraction

induced by acetylcholine was regarded as 100%

activity.

62

CONTROL

SYN 4.13x10-5M

EPI 1.2x10-7M

3.0

2.0

1.0

0

(4)

(4)

(4)

(4)

1 3 10 3010-5M ACh

Fig. 20 Relaxation effect of synephrine tartrate( SYN)

and epinephrine( EPI) on the stimulatory effect

of acetylcholine on isolated rat uterus. The

maximum peak contraction was measured as response.

CONTRACTION(g-force)

63

Significant spontaneous contraction occurs

in diestrus rat uterus. Synephrine tartrate was used in

this experiment, it exhibited a relaxation effect on

the contracting uterus. Synephrine tartrate was tested

between the doses of 0.6 ig/ml and 20 ig/ml( 1.2 x 10-6M

to 4.13 x 10-5M). The action of synephrine tartrate was

inhibited by 0.6 pg/ml( 2 x 10-6M) of propranolol

hydrochloride. The results are presented in Fig. 18.

Uterine contraction was caused by the addition

of acetylcholine. Synephrine tartrate( 1.2 x 10-7M to

1.2 x 10-5M) was added before acetycholine. The%

activity decreased was presented in Fig. 19. The relaxation

effect of epinephrine is also presented. In Fig. 20, the

displacement of acetylcholine standard curve by synephrine

and epinephrine is reported. Acetylcholine was added 2 minutes

after addition of synephrine or epinephrine.

3.3 EFFECT OF DHE ON ACETYLCHOLINESTERASE ACTIVITY

As shown in Fig. 21 and 22, DHE inhibited

acetylcholinesterase activity from both bovine erythrocyte

and rat uterus. The Michelis-Menten constant of bovine

erythrocyte acetylcholinesterase for the hydrolysis of

acetylthiocholine, as determined by Lineweaver-Burk plot,

was 6.25 x 10-5M. The K of DHE is 1.13 x 10-5M Fig. 22)

64

100

80

60

40

20

0.33 ug/ml DHE

Fig. 21 Dose-dependent inhibitory effects of DHE on rat

uterine. ace.tylcholines terase. Assays were made at

37°C and pH 7.6 on the hydrolysis of 5x10-4M

acetylthiocholine.

1.0 3.310

ACTIVITY

65

CONTROL

DHE

DHE

1.4

1.2

1.0

0.8

0.6

0.4

0.2

10

1 2

ACETYLTHIOCHOLINE

66

Fig. 22 Lineweaver-Burk plot for the inhibition of

bovine erythrocyte acetylcholinesterase by DHE

Assays were made at 37°C and pH 7.6 on the

hydrolysis of acetylthiocholine.

67

4. DISCUSSIONS

4.1 SUITABILITY OF THE ANIMAL MODEL

Fertility control include methods of contraception

( preventing ovulation and fertilization), interception

( anti-implantation) and early abortion. In this study,

special emphasis is put on finding herbs that can affect

the fertilization and implantation process. Through an

increase in uterine motility, uterine stimulants may

interfere with these processes. For instance, PGF2a has

been advocated to prevent implantation( Karim and

Rao, 1976 Labhsetwar, 1971a). At the same time, PGF2

is a powerful uterine stimulant( Karim,. 1971). This

uterotonic property may contribute to the anti-implantation

effect of PGF2a, although the disturbance of hormonal

pattern( luteolytic effect) by PGF2a may not be ignored

( Labhsetwar and Watson, 1974 Labhsetwar, 1971b Challis,

Davies and Ryan, 1973).

While our final objective is to select plants

for fertility control, uterotonic assay is used as a preliminary

screening for this purpose. Bioasssy models for anti-implantation

or induced abortion is time consuming and laborious. It

does not permit the evaluation of a large number of plants

in a short time. In the present study, uterotonic activity

was tested in vivo by measuring the intrauterine pressure

68

and in vitro by measuring the tension of the isolated

uterine muscle strip. The in vitro method is easier to

handle and the experimental conditions can be totally

under control, but it has many drawbacks. For example,

there are marked changes in ion composition of the uterine

tissue during excision the loss of potassium and acquisition

of sodium ions occur as,a result of the surgical trauma

( Bohr, 1964). Muscle tone, or tonus, is the resistance of

a muscle to stretch. Denervation of the excised uterus

completely abolishes this tonicity. Therefore, the in vitro

result reveals at best the contractile property of the

myometrium at the tissue level, it cannot be extrapolated

to the intact system.

