NL8003871A - PHARMACEUTICAL COMPOSITION USING HEDERA HELIX EXTRACTS AS AN ACTIVE COMPONENT AND METHOD FOR EXTRACTING THE EXTRACTS. - Google Patents

Description

! * -1- 21371 / Vk / mv $

Applicant: Synthélabo, Paris, France

Short designation: Pharmaceutical composition with extracts of Hedera Helix as active component, and method for extracting the extracts.

The invention relates to a pharmaceutical composition. In particular, the invention relates to pharmaceutical compositions containing extracts from a climbing plant and to a method of recovering the extracts based on hederasaponin C by starting from this plant. Extracts of Hedera and in particular of Hedera Helix have already been prepared by extraction with water and / or alcohol.

The pharmaceutical composition according to the invention is characterized in that it contains, as the main active ingredient, an extract of Hedera Helix with about 90% or 60% hederasaponin C and the saponification product of hederasaponin C, namely -hederine.

The method according to the invention relates to the preparation in the first instance of an extract of saponins with a purity of 60%. To this end, a leaching of the powder of Hedera is first effected with the aid of acetone or with a solvent having a dielectric constant equal to that of acetone, such as a mixture of ethyl acetate / alcohol, in particular methanol or ethanol. The powder taken up by the solvent is then dried. A second leaching is then effected with pure methanol. The active carbon treated solution is evaporated and then filtered.

Evaporation of the filtrate is carried out under reduced pressure. Ethyl ether or a mixture of solvents of the same polarity as that of the ether, for example a mixture of chloroform and ethyl acetate, is added to the filtrate in order to precipitate the saponification extracts. This extract is taken up in pure methanol, filtered and dried under reduced pressure or by spraying. The product thus obtained is a complex of saponin containing about 60% Hederasaponin C.

This complex has a yellow color and is very soluble in water and methanol and only slightly soluble in ethanol. When a thin layer chromatogram is made with silica gel with a solvent of a mixture of benzene / methanol / acetic acid (65:35:10) as solvent, a solution of 1% vanillin in sulfuric acid is seen as running liquid, clearly different colored traces of which in particular those of hederasaponine G is colored brown-black.

According to the method of the invention, an 8003871 if ir -2-21371 / Vk / mv second time, an extract with a purity of 90% hederasaponin C is prepared.

To this end, the first extract with 60% hederasaponin C is passed over a column of aluminum oxide with pure methanol. Thus, with an eluate containing at least 90% hederasaponin C corresponding to formula 5, 1, indicated on the formula sheet. According to the process of the invention, finally, prepare s--hederine or its alkaline salt by starting from the aforementioned extract by saponification with sodium hydroxide or potassium hydroxide corresponding to formula 2, indicated on the formula sheet.

The three substances obtained at each step of the process according to the invention are further studied with regard to the antifungal and antiparasitic activity in vitro and in vivo.

The invention is further elucidated by means of the following example.

Example I

25 kg of Hedera Helix are treated with 120 liters of acetone.

The mixture is evaporated and dried to obtain a viscous mass which is dark green in an amount of 1000 g. This mixture is dried. 110 liters of pure methanol are added to this.

The mixture is then evaporated under reduced pressure to an amount of 25 liters. This mixture is stirred with 100-200 g of neutral active carbon. The mixture is then filtered and the filtrate is evaporated under reduced pressure to about 10 liters. Then 20 liters of diethyl ether are added to 10 liters of the filtrate. A precipitate is thus obtained. 20 liters of diethyl ether are added to the filtrate which is evaporated to 6 liters. Thus, a precipitate is formed which is combined with the precipitate. The precipitates are taken up again in 10 liters of pure methanol, the mixture is filtered and dried under reduced pressure. Thus, 1.5-2 kg of an extract containing 60% hederasaponin C is obtained.

Example II

The separation over an alumina column to obtain the extract containing 90% hederasaponin C, which operation is carried out in the following manner.

1 kg of neutral aluminum oxide are dispersed in pure methanol. This mixture is placed in a column and eluted with methanol until the eluate is completely clear. Then 300 g of the first extract are added and it is diluted in 2 liters of pure methanol. This 8 0 0 3 8 7 1 * -3- 21371 / Vk / mv% is eluted with pure methanol and 4 liters of eluate are collected and evaporated under reduced pressure. This gives 190 g of extract containing at least 90% hederasaponin C.

Example III

The extract with 90% hederasaponin C is treated in an aqueous mixture with 2N 'and NaOH or KOH at an elevated temperature for 1 hour. The mixture is acidified and the precipitate washed. The precipitate is taken up in pure methanol, filtered and dried. Thus t (-hederine.

