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