WO2006111251A2

WO2006111251A2 - Hydro-alcoholic extract of butea frondosa flowers, a process for its preparation, pharmaceutical compositions containing it and its use for the treatment of ulcer

Info

Publication number: WO2006111251A2
Application number: PCT/EP2006/002839
Authority: WO
Inventors: Andrea Giori; Federico Franceschi
Original assignee: Indena SpA
Current assignee: Indena SpA
Priority date: 2005-04-19
Filing date: 2006-03-29
Publication date: 2006-10-26
Anticipated expiration: 2007-10-19
Also published as: WO2006111251A3; ITMI20050691A1

Abstract

A hydro-alcoholic extract of Butea frondosa flowers for the treatment of ulcer, more particularly a purified hydro-ethanolic extract of Butea frondosa flowers, the process for its preparation and its use for the treatment of ulcer.

Description

HYDRO-ALCOHOLIC EXTRACT OF BUTEA FRONDOSA FLOWERS, A PROCESS FOR ITS PREPARATION, PHARMACEUTICAL COMPOSITIONS CONTAINING IT AND ITS USE FOR THE TREATMENT OF ULCER

DISCLOSURE

The present invention relates to a hydro-alcoholic extract of Butea frondosa flowers for the treatment of ulcer, more particularly to a purified hydro-ethanolic extract of Butea frondosa flowers, the process for its preparation and its use for the treatment of ulcer.

Butea frondosa (Koen. Ex Roxb), belonging to the class of Fabaceae, is a deciduous tree growing in the Indian subcontinent. It is also known under the name dhak or palas, and is used in Indian traditional medicine for the treatment of various conditions. Previous pharmacological studies evidenced that the aqueous extract of leaves and roots has eye anti-inflammatory activity (S. A. Mengi et al., Indian Journal of Pharmacology, 1995, 27,116-119), the seeds methanolic extract has anti-helminthic activity (D. Prashanth et al., Fitoterapia, 2001, 72, 421-422), while the triterpene fraction extracted from the flowers exerts anticonvulsive activity (V. S. Kasture et al., Pharm. Biochem. and Behaviour, 2002, 72, 969-972). Furthermore, isobutrin and butrin isolated from the methanol extract of the flowers proved active as in vitro anti-hepatotoxic agents in rat hepatocytes cultures (H. Wagner et al., Planta Medica, 1986, 2, 77-79). The flowers ethanolic extract is also known to have antiestrogenic activity (K.R. Laumas et al. 1966, 4, 246-256).

It has now surprisingly been found that the purified hydro-alcoholic extract of Butea frondosa flowers has remarkable anti-ulcer activity.

More particularly, the present invention relates to the use of purified hydro-ethanolic extract of Butea frondosa flowers for the treatment of ulcer. According to the invention, said purified extract is characterized by:

• (butrin + butin + isobutrin + butein) total content: 20-90%;

• butrin (main component) content: 10-70%; • (isobutrin + butein)/(butrin + butin) ratio ranging from 0.3 to 3.

The process for the preparation of the purified hydro-ethanolic extract of the invention comprises the following steps: a) grinding of Butea frondosa dry flowers; b) extraction in a percolator with ethanol/water mixtures; c) recovery of the solvent and extraction with ethanol/water fresh mixture, repeating such procedure 5 times; d) filtration of the percolates and subsequent concentration under reduced pressure to obtain a dry residue; e) dissolution of the dry residue in water; f) defatting of the resulting dark solution with hexane; g) removal of the organic phases; h) extraction of the aqueous phase with butanol; i) concentration of the combined organic phases, under reduced pressure; j) drying of the resulting dark syrup under vacuum at 60⁰C for 24 hours; k) grinding of the resulting solid from j).

Alternatively to steps f)-h), the dark solution from step e) is eluted with water on an adsorption resin column, the resulting fractions are removed, and one or more elutions with ethanol/water mixtures are effected; the combined organic phases are subsequently subjected to the above steps i), j) and k).

