NO791704L - PROCEDURES FOR THE PREPARATION OF THERAPEUTICALLY ACTIVE NATURAL TERPENES - Google Patents

Description

Process for the production of therapeutically active natural terpenes

The present invention relates to the production of natural terpenes with anti-psoriatic activity by extraction from root stems and leaves of various ferns.

Psoriasis and parapsoriasis are pathological symptoms that manifest on any part of the body's surface and are capable of attacking it completely, i.e. from head to toe. This indicates that the severity of these diseases is therefore dependent on the extent as well as the depth to which the psoriatic process affects the skin layers. It manifests itself on the skin, but no dermatologist or general practitioner can overlook the so-called complications of psoriasis which, above all, manifest mainly at the level of the joints, tissues, such as the hepatic and renal tissues, and lead to the psoriatic patient showing signs of diabetes, gout and even renal insufficiency, when this disease is in an advanced stage, and there is a marked weakness in the patient.

It is important to point out that a genetic transmission of a recessive character takes place, a very important detail that explains its daily increasing occurrence in the human race. In this regard, there are references from the World Health Organization which assume that the number of people suffering from psoriasis with a clear activity of the disease in its various clinical forms can be estimated at 1% of the population.

Despite previous knowledge of this subject, the therapeutic attempts to cure or improve this pathological disease have been practically zero: Certain drugs of the cytostatic group have. have shown some relative effectiveness in treatment, but they are highly toxic, and patients cannot be treated more than twice with them because of their toxicity.

In short, at the moment there are only palliatives which, together with the fact that it is taken for granted that psoriasis cannot be cured, keep these patients with a very limited life in their daily activities.

It has now been shown that natural terpenes isolated from root stems and leaves of Dryopteris crassirhizoma, Polypodium vulgare, Linn, Polypodium leucotomos, Phlebodium decumanun J. Smith, Polypodium decumanum, Cyathea taiwaniana and root stems of Polypodium aureum Linn and Polypodium triseriale, have very useful properties in the treatment of psoriatic and parapsoriatic patients of both sexes and of all ages. These terpenes include the fern and its isomers, as well as other terpenes. A group of 152 patients suffering from psoriasis were treated orally with a daily dose of 200 mg, and this caused the disappearance of more than 80% of the cases within a period of 1 to 3 months. In a series of double-blind trials, Fernen proved to be effective in comparison with the placebo of the same size and shape. The fern is particularly useful in any type of psoriatic or parapsoriatic lesions, including the pustular palmar-plantar form of this disease.

The fern does not alter fertility in rats or mice, and it has no teratogenic effect on rats and mice, according to studies conducted and the "Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human use" Food and Drug Administration of the United States ( I966).

The fern shows no carcinogenic activity in studies carried out on rats, after being administered daily at 1 mg/kg body weight, orally, for 20 weeks and according to the observations at the end of 7 weeks with and without the introduction of glass particles into the urinary bladder. The methods used in these investigations are described by Jull, J. W. in Brit. J. Cancer; 5, 328 (1951) and the statistical evaluations used are described in Arcos et al., ChemicalInduction of Cancer., page 416, Academic Press Inc., New York (1963).

The mechanism of action of the fern is apparently linked to the membrane defect which causes the altered regulation of cell growth in the epidermis (Orfanos, C. E. and Mahrle G. Dermatologic 151, 199-215 (1976)).

The fern has no anti-inflammatory or anabolic steroidal activity. It is essentially non-toxic up to 1 g/kg body weight.

Pure fern was isolated earlier by Ageta, H. and co-workers (Tetrahedron Letters No. 22, 1442-50 (1963)) in very small. quantities and on a laboratory scale.

The method according to the invention allows the extraction, with industrial yields, of the active terpenes from the previously mentioned parts of the plant, in useful quantities to be used in the manufacture of pharmaceutical preparations. To achieve an optimal yield, the leaves should be collected after the spore housing has already developed.

