Stephania cepharantha extract, its method of preparation and its use as pharmaceutical

This invention is concerned with the preparation of an extract of Stephania cepharantha which is useful in therapy, in particular as bacteriostatic and antiphlogistic and antalgic agent. A therapeutical composition is proposed which contains, in association with a physiologically acceptable excipient, a pharmaceutically effective amount of said Stephania cepharantha extract.

BACKGROUND OF THE INVENTION AND PRIOR ART 
Stephania cepharantha is a plant of the Menispermaceae family which grows 
in China and Japan. It has been disclosed by R. R. IS and H. MOYSE in 
"Matiere Medicale" vol. 2, page 178 (Masson ed., Paris 1967). 
It is also known, in particular from the R. R. IS and H. MOYSE 
publication, that root extracts of Stephania cepharantha prepared by 
extraction with a water-alcohol mixture [such as a water-ethanol (50:50) 
v/v mixture] at 60.degree.-100.degree. C., have been proposed in folk 
medicine in the treatment of tuberculosis and leprosis. 
It has now been found that the stem extract of Stephania cepharanthais (i) 
different from the previous root extracts of the very same plant, and (ii) 
useful in therapy, in particular in the treatment of infectious diseases 
in view of its bacteriostatic, antalgic and antiphlogistic properties. 
SUBJECT OF THE INVENTION 
The subject of the invention is to propose a method of extraction of stems 
of Stephania cepharantha in order to obtain a new extract which is useful 
in the treatment of human beings suffering, in particular from infectious 
diseases.

