HK1122491B - Salix extract, its use and formulations containing it - Google Patents

Description

Salix extract, its use and formulations containing it

Summary of The Invention

The present invention relates to Salix (Salix) extracts obtainable by fractionation on a resin and to a process for their preparation.

The extracts of the invention are characterized by a high content of salicin derivatives, a reduced content of high molecular tannins and a content of proanthocyanidins (proanthocyanidins) sufficient to inhibit some of the tissue metalloproteinases. The product is formulated in oils rich in omega-3 and omega-6 acids, which allow better absorption of the active ingredients and also enhance their action in a synergistic manner.

Background

Extracts of different kinds of bark and branches of salix have been used for the treatment of rheumatoid forms of joints and gout for a long time. However, at the end of the 19 th century, salix extracts were essentially discarded when aspirin was synthesized by acetylation of salicylic acid (which is obtained by oxidation of compounds present in salix). However, aspirin and salix extracts differ greatly in their mechanism of action and activity on bone joints. The extract acts on the enzyme COX 2, whereas aspirin acts mainly on COX 1, which is associated with well-known side effects on the gastrointestinal tract and blood coagulation, which severely limit its long-term use, which is necessary in the case of chronic-degenerative pathologies such as arthrosis and rheumatoid arthritis.

It is known from the literature that salix extracts have a very variable content of salicin derivatives, generally not exceeding 15%, and a tannin content of 8-20%. As with all gallic and catechin tannins, tannins present in salix extracts have a strong affinity for proteins and proteoglycans, which are associated with tissue damage in the case of long-term treatment.

Therefore, there is a need for suitable methods which can be easily applied industrially and which provide extracts with a standardized content of active components.

Disclosure of Invention

The present invention relates to a process for preparing novel salix extracts characterized by a high salicin derivative content, a reduced high molecular tannin content and a proanthocyanidin content sufficient to inhibit some tissue metalloproteinases.

It has been surprisingly found that extraction of willow bark or branches under suitable conditions and specific purification of the resulting extract provides an extract having a salicin derivative content of up to 50%, a tannin content of not more than 5% and an oligomeric proanthocyanidin content of more than 5%.

The advantages of using salix extracts, in particular the extract of the invention, compared to salicin derivatives alone, are linked to the presence of proanthocyanidins, which are strong radical scavengers and metalloproteinases (which are via leukocytes Il in arthritic conditions)₁Is activated) is activated.

The process of the present invention for preparing salix extracts differs from the prior art processes in terms of the salicin and derivatives content of the extract and in the use of a matrix that selectively reduces the tannin content while retaining therapeutically useful proanthocyanidins in the extract.

The method of the invention comprises four main steps:

a) extracting the branches and bark of Salix with a suitable solvent which dissolves the desired product (total extract);

b) removing water insoluble (or poorly soluble) tannins;

c) removing water soluble tannins;

d) the salicin derivative is increased by purifying on an adsorption resin column.

Step (a) is carried out by extracting the plant material consisting of plant bark and branches with a C1-C3 alcohol, preferably ethanol, or acetone or a mixture of these solvents, or an aqueous solution of these solvents, or water alone. Preferably 30% v/v water-ethanol solution.

The extraction temperature may be 10 ℃ to 80 ℃, preferably 25 ℃.

Step (b) allows the removal of water-insoluble, in particular high molecular tannins from the extract.

Step (c) allows the removal of the majority of the water-soluble tannins from the extract. This is an optional step which is carried out to remove any tannins still present in the extract after step (b). These metabolites can be removed by using polyvinylpolypyrrolidone (PVPP).

Step (d) allows the fractionation of the extract to remove most of the unwanted metabolites (sugars, etc.) while retaining the desired secondary metabolites, i.e., salicin derivatives and oligomeric proanthocyanidins. This step consists in carrying out chromatographic separations by adsorption on polymeric resins. Examples of suitable resins for this purpose are Styrene-DVB resins such as Amberlite HP20^_Or Rohm and Haas XAD1180^_And acrylic resins such as Rohm and Haas XAD7HP^_。

During the column fractionation step with a suitable solvent mixture, the free salicin can be separated from its derivatives, obtaining a fraction enriched with free salicin and with a reduced amount of its derivatives and a fraction with a completely different composition.

The total extract obtained from the bark and branches of Salix with 30% ethanol is concentrated to a dry residue of 5% to 50% w/w, preferably 25% w/w, and left without stirring at a temperature of 1 ℃ to 20 ℃, preferably at 4 ℃, for a period of 1 hour to 24 hours, preferably 16 hours.

The resulting suspension was centrifuged at 4 ℃ to remove the residual precipitate containing the polymeric derivatives and tannins from the clear aqueous solution.

The water-insoluble or poorly soluble tannins contained in the total extract are removed by water purification, which can be further improved by an optional treatment (step c) with polyvinylpolypyrrolidone (PVPP). Part of the water purification (step b) removes only part of the tannins (more than 50% w/w of the tannins present), whereas the PVPP purification removes residual tannins within a value below 5% by weight of the final extract.

