WO1997023233A1

WO1997023233A1 - Biologically active compositions and their use

Info

Publication number: WO1997023233A1
Application number: PCT/NL1996/000495
Authority: WO
Inventors: Eleonora Fedorovna Stepanova; Alexander Sergeevich Saraf
Original assignee: Parbold Investments N.V.; Van Der Beek, Janco, Jan, Wigbold
Priority date: 1995-12-21
Filing date: 1996-12-20
Publication date: 1997-07-03
Also published as: NL1001972C2; AU1153997A

Abstract

The present invention relates to various biological compositions that can be extracted from the grass Glycerrhiza glabra L. Different extraction methods lead to different types of compositions having each a specific spectrum of activities. The activities of the various compositions comprise anti-fungal, anti-bacterial, anti-viral, anti-inflammatory, anti-tumor, anti-protozoan and anti-allergic activities.

Description

BIOLOGICALLY ACTIVE COMPOSITIONS AND THEIR USE

The present invention relates to various biologically active compounds and compositions, having therapeutical and/or prophylactic properties.

Surprisingly, it has been found according to the invention that plant material originating from Glycerrhiza glabra L. comprises a wide variety of compounds that proved to have a broad range of biological activities. Glycerrhiza glabra L. is a grass that is particularly abundant on natural and cultivated land in Russia, some CIS countries and a few other states. The active compounds and compositions are found both in the leaves of the grass, and m the roots.

The compositions of the invention may be isolated in various ways. The different compositions obtained by performing the different methods also have differing properties and activities. All compositions and compounds have their origin, namely the plant Glycerrhiza glabra L. , in common.

In a first embodiment the grass without the roots is extracted with methylene chloride under vacuum, after which the extraction agent is evaporated. The remaining material is a resinous dark-green substance further identified in this application as "Glycefungin" .

In a second embodiment a composition named "Esefsol" is prepared by drying the raw material remaining after isolation of Glycefungm to completely remove the methylene chloride and subsequently extracting this dried raw material by means of extraction with 70% acetone. After evaporation and drying Esefsol is obtained as a light, sandy material.

Subsequently the raw material remaining after complete removal of acetone s extracted with hot water at an elevated temperature, preferably between 80 and 90°C. The extraction is repeated twice, after which the extraction agent is evaporated off and the material thus obtained is dried further. The resulting material is an aromatic, thick, viscous, dark-brown mass, named "Vodexol". This constitutes the third embodiment of the invention.

A polysaccharide complex, named "Polysol", is obtained by first separating lipophilic and phenolic components from a new batch of chopped grass by means of methylene chloride. After complete removal of the methylene chloride by drying, the grass is further extracted with an alcohol, such as 82% ethylic spirit and dried. Then, the extract is concentrated under vacuum. Precipitation is conducted from the concentrated extract by means of pure spirit. The obtained precipitate is separated and purified by reprecipitation and washing with acetone. After drying and pulverization Polysol is obtained.

By extracting the grass with pure water-free acetone a composition is obtained, further referred to as "Glycacet" .

Chloroform extraction of the grass leads to a composition named "Glycochlor" .

Further characterization of the various compositions and details on their preparation methods are given in the Examples.

Extensive research led to the conclusion that the compounds and compositions that may be isolated from the leaves have fungicidal, anti-bacterial, anti-viral, anti- inflammatory, anti-tumor, anti-protozoan, anti-allergic, immune-stimulating and other effects.

The applications for the compositions and compounds are numerous .

Among these is the treatment of fungal infections and diseases caused by for example Trichophvton rubrum, T. interdiqitale. T. gypseum, Microsporum cams, Candida albicans. Rhizopus orvsae, Fusarium heterosporum. Penicillium. Aspergillus etc.. The activity of some of the compositions of the invention, in particular of Glycefungin exceeds the activity of the known anti-fungal agents Nystatin, Levoπn and Clotri azol .

Some of the compositions, Glycefungin, Glycacet and Glycochlor in particular, have anti-bacterial properties. Glycefungin is found to be active against Staphylococcus aureus and S. epidermis, while Glycacet is also active against S . aureus and Bacillus polymyxa.

Anti-viral and anti-protozoan effects are in particular displayed by Glycefungin. This composition may for example be used in disorders of the cervical vagina, caused by the virus Herpes simplex, and the protozoans Chlamidium, Gardnerella. Some of the compositions inhibit HIV-1, in particular its absorption on lymphoblastoid cells. Anti-tumor effects have been demonstrated on the experimental models lymphocytic leukaemia P-388, Erlich's tumor, sarcoma 37, adenocarcinoma of the lactic gland Ca- 755, melanoma B-16, sarcoma 180, especially for Glycefungin. Among the other effects are anti-inflammatory activity, immune-stimulating activity and anti-allergic effects found in various compositions.

Furthermore, Glycefungin may be used as a prophylactic and curative agent in dermatology to treat candidosis, rubrophytosis, microsporia, other fungal and bacterial diseases, atopic and contact dermatitis, skin cancer, inflammatory and allergic disorders of the skin and mucous membranes, in gynaecology for treatment of intravaginal viral and bacterial infections, in oncology for treating various types of tumours, as a desinfective agent epidemiology, for the treatment of paradontosis, gingivitis, stomatitis and other intraoral diseases in stomatology, in otorhinolaryngology for treating infectious rhinitis, tonsillitis, laryngitis etc.

In the food industry Glycefungin may be used as a bioactive supplement (having for example immunostimulating effects, or as an anti-tumour agent, antioxidant etc.), natural preservative etc..