The in vivo method reveals the uterine activity

under normal physiological conditions. The drug reaches

the target organ through systemic route, with or without

prior metabolic transformation. But in vivo study suffers

from the multiplicity of variables which are difficult

to control. Many endogenous uterotonic substances such

as oxytocin, serotonin and catecholamines may interfere

with the drug effect. Nevertheless, if the interfering

factors are fully taken into account, in vivo experiments

usually yield more meaningful results in a physiological

sense.

69

4.2 UTEROTONIC EFFECT OF EVODIA RUTAECARPA ALKALOIDS

There is no previous report of uterotonic

compounds isolated from the fruits of Evodia rutaecarpa

A degradation product of rutaecarpine, rutamine, of

unknown structure was reported to be uterotonic, but

detail information was not available( Tsun, 1936).

In the present study, the uterotonic effect

of DHE is demonstrated both in vivo and in vitro with

rat uterus. The effect of DHE is not inhibited by

atropine at 3 x 10-3M but inhibited by methysergide

at 3 x 10-9M. It shows that DHE is probably a serotoninergic

agonist on uterine contraction.

At high doses such as 3.6 jig/ml or above, it

is very difficult or even impossible to wash away the

uterotonic effect of DHE. There are two possible explanations

for this phenomenon first, DHE may bind to the cell membrane

irreversibly second, DHE may penetrate into the cell

and the uterotonic effect is caused by a post-membranous

event.

The uterotonic potency is the same with the

natural compound isolated in this study and the authentic

preparations of DHE supplied by Japanese scientists

( King et at 1979).

70

Other compounds from Evodia rutaecarpa are

also tested. Evodiamine•HC1 is completely devoid of

uterotonic activity up to 20 jig/ml. Rutaecarpine•HC1

shows some activity at a concentration less than 1 pg/ml.

Since rutaecarpine•HC1 is only sparingly soluble in water

( less than 50 jig/ml) it is very difficult to estimate

the exact final concentration in the bioasssy. Other

soluble salts of rutaecarpine should be prepared for better

assessment of its uterotonic property.

DHE, rutaecarpine and evodiamine are all indole

alkaloids. They have the same nucleus and vary only at

the N position( Fig. 1). The differences in uterotonic

activity among them may be due to this variation.

N,N-dimethyl-5-methoxytryptamine has been

isolated from Mucuna pruri ens( Leguminosae)( Ghosal,

Singh and Bhattacharya, 1971 ).It is an analogue of

5-hydroxytryptamine( serotonin). The uterotonic activity

of N,N-dimethyl-5-methoxytryptamine was observed at 0.1 iig/ml

but was less potent than serotonin( Fig. 14 and 17).

Its uterotonic action was inhibited by 1 x 10-9M methysergide.

Synthetic synephrine was commercially available

( Sigma Co.) and used as vasopressor before it was

purified from plant sources. The relaxation effect was

demonstrated both in vivo and in vitro in rat uterus.

Its relaxation effect was suppressed by propranolol which

71

indicated that synephrine is a -adrenergic agonist( Fig. 18)

The potency of synephrine is far less than that of

epinephrine( Fig. 19 and 20). as far as uterine relaxation

is concerned.

4.3 POTENTIATION EFFECT OF DHE ON UTERINE STIMULANTS

A subthreshold dose( 0.33 jig/ml) of DHE was

shown to potentiate a number of uterine stimulants. The

potentiation effect is probably not related to the

protection of the stimulants from enzyme degradation.

Enzymatic assay revealed that 0.33 iig/ml of DHE inhibited

only 10% of the activity of acetylcholinesterase

( Fig. 21). As shown in Fig. 22, the KM of acetlcholinesterase

for the hydrolysis of acetylthiocholine iodide is 6.25 x 10-5M

while the Ki of DHE on acetylcholinesterase is 1.13 x 10-5M.

The inhibitory effect of DHE is so weak that it cannot produce

any significant effect on the potentiation of acethylcholine.

On the contrary,- 0.3 pg/ml of berberine( Sigma Co.) which

inhibits 10% of the activity of acetylcholinesterase does

not yield any significant potentiation effect on acetylcholine

on uterine contraction.

DHE also has a potentiation effect on serotonin,

oxytocin and barium chloride on uterine contraction. The

action of barium ion in skeletal muscle contraction

is the same as calcium, i.e., direct binding to the troponin

molecule and initiation of the contraction process

72

( Webber and Murray, 1973). The action of barium ion

on smooth muscle may be similar. The precise manner in

which DHE can potentiate.. these agonists action is not

well understood at the moment. It is therefore impossible

to afford a definite conclusion based on the present

limited data. However, some possible mechanisms can be

speculated. Since calcium ion is thought to be the initiator

for muscle contraction, DHE may act on the cell membrane

and increase the permeability to calcium ion. Facilitation

of calcium ion influx results in larger contractions.