Thus, the clear aqueous solution obtained from step b) is treated with PVPP (1-50% w/w, preferably 1: 30, most preferably 1: 20 based on the dry residue of the treated aqueous extract) while maintaining stirring for 1 or more hours.

The solution was filtered from the PVPP and adsorbed tannins. The aqueous solution is then adsorbed on a resin and the support is washed thoroughly with water to remove unwanted soluble species. The solution that did not remain was discarded.

The product is eluted with a water-alcohol solution (C1-C3 alcohol, preferably ethanol) having a water content of 50% v/v to 0% v/v, preferably 10% v/v. Alternatively, a water-acetone solution may be used in which the water content is from 50% v/v to 0% v/v, preferably 10% v/v. The water-ethanol solution is concentrated to dryness or to atomization (atomized). The extract obtained can be formulated in the form of a conventional pharmaceutical solid or as an oily suspension in capsules, in particular in oils rich in omega-3/omega-6 polyunsaturated fatty acids; particularly preferred are the Enothera biennis and fish oils and their derivatives.

The active dose for the treatment of arthrosis and rheumatoid arthritis in humans is between 100 and 1000mg per day, depending on the severity of the disease to be treated.

The present invention is described in more detail in the following examples.

Example 1

Extracting the branches and bark of Salix with water-ethanol solution (step a):

in this step, a total extract is prepared, which can be used as a starting material for subsequent column chromatography separation.

In a static percolator, 1000 g of willow branches and bark were covered with 1.5 l of 30% v/v ethanol at 20 ℃ for 4 hours. After 4 hours, the exudate is recovered and extracted 6 more times under the same conditions, but using 1 liter of solvent per extraction, obtaining about 7 liters of total exudate. The combined exudates were filtered to remove impurities and plant residues. This solution (product 1) had a total dry residue of 154 g, a yield of 15.4% w/w compared to the starting material.

Free salicin HPLC content 4.63%; the total salicin HPLC content was 15.4% w/w. The tannin content was 16.26% w/w.

Example 2

Extracting the branches and bark of Salix with water-acetone solution (step a):

in this step, a total extract is prepared, which can be used as a starting material for subsequent column chromatography separation.

1000 g of willow branches and barks are extracted with 1.5 l of 80% v/v acetone at 20 ℃ for 4 hours in a static percolator. After 4 hours, the exudate is recovered and extracted 6 more times under the same conditions, but using 1 liter of solvent per extraction, obtaining about 7 liters of total exudate. The combined permeate was filtered hot and concentrated at 60 ℃ under reduced pressure using a rotary evaporator. The extract had a total dry residue of 143 grams, with a yield of 14.3% w/w compared to the starting material.

Free salicin HPLC content 4.3%; the total salicin HPLC content was 15.7% w/w. The tannin content was 15.42% w/w.

Example 3

Extracting the branches and bark of Salix with water (step a):

in this step, a total extract is prepared, which can be used as a starting material for subsequent column chromatography separation.

In a static percolator, 1000 g of willow branches and barks were covered with 1.5 l of water at 20 ℃ for 4 hours. After 4 hours, the exudate is recovered and extracted 6 more times under the same conditions, but using 1 liter of solvent per extraction, obtaining about 7 liters of total exudate. The combined filtrates were filtered off with suction and concentrated at 60 ℃ under reduced pressure using a rotary evaporator. The extract had a total dry residue of 167 grams, a yield of 16.7% w/w compared to the starting material.

Free salicin HPLC content 3.94%; the total salicin HPLC content was 13.6% w/w. The tannin content was 6.8% w/w.

Example 4

Extraction of willow branches and bark with methanol (step a):

in this step, a total extract is prepared, which can be used as a starting material for subsequent column chromatography separation.

In a static percolator, 1000 g of willow branches were covered with 1.5 l of methanol at 20 ℃ for 4 hours. After 4 hours, the exudate is recovered and extracted 6 more times under the same conditions, but using 1 liter of solvent per extraction, obtaining about 7 liters of total exudate. The combined filtrates were filtered and concentrated at 60 ℃ under reduced pressure using a rotary evaporator. The extract had a total dry residue of 101 grams, a yield of 10.1% w/w compared to the starting material.

Free salicin HPLC content 5.9%; the total salicin HPLC content was 19.9% w/w. The tannin content was 14.5% w/w.

Example 5

Purification of extracts of branches and bark of salix (step b): removing water insoluble matters:

the solution 1 obtained at the end of the treatment described in example 1 (step a) was concentrated at 60 ℃ under reduced pressure using a rotary evaporator to obtain an aqueous suspension with a dry residue of 25% w/w of the total aqueous suspension, the total weight of the solution being 615 g.

The resulting aqueous suspension was cooled at 4 ℃ and allowed to stand for 16 hours, after which the quiescent cold aqueous suspension was centrifuged at 3000g for 20 minutes to separate the precipitated residue from the clear aqueous solution. The precipitate with 16.3g of dry residue was rich in tannin and high molecular products, which were removed.

The resulting clear solution (solution 2) had a dry residue equivalent to 137g of partially purified extract with a free salicin HPLC content of 5.0% and a total salicin HPLC content of 16.7% w/w. The tannin content was 6.9% w/w.