Glycefungin and Esefsol are also very useful as an additive in cosmetic compositions, such as lotions, soaps, creams, sprays etc.. The effect of Glycefungin is antifungal and antibacterial and it is therefore very useful in the treatment of acne and some other skin disorders that may be caused and/or complicated by fungi, staphylococci etc.. Vodexol may be used in anti-acne, anti-wrinkle, and nutritive creams and lotions and is very useful in anti- dandruff shampoo. A combination composition comprising Glycefungin, Vodexol and Esefsol is very effective in treating acne vulgaris, acne juveniles, acne rosaceae, atopic and contact dermatitis and skin candidosis. All the cosmetic compositions may also be used for therapeutic purposes in dermatology and as a prophylactic composition in cosmetology. The compositions of the invention may also be used in veterinary practice, for example for the treatment of infectious and inflammatory skin diseases or for treatment of fungal, protozoan, viral and bacterial diseases.

The compositions of the invention may be used to impregnate various textiles and fibers for manufacturing clothing, foot inner soles for shoes and sportswear, sanitary napkins and tampons with anti-fungal, anti¬ bacterial, anti-viral effects for use as protection, for treating perspiring feet, in surgery and gynaecology etc.. The invention also relates to the methods for preparing the various compositions and to the different applications.

Suitable extraction agents are selected from the group consisting of methylene chloride, water, a mixture of 70% acetone and 30% water, acetone, chloroform, or combinations thereof. Some other products were derived from Glycerrhiza glabra L. grass by the extraction with glycerine, polyethylenoxide-400, olive oil, diethylic ether, petroleic ether. The present invention will be further elucidated in the following Examples, which are given for illustration purposes only and are in no way intended to limit the scope of the invention. EXAMPLES EXAMPLE 1 Glycefungin

1. Preparation The grass of Glycerrhiza glabra L. was chopped by a combined procedure using a rolling crusher for 2 passages and a mill DKJ with a screen of 0.2 mm. The material thus obtained is extracted by methylene chloride under the vacuum by means of the filtration principle based on a continuous flow of fresh extraction agent during 2 hours.

The ratio between raw material and extraction agent is 1:7. Subsequently, the extract is evaporated under vacuum in a Simax apparatus to a residual moisture content of 22-23%. After that it is dried in a vacuum-drying box under a temperature of 50-55°C. The material obtained is a resinous dark-green substance having a moisture content of 14-16%.

2. Chemical composition The chemical composition of the obtained material was determined as follows.

Beta-sitosterin, terpenoids, chlorophyll, resins, fatty acids are discovered by a qualitative analysis of the composition by means of paper chromatography methods, thin layer chromatography (TLC) on silufol plates using reference molecules and qualitative chemical reactions.

The paper chromatography analysis on steroids and terpenoids was conducted using chloroform as a mobile phase and methanol-formamide (1:1) as stationary phase. The saturated solution of antimony(III) chloride in chloroform was taken as the developer. For TLC a chloroform-methanol- water (65:35:10) system was used. Chromatograms were developed with a 1% solution of Vanillin in concentrated sulphuric acid. The quantity of resins in Glycefungin was carried out by a gravimetric assay based on dissolving the material in boiling alcohol.

The content of unsaturated fatty acids was determined by heating a precisely weighed amount of pulverized extract with a thirty fold quantity of chloroformum for complete dissolution. Subsequently the homogenic solution was washed with water in a dividing funnel. Then a 10% sodium hydroxide solution was added to the washed chloroformum solution, still in the dividing funnel. The different phases were contacted during 5 min. Then, the alkaline solution containing the fatty acids were poured off. The separation was repeated twice. The combined alkaline solutions were washed twice with chloroformum and the pH adjusted to 2 using a dilute solution of hydrochloric acid. The acidic solution thus obtained was extracted three times with chloroformum. The chloroformum fractions were combined, 50 ml of purified water, 50 ml of 0.1 mol/1 iodo onochloride solution were added and left during 1 hour with periodically mixing and shaking. Then 10 ml of 10% solution of patassium iodide was added. Discharged molecular iodine was titred with 0.1 mol/1 solution of sodium thiosulfate with starch as an indicator. The control experiment was carried out simultaneously. The content of unsaturated fatty acids was determined by calculation on the oleinic acid content .

The quantity of chlorophyll in the composition was determined by spectrophotometry (285 nm) and photocolorimetric assays. As a standard a mixture of chlorophyll (85 mg/1) was used.

The shelf-life of the composition is at least 3 years.

The following components were detected: 1. Chlorophylls A and B 6-8%

2. Phytosterols : beta-sitosterin and sigmasterin 2.0-

2.5%

3. Phospholipids (lecithin, choline) 1.0%

4. Unsaturated fatty acids (oleinic, linolenic acids) 7.1% 5. Coumarines 1.8%

6. Resins are represented by resin acids with the diterpenoid structure and hydrocarbons of diterpenoid series. The general quantity of the resins is about 4%

7. Carotinoids (beta-carotin) 10-

12%

3. The biological properties 3.1. General effects

The acute toxicity of Glycefungin was determined by the method of Wilcokson, Litchfield at the Toxicology Department of the Russia National Research Center of Biological Active Compounds (St.Kupavna) . The LD₅₀ of the composition was established on white mice of unknown strain by means of intraperitoneal and oral administration and was found to be more than 5000 mg/kg.

The study of the irritation-inducing and allergenic effects was carried out at the laboratories of the State Pyatigorsk Pharmaceutical Academy. It was revealed that 3% ointment with glycefungin does not irritate and sensitize the skin during local application in guinea pigs.