10 The extracts of the Hedera Helix obtained according to the

method according to the invention which extracts have a purity of 60% with respect to hederasaponine C, and with 90% hederasaponine C

and 0 (-hederine) have been found to be active substances as antiparasitic agents and antifungals.

The toxicity of the extracts has been determined on mice by intraperitoneal administration. The DL 2 value after 24 hours ranged from 2000 mg to 3200 mg. The DL.0 value after 8 days ranged from 1500 mg to 2500 mg.

The anti-parasitic activity.

The anthelmintic activity and the protozoic activity of the extracts according to the invention have been further investigated.

1) The anthelmintic activity towards cestodes, nematodes and trematodes was determined in vitro and in vivo.

1a) The activity towards cestodes has been further investigated with respect to the Taenia of mice.

In vitro experiment:

The Hymenolepis nana ver. fraterna were recovered by shredding the small intestine of parasitized mice and placed in an environment of Sen and Hawking in a drying oven at 37 ° C. The experiment consists in that the different solutions of the products with the worms that have survived and the lethal dose is determined after 24 hours. The procedure is such that a number of untreated worms must remain alive for several days and a number of treated worms with the comparative products, helenine and santonine.

In vivo determinations.

This assay consists in introducing variable dosages of the product to be tested into the mouse on which 8003871 -4- 21371 / Vk / mv has been parasitized and examining the excretion of eggs of Hyraenolepis in faeces.

Recovery or healing is determined by counting eggs per gram of faeces. This count should be negative at the end of 5 treatment. To ensure that the termination of the parasitization is complete, a distribution of the small intestine of the mouse is effected after the autopsy to ensure that no worms have been left behind or that all have died. The comparative agents used in the test performed in vivo are mepacrine and helenine. »10 The results of the in vitro experiments are expressed in lethal doses (DL-100) after 24 hours corresponding to the amounts of the products which are sufficient to kill 100% of the cesto-des (tapeworms) after 24 hours.

The results of the in vivo experiments indicate the percentage of the suppression of the parasitization obtained in equal amounts. These data are summarized in Table A.

TABLE A

20 in vitro - substance__DL-100 (after 24 hours) helenine (for comparison) 50 jug / ml santonine (for comparison) 100 jug / ml of-hederine 100 Jug / ml extract with 90% 1 mg / ml 25 extract with 60% 5 mg / ml _____ ____ i V ____________ in vivo - substance__dose (mg / kg) deparasitization% mepacrine 220 100 30 helenine 300 60 ^ -hederine 400 20 extract with 90% 400 30 extract with 60% 400 60 35 1b) The nematocidal activity has been studied in Syphacia obvelata (parasites occurring in mice) in mice.

j. Experiments in vitro and in vivo have been performed 8003871-521371 / Vk / rav in the same manner as used for the study of the activity of the taenifugum (tapeworm killer or repellant).

The comparative products are santonine and helenine in vitro and piperazine and helenine in vivo. The results obtained are shown in Table B.

TABLE B

I - I »I. I I - I I !! I l | III mill II .I 11. - I - I I I

In vitro substance__PL-100 (after 24 hours) 10 helenine 10 jug / ml santonine 100 jug / ml 0 (-hederine 1 mg / ml extract with 90% 5 mg / ml extract with 60% 5 mg / ml 15 ___

Ln vivo substance dosage (mg / ml deparasitization (%) piperazine citrate 200 I 9Ö 20 helenine 300 80 0 (-hederine 400 10 extract with 90% 400 30 extract with 60% 400 70 25 1c) Activity relative to trematodes (women ) has been studied against liver bones in vitro and in vivo.

The assay performed in vitro consisted in that liver bones, in particular Fasciola hepatica and Diorocoeslium lancéolatum 30, were collected immediately after killing sheep and bovine animals infected with parasites by separating the bile ducts and liver from these animals. The worms are transferred directly into a Benex survival medium modified by Cavier in an incubator at 37 ° C. The products to be tested are applied in different concentrations and the lethal dose (DL-100) is determined after 24 hours. The comparative substances are helenine and santonine.

In vivo assays on bovine animals with parsites.

8 0 0 3 8 7 1 First, the number of eggs of the liver bones -6- 21371 / Vk / mv r in the faeces and the degree of contamination of the sheep to be treated are regulated.

Then, with the aid of a gun, a certain dose is applied down the throat of sheep, with three consecutive different products being applied at eight-day intervals. The duration of the treatment is therefore three weeks.