The present method includes the following steps:

Drying

The root stalks are cut into strips of 2 - 3 cm, the leaves are dried as they are collected. Drying takes place continuously, as the material is fed into a rotating metallic wire mesh cloth with a width of approx. 5 m and 25 m in length, which are moved in a warm room at a temperature not exceeding 80°C. By adjusting the speed of the rotating metallic wire cloth, approx. half a ton of moist material is dried per hour.

Granulation

The dry material (remaining moisture below 5%) is granulated in a disc mill set to give particles of up to 2 mm in diameter (ASTM sieve 10) with a density of approx. 0.36.

Extraction- evaporation

Extraction can be carried out with any solvent (dielectric constant of 1.890 to 9.08), preferably methanol in a ratio of 1:4 by volume.

Cleaning

1. The semi-solid residue of the extract is taken up in n-hexane/water (10:4) and after being left overnight in a separator, the salts and sugars are eliminated with the water phase. 2. To the n-hexane solution is added 1/10 of its volume of 10-20% KOH in ethanol, and after occasional stirring it is obtained

stand for 6 hours. The n-hexane phase is neutralized by washing with water.

3. After evaporation, the residue is taken up in three times its volume of 96% ethanol and is allowed to stand until complete precipitation, usually 48 hours at room temperature. 4. The precipitate is filtered off on glass filters with an average porosity, under suction, it is washed with suitable amounts of 96% ethanol until it is almost colorless and dried under vacuum over KOH or concentrated sulfuric acid. 5. The precipitate is dissolved in n-hexane to obtain an ID weight% solution at a room temperature of approx. 20<o>C» 6. The solution is chromatographed in an SI-60 Geduran column (0.063 - 0.2 mm, Merck) of 5 x 100 or 10 x 200 cm, depending on the amount of material (500 g to 1 kg), eluting with n-hexane. The fernenet is eluted in the first 1-liter fraction (with a view to

2 litres).

7. After evaporation to a concentration of approx. 10%, the n-hexane solution is allowed to crystallize spontaneously at room temperature. The crystals are filtered off under suction, and the mother liquors are evaporated again to obtain another crystalline mass. 8. Another method consists in using neutral Alo0o columns (activity) (5 x 100 cm) and eluting with n-hexane. In this case, the eluate extracts only the fern. 9. Recrystallization takes place in hot, absolute ethanol.

The pharmaceutical preparations produced according to the invention are produced in conventional dosage forms by incorporating the fernene in pharmaceutical carriers, in accordance with accepted pharmaceutical practice. The active ingredient, i.e. the fern, will be present in the preparations in sufficient quantities to produce an anti-psoriatic activity.

The preparations of the compounds prepared according to the invention preferably contain from approx. 1 to 100 mg of active ingredient per unit dose. The pharmaceutical carrier can e.g. be solid or liquid. Examples of solid carriers are lactose, magnesium stearate, alba terra, sucrose, talc, stearic acid, gelatin, agar, pectin, gum arabic, aerosil and the like. Examples of liquid carriers are alcohols (such as ethanol or propylene glycol), water containing a solubilizing agent such as polyethylene glycol, peanut oil, olive oil and the like. The carrier or diluent may contain a retarding agent, such as glycerol mono-stearate or glyceryl distearate, alone or with a wax.

A wide range of pharmaceutical forms can be used. Thus, if a solid carrier is used, the preparation can be transferred to

tablets, are introduced into a hard gelatin capsule in the form of a powder or grain, or it may take the form of a lozenge. The amount of solid carrier can vary within wide limits, but is preferably from approx. 25 mg to 250 mg. If a liquid carrier is used, the preparation may take the form of a syrup, emulsion, soft gelatin capsule, suspension or liquid solution, or a sterile injectable form for parenteral use, e.g. in an ampoule.