3.2. Antifungal effect

The experiments to establish antifungal effects were performed at the Mycology Department of the Central Skin Venerology Institute (Moscow) . It was found that Glycefungin possessed potent fungicidal and fungistatic effects which were revealed by disk's method on the test- cultures Rhizopus orysae, Fusarium heterosporum, Candida albicans . For this, 1 ml of a suspension with spores of the testfungi in a concentration of 1 mln/ml was sown on Petri dishes with Chapek ' s medium. Paper disks (10 mm diameter) were soaked with 5% and 10% alkali solutions of Glycefungin, dried during 3 minutes, and put on the surface of the media with fungus cultures. The dishes were incubated for 7 days at 28°C in a thermal box. The effect of the drug was estimated by measuring the size of the growth delay zone. As references Levorm and Nystatin were used. Glycefungin surpassed these compositions by 5-12 times in activity.

Ointments containing Glycefungin were prepared by mixing the following components: polyethylenoxide-400 45 parts polyethylenoxide-1500 33 parts dimethylsulfoxide 17 parts aerosil 2 parts Glycefungin 3 parts .

The ointments with Glycefungin were tested in the concentrations 1.5; 3.0; 5.0; and 10.0%. The antifungal action was studied in two stages (as a reference composition Canesten (Clotrimazol) was used) : 1. By the agar-diffusion method in vitro on the dermatophyte cultures Trichophyton rubrum, T. interdigitale. T. gypseum, Microsporu canis, Candida albicans, the mouldy fungi Penicillium and Aspergillus (8 test cultures of each) . 2. On an experimental Trychophvton model in vivo in guinea-pigs. It was established that the application of an ointment with 3% Glycefungin during 14 days led to the complete suppression of Trychophvton fungi development. This result is comparable with Canesten (Clotrimazol) .

The ointment is thus recommended not only for prophylaxis but also for therapy.

The Glycefungin-based ointment was also test on a limited scale in humans on 20 patients with dermatophytosis and demonstrated an antifungal, anti-itching and keratolytic effect . The most effective use of Glycefungin is the treatment of glabrous skin candidosis, rubrophytosis etc.. The composition is well absorbed by the skin and has no side effects (no local irritation and sensitization) .

The composition was also studied for possible applications in cosmetics. As a result recipes for creams, lotions and sprays were developed, which potently suppressed the growth of Trychophyton rubrum, T. gypseum (1.0-3.7 mm. inhibition growth zones) in vitro and cured foot mycosis.

3.3. Antibacterial effect

The anti-bacterial effect was tested at the Institute of Medical and Biological Problems (Moscow) by the Disk's method on test-cultures of Staphylococcus aureus. S . epidermidis, Pseudomonas aeruginosa. Bacillus licheniformis.

A 5-10% water-alkali solution of Glycefungin produced a broad spectrum bacteriostatic effect with respect to Staphylococcus (3.0; 5.5; 2.0; 3.0 mm inhibition growth zones) .

3.4. Anti-inflammatory effect

It was demonstrated at the Pharmacology Dpt . of Perm State Pharmaceutical Institute that an ointment containing 3% Glycefungin inhibited acute inflammatory reaction (rat paw oedema) on 32.6% of the rats tested.

3.5. Anti-tumor effect

The anti-tumor effects was studied at the Research Institute of Tumor Diagnostics and Treatment of the Russian National Oncological Center (Moscow) .

The composition was investigated in a wide range of doses both for intraperitoneal and oral administration on 6 different tumours implanted in mice-hybrid first generation BDFl (C57B1/6 x DBA/2) and SHK) . The tumors evaluated comprise lymphocytic leukaemia P-388, Erlich's tumor, Sarcoma 37, adenocarcinoma of the lactic gland Ca- 755, melanoma B-16, and sarcoma 180. It was found that Glycefungin suppresses 5 of the 6 tumors tested. It inhibited the growth of sarcoma 37 in 59-63% of the animals in a range of doses 6-100 mg/kg when treated both intraperitoneally and orally during 5-10 days, once a day. 29-43% of the animals were cured.

The composition did not inhibit sarcoma-180 growth, but cured the animals in 62.5% cases. It suppressed the growth of carcinoma 755 and melanoma B-16 in 56% and 52% of the animals, respectively, prolonged the animal's life with Ca-755, melanoma B-16, Erlich's tumour in 36%, 34% and 40% of the cases, in comparison with the control animals who were not treated with the composition.

The data obtained indicate not only the direct action of the composition on tumor cells, but its potential stimulating effect on the immune system. 3.6. Anti-HIV effect

This effect was demonstrated at the Laboratory of Associated Infections of the Epidemiology and Microbiology Research Institute (Moscow) . The inhibition of HIV-1 adsorption on lymphoblastoid cells MT-4 was studied by the indirect immuno-fluorescent method after 7 days incubation of the cells with Glycefungin. Azidothymidin was used as a control .

The HIV-l virus (HTHIV27 species) was cultured in this laboratory and is the subject of the Russian Patent granted on application N 94002006/13, filed January 31, 1994. It was discovered that the composition possesses very potent anti-HIV activity comparable with Azidothymidin. 100% inhibition of HIV-l was effected by a concentration of 1 μg/ml (1 gamma) . The composition does not possess cytotoxic effect .

4. Possible applications for use in humans 4.1. Anti-bacterial and antifungal fibers It is possible to impregnate fabrics with a water- alkali solution of the composition to obtain a cloth having an anti-bacterial and antifungal effect.

Fibers suitable for clothing (coarse calico, felt, viscose, etc.) were impregnated with Glycefungin in different concentrations. The tests were carried out at the Textile Academy (Moscow) . The impregnated fibers can be manufactured at textile or man-made fibre factories based on a continuous production cycle.

Fibers impregnated with Glycefungin were studied in terms of their effect on pathogenic flora m humans and the capacity to retain the effect after different types of processing. Treatments tested were 10-20 washing in alkali and neutral medium, gamma radiation at 25 kGr, 10 times autoclaving during 30 mm under 1.5 atm., storage during 3 months. Neither of these treatments changed their properties.