The weight yield compared to the starting material was 13.7% w/w.

Example 6

Purification of extracts of branches and bark of salix (step c):

the clear aqueous solution obtained at the end of the partial purification process of step b with 137g of dry residue (example 5, solution 2) was subjected to a treatment to remove the water-soluble tannins.

To the solution was added 14g PVPP, corresponding to about 10% w/w of the dry residue of the treated extract. After stirring at room temperature for 1 hour, the PVPP was separated from the solution by centrifugation.

The resulting solution (solution 3) had a dry residue equivalent to 125g of partially purified extract with a free salicin HPLC content of 5.3% and a total salicin HPLC content of 8% w/w. The tannin content was 1.2% w/w.

The weight yield compared to the starting material was 12.5% w/w.

Example 7

Chromatographic purification of extracts of branches and bark of salix (step d):

the aqueous solution from step c (example 6, solution 3) was loaded in a solution containing 1250ml of Rohm and Haas XAD1180 adjusted with water^_On a chromatographic column of the resin. The water-alcohol solution is adsorbed onto the resin while the non-retained solution that flows out of the column is discarded. The resin was then washed with 1.25 liters of water, as wellThis solution is removed because the content of the desired component therein is very small. These discarded aqueous solutions (product 1) had in fact 52.6 g of total dry residue with an HPLC content of free salicin of 0.87% and a total salicin of 9.2% w/w.

The column was eluted with 3.75 liters of 90% v/v aqueous ethanol. The resulting eluate was recovered and dried at 60 ℃ under reduced pressure to give 72.4 g of dry product (product 2), corresponding to a yield of 7.2% w/w compared to the starting material. The HPLC content of free salicin is 8.95%, and the HPLC content of total salicin is 30.0% w/w. The oligomeric proanthocyanidin content was 11.2% w/w and the tannin content was 2.1% w/w.

Example 8

Chromatographic purification of extracts of branches and bark of salix (step d) and isolation of free salicin from its derivatives:

the aqueous solution from step c (example 6, solution 3) was loaded in a solution containing 1250ml of Rohm and Haas XAD1180 adjusted with water^_On a chromatographic column of the resin. The water-alcohol solution is adsorbed onto the resin while the non-retained solution that flows out of the column is discarded. The resin was then washed with 1.25 liters of water and the solution was also removed because the content of the desired component therein was very small. These discarded aqueous solutions (product 3) had in fact 51.7g of total dry residue with a free salicin HPLC content of 1.34% and a total salicin HPLC content of 1.41% w/w.

The resin was washed with 2.5 liters of 10% v/v aqueous ethanol to obtain a solution (product 4) with 17.1g of dry residue (yield 1.7% w/w compared to starting material). The HPLC content of free salicin is 34.6%, and the HPLC content of total salicin is 34.9% w/w.

The column was eluted with 3.75 liters of 90% v/v aqueous ethanol. The resulting eluate was recovered and dried at 60 ℃ under reduced pressure to give 43.2 g of dry product (product 5) corresponding to a yield of 4.3% w/w, free salicin HPLC content of 0.45% and total salicin HPLC content of 39.7% w/w compared to the starting material.

Example 9

Extract preparation in the form of soft gelatin capsules

Salix rubra extract formulation in the form of an oily suspension for use in soft gelatin capsules.Unit composition:

salix rubra extract of example 7 250mg

Glycerol monostearate 30mg

Soybean lecithin 10mg

Proper amount of Enothera biennis oil to 700mg

Preparation:

1) the Enothera biennis oil was heated at about 70 ℃ with stirring and the glycerol monostearate was melted therein.

2) To the resulting solution was added soybean lecithin.

3) The Salix ruba extract was dispersed in the resulting solution and a suitable stirring system was used to facilitate uniform distribution.

The resulting solution was gradually cooled under stirring.

Claims (9)

1. A method of extracting branches and bark of salix comprising:

a) extracting the branches and bark of Salix with a suitable solvent for dissolving the desired product, wherein the suitable solvent is selected from C1-C3 alcohol or acetone or mixture of these solvents or aqueous solution of these solvents or water alone;

b) removing water-insoluble or poorly soluble tannins by water purification;

c) removing the water-soluble tannin with polyvinylpolypyrrolidone;

d) purifying on adsorption resin column to increase salicin derivative content.

2. The method of claim 1, wherein the extraction solvent is ethanol.

3. The method of claim 1, wherein the extraction solvent is a 30% v/v water-ethanol solution.

4. The method of claim 1, wherein the extraction solvent is acetone.

5. The process of claim 1, wherein the extraction solvent is 80% v/v aqueous acetone.

6. The method of claim 1, wherein the extraction is performed at a temperature of 10 ℃ to 80 ℃.

7. The method of claim 6, wherein the extraction is performed at 25 ℃.

8. The process of any one of claims 1 to 7 wherein the adsorbent resin is a styrene-divinylbenzene or acrylic resin.

9. Use of an extract obtained according to the method of any one of claims 1 to 8 for the preparation of a medicament for the treatment of rheumatoid arthritis.