Secondly, as mentioned before, DHE may enter the cell and

exerts its action. e.g. facilitate the binding of calcium

ion to the contractile mechanism. Thirdly, DHE at low

concentration may induce subthreshold depolarization which

is not reaching the firing level, but can facilitate the

depolarization by other uterine stimulants.

In order to elucidate the mechanism of action

of DHE, radioactive DHE should be prepared to study the

action site of DHE. Studies on the ion influx and the cell

membrane potential seem inevitable for the investigation

of the potentiation effect of DHE.

4.4 PHARMACOLOGICAL EFFECT OF EVODIA RUTAECARPA IN

ETHNOMEDICAL PREPARATION

Both uterine stimulants and relaxant were found

in the unripe.fruits of Evodia rutaecarpa, i.e.,. DHE,

73

rutaecarpine and N,N-dimethyl-5-methoxytryptamine are uterine

stimulants synephrine is uterine relaxant. It is interesting

to know which predominant effect is referred to in the

ethnomedical information.

Since synephrine and N,N-dimethyl-5-methoxytryptamine

were purified from the fruits of Evodia rutaecarpa of

Japanese origin, it is necessary to employ the same procedure

for the purification of these two compounds from Evodia

rutaecarpa of Chinese origin in order to see whether it

contains the same compounds. It is well recognized that

geographic variation can alter the chemical constituents

and the pharmacological properties of the same species.

e.g., chemical constituents are different for Chelidonium

majus between the Chinese origin and the American origin

( Lam, 1977b).

Furthermore, two factors contribute to the

uterotonic effect of Evodia rutaecarpa. First, the

relative composition of the individual compounds second,

the relative potency of the compounds. It is very

difficult to assess the activity by just calculating the

sum of the effects of the individual compounds. The

best evidence is that the ethnomedical extract( aqueous

decoction) has been demonstrated to be uterotonic in vivo

in rat. It is well correlated to the therapeutic use of

Evodia rutaecarpa as a treatment for postpartum hemorrhage,

74

In another prescription Tso Chin Wan (%L r

which is used for the treatment of gastrointestinal diseases

such as dyspepsia. Evodia rutaecarpa itself is used as

stomachic and deobstruent. These effects may be due to

the presence of DHE and N,N-dimethyl-5-methoxytryptamine

in it. Since DHE and N,N-dimethyl-5-methoxytryptamine

are serotoninergic agonist, they are able to contract the

intestinal tract thus cause the stomachic and deobstruent

effects.

4.5 PROSPECT OF USING DHE AS ANTIFERTILITY AGENT

The uterotonic effect of DHE has been demonstrated

both in vivo and in vitro. It is worthwhile to evaluate further the

potentials of DHE as an antifertility agent in anti-implantation

and early abortion.

There are two ways to disturb implantation, one

can disrupt the luteal function or interfere with the ovum

or embryo transport. The former includes three approaches

1) by inhibiting the early luteotrophic activity of the

blastocyst, 2) by interfering with progesterone receptors

in the endometrium and 3) by using prostaglandins, steroids

and other compounds acting directly on the corpus luteum.

Evidences showing that the primary function in

transport of sperms, ova and embryos is due to the segmental

and peristaltic contraction of layers of smooth muscle fibres

75

Delivery of the embryos to the uterus at specifically the

right time is required for assuring implantation. DHE

is thought to be a serotoninergic agonist on uterine

contraction, it is reasonable to investigate the stimulatory

effect of DHE on the smooth muscle contraction of the

oviduct.

In early abortion usually surgical method such

as vacuum curettage is used. Abortion can also be induced

by using drugs that can inhibit the corpus luteum function

and increase the myometrial activity. For the latter

purpose prostaglandin is frequently employed. But

prostaglandin must be used at a high dose that can elicit

severe systemic side effects such as actions on gastro-

intestinal tract and cardiovascular system. DHE is a uterine

stimulant which can theoretically disturb the pregnancy

process.

Furthermore, since DHE can potentiate the action

of other uterine stimulants, suggesting that it can be used

synergistically with other drugs for antifertility purpose.

For example, use in combination with prostaglandins so as

to potentiate its action and decrease the therapeutic dose

of prostaglandins.

4.6 APPROACHES TO THE STUDY OF MEDICINAL HERBS

Before the advent of modern medicine, medicinal

76

plants have been used for the treatment of various diseases

for thousands of years by all kinds of people. Although

modern western medicines are effective for many diseases,

they are expensive and may be a burden for most developing

countries. Herbal medicines have the characteristics of

being cheap, nontoxic, readily available, having a wide

spectrum of bioactivity and ethically acceptable by many

people.