The activity of the fibers were tested on 12 test- cultures including S. marcescens. E. coli, P. aerugmosa. B. subtilis. S. aureus etc. The most effective were the fibers impregnated with 0.02% and 0.04% water-alkali solutions of Glycefungin relating to B. subtilis and S.aureus.

Antifungal foot inner soles impregnated with the 2.5; 5.0; and 10% water alkali solutions of Glycefungin were made on the base of different materials to prevent bad footwear and feet odour. They successfully passed laboratory clinical tests on dermatomycosis patients at the Central Research Skin Venerology Institute (Moscow) . In vitro and in vivo tests demonstrated that inner soles impregnated with the composition solution have antifungal effect against Trychophvton rubrum, the main cause of foot mycosis, Trvcnophvton gypseum and Microsporu cams. There inner soles remove bad odour, soothe itching skin, do not cause irritation, and can be used successfully to prevent mycosis of feet.

Inner soles impregnated with Glycefungin were tested at the Institute of Physical Culture (Moscow) which recommended that the internal surface of sports footwear be impregnated with Glycefungin solution during the production process (Opinion of the Russian Olympic Committee from 12 November 1992) .

4.2. Clinical Trials in Gynaecology The tests were performed at the 15th Women's

Consultation Center (Moscow) .

27 women suffering from vagina infections were investigated and treated with a 10% oil solution of Glycefungin (10 treatments) . The composition was applied locally in a form of irrigations and tampons impregnated with the composition.

The bacterial and viral content of the cervical channel of vagina and uretrum were analyzed by the direct immune-fluorescent method before and after treatment. It was revealed that the composition does not irritate the mucous tissue of the vagina, prevents the development of Candidosis, effectively suppresses the clinical symptoms of infection inflammations (excretions, itch, pain) , and normalizes the pacteriogram The prevalent effect of the composition was shown in the treatment of patients with cervical vagina disorders caused by Herpes simplex virus, and the protozoans Chlam dium, Gardnerella which were completely decreased by the oil solution of Glycefungin during 10 treatments. The combination of the composition with some drugs (Polygenax, Macmiror, Dardum, Fortum, Ciprobay) brought the entire curing of mixed vaginal infections .

5. Veterinary applications

5.1. Trials in veterinary clinics

An ointment with 10% Glycefungin was studied at the Pyatigorsk Veterinary Station in 30 cats and 25 dogs suffering from microspoπa. It was established that the composition applied during 7 days (3 times a day) completely cured the symptoms of microspona. Affected skin zones were restored, the hair grew actively. The composition is recommended in veterinary practice as antifungal curing agent.

5.2. The effect on Leptospira, Salmonella and air microflora

The trials to test this effect were conducted at the State Russian Scientific Research Institute of Control, Standardisation and Certification of Veterinary Drugs.

The tests were performed on virulent production series of Leptospira of 6 serological groups: Pomona, Tarassovi, Grippotyphosa, Icterohaemorragi, Ca cola, Sejroe. These groups are used for the production and control of vaccines against animal leptospirosis.

The group of Salmonella enteritidis and air microflora Staphylococcus album, S. aureus, Sarcma was also used.

10% Glycefungin as a water-alkali solution was used for study on Leptospira. The control was a 0.4% water- alkali solution. Glycefungin' s solution was added in consistently decreasing concentrations (from 10 ' to 10 ^l ) to the special water-serum nutritive medium for leptospira cultivation. To each line of consecutive dilutions the different series of Leptospira were added in a concentration of 10%. The sowings were cultivated during 10 days at 28°C. On days 3, 5, 7 and 10 the growth was determined in a Petrov-Chausser' s calculating camera with the help of a microscop .

Glycefungin was dissolved in DMSO and in 0.4% sodium hydroxide solution which were used for the study on Salmonella and air microflora. The control solution was the same solvent without composition.

It was shown that Glycefungin in a range of concentrations from 10^"1 to 10^"10 in 0.4% water-alkali solution and from 2*10^"3 to 2*10^"12 in DMSO solution does not display the bactericidal and bacteriostatic effect on Leptospira and Salmonella. The composition in a concentration of 10^"1 dissolved in 0.4% water-alkali solution suppresses the growth of Staphylococcus and Sarcma. These results showed the real possibilities of 10% Glycefungin in a water-alkali usage form as a biosupplement to the special nutritive media for Leptospira cultivation during vaccine manufacturing at the biofactories . The selective bacteriostatic effect of 10% Glycefungin solution on air microflora and the lack of its influence in Leptospira growth in vitro are a very beneficial property of Glycefungin for preventing the appearance of adverse microflora growth in retorts with pure vaccine cultures.

The additional tests carried out at the Epidemiology and Microbiology Research Institute (Moscow) with Glycefungin showed that at different doses (1-100 mg/kg, p.o.) it protects the animals (mice, rats) from a death caused by some infectious agents (Salmonella typhi . etc. ) .

The immune-stimulating effect of Glycefungin was revealed by agar-free local haemolysis in erythrocyte monolayers. The composition at a dose of 10 mg/kg significantly increases haemagglutinin titres in mice blood serum and the number of antibody-producing cells in the rat spleen. 6. Therapeutical compositions

The composition can be used in dermatology, gynaecology, oncology, epidemiology as a potent nontoxic plant agent with a wide range of biological activities, for prophylaxis and treatment of bacterial, protozoan, fungal and viral infections, different kinds of tumours, local inflammations, immune disorders.

The therapeutical composition could be applied in veterinary practice for local usage (ointments, oil solutions, creams, sprays, etc.) and for use in humans, for example cosmetology (as a bioactive concentrate added to lotions, soaps, sprays, creams, etc.) , for clothes, napkins, tampons and footwear production impregnated with the composition, and in the food industry as a natural preservative.