During the treatment time, the reduction of the eggs in the faeces is monitored until they are gone. To ensure the cure, the sheep are killed and the disappearance of the liver bones in the liver is examined in the bile, which provides irrefutable evidence that the products tested have a killer effect. The results obtained are shown in Table C.

TABLE C

15 __ in vitro a) compared to Fasciola hepatica DL-100 (after 24 hours) substance ___________________ ________________________ santonin · 100 jig / ml 20 helenine _ 10 ^ g / ral oi-hederine 5 jug / ml extract with 90% 5 "mg / ml extract with 60% 1 mg / ml b) relative to Dicrocoelium DL-100 (after 24 hours) 25 lanceolate. dust __; __ santonine 50 / ag / ml helenine 10 µg / ml o (-hederine 10 µg / ml 30 extract with 90% 5 mg / ml extract with 60% 500 µg / ml

In vivo, at 8-day intervals, three treatments were performed with 800 mg / kg in sheep containing parasites of Dicrocoelium lanceolatum (liver fluke). With Q (-hederine) there is a reduction in the number of eggs in the faeces, while with the two extracts with 60% and 90% of hederasaponin C there is a total disappearance of the 8003871 * -7- 21371 / Vk / mv. Eggs. The post-autopsy control indicates that there has been an effective disappearance of the liver fluke or that the liver flukes have been killed when the treated animals have had administrations of the two extracts at 60% and 90%.

2) The activity against unicellular organisms (protozao) has been investigated with regard to monocellular organisms present in the intestines and with regard to Trichomonas intestinalis.

The activity of the substances was investigated in two experiments in which the inhibition on growth was determined by starting from two cultures.

The minimum inhibitory concentration (CMI) was determined, which means the smallest amount of the product which, when added to the culture mixture before seeding, completely stopped culture development after a contact time of 72 hours at a temperature of 37 ° C .

The lethal activity was determined in a culture for 48 hours. - The smallest amount of the product to be tested is determined, which, after being added to a culture for 2 days as it grows, is capable of killing all protozoan single-cells (amiben or 20 Trichomonas) after an incubation period of 48 hours at a temperature of 37 ° C.

These two experiments were performed in parallel with a comparative, known product, metronidazole.

For these tests, a determination of the activity of the products in which retrocultures are prepared is effected by starting from mixtures of which the protozoan single-cells are killed. These negative retro cultures are also used to confirm the activity of the products investigated. The results obtained are summarized in Table D.

30 TABLE D

_ dust__CMI

metronidazole 5 µg / ml OWhederine 50 µg / ml 35 extract with 90%> 10 mg / ml extract with 60%. i 10 mg / ml 8003871 r -8- 21371 / Vk / mv *

Fungicidal action. The fungicidal activity has been investigated with regard to dermatophytes.

1). The activity towards yeast (Candida albicans) has been investigated in vitro and in vivo.

For the in vitro determination, a culture of the yeast Candida albicans on Sabouraud liquid was prepared with a doubled concentration of a suspension of 10 cells. A certain amount of the inoculum medium was contacted with an increasing dose of the diluted products under investigation. The assay was performed after a storage time of 24 hours at a temperature of 37 ° C. The test tubes in which the products were stirred remained clear. They determined.

the fungicidal activity by the retro cultures which had to remain negative 72 hours after incubation in an incubator at a temperature of 37 ° C. The substance used for comparison was amphotericin B.

In vivo, the study was conducted in mice.

After dorsal epilation, the mice were exposed to UV irradiation to effect an inflammatory response and inflammation was effected under the influence of Candida albieans sous-cutane by introducing 0.25 ml of a diluted culture of Candida albieans diluted to 1/10 .

Two days later, treatment began in which the mice were divided into different groups receiving different concentrations of the products to be tested by artificial feeding using a gavage. This treatment was performed in parallel with a comparative substance, amphotericin B. The treatment time was 10 days.

The cure was manifested by the gradual disappearance of the abscess caused by the yeast Candida. The untreated blank animals maintained the abscess for more than 1 month. The results obtained are expressed in minimum inhibitory concentrations (CMI) for the in vitro test and these data are summarized in Table E.

8 0 0 3 S 7 1 -9- 21371 / Vk / mv «

TABLE E

Substance _CMI__ 5 amphotericin B 2.5 / µg / ml ^, - hederine 500 μg / ml extract with 90%> 50 mg / ml extract with 60%) 50 mg / ml 10

The test performed in vivo determined whether or not the abscess that had been caused subcutaneously in mice was resolved. The results obtained hereby were that the abscess remained in the untreated animals for more than 1 month. The abscess remained in animals treated with 2.5 mg / kg amphotericin B but a new treatment for 10 days caused the abscess to disappear while the subcutaneous administration of substances of the invention such as Ή-hederine, the extracts by 90% and 60% hederasapo-nine C at a dose of 50 mg / kg per day for 10 days effected the disappearance of the abscess in all cases.