The pharmaceutical preparations according to the invention in suspensions or liquid solutions do not include the simple suspension or liquid solutions of the active ingredient in the usual solvents which are not suitable for internal administration to produce the desired pharmacological activity.

A unit dose in the form of tablet, capsule, lozenge, liquid suspension or sterile injectable liquid for internal administration to produce an anti-psoriatic activity, consisting of a pharmaceutical carrier and the fern in an amount sufficient to produce it. said activity also constitutes a goal of the present invention.

Administration can take place orally or parenterally, preferably orally. The active ingredient will preferably be administered in a daily dose that amounts to approx. 100 mg to 6oo mg, and even better from approx. 50 to 300 mg. It is advantageous to administer doses corresponding to 1 to 4 times daily. When administered as described above, an anti-psoriatic activity is achieved.

Since psoriasis is a chronic, insidious process with a genetic etiopathogenesis, it is advantageous to use the capsule or tablet form to maintain stable blood concentrations and which allows a continuous effect of the drug. Undoubtedly, when severe outbreaks of psoriasis induced by other drugs are treated, as in the case of steroids or by the disease itself, intravenous injection or continuous dropwise feeding of serum may

used to prevent the crisis.

It should also be pointed out that the form of drops or emulsion has been used for children for obvious reasons, and in the case of adults with problems in the upper digestive tract, suppositories can be used.

The pharmaceutical preparations are prepared by conventional methods such as mixing, granulation and pressing, when necessary, or by mixing and dissolving the appropriate ingredients for the desired preparations.

The invention is illustrated by the following examples.

Example 1

12 kg of dried leaves, granulated in a No. 10 sieve (2.5 mm) with 10 liters of 96% ethanol at 70°C were extracted for 1.5 hours. The extract was filtered and washed with 10 liters of 96% ethanol in a filter. The filtered extract was evaporated to dryness under a moderate vacuum (20-30 mm Hg).

The residue was taken up in a mixture of n-hexane/water

(15:10 liters) and set aside overnight in a separator. The water phase was removed, and the hexane phase was clarified with a 10µg solution of KOH in 90% ethanol (5 litres). The hexane phase thus clarified was neutralized by washing with water and evaporated to dryness.

The residue was dissolved in 500 ml of 96% ethanol while heating, and the solution was left for 48 hours. It was filtered and dried (yield approx. 50 g).

The dry precipitate was dissolved in 1 liter of n-hexane and chromatographed in an SI-60Geduran column (100 x 10 cm) eluting with portions of 1 liter of n-hexane.

The first 1-liter fraction was evaporated, and the residue was crystallized in hot hexane, benzene, or absolute ethanol (yield 12 g).

Example 2

36 kg of dry, whole plant, granulated in a disc mill to pass a No. 10 sieve (2.5 mm) was continuously extracted with n-hexane at room temperature, the hexane solution was evaporated to approx. 10 liters and was clarified in a separator with 1 liter of 10% KOH in 90% ethanol overnight. The hexane phase was neutralized by

washing with water and was evaporated to dryness.

The residue was taken up in hot benzene in a sufficient amount to give a saturated solution (about 1 liter). The precipitate was filtered off when cooled and crystallized in hot benzene or hot absolute ethanol.

The crystalline material was dissolved in 500 ml of n-hexane and chromatographed on a 50 x 50 cm SI-60 Geduran column (Merck-Darmstadt) eluting in portions of 0.5 L. The first half-liter fractions of eluate were evaporated and crystallized in hot hexane, hot benzene or hot absolute ethanol (yield 38 g).

Example 3

12 kg of dry rhizome, granulated in a disc mill to pass a No. 10 sieve (2.5 mm) was continuously extracted with dichloromethane at room temperature. The dichloromethane was evaporated to dryness, and the residue was taken up in 5 L of n-hexane.

The hexane solution was clarified with 1 liter of 10% KOH in 90% ethanol overnight, and the hexane phase was then neutralized by washing with water and evaporated.