EXAMPLE 2 Vodexol 1. Preparation

The chopped raw grass (at least 1.0 mm) was first processed by methylene chloride and then with 70% acetone. Subsequently the thus obtaien material was extracted during 1 hour with hot water at a temperature of 80-90°C. The ratio between extraction agent and raw material is 1:8. The extract obtained was poured off and a second extraction was performed under analogous conditions, except for the ratio which was 1:5. Then the extraction was repeated a third time (ratio is 1:5) . The obtained second and third extracts were added to the first one and evaporated in a vacuum apparatus to a final moisture content of 30-32%.

The quantitative yield of the product is 35.5%. The product is a very aromatic thick mass that may be pulled in dark-brown threads.

2. Chemical composition

The qualitative analysis of the product was conducted by the use of chromatography. The presence of 4 glycosidic terpenoids was established. TLC was carried out on inactivated aluminum oxide, the mobile phase is chloroform and benzolacetic acid (90:10, 98:2, 97:3) . As the developer an iodine spray was used. The quantitative determination of saponins was carried out by a gravimetric assay with precipitation of diethylic ether from alcohol solution. The saponin content is 29.8%.

Furthermore, the product was analyzed on flavonoids content. The investigation was carried out by means of a two-phase chromatography method (Zaprometov M.N. Phenolic compounds and its investigation method. In: "Biochemical methods of plant physiology". Moscow, 1971, pl86-207. The flavonoids, such as astragalin, kempferol, lsoquercet etc., were used as references. The mobile phase was either water/acetic acid (85:15) or n-butanol/acetic acid/water (4:1:2) .

The qualitative analysis of the ammo acids was performed by paper chromatography on "Filtrak" (Number 1) paper using 0.5 g Vodexol in 5 ml purified water. The mobile phase used was n-butanol/acetic acid/water (4:1:2) . As a reference standard samples of amino acids, such as arginine, asparaginic acid, alanme, valine, serine, glycine etc. were used. A 0.2% alcohol solution of ninhydπn was the developer. 7 amino acids were detected. By the ninhydrin method of quantitative determination 64.5 mg ammo nitrogen was detected in 100 g of the product.

The quantitative determination of oxycmnamic acids was carried out by ion exchange chromotagraphy assay with further titration. Ionic exchange was performed on cationite KU-2 which has an adsorbdtive value on sodium chloride of 1.8 mg/equiv., on sodium hydroxide of 8.0 mg/equiv. A precisely weighed amount of Vodexol was put m a measuring retort and added was water to a limited volume. The determined volume of solution was taken and passed through the column filled with cationite KU-2 (V = 2 ml/min) . Then the column was washed with a 6-fold volume of water. Eluate and passed water were combined, 1 ml bromothymol dark-blue was added and titred with 0.1 N solution of sodium hydroxide to a dark-blue colour.

The control test was performed simultaneously where the same quantity of water was titred.

The quantitative determination of monosaccharides was carried out as follows. A precisely weighed amount of Vodexol was worked with 80% ethylalcohol in a ratio of 1-50 on a boiling water bath a retort with reverse refrigeration during 1 hour for the separation of alcohol- soluble sugars. The obtained alcohol extract after cooling was separated from the precipitate. The non-carbohydrate compounds were removed from the filtrate by a 10% solution of Pb(CH₃COO), (lead acetate) The filtered alcohol extract was then passed through a layer of sodium sulfate, dried and the rest was dissolved in a 5-fold volume (relating to Vodexol mass) of methanol. Sugars were precipitated from the methanolic solution with an equal volume of acetone. The sediment was separated and analysed by chromatography assay. The identification of free monosaccharides was carried out by a chromatography assay on FN-11 paper in the following systems: n-butanol-pyπdme-water (6:4:3) , ethylaccetate - acetic acid - formic acid - water (18:3:1.4) , and also by thin layer chromatography on Silufol UF-254 plates The chromatography was conducted in the following systems ethyl - acetate - pyridine - water - n-butyl alcohol - acetic acid (5:4-4:10:2) . As developing reagents in both cases aniline phtalic reagent (for hydrogenated sugars) , and Bonner's reagent (for unhydrogenated sugars) were used. The following monosaccharides were identified: glucose, galactose, arabinose, xylose, galacturonic acid.

The product was found to have the following composition:

1. Triterpene saponins (glycerretic acid, 24- oxyglycerretic acid, uraletic acid)

(total quantity: 29.8%)

2. Flavonoids (isoquercitm, astragalin, kempferol and gliphosid) (total quantity: 6.5%) 3. Oxycinnamic acids (ferrulic, chlorogenic, caffeinic acids) (total quantity: 4.82%) 4. Pectinic substances

(total quantity: 40.5%)

5. Polysaccharides

(total quantity: 24.61%) monosaccharides (glucose, galactose, arabinose, xylose, galacturonic acid)

(total quantity: 7.6%)

6. Amino acids (valine, serine, glycine, alanine, asparagine, arginine, asparaginic acid) .

(total quantity: 1.7-2.0%)

3. Biological properties

3.1. General

Acute toxicity is very low. LD_5C was calculated with the help of Kerber's method using white mice. LD₅₀ for oral administration was 4666 mg/kg, and for intraperitoneal administration 4833 mg/kg.

3.2. Anti-inflammatory effect

The experiments were conducted at Pyatigoresk State Pharmaceutical Academy. It was revealed that the composition at a dose of 50 mg/kg, p.o., inhibited acute oedema of rat paw, which was induced by a 10% solution of kaolin, in 22% of the cases. Orthophen was used as a standard. The composition inhibited the inflammatory reaction at 10 mg/kg in 18.5% of the cases.