The therapeutic value of herbal medicines lies

in its chemical constituents. The purification of the

active ingredient from plant sources is a complicated work.

A simple way to do it is to screen by a large number of

bioassay methods such as uterotonic assay, hypotensive

assay etc. But it is an extremely laborious and uneconomical

task.

Another approach is to preselect a small number

of potentially physiologically active plants based on

written or daily ethnomedical information. By careful

searching of ancient medical literatures and modern

international publications, one can formulate some hypotheses

to correlate the physiological changes caused by the administration

of the drugs. Then it will be possible to design some

bioassay methods to monitor the drug effects for further

studies.

77

All known chemical constituents of medicinal

plants can be found from international phytochemical

references. Many groups of phytochemicals have some inherent

pharmacological effects, e.g., the estrogenic effect of

isoflavones.

Based on literature information, one may be able

to prejudge which groups of chemicals should be used and

isolated thus avoiding the proliferation of fractions in a

routine phytochemical analysis. With a suitable bioassay

method, this forms the basis of a functional approach to

phytochemical studies. Functional phytochemistry is made

through the combination of knowledge in ancient medical

documentations, pharmacology and modern phytochemical

techniques.

Lastly, some other criteria should also be

satisfied in order to choose an appropriate Chinese drug

for laboratory studies. It first concerns the toxicity of

the plant and the second is the sources of authentic supply

The present study follows closely these guiding

principles. According to the pharmacopoeias in Chinese

medicine, the fruit of Evodia rutaecarpa is thought- to

contain uterotonic compounds. Chemical studies show that

it contains a lot of alkaloids. It is well known that many

alkaloids are uterine stimulants. Then, special effort

was put on the isolation of the alkaloid fraction. With the

78

help of an appropriate bioassay method, the uterotonic

compounds are isolated and identified.

At this stage, it is difficult to speculate on

the application of DHE as an antifertility agent. Other

more relevant bioassay methods should be employed to evaluate

its potential. Furthermore, other systemic pharmacological

effects and toxicity of DHE should be tested, before we can

consider DHE seriously as a new fertility regulation agent.

Since DHE is a serotoninergic agonist on uterine contraction

andserotoni:n is a neurotransmittor in the mammalian central nervous

system, it is necessary to examine the permeability of the

blood-brain barrier to DHE and its effect on the central

nervous system. Drugs affecting the central nervous system

must not be used as antifertility agents.

The significance of the present study is an

exercise to seek a scientific rationale for the use of

Chinese medicinal herbs. By the combination of information

study, phytochemical processing and pharmacological test,

a functional approach to examine the chemical basis of the

therapeutic value of Chinese medicinal material is

demonstrated.

79

5. SUMMARY

1. DHE was isolated and identified in the unripe fruit of

Evodia rutaecarpa. The uterotonic effect was demonstrated

both in vivo and in vitro with rat uterus at a dose

range of 1.8 x 10-6M to 2.0 x 10-5M.

2. The uterotonic effect of DHE was not inhibited by

atropine but inhibited by methysergide. It indicated

that DHE is probably a serotoninergic agonist on uterine

contraction.

3. The potentiation effect by subthreshold dose of DHE on

acetylcholine, serotonin, oxytocin as well as barium

chloride was demonstrated in isolated rat uteri in vitro.

4. The potentiation effect of DHE is probably not due to

the inhibition of the lyric enzymes of the uterine

stimulants. Enzymatic studies show that 0.33 ug/ml of

DHE inhibited only 10% activity of acetylcholinesterase,

5. The other alkaloids from the fruit of Evodia rutaecarpa

were also tested. Rutaecarpine•HC1 was uterotonic while

evodiamine•HC1 was devoid of any uterotonic activity.

6. N,N-dimethyl-5-methoxytryptamine and synephrine were

isolated from the fruit of Evodia rutaecarpa of Japnaese

origin. N,N-dimethyl-5-metfioxytryptamine stimulated

the isolated rat uteri and was demonstrated to be a

80

serotoninergic agonist. Synephrine was an analogue of

epinephrine which exerted a relaxation effect on

contracting rat uteri.

7. The present study indicates that ethnomedical information,

aided by modern biomedical methodology, can lead to the

development of new therapeutic agents.

81

6. REFERENCES

Acton, H.W. (1921)' The action of quinine on pregnant uterus

Lancet, 1921(1): 526-8.