2. The activity against infections caused with dermatophyllis has been further investigated in vitro for Microsporum canis. Culturing of dermatophytes was accomplished as follows. The seed medium is a dilution to 1/10 in sterilized water from a 7 day culture. 0.1 ml of this diluted liquid was added to each tube, as was 1 ml of Sabouraud medium at double concentration and 1 ml of diluted products. The fungicidal activity was determined at the end of 7 days. Growth inhibition was investigated in the different tubes. When such inhibition was observed, the experiment was continued with a retro culture to check the assay, which was done 15 days later. The negative result of this retro culture then confirmed the fungicidal effect of the product. The substance used for comparison was amphotericin B. The results of the substances tested are that the CMI value • for amphotericin B is 2.5 Jig / wl and for tf-hederine the CMI value is 50 pg / ml.

The results of these experiments indicate that the substances according to the invention have a pharmaceutical effect with an antiparasitic fungicidal activity. The substances according to the invention, namely C 1 -hederine, the extract of Hedera with 90% hederasaponine C and the extract of Hedera with 60% hederasaponine C, can be used for the treatment of parasitic and fungal diseases for the treatment of humans and animals.

The substances can be administered in a form suitable for medical purposes such as by topical, oral or parenteral administration together with suitable excipients for example in the form of tablets, capsules, capsules which can be taken or injected , lyophilized powders, creams, lotions and the like. For example, the gelatin capsule (gelule) can be prepared starting from the following substances: 200 mg extract with 60% hederasaponin C 20 mg mannitol 15 35 mg microcrystalline cellulose and 5 mg magnesium stearate, for a gelatin capsule.

conclusions 8003871

Claims (2)

Pharmaceutical composition, characterized in that it contains, as the main active ingredient, an extract of Hedera Helix with about 90% or 60% hederasaponin C or the saponification product of hedera-saponin C, namely ζ ^ -hederine. Medicament with an anti-parasitic and fungicidal action, characterized in that it contains as active substance the compounds as defined in claim 1. 3. Pharmaceutical compositions with an anti-worm effect, characterized in that this composition as active component contains substances as claimed in claim 1, 4. Pharmaceutical composition with an activity against worms, for oral administration, characterized in that it contains as an active ingredient an extract of Hedera Helix with about 90 or 60% hederasaponin C. 5. Pharmaceutical compositions having an anti-cellular activity, characterized in that this active ingredient composition contains an extract of Hedera Helix with about 90 or 60% hederasaponin C or the saponification product of hederasaponin C-hederine. 6. Pharmaceutical compositions having a fungicidal activity, characterized in that they contain as an active ingredient an extract of Hedera Helix with approximately 90% or 60% hederasaponin C, or the saponification product of hederasaponin C, o-hederine. 7. Pharmaceutical compositions having a fungicidal action, characterized in that they contain, as active ingredient, an extract of Hedera Helix with approximately 90% or 60% hederasaponin C. Pharmaceutical compositions according to claims 1-7, characterized in that these formulated so that they can be administered orally in the form of a gelatin capsule, administered parenterally in the form of an injectable solution, optionally lyophilized, or applied topically in the form of an ointment, lotion or solution. preparation of an extract of Hedera Helix, characterized in that a leaching of the HSdera powder is effected with the aid of acetone or ee n solvent which has a dielectric constant equal to that of acetone, the powder leached with the 3 0 0 3 S 7 1 «-12- 21371 / Vk / inv solvent is dried, a second leaching is effected with pure methanol the solution obtained, after treatment with neutral activated carbon and filtration, is evaporated, the filtrate is evaporated under reduced pressure, diethyl ether 5 or a mixture of solvents with a polarity equal to that of the ether are added to the filtrate. to precipitate the saponification extract, which is taken up again in pure methanol, filtered and dried, after which an extract is obtained with 60% hederasaponine C, which is then passed over a column of alumina by eluting with 10 pure methanol, so that the extract is finally obtained. obtains hederasaponine with approximately 90% C. Eindhoven, June 25, 1980 8003871 cr - 21371 / Vk / mv FORMULA SHEET 1. CH3 C33 COQ ^ CH II 1 J 3 dGlu 1. Rhamm. -I. Arab in. -Cr j \ 1.Rhamm. CH 3 CH 2 OH H C “CH, 3> x 3 2. CH3 CH3 ^ COO (H Of Me) cii3 1.Rhamm.-1.Arabin. -Ö hoch2 ch3 8003871