WO1997040844A2

WO1997040844A2 - Anti-hiv activity of medicinal plant extract preparation

Info

Publication number: WO1997040844A2
Application number: PCT/HU1997/000015
Authority: WO
Inventors: János ARADI; Ferenc TÓTH; László FÉSU^'S; Szilvia TO^'KÉS; József TO^'ZSÉR; Károly Nagy; Iván URAY; Péter VÁRNAI; Irén MEZO^'
Original assignee: Aradi Janos; Toth Ferenc; Fesus Laszlo; Tokes Szilvia; Tozser Jozsef; Nagy Karoly; Uray Ivan; Varnai Peter; Mezo Iren
Priority date: 1996-04-26
Filing date: 1997-04-16
Publication date: 1997-11-06
Also published as: WO1997040844A3; HUP9601096A2; AU2519597A; HU9601096D0

Abstract

The invention relates to medicinal plant extract preparation and its compounds with therapeutic and curative anti-HIV and anti-apoptotic effects. The medicinal plant extract preparation is: an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material, or an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material and any other compounds with known anti-HIV activity, preferably 2'-deoxy-3'azidothimidine, or chelidonoine and/or chelerythrine and/or sanguinarine or a mixture of these compounds with 2'-deoxy-3'-azidothimidine, or an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material and chelidonoine and/or chelerythrine and/or sanguinarine or any composition of these compounds with 2'-deoxy-3'-azidothimidine, or the above preparations and/or compounds further supplemented with known medicinal auxiliary and additive materials.

Description

ANTI-HIV ACTIVITY OF MEDICAL PLANT EXTRACT PREPARATION AND ITS COMPOUNDS WITH THERAPEUTIC AND CURATIVE EFFECT

The subject of the invention are medicinal plant extract preparation and its 5 compounds with therapeutic and curative anti-HIV and anti-apoptotic effects. The Chelidonium majus, greater celandine, is a well known herb, containing alkaloids, like chelidonine, chelerythrine, sanguinarine, protopine and also malic acid, citric acid, various salts and natural resins. The whole plant or the roots can be used as drug.

10 The alcoholic tincture or aqueous extract prepared from the plant is utilized as therapeutic agent for a long time.

It was successfully utilized in human therapy against some liver and bile disease and also against goat. It proved to be useful in veterinary medicine against distension and against some other ailments. Details of the

15 above mentioned effects are discussed in the book entitled "Therapeutic effects of herbs" pp. 134-135, written by Aladar Bela Varrό (Kaposvar, 1991). It is also known that the thiophosphoric acid derivatives of the alkaloids isolated from Chelidonium majus exert antitumor effect.

These facts are detailed in the following articles: Drugs Exptl. Clin.

20 Res. XVIII (Suppl.) 17-20 (1992) and Pol. J. Pharmacol. Pharm. 44, 227-239 (1992).

In our experimental work it was found that both the alcoholic and aqueous-alcoholic extract of Chelidonium majus has potent anti-HIV activity, suφrisingly, this interesting effect of the extracts was not known before.

25 It was shown in our laboratory that the extract of the Chelidonium majus synergistically potentiate the activity of some therapeutically utilized antiviral compounds and the alkaloids of the herb, including chelidonine, chelerythrine and sanguinarine, also exerted potent anti-HIV activity. Similarly, the extract of the Chelidonium majus enriched with the above mentioned alkaloids shows potent anti-HIV activity.

It was also observed in our laboratory that the above products not only potent anti-HIV agents but also affects the apoptosis (cell death), decreasing it.

Consequently, the subject of the invention are medicinal plant based preparation and its compounds with therapeutic and curative anti-HIV and anti-apoptotic effects.

The preparations of this invention are characterized is by the followings: an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material, or- an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material and any other compounds with known anti-HIV activity, preferably 2'-deoxy-3'-azidothimidine, or- an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material and chelidonine and/or chelerythrine and/or- sanguinarine or a mixture of these compounds with 2'-deoxy- -3'-azidothimidine- an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material and chelidonoine and/or chelerythrine and/or sanguinarine or any composition of these compounds with 2'-deoxy- -3'-azidothimidine, and further supplemented - in given case with known medicinal auxiliary- and additive materials . The above mentioned effects of the preparations, made according to this invention, were not known up to the present. The extract is prepared from the whole plant, or expediently from roots. The diluted solution, which is formed on extraction, is evaporated, or if required in some cases, lyophilized. The chelidonin, chelerythrine and sanguinarine may be isolated from the extract with preparative HPLC. The effects of the products and compounds prepared according to the invention, and the mode of the preparation are shown in the following examples.

Example 1

The dirt was removed from freshly picked roots of Chelidonium majus by washing and then dried partially at room temperature for 1-2 hours. The roots were cut up to small pieces and 100 part (mass) cut root was mixed with 250 part (mass) of 96% alcohol. The mixture was kept at room temperature in dark for 9 days and mixed up twice daily. The solid was separated from the liquid, Extract I, containing 1,5% (w/w) dry material. This product was partially evaporated at 45°C in vacuum, then the residue was mixed with two parts (mass) of alcohol. The mixture was filtrated. The filtrate contains 6,2% (w/w) dry material,Extract II.