Allan, W.M. and Reynolds, S.R.M. (1953)' Physiology of the

corpus luteum: X. The comparative actions of crystalline

progestine_.and crude progestine on uterine motility in

unanaesthetized rabbits' Am. J. Obst. Gynec., 30 309-12

Anon, (1959)

Sian Yixue Yuan Xue Bao 8: 104-6( In Chinese)

through中藥臨床應用， 廣東人民出版社 ( 1975)

Anon, (1961) 中藥在人和動物體內利尿作用的研究

Zhong Hwa yixue Zas shi, 1: 7, through

(1975)

Ashina, Y. and Fujita, A. (1921)' Constituent of rutae'carpine

J. Pharm. Soc. (Japan) 476: 863-9

Ashina, Y. and Ishio, M. (1915)' Evodine, a crystalline

constituent of the fruit of Evodia rutaecarpa

J. Pharm. Soc. Japan, 404: 1148-52

Ashina, Y and Kashiwaki, K. (1915) Chemical constituent

of the fruits of Evodia rutaecarpa' J. Pharm. Soc.

Japan, 405: 1293-6

Bagby, R.M. et al., (1971)' Contraction of single smooth

muscle cells from Bufo marinus stomach Nature (London)

234: 351-2

Bengtsson, L.P. (1968)' The sponge-tipped catheter-

A modification of the open end catheter for recording

myometrial activity in vivo' J. Repro. Fert. 16: 115-8

Bohr, D.F. (1964)' Electrolytes and smooth muscle contraction

Pharm. Rev. 16: 85-111

Bray, D. and Thomas, C. (1975) The actin content of

fibroblast' Biochem. J., 147: 221-8

吳 茱 萸 對 心 臟 及 血 壓 的 作 用

中 藥 臨 床 應 用

廣 東 人 民 出 版 社

82

Breger, A. (1960) In 'Medicinal Chemistry' P. 722, ed. 2

Wiley-Interscience, N.Y.

Carroll, W.R. (1945)' Variations in the water content of

the rat's uterus during contineous estrogenic treatment

Endocr., 36: 266-71

Carsten, M.E. and Katz, A.M. (1964) Actin: A comparative

study' Biochem. Biophy. Acta, 90: 534-41

Challis, J.R.G., Davies, I.J. and Ryan, K.J. (1973)

The relationship between progesterone and prostaglandin F

concentrations in the plasma of pregnant rabbits

Prostaglandins, 4(4): 509-16

Chen, A.L. and Chen, K.K. (1933)' The constituents of

Wu Chu Yu( Evodia rutaecarpa)

J. Am. Pharm. Assoc. 22: 716-9

Cheng, M. F. (1952) 普 通 中 药 在 試 管 內 対 対 致 病 性 及 非 致 病 性

真 菌 的 抗 真 菌 力

38(4): 315-8, through (1975)

(1820-1961) P.337 p.337

Chu, J.H. (1951) 'Constituents of the Chinese drug Wu

Chu Yu, Evodia rutaecarpa' Sci. Rec. (China) 4. 279-84

Csapo, A.I. (1955) In' Modern Trends in Obstetric and

Gynecology'( K. Bowes ed.) P.20, Bulterworth, London

Csapo, A.I. (1956a)' The mechanism of effect of the ovarian

steroids' Recent Prog. Horm. Res. 12:404-31

Csapo, A.I. (1956b)' Progesterone block' Am. J. Anat. 98273-91

Csapo, A.I. (1959) In 'Cell, organism and Milieu'( D. Rudnick

ed.) P. 107, Ronald. Press, N.Y.

Csapo, A. I. (1961a)' The in vivo and in vitro effects

of estrogen and progesterone on the myometrium' in

'Mechanism of action of steroid hormones' P. 126

Pergamon Press, Oxford

劉 寿 山 中 药 研 究1975

文 献 摘 要 科学出版社

Zhong Hwa Yixue Zasshi

83

Csapo, A.I. (1961b)' Defence mechanism of pregnancy' in

' Progesterone and the defence mechanism of pregnancy

P. 3-27, Ciba Fdn. Study Group No. 9 Eds. G.E.W.

Wolstenholme and M.P. Cameron, Little, Brown and Co.,

Boston

Csapo, A.I. (1962) Smooth muscle as a contractile unit

Physiol. Rev. Suppl.5, P.7-33

Csapo, A.I. and Takeda, H.(1965)' Effect of progesterone

on the electric activity and intrauterine pressure

of pregnant and parturient rabbits' Am. J. Obst.