Vodexol was studied in local application in the form of ointment. 0.5% Prednisolon ointment was applied as a standard drug. The studied samples do not differ from the standard drugs with respect to activity. Vodexol showed 16% inhibition, whereas the Prednisolon ointment showed 4.4% during the first 2 hours. Afterwards the results were 21-26% and 20-29%, respectively) . 3.3. Anti-allergic effect

This effect was demonstrated on the rat passive cutaneous anaphylaxis (RPCA) mediated by IgE-antibodies (type I hypersensitivity reaction) . The composition at a dose of 50 mg/kg, p.o., inhibited RPCA in 58.4% of the cases (single adminstration) and in 71.2% of the cases (5 admininistrations before antigen challenge) and surpassed the standard drugs (Diasolin and Glyceram) in activity.

3.4. Immune-stimulating effect

The study was performed at the Pyatigorsk State Pharmaceutical Academy. It was demonstrated by agar-free local haemolyses in erythrocyte monolayers that the composition at a dose of 50 mg/kg significantly increases haemagglutinin titres in mice blood serum and the number of antibody produced cells in the rat spleen.

3.5. Anti-HIV tests

It was shown that the composition inhibits the virus HIV-l absorption (HTHIV27) on lymphoblastoid cells MT- 4 at 100% in a concentration of lOOμl/ml (100 gamma) . These trails were conducted at the Laboratory of Associated Infections of the Epidemiology and Microbiology Research Institute (Moscow) .

4. Cosmetic compositions

Cosmetic remedies were developed on the base of the composition (anti-acne cream, anti-wrinkle cream, and nutritive cream; anti-dandruff shampoo with hyposensitization and anti-inflammatory properties, sprays) and limited clinical trials were performed.

A shampoo containing the composition has anti¬ inflammatory, wound-healing, anti-allergic and tonic effects. It provides softness to skin and hair, gives it a pleasant smell, removes itch and prevents the dandruff appearance. The concentration Vodexol in the shampoo is 0.5%, 1.0% and 2.0%. The nutritive cream containing the composition has anti-inflammatory and softening effects, the skin becomes very smooth and velvety.

5. Therapeutical and cosmetic compositions

The Vedexol composition could be used as an anti¬ inflammatory, anti-allergic agent in medicine and cosmetology.

EXAMPLE 3 Esefsol

1. Preparation

The raw material left after extraction by methylene chloride, was extracted under vacuum with 70% acetone until depletion of the raw material. The depletion was monitored by specific reactions on triterpenes . The extraction time is 3 hours. The extract was evaporated to 1/4 of the volume in a vacuum apparatus (type "Simax") . The residue was dried in a special disperse drying box having an entrance temperature of 160-180°C and an exit temperature of 110-120 °C. The drying time is 1 hour. The substance obtained is a light-sandy powder.

2. Chemical composition A qualitative analysis was carried out by means of paper chromatography in a system of n-butylic alcohol/ ethylic alcohol/lN ammonium (6:1:3) .

As the developers for triterpenoids a 1% alcohol solution of silicon-tungsten acid and a 0.1% methylene- violet solution were used. The reference was a glycerrizinic acid solution.

The presence of flavonoids was qualitatively examined by means of paper chromatography in the systems dilute acetic acid/water (85:15) and n-butanol/acetic acid/water (4:1:2) . After that the chromatograms were examined in ultra-violet light and processed with ammonium sprays. As the developer rutin-standard was used. Quantitative analysis of flavonoids was conducted by spectrophotometric assay (λ-374 nm) , rutin solutions were used as the standards. The composition must contain not less that 14% of flavonoids. Quantitative analysis of saponins was also carried out by spectrophotometric assay. For calibre diagram construction the solution of glycerrizinic acid (λ-258 nm) was used. The saponins quantity is 24%.

The standardization of Esefsol on triterpene saponins was performed with the help of reverse phase HPLC. The division of components was conducted in a multiplan regime of detection (in an interval from 210 to 250 nm) . The aglycons of triterpenoids were determined after acidic hydrolysis in methanol-glycerretic acid, uralemc acid, 24-oxyglycerretιc acid (the fourth spot in chromatograms, 11, 13 (18) -dιen-3 , 2 , 4 , diol-30ιc acid) .

Quantitative content of aglycones after hydrolysis is 6.6%. The absorption spectrum of terpenoids was taken a flow stop regime (range 210-250 nm) . The number of peaks, its extent of dilution and asymmetric coefficient were used as optimized criteria. Marking off the substance content (280 nm) was conducted by the method of inner normalization in automatic regime without background correction. 4 Saponins were found, the relative content of which was determined with an accuracy of +3%. The relative triterpenoid content is 24% (the mean of 16.3%; 39.4%; 21.8%; and 22.5%) .

The following components were detected:

1. Triterpenoids (glycerretic acιd-16.3%, uralemc acιd-39.4%, 24-oxyglycerretic acιd-21.8%

(total quantity: 24%) .

2. Flavonoids (rutin, kempferol, astragalin, nicotiphlorm

(total quantity: 14.3%) .

3. Biological properties 3.1. General Acute toxicity is low, the LD₅₀ was more than 5000 mg/kg, no allergenic and local irritating properties were observed.

3.2. Anti-inflammatory effect

The experiments were carried out in white rats 10% Caolm solution induced acute inflammatory reaction after subplantar injection An ointment of 5% of the composition (based on lanolme and vaseline 1.1) was locally applied. The composition reduced the rat paw oedema during the first hour with 11.2%, durmg the second with 11.9%, during the fourth with 20.1%, and durmg the fifth with 26.6%

3.3. Hepatoprotective effect

This was revealed in experimental hepato-dystrophy induced by 50% oil solution of CCL4. The composition at a dose of 25mg/kg, p.o., statistically significant reduced cholesterin level in blood with 50%, and triglyceride with 60% It increased the total protein with 12.5%, and hepatic glycogen with 400%. The composition surpassed Legalon on activity The tests were carried out at the Pyatigorsk State Pharmaceutical Academy.