Example 2

The procedure was completed according to the Example N°. 1 but instead of the roots of Chelidonium majus the whole plant was utilized. The dry material content of Extract I was 1,89% (w/w), the Extract II contained 6,5% by weight of dry material. Example 3

To study the anti-HIV effect of the drugs stock solutions were prepared, as follows. Extract 1 and Extract II was evaporated in vacuum and the dry residue dissolved in DMSO in a final concentration of 100 mg/ml. These stock solutions were diluted with RPMI to get a final drug concentration of 100 μg/ml, and used in the treatment of infected cells. H9 cells were infected with HIV, and used in this study.

The drug solutions were added to the tissue culture (10^ cells/ml) at 1 or 6 hours prior infection, at the viral infection (0 time), 24 and 48 hours after infection.

The final drug concentration was 10 μg/ml at each addition. The viruses were HIV-1 IIIB, produced by H9 cells (2xl0⁵ IU/ml). The infection was completed in 2 hours at 37 °C, in CO2 incubator. When the infection of the cells was finished the cells were washed thoroughly with PBS the 0,5 million cells were seeded into 24-well plates and 2 ml medium was added into each well. The culturing was performed in CO2 incubator. The infected cell cultures were treated with Extract I and Extract II as mentioned above, appropriate control tissue cultures were also run, without drug.

The results were as follows.

In both cases (the first treatment at -6 hour and at -1 hour) the viral replication was inhibited by the Extract I until the 5th day. On the 5th day, 30% inhibition was observed in the viral -infected cultures where the first treatment was at -1 hour and 90% inhibition was observed in the viral- infected cultures where the first treatment was at -6 hour. The Extract II inhibited the virus replication 45%, when the first treatment was performed at -1 hour, and showed 90% inhibition when the first treatment was 6 hours before infection. Example 4

In this experiment MT4 cells were infected with HIV. The Extract I was added to the tissue cultures 4 hours prior infection, 24 and 48 hours after viral infection. In Experiment A the final concentration of the extract (dry material) was 1 μg/ml. In Experiment B the final concentration of the extract (dry material) was 3 μg/ml. Control cultures were also prepared without drug. The virus concentrations were measured on the 5th day by determining the reverse transcriptase activity of the cell free medium. In Experiment A the amount of virus was 20,2%, in Experiment B the amount of virus was 4,4% ot fhe controls.

Example 5

This example is similar to the N°. 4, but in Experiment A: the extract is replaced with 1 μg/ml AZT; in Experiment B: 1 μl/ml AZT 1 μg/ml dry- material of extract II is combined. In both cases, the active compound and mixture is given to the tissue culture only once, after the viral infection. The concentration of produced virions were determined on the 13th day of postinfection measuring the activity of reverse transcriptase in the cell free medium. In Experiment A the reverse transcriptase activity was 19,9% compared to the control, in Experiment B this value was only 1,4%, representing 98,6% inhibition.

Example 6

MT4 cells were infected with HIV-1 IIIB and the virus replication was inhibited using the following agents: chelidonine (I), chelerythrine (III), sanguinarine (II). The inhibition of virus replication was followed by determining the reverse transcriptase activity as above, on 5th and 8th day after viral infections.

The control values (without drugs) were on 5th day: 153 824 cpm on 8th day: 56 266 cpm.

The results are summarized in the following table.

The results, summarized in the above table, clearly indicate that the compounds No I. II and III. are active as single agents at a concentration as low as 1 μg/ml, exerting potent anti-HIV activity compared to the controls.

Claims

1. Medicinal plant extract preparation and its compounds based on medical plant with therapeutic and curative anti-HIV and anti-apoptotic effects characterized by that the preparation is an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9% by weight of dry material, or- an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9 % by weight of dry material and any other compounds with known anti-HIV activity, preferably 2'-deoxy-3'azidothimidine, or- chelidonoine and/or chelerythrine and/or sanguinarine or a mixture of these compounds with 2'-deoxy-3'-azidothimidine, or- an alcoholic or aqueous extract of Chelidonium majus containing 0,5-99,9% by weight of dry material and chelidonoine and/or chelerythrine and/or sanguinarine or any composition of these compounds with 2'-deoxy-3'- azidothimidine, or- the above preparations and/or compounds further supplemented with known medicinal auxiliary- and additive materials.

2. Preparation according to the Claim 1. characterized by that it is an alcoholic extract of the root of Chelidonium majus containing 1-10% by weight of dry material.

3. Preparation according to the Claim 1. characterized by that it is a mixture of the alcoholic extract of the root of Chelidonium majus containing 1-10%) by weight of dry material and 2'-deoxy-3'-azidothymidine.

4. Preparation according Claim 1. characterized by that it contains chelidonoine and/or chelerythrine and/or sanguinarine or any composition of these compounds with 2'-deoxy-3'-azidothimidine.