DETAILED DESCRIPTION OF THE INVENTION 
According to the present invention the stems of the plant are extracted 
with at least one solvent. The product thus obtained is then purified, if 
necessary, and then recovered according to a method known per se. 
The extraction can be carried out by using, per liter of solvent, 30 to 150 
g of ground dry plant, Solvents which can be used include water, alcohols 
(such as methanol, ethanol, propanol and isopropanol), ketones (such as 
acetone, methyl ethyl ketone and methyl propyl ketone), ethers (such as 
dimethyl ether, diethyl ether and diisopropyl ethyl), esters (such as 
ethyl acetate), hydrocarbons (such as pentane, hexane, cyclopentane, 
cyclohexane, petroleum ether and benzene), halogenated hydrocarbons (such 
as chloroform and methylene chloride), and mixtures thereof. 
BEST MODE 
The preferred solvents in order to extract the Stephania cepharantha stems 
are chloroform, methanol and a methanol-water (75:25) v/v mixture. 
The extract, which is obtained by extraction of one solvent then 
evaporation to dryness under reduced pressure, generally contains alkaloid 
components and non-alkaloid components. The preferred methods for 
recovering the alkaloids are given in examples 2 and 3 hereinafter, the 
method of example 3 enabling to isolate both the alkaloid and non-alkaloid 
components. 
EXAMPLE 1 
Preparation of the total extract 
140 g of ground and dried stems of Stephania cepharantha are extracted with 
3 liters of methanol in a Soxhlet apparatus for 4 hours. The insoluble 
material is discarded and the methanol solution is evaporated to dryness 
under reduced pressure to obtain a dry product which is taken up in the 
minimal amount of water and then lyophilised. 85 g of total extract (which 
is coded as AJ-01) are obtained. The yield is 42.5% by weight with respect 
to the starting plant material. 
EXAMPLE 2 
Extraction of the alkaloid components. 
(a) Extraction 
1.2 kg of dried (at 37.degree. C. in an oven) and ground stems of Stephania 
cepharantha are treated with 1.2 liters of NH.sub.4 OH (containing 150 
g/liter of NH.sub.3) and extracted with 9 liters of CHCl.sub.3 in a 
Soxhlet apparatus. The chloroform solution which is recovered is then 
concentrated to 3 liters under reduced pressure and extracted with 
acidulated water (5 times 500 ml of water containing 20 g/liter of citric 
acid). After adding NH.sub.4 OH up to pH8 the free bases contained in the 
solution are extracted with chloroform (5 times 200 ml). The organical 
phase is dried over anhydrous sodium sulphate and evaporated to dryness 
under reduced pressure to give 11.76 g of a dried product called total 
alkaloid extract. Yield 9.8% by weight with respect to the starting plant 
material. 
(b) Purification 
The total alkaloid extract is subjected to an extraction with methanol in a 
Soxhlet apparatus. The methanol solution is evaporated to dryness under 
reduced pressure. The evaporation residue is treated with water containing 
20 g/l of citric acid and the insoluble is discarded by filtration. 
NH.sub.4 OH is added to the aqueous phase up to pH 8 which is then washed 
with chloroform (3 times 200 ml). After acidification (with citric acid) 
the quaternized alkaloids are precipitated by means of the Mayer reagent 
in order to give, after filtration and drying alkaloid iodomercurates (51 
g) which are transformed into chlorides of quaternized alkaloids, by 
chromatography with an ion exchange resin (Amberlite IRA 400 in the 
chloride form). After elution and lyophilisation the alkaloid components 
are obtained. 
EXAMPLE 3 
Extraction of the alkaloid and non-alkaloid components. 
100 g of dried (at 37.degree. C. in an oven) and ground stems of Stephania 
cepharantha are extracted with 1 liter of a boiling methanol-water (75:25) 
v/v mixture. After filtration of the insoluble, the filtrate is 
concentrated to 250 cm.sup.3 under reduced pressure. The aqueous phase 
which is obtained is filtered and chromatographied on a non ionic resin 
(300 ml of Amberlite XAD-2). 
The filtrate, which includes the non adsorbed material, is lyophilised. The 
product thus obtained contains the non-alkaloid components. 
Elution of the adsorbed material with an ethanol-water (90:10) v/v mixture 
gives after concentration (under reduced pressure) then lyophilisation a 
product containing the alkaloid components. 
The extracts of Stephania cepharantha according to the invention have been 
tested with respect to their pharmacological properties. The assays 
concerning AJ-01 (the extract of example 1) are summed up hereinafter. 
(1) Toxicity 
AJ-01 administered to animals by i.v. and i.p. routes in solution in 
physiological serum (water containing 9 g/l of NaCl) is well tolerated. 
LD-0 (i.e. the maximum non-lethal dose) by i.v. route and i.p. route in 
mice is higher than 500 mg/kg. 
(2) Bacteriostatic activity 
AJ-01 exhibits a bacteriostatic activity against gram (+) and gram (-) 
bacteriae. For instance the MIC (minimal inhibitory concentration) values 
of AJ-01 are 2 mg/ml on Staphylococcus aureus London and 44 mg/ml on a 
strain of Proteus (Proteus 1557 of the catalogue of the collection of the 
"Centre International de Distribution de Souches et d'Information sur les 
Types Microbiens" of Lausanne). 
(3) Antiphlogistic activity 
The antiphlogistic activity of AJ-01 was studied on female rats (weighing 
100 g) according to the carrageen oedema test. The results given in table 
I for AJ-01 and phenylbutazone (a reference product) show that AJ-01 
inhibits in a statistically significant manner the development of the 
carrageen oedema at 100 mg/kg, the action being maximum 3 hours after 
administration. 
(4) Antalgic activity 
The study was carried out according to the method of Koster by i.p. 
administration to male mice of 0.2 ml of an aqueous solution of acetic 
acid (30 g/l) 30 minutes after i.p. or oral administration of products 
(AJ-01 and aspirin as a reference substance) to be tested. The results are 
given in table II wherein the number of crampings and the percentage 
variation with respect to the control animals have been given. 
The results of the pharmacological assays point out that stems extracts of 
Stephania cepharantha according to the invention such as AJ-01 are useful 
for the treatment of human beings suffering from infectious diseases, 
algiae and inflammation. 
The invention includes within its scope a therapeutic composition 
comprising, in association with a physiologically acceptable excipient, a 
pharmaceutically effective amount of an extract of the invention. 
Such a composition can be administered orally in the form of dragees, 
pills, syrups and potable ampoules, locally in the form of ointments, or 
sprays or by injection. 
TABLE I 
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CARRAGEEN OEDEMA TEST 
Number of 
Plethysmographic measure of oedema volume after 
administration 
Product 
animals 
of carrageen and product to be tested at instant T.sup.a 
(dose) per batch 
T + 1h 
T + 2h 
T + 3h T + 4h 
T + 5h 
__________________________________________________________________________ 
control 
10 4.3 .+-. 1.3 
7.2 .+-. 1.6 
10.6 .+-. 2.1 
12.7 .+-. 2.2 
13 .+-. 1.3 
Phenybuta- 
8 4.6 .+-. 0.3 
3.8 .+-. 0.7 
5.2 .+-. 1.5 
7.2 .+-. 1.6 
8.3 .+-. 1.5 
zone (+7%) (-48%) 
(-51%) (-43%) 
(-36%) 
(20mg/kg) 
AJ-01 10 4.0 .+-. 0.4 
5.9 .+-.1.4 
10.6 .+-. 1.4 
12.7 .+-.1.1 
12.7 .+-. 1.1 
20 mg/kg) (-8% (-18%) 
(0%) (0%) (-2%) 
AJ-01 8 3.6 .+-. 1.6 
5.5 .+-. 0.8 
7.3 .+-. 1.9 
10 .+-. 1.2 
11.5 .+-. 1 
(100 mg/kg) (-16%) 
(-24%) 
(-31%).sup.b 
(-21%).sup.b 
(-12%) 
__________________________________________________________________________ 
Notes: 
.sup.a The percentage variation with respect to control animals is given 
in brackets. 
.sup.b Statistically significant (p&lt;0.05). 
TABLE II 
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ACETIC ACID TEST 
Variation with 
Product Number of Number of respect to 
(dose) animals crampings control animals 
______________________________________ 
control 10 28.1 .+-. 6.8 
-- 
Aspirin 10 17.7 .+-. 5.0.sup.a 
-37% 
200 mg/ 
kg 
p.o. 
AJ-01 10 25.0 .+-. 7.2 
-11% 
100 mg/ 
kg i.p. 
AJ-01 10 19.1 .+-. 4.0.sup.a 
-32% 
250 mg/ 
kg 
i.p. 
______________________________________ 
note: 
.sup.a statistically significant (p&lt;0.05)?