Gynec. 91:221-31

Devine, C.E. and Someyo, A.P. (1971) Thick filaments in

vascular smooth muscle' J. Cell Biol. 49:636-49

Dousa T.P. (1977)' Cyclic nucleotides in the cellular

action of neurohypophyseal hormones' Federation

Proc. 36:1867-71

Ebashi, S. (1969)' Comparative aspect of regulatory

structure properties of muscles, with particular

reference to troponin' In symposium on vascular

neuroeffector system. Interlaken, August

Elliot, G.F. (1967)' Variation of the contractile

appararus in smooth smooth and striated muscles,

X-ray diffraction studies at rest and in contraction

J. Gen. Physiol., 50(6):171-84

Ellman, G.L. et al., (1961)' A new and rapid colorimetric

determination of acetylcholinesterase activity

Biochem. Pharm., 7:88-95

Elftmen, H. (1963) 'Estrogen induces changes in the golgi

apparatus and lipid of the uterine epithelium of

the rat in the normal cycle: Anat. Rec. 146:139-43

84

Engle, E.T. and Smith, P.E. (1938)' The endometrium of

the monkey and estrogen-progesterone balance

Am. J. Anat., 63:349-65

Flemming, S., Tweedle, D.N. and Roddick, J.W. (1968)

' Ciliated endometrial cells' Am. J. Obst. Gynec.

102:186-91

Farnsworth, N.R. et al., (1975)' Potential value of plants

as sources of new antifertility agent I

J. Pharmaceutical Sci. 64(4) :535-98

Fujita, A. and Akatsuka, M. (1949)' Obakulacton. V. Evodin

J. Pharm. Soc. Japan, 69:322-5

Ghosal, S., Singh, S.S. and Bhattacharya, S.K. (1971)

' Alkaloids of Mucuna pruricus, chemistry and pharmacology

Planta Med., 19(3): 279-84

Goodall,F.R. (1965)' Degradation enzymes in the uterine

myometrium of rabbits under different hormonal condition

Arch. Biochem. Biophys., 112:403-10

Goodall, F.R. (1966)' Progesterone retards postpartum

involution of the rabbit myometrium' Sci., 152:356-8

Grosselin-Rey, C. et al., (1969)' Amino acid analysis and

mapping of bovine carotid actin' Biochem. Biophys.

Acta, 175:165-73

Hamilton, W.J. and Harrison, R.J. (1951)' Cyclic changes

in the uterine mucosa and vagina of the goat

J. Anat., 85:316-24

Hanson, J. and Lowy, J. (1963) The structure of F. actin

filaments isolated from muscles' J. Mot. Biol.,6.46-60

Hanson, J, and Lowy, J. (1964)' The problem of the location

of myosin in vertebrate smooth muscle'

Proc. Roy. Soc. Ser, B. 160:523-4, through biol. Abstr.

45712 (1965)

85

Hanson, J. and Huxley, H.E. (1955)' The structural basis of

contraction in striated muscles' Symp. Soc. Exp. Biol.

9:228-64, through' Biology of the uterus Wynn ed)

Plenum.

Hirose, Y. (1963)' The structure of evodol, a principle of

Evodia rutaecarpa' Chem. Pharm. Bull. 11:535-6

Hirose, Y. (1971)' Studies on components of Evodia Fructus.

III. Structure of rutaevin and dehydrolimonin.

Chem. Pharm. Bull. (Tokyo), 19(6):1268-9

Hirose, Y. et al.,(1969)' Studies on components of Evodia

Fructus. II.' Shoyakugaku Zasshi, 21(2):126-7

Huxley, H.E. (1971)' The structural basis of muscle

contraction' Proc. Roy. Soc. Ser. B, 178:131-49

through Biology of the uterus (..Wynn ed.) Plenum

Huxley, A.F. (1974)' Muscular contraction' J. Physiol.

243:1-44

Ichikawa, s. and Bortoff, A. (1970)' Tissue resistance of

the progesterone-dominated rabbit myometrium

Am. J. Physiol., 219:1763-7

Imaseki, I., Kitabatake, Y. and Taguchi, H. (1961)

Studies of effect on berberine alkaloids in intestine

and uterus in mice Yakugaku Zasshi, 81:1281-4

Josimovich, J.B. (1973) In' Uterine Contraction-Side effects

of steroidal contraceptives'P. 301-80, John Wiley and

Sons, USA

Kamikado, T. et al., (1976)' Isolation and structure

elucidation of three quinolone alkaloids from Evodia

rutaecarpa' Agric. Biol. Chem., 40(3):605-9

Karim, S.M.M. (1971)' Once-a-month vaginal administration

of prostaglandins E2 and Fla for fertility control

Contraception 3:173-83

86

Karim, S.M.M. and Rao, B. (1976)' Prostaglandins in human

reproduction' In' Obstetric and Gynaecological use

of prostaglandins'( S.M.M. Karim ed.) P. 1-21,

Proc. Asian. Fed. Obstet. Gynae. Intercongress, M.T.P.