3 4 Anti-HIV tests

It was shown that the composition at a concentration of 50 ug/ml (50 gamma) inhibited the adsorption of virus HIV-l (HTHIV27) on lymphoblastoid cells MT-4 with 100%. The trials were performed at the Laboratory of Associated Infections of the Epidemiology and Microbiology Research Institute (Moscow) .

3.5 Hypolipidemic effect

Flavonoids display hypolipidemic effects at the conditions of "twm-60 probe". Hyperlipemia m rats was induced by intraperitoneal injection of twm-60 (80-200 mg/100 kg body mass) . The so-called "Vitamin model" was also used oral administration during 4 days of 2 ml alcohol and oil solution mixtures of vitamin B2 at a dose 200 mg/kg (80000 ED/llOg body mass) .

Cholesterme (2.54+0.11) and triglycerides (2.03+0.15) contents were determined with well-known methods. Polysponin was used as the standard (2.49+0.04 and 1.04+0.14) .

The decrease of cholesterine in blood with 50% and of triglycerides with 60%, and a total protein increase of 12.5% were demonstrated in rats with toxic hepatitis induced by oral administration of vitamin at 25 mg/kg. The lowering of cholesterme with 23%, and of triglycerides with 41% in comparison with the control was shown at hepatic tissue. The enzyme activity was reduced with 35.2% and 46.1% and the glycogen level in liver was increased with 400% n comparison with the control.

4. Therapeutical compositions

The Esefsol composition could be used in medicine as hypolipidemic, hepatoprotective, lipid metabolism modulator agent.

It would also be expedient to use the composition in cosmetology as a biologically active component m creams w th anti-inflammatory, capillary protective properties (in liposomal structures) , shampoos (a good solubilizing effect is provided by triterpenoid saponins) .

EXAMPLE 4

Polysaccharide complex (Polysol)

1. Preparation Specimens of the raw material chopped to 1mm with a mass of 250g were loaded in a laboratory extractor (filtration type) . The separation of lipophilic and phenolic substances from the raw material was performed.

Removal of the fat was conducted by adding a seven-fold quantity of methylene chloride relative to the mass of raw materials. The extract obtained (1.5 1) was used for the production of Glycefungin. The grass deprived of the fat, was unloaded from the extractor and dried on the air at 20°C till the complete removal of the extraction agent from the raw material. After that the dried raw material was loaded in an extractor and worked with 82% aqeous ethanol to obtain a alcohol-soluble sugar fraction. This fraction was separated from non- carbohydrate (phenols) components with the help of a 10% solution of Pb(CH₃COO)₂ and sodium sulphate. Precipitation was conducted and filtration of the sediment performed from the sodium sulphate. The sediment was separated, the filtrate was condensed to 100 ml and dissolved in a 5-fold quantity of methanol. Sugars were precipitated by acetone (1:3) . The sediment after half an hour settling was filtrated under vacuum, washed with acetone and dried, burned on the air, after that in vacuum-drying box to constant mass at 105°C to completely remove the acetone. The yield of this fraction is 1.2%.

The rest of the raw materials were dried twice and extracted two times with hot water (1:20) on a boiling bath for the separation of water-soluble polysaccharides. A water fraction was condensed under vacuum to 1/2 volume. A 3-fold volume of methanol was added to the extract . The obtained sediment is a complex of water-soluble polysaccharides (yield: 6.5%) .

2. Chemical composition.

The quantitative characterization of the product was carried out by gravimetric determination of water- soluble polysaccharides in the complex. For this a precisely weighed amount of Polysol was dissolved in water while heating on a boiling bath till disappearance of the solution's turbidity. The hot solution (90°C) was poured at portions in strong ethylic spirit during constant mixing. The obtained sediment was separated by filtration under vacuum (at a residual pressure of 13-16 kPa) through glass filter PDR-16 and dried to a constant weight. The sediment on the filter was washed with 70% ethylic spirit, then with water-free acetone and brought to a constant weight in a drying case .

The investigations of the polysaccharides were carried out after hydrolysis in 10% sulphuric acid solution at 100 ± 5°C. Identification of free monosaccharides was conducted by a chromatography method (on paper FN-11) m the system n-butanol/pyridme/water (6:4:3) and by a thin layer chromatography in the system ethylacetate/pyridme/water/n- butyllic alcohol/acetic acid (5:4:4:4:10:2) . The developer is anilme-phtalic reagent and

Bonner's complex. Polysaccharides were determined by a gravimetric method, hydrogenated and acidic sugars by a spectrophotometric assay with picric acid and carbozol . For the determination of the monosaccharide content of water soluble polysaccharides the hydrolysis was conducted m a 10% solution of sulfuric acid at 102 ± 2.5°C The identification of monosaccharides was performed by chromatography assay as described earlier. The quantitative content of hydrogenated and acidic sugars was determined by a spectrofoto etry assay on apparatus SF-42. For the quantitative estimation of hydrogenated sugars the reaction with picrimc acid was used, and for acidic sugars one with carbazol .

Proteinic complex was analysed by Louri ' s method. Ash content of water soluble polysaccharide fraction was calculated by Pharmacopeia's method.

Obtained data are represented at the table 1

Table 1

Total Monosaccharide Content Quantity of sugars, % Quantity Ash amount of of content % poly¬ proteinic neutral acidic hydro¬ acidic saccharides, compounds genated

% % 35.5-40.0 galactose, galacturonic 3.5-3.9 arabinose, acid 12.6-13.0 11.0-11.4 * glucose. 22.2-22.4 xylose

- glucose - 29.97 - 16.9-17.2 -

Water soluble polysaccharides relate to the group of glucuronaglycones . Free sugars are represented by glucose, galactose and saccharose.