Some examples of the preparation of the extract according to the invention are reported in the following. Example 1 : Preparation of the concentrated aqueous extract

1000 g of Butea frondosa ground dry flowers are placed in a percolator.

The material is covered with an ethanol/water 4: 1 v/v mixture and the whole is left at 2O⁰C for 3 hours. The solvent is then percolated and the mass is covered with a fresh portion of ethanol/water 4:1 v/v mixture. The procedure is repeated 5 times totally using approx. 19900 ml of solvent. The 5 percolates are filtered, combined and concentrated under reduced pressure to remove ethanol, thereby obtaining the dry residue (233 g) suspended in water

(2330 ml). When a suspension is obtained, the solid, which contains no substances which will be titrated in the purified extracts described in the following, is filtered and discarded.

Example 2: Preparation of the total extract

The dark solution obtained in Example 1 is concentrated under reduced pressure until obtaining a dark syrup which is dried under vacuum at 60⁰C for 24 hours. The resulting pale brown solid (233 g; yield: 23.3% w/w) is ground to obtain a yellow powder.

The product is characterized by a butein, isobutrin, butin and butrin total content of 23.7% w/w (butein: 0.59% w/w; isobutrin 8.02% w/w; butin 1.46% w/w; butrin 13.6% w/w). Example 3: Preparation of the purified extract by liquid-liquid

(water/butanol) extraction

The dark solution obtained in Example 1 is defatted with hexane (2 x 1160 ml). The organic phases are separated and discarded. The aqueous phase is then extracted with butanol (7 x 1160 ml). The organic phases are combined and concentrated under reduced pressure until obtaining a dark syrup which is dried under vacuum at 60⁰C for 24 hours. The resulting pale brown solid (100 g; yield: 10% w/w) is ground to obtain a yellow powder.

The product is characterized by a butein, isobutrin, butin and butrin total content of 38% w/w (butein: 1.8% w/w; isobutrin 12.6% w/w; butin 4.5% w/w; butrin 19.1% w/w).

Example 4: Preparation of the purified extract using an adsorbption resin The dark solution obtained in Example 1 is placed on a column containing 2330 ml of Amberlite XAD 1180 resin, Rohm & Haas, and eluted with water (9320 ml, i.e. 4 bed volumes); the eluted water fractions are discarded. After that, elution is continued with an ethanol/water 7:3 v/v mixture (4660 ml, i.e. 2 bed volumes); the hydro-alcoholic fractions are combined and concentrated under reduced pressure until obtaining a dark syrup which is dried under vacuum at 60⁰C for 24 hours. The resulting pale brown solid (90 g; yield: 9% w/w) is ground to obtain a yellow powder.

The product is characterized by a butein, isobutrin, butin and butrin total content of 50.5% w/w (butein: 1.7% w/w; isobutrin 15.7% w/w; butin 6.3% w/w; butrin 26.8% w/w).

Example 5: Preparation of the high titre extract by fractionation on an adsorbption resin (fraction A and fraction B)

The dark solution obtained in Example 1 is placed on a column containing 2330 ml of resin Relite SP 207, Resindion, and eluted with water (23300 ml, i.e. 10 bed volumes ); the eluted water fractions are discarded.

After that, elution is continued with an ethanol/water 3:7 v/v mixture (6990 ml, i.e. 3 bed volumes ); the eluate A is concentrated under reduced pressure until obtaining a dark syrup which is dried under vacuum at 60⁰C for 24 hours. The resulting pale brown solid (39.2 g; yield: 3.9% w/w) is ground to obtain a yellow powder. The product only contains 2 out of 4 components, namely isobutrin (20.4% w/w) and butrin (61.2% w/w), the total content being

81.6% w/w.