Lancaster

King, C.L. et al., (1979) Uterotonic effect of Evodia

rutaecarpa'( submit for publication)

Knaus, H.H. (1926) Action of pituitary extract upon the

pregnant uterus of the rabbit' J. Physiol., 61:383-97

Kohorn, E.I. and Tchao, R. (1969) Conversion of proliferation

endometrium to secretory epithelium by progesterone in

organ culture' J. Endocr., 45:401-5

Kong, Y.C. et al.,(1976a)' Potential antifertility plants

from Chinese medicines' Am. J. Chi. Med., 4(2):105-28

Kong, Y.C. et al.,(1976b)' Isolation of the uterotonic

principle from Leoniurus artemisia, the Chinese motherwort

Am. J. Chi. Med. 4(4):372-82

Kovalenko, V.N. (1946)' Pharmacochemical and pharmcological

properties of the alkaloid dictamine from Dictaminus

albus Turkestanicus' Farmatsiya( Moscow) 9:20-28

through Chem. Abstr., 416989c (1947)

Kurona, G. et al., (1973)' Studies on fatty acids from fruit

and seed oils. IV. Analyisis of fatty acid composition

in the fruits of Evodia rutaecarpa of Chinese origin

Yakugaku Zasshi, 93(5):691-2

Labhsetwar, A.P.(1971a)' PGF2a and the implantation process

in the rat' J. Endocr., 50:353-4

Labhsetwar, A.P. (1971b)' Luteolysis and ovulation induced

by PGF2a in the hamster' Nature (London), 230:528-34

87

Labhsetwar, A.P. and Watson, D. (1974)' Temporal relation-

ship between secretory patterns of gonadotrophines,

estrogen, progestines and prostaglandin F in peri-

parturient rats' Biol. Repro., 10:103-10

Lam, K.S. (1977a) In' Phytochemistry of Chinese medicinal

herbs' P. 764 Scientific Press, Peking

Lam, K.S. (1977b) In' Phytochemistry of Chinese medicinal

herbs' P. 758, Scientific Press, Peking

Li, M.T. and Huang, H.I. (1966)' Studies on the chemical

constituents of the Chinese drug, Shih-Hu( Evodia

rutaecarpa)' Yao Hsueh Hsueh Pao 13(4):265-72

Li, S. C. (1593) Compendium of the matenia medica

Manske, R.H.F. (1955)' Uterine stimulants'

Alkaloid, 5:179-82

Mester, I. (1973)' The occurence of alkaloids in rutaceae

Fitoterapia( Milano) 44:123-53

Mester, I. (1977)' The occurence of alkaloids in rutaceae,

Addendum I' Fitoterapia( Milano) 6:268-78

Munsick, R.A. (1973)' Bioassay of oxytocin' In' Uterine

contraction-Side effects of steroidal contraceptives'

P. 83-100, John Wiley and Son, USA

Nam, T.H. et al., (1957)' The antibacterial effect of

berberine and its action mechanism' Acta Phsiol.,

21(3) :213-23, through中 药 研 究

(1820-1961)

Needham, D.M. and Williams, J.M. (1963a)' The protein of

the dilution precipitate obtained from salt extracts

of pregnant and non-pregnant,uterus' Biochem. J.

89:534-45

李 时 珍 ， 夺 草 纲 目

劉 寿 山1975

文 献 摘 要科 学 出 版 社

88

Needham, D.M. and Williams, J.M. (1963b)' Salt soluble

collagen in extracts of uterus muscle and in fetal

metamyosin' Biochem. J. 89:546-52

Needham, D.M. and Shoenberg, C.F. (1967) Biochemistry

of the uterus in' Cellular Biology of the uterus

P. 291-352, R.M. Wynn ed., Appleton-century-Crofts,

N.Y.

Nilsson, 0. (1958a)' Ultrastructure of mouse uterine surface

epithelium under different estrogen influences.

1) Spayed aminals and estrus animals' J. Ultrastr. Res.

1: 375-96

Nilsson, 0. (1958b)' Ultrastructure of mouse uterine surface

epithelium under-different estrogen influences.

2) Early effect of estrogen administered to spayed

animals J. ultrastr. Res. 2:73-95

Nilsson, 0. (1958c) Ultrastructure of mouse uterine surface

epithelium under different estrogen influences.