The residue was taken up in 1 liter of n-hexane and chromatographed in a silica gel-60 column (100 x 10 cm) with n-hexane. The first two eluate fractions were evaporated, and the residue was crystallized in hot n-hexane, hot benzene or hot ethanol (yield 25 g).

Example 4

By refluxing for 1 hour, 12 kg of granulated and dried whole plant (grain size .2.5 mm or No. 10 sieve) was extracted with 100 1 methanol at 70°C. The extract was filtered and washed with 20 1 methanol in a filter.

The methanol extract was evaporated, and the residue was taken up

in a mixture of n-hexane/water (3:2), (ie 30 to 20 liters) and was left overnight in a separator. The water phase was removed, and the hexane phase was clarified with 10 liters of 20% KOH in 90% ethanol, it was neutralized by washing with water and evaporated to dryness.

The residue was dissolved in 1 liter of hot 96% ethanol and left at room temperature for 48 hours.

The precipitate was filtered off and dried under vacuum over KOH.

The dry precipitate was dissolved in 1 liter of n-hexane and chromatographed on neutral Al 2 O 2 (50 x 5 cm) eluting with n-hexane.

The first five liters were evaporated, and the material was crystallized in n-hexane, cyclohexane, hot benzene and hot absolute ethanol (yield 12 - 13 g).

The following examples of pharmaceutical preparations are not limiting, but merely illustrative of the invention.

Example 1

The ingredients were mixed, sieved and introduced into hard gelatin capsules. A capsule prepared as described above is administered three times per day.

Example 2

The fern and the sucrose were mixed and granulated with a solution of 10% gelatin. The granules were sieved, dried and then mixed with starch, talc and stearic acid. They were charged and transferred to tablets.

Example 3

The ingredients were mixed and introduced into soft gelatin capsules.

Claims (2)

1. Process for obtaining natural terpenes with an anti-psoriatic activity, by extracting root stems and leaves of Dryopteris crassirhizoma, Polypodium vulgara Linn, Polypodium leucotomos, Phlebodium decumanun J. Smith, Polypodium decumanum, Cyathea taiwaniana and root stems of Polypodium aureum Linn and Polypodium triseriale, characterized by: a) the rhizomes are cut into strips of 2 - 3 cm and the root-stem strips and leaves are subjected to a continuous drying treatment over a rotating metallic wire mesh cloth at a temperature not exceeding 70°C, b) the dried material with a residual moisture below 5% is granulated in a disc mill to form particles up to 2 mm in diameter and with an approximate density of 0.36, c) where extracted with a solvent with a dielectric constant from 1.890 to 9.08, d) the solvent is evaporated, and e) the residue is cleaned. 2. Method according to claim 1, where the cleaning of the residue is characterized by the fact that it comprises the following steps: i) the semi-solid residue from the extract is collected in hexane/water (10:4)', left overnight in a separator and the water phase separated ii) to the n-hexane solution is added 1/10 of its volume of 10-20% KOH in ethanol, where it is stirred occasionally, left for 6 hours and neutralized by washing with water; iii) the solvent is evaporated, and the residue is collected in three times its volume of 96% ethanol, and set aside until the sweeping is complete, iv) the precipitate is filtered off through glass filters of average porosity, under suction, washed with sufficient amounts of 96% ethanol until it is almost colorless, and dried under vacuum over KOH or concentrated sulfuric acid; v) the precipitate is dissolved in n-hexane to obtain a 10% by weight solution at a room temperature of approx. 20°C, vi) the solution is chromatographed in an SI-60 Geduran column (0.063 0.2 mm, Merck) or a neutral A^O^ column (activity), eluting with n-hexane, vii) where it is evaporated up to a concentration of approx. 10%, the n-hexane solution is allowed to crystallize at room temperature, the crystals are filtered off by suction, and the mother liquors are evaporated again to obtain another crystalline mass, viii) where recrystallized in hot, absolute ethanol.