The column is then eluted with an ethanol/water 9: 1 v/v mixture (6990 ml, i.e. 3 bed volumes ); the eluate B is concentrated under reduced pressure until obtaining a dark syrup which is dried under vacuum at 6O⁰C for 24 hours. The resulting pale brown solid (32 g; yield: 3.2% w/w) is ground to obtain a yellow powder. The product is characterized by a butein, isobutrin, butin and butrin total content of 51.8% w/w (butein: 3.8% w/w; isobutrin 34.7% w/w; butin 8.6% w/w; butrin 4.7% w/w).

PHARMACOLOGICAL EXPERIMENTS

Anti-Ulcer Activity

The anti-ulcer activity of the extract of Example 1 was tested on two experimental models.

1. Pylorus-ligation ulcer

Rats fasted for 48 hours where subjected to pylorus ligation under ether anesthesia, then given 125-250-500 mg/kg of the extract of Example 1 and

100 mg/kg of ranitidine. The animals were killed after 6 hours and the stomachs were opened along the great curvature and scored for the presence of ulcers in the glandular part. Results are reported in the following Table 1.

2. Ethanol ulcer

Gastric lesions were induced by oral administration of 1 ml of absolute alcohol to rats fasted for 24 hours. The animals were treated 1 hour before the administration of the tested compounds, then killed in ether excess, 1 hour after the alcohol administration. Stomachs were opened along the great curvature and scored for the presence of ulcers in the glandular part. Results are reported in the following Table 2.

The present invention also relates to compositions for the treatment of ulcer, which can be administered through the oral route, containing the extracts described above. Said compositions will be prepared according to conventional methods well-known in pharmaceutical technique, such as those described in "Remington's Pharmaceutical Handbook", Mack Publishing Co., N. Y., USA, together with suitable excipients commonly used in the technique.

Claims

1. Purified hydro-alcoholic extract of Butea frondosa flowers, characterized by: • (butrin + butin + isobutrin + butein) total content: 20-90%;

• butrin (main component) content: 10-70%;

• (isobutrin + butein)/(butrin + butin) ratio ranging from 0.3 to 3.

2. Pharmaceutical compositions comprising a hydro-alcoholic extract of Butea frondosa flowers.

3. Pharmaceutical compositions as claimed in claim 2, in which the hydro-alcoholic extract is a purified hydro-ethanolic extract of Butea frondosa flowers.

4. Pharmaceutical compositions as claimed in claim 3, in which the purified hydro-ethanolic extract of Butea frondosa flowers is characterized by: • (butrin + butin + isobutrin + butein) total content: 30-60%;

• butrin (main component) content: 10-30%;

• (isobutrin + butein)/(butrin + butin) ratio ranging from 0.3 to 0.9.

5. A process for the preparation of the extract according to the above claims, characterized by the following steps: a) grinding of Butea frondosa dry flowers; b) extraction in a percolator with ethanol/water mixtures; c) recovery of the solvent and extraction with ethanol/water fresh mixture; d) filtration of the percolates and subsequent concentration under reduced pressure to obtain a dry residue; e) dissolution of the dry residue in water; f) defatting of the resulting dark solution with hexane; g) removal of the organic phases; h) extraction of the aqueous phase with butanol; i) concentration of the combined organic phases, under reduced pressure; j) drying of the resulting dark syrup under vacuum at 60⁰C for 24 hours; k) grinding of the resulting solid from j).

6. A process as claimed in claim 5 in which, alternatively to steps f) to h), the dark solution from step e) is eluted with water on an adsorption resin column, the resulting fractions are removed, and one or more elutions with ethanol/water mixtures are effected; the combined organic phases are subsequently subjected to the above steps i), j) and k).

7. A process as claimed in claims 5 and 6, in which the hydro-ethanolic mixture consists of an ethanol/water 4:1 v/v mixture.

8. A process as claimed in claim 6, in which the adsorption resin used is Amberlite XAD 1180.

9. The use of the extract according to the above claims for the treatment of ulcer.