3) Late effect of estrogen administered to spayed animals

J. Ultrastr. Res., 2:185-99

Panashchenko, V.A. (1967)' Furccoumarins as potential

general contraceptives' Farmakol alkaloidov Glikozidov,

226-9, through Farnsworth, N.R. et al.,(1975)

J. Pharmaceutical Sci. 64(4):535-98

Perry, S.V. and Corsi, A. (1958)' Extraction of proteins

other than myosin from the isolated rabbit myofibril

Biochem. J. 68:5-12

Reynolds, A.K. (1940) 'The pharmacological actions of

cularine' J. Pharrnacol. 69:112-16

Reynolds, S.R.M. (1965)' Physiology of the uterus' P.127

2nd ed., Hafner, N.Y.

89

Rice, R.V. et al.,( 1970)' The organization of contractile

filaments in a mammalian smooth muscle' J. Cell. Biol.

47:183-96

Schofield, B.M, (1957)' The hormone control of myometrial

function during pregnancy' J. Physiol. 138:1-10

Schultz, R.H. et al., (1969)' A karyometric study of

epithelial cell lining the glands of the bovine

endometrium' J. Reprod. Fert. 19:169-71

Sheng, P.K. and Tso, T.C. (1955)' A comparative study of

nucleotropomyosins from different sources' Sci.Sinica.

4:157-76, through 'Biology of the uterus'( Wynn ed.)

Plenum.

Shoengberg, C.F. (1969)' A study of myosin filaments in

extracts and homogenates of vertebrate smooth muscle'

Angiologica, 6:233-46

Shoengberg, C.F. et al., (1966)' A biochemical and electron

microscopic study of the contractile proteins in

vertebrate smooth muscle' Biochem. Z. 345(1):255-66

through Biol. Abstr. 92038 (1966)

Shoengberg, C.F. and Needham, D.M. (1976)' A study of the

mechanism in vertebrate smooth muscle' Biol. Rev. 53-104

Sizov, P.I. (1969)' Experimental parturifacient action of

pachycarpine,brevicilline and rhalictrimine'

Zdravookhr. Beloruss., 15(11):44-6 through Chem. Abstr.

72:119957u (1970)

Small, J.V. (1974)' Contractile unit in vertebrate smooth

muscle' Nature (London), 249:234-7

Smith, D. S. (1966)' The organization and function of the

sacroplasmic reticulum and T system of muscle cells

Prog. Biophys. 16:107-42

90

Szent-Gyeorgui, A.G. (1951)' A new method for the prepara-

tion of actin' J. Biol. Chem. 192:361-9

The encyclopedia of Chinese Medicine (1977) P.1118

People's Press, Shanghai

Tsao, Y.L. (1957)' The antibacterial effect of the extracts

of Chinese medicinal herbs' Zhong Hwa Pi Fu Kao Zasshi,

4:286-292, through

P. 337,科 學 出 版 社

Tschesche, R. and Werner, W. (1967)' Evocarpine, a new

alkaloid from Evodia rutaecarpa' Tetrahedron 23(4):

1873-81

□ 糖 提 化 學 □ 研 究 國 藥 之 □ □T sun, K. F. (1936)

劉 壽 山

Zhong Hwa Yixue Zasshi 22(6)397-413, through

(1975) P.337

Vibert, P.J. (1972)' Structural changes in actin-containing

filaments of muscle' J. Mol. Biol. 71:757-67

Webber, A. and Murray, J.M. (1973)' Molecular mechanisms in

muscle contraction' Physiol. Rev. 53:612-73

Welch, A.D. and Henderson, V.E. (1934)' Hydrastine,

biculline and adlumine' J. Pharmcol. 51:482-91

Wilson, J.D. (1963)' The nature of the RNA response to

estrodiol administration by the uterus of the rat

Proc. Natn. Acad. Sci. USA 50:93-100

World Health( The magazine of the world health organization)

August-September,l978, Research in family planning

Wu) W.X., Xu, B.S. and Xiao, S.C. (1948)' 國產沿蟲藥物

Zhong Hwa Yixue Zasshi 34(10):

437-41, through (1975)

P. 336

劉 壽 山1975

中 藥 研 究 文 獻 摘 要

藥 理 研 究 初 步 報 告

中 藥 研 究 文 獻

摘 要

91

Yamazaki, M. and Kawana, T. (1967)' Isolation of hydroxy-

evodiamine (rhetsinine) from the fruits of Evodia

rytaecarpa' Yakugaku Zasshi 87(5):608-10

Zhu, Z.H. (1350) Dan Qi Xin Fa revised by C. Cheng,

Zhvaniya, G.P. (1970)' Effect of pachycarpine on the

electrical activity of the womb of nongravid rabbit

Soobshch Akad.Nauk. Gruz. SSR, 58(2):433-6, through

Chem. Abstr. '73, 64858a (1970)

1460-1490)朱 農 享 ： 丹 溪 心 清 ， 程 充 校