The composition of Polysol is as follows: 1. monosaccharides (glucose, galactose, arabinose, xylose, galacturonic acid) (quantity: 16.1%) ; 2. polysaccharides

(quantity: 35.56%) ; 3. hydrogenated sugars

(quantity: 12.65%) ; and 4. acidic sugars (quantity: 3.44%) .

3. Biological properties. 3.1. Antitumour effect

The composition at a dose of 10-15 mg/kg by means of intraperitoneal injection during 5 days suppressed the growth of sarcoma 37 with 59%. At the same doses the composition administered during 10 days inhibited the growth of melanoma B-16 with 54% and increased the life time of cured animals on 25% and also with Erlich's tumour on 38%. The composition at 100 mg/kg during 10 days administration increased mice life time with melanoma B-16 on 36% and with ascites Erlich's tumour on 66%.

EXAMPLE 5 Glycacet

1. Preparation This composition was derived by the extraction of chopped grass (0.2 mm size, method the same as described above) with water-free acetone (ratio between raw material and extraction agent is 1:7) . The extraction agent was evaporated under vacuum and the extract was dried at 50- 55°C.

2. Chemical Composition

The chemical composition was determined as described in Example 1.

The carotenoid content of Glycacet was carried out by high performance liquid chromatography (HPLC) assay on MILIOHROM-E apparatus. The column of the apparatus was filled by Sιlasorb-600. As a mobile phase heptan-isopropanol (8:2) were used at 326 nm (wavelength) . The standard was a 0,002% solution of pure beta-caroteinne propanol. The calculation was performed by the calibration method.

The determination of isoflavonoids was performed by thin layer chromatography assay m a fixed layer of silicagel in a system of chloroformum saturated with water; chloroformu -ethanol (3.1) and also by paper chromatography on paper mark M in the system: n-propanol-acetic acid - water (1:1:7) ; n-butanol - acetic acid - water (4:1:5) . Two spots were discovered. One of them was characterized as prunetm, the second as phytoalexm.

The quantitative determination of isoflavonoids was performed on Wilson's method in A. Guseva' s modification, based on the appearance of yellow coloured complexes with boric acid in a ratio of 1:1. Citric acid was used as a supplement which stabilizes and increases the colour. Further analysis was carried out by a photometric assay on polyphotometer system LABOR (Hungary) with a green light filter.

The determination of phytosterines was performed by thin layer chromatography assay m a fixed layer of silicagel in the system of. chroformum - methanol - water (65:30:10) . After drying the chromatogram was developed with Sanye reagent (5% spiritous solution of vanilme) , heated at 110°C during 10 min in a drying case, and was then sprinkled with 50% solution of sulfuric acid and heated again.

2 spots were determined, one of them corresponded to the standard -beta-sitosterin (yellow-brown coloured spot) .

The quantitative analysis of phytosterins was performed as follows: 10% aceton solution of fraction in concentrations of 1.0; 1.5; 2.0; 2.5; 3.0 μl and 0.1% solution of betasitosterin were put on a chromatographic plate "Sorbfil". Chromatography was realised in a system of heptanisopropanol (9:1) . Then they were developed in iodine vapours .

The composition was as follows:

1. chlorophyll A and B (quantity: 4.0-4.5%)

2. resins (resin acids of diterpene series, hydrocarbons)

(quantity: 2.0-2.5%)

3. unsaturated fatty acids (oleinic, linolenic, palmitinic)

(quantity: 36.29-40.11%)

4. carotinoids (quantity: 0.27-0.28%)

5. isoflavonoids (quantity: 3.22-3.26%)

6. sitosterins (beta-sitosterine, stigmasterine) (quantity: 1.9-2.1%)

3. Biological properties The tests to determine the biological activity were carried out at the Institute of Medical and Biological Problems (Moscow) and the Central Skin Venerology Institute (Moscow) . It was discovered that Glycacet possesses a potent anti-bacterial and anti-fungal activity. The experiments were performed in vitro by the agar diffusion method on the cultures of dermatophyte, mouldy fungi, Staphylococcus aureus. Bacillus polvmyxa. It was revealed that the extract leads to delayed growth zones of pathogenic fungi (Trichophvton rubrum - 0.3mm, T. interdigitale - 2.6mm, T. gypseum - 2.6mm, Microsporum canis - 3.2mm) and bacteria (Staphylococcus aureus - 4.0mm, B. polvmyxa - 5.0mm) .

EXAMPLE 6

Glycochlor

1. Preparation

The composition was derived from the chopped (0.2 mm size) grass by extraction with chloroform (ratio 1:7) . The extraction agent was evaporated under vacuum, then sodium hydroxide (0.4% solution) was added to the extract at 50°C and it was dried.

2. Chemical composition

The chemical composition was determined as described in Example 1. The determination of sodium salts of unsaturated fatty acids in chloroformum fraction was conducted by titremetric assay. A precisely weighed amount of the extract was dissolved in 50 ml of pure ethyl alcohol while heating. After complete dissolution of it the solution was developed with heptane in a dividing funnel. The washed ethanoiic solution was diluted with water to 100 ml volume, ^• 25 ml of chloroformum was added and the resulting solution was titred with 0.1 mol/1 solution of HCl (hydrochloric acid) .

For the determination of equivalent point the potentiometer was used.

The composition was as follows: 1. Sodium salt of chlorophyll 4.0-4.2%

2. beta-sitosterin 0.02%

3. sodium salts of unsaturated fatty acids (palmitates, oleates) 33.9-37.5%

3. Biological properties

The potent antibacterial and antifungal effects were demonstrated at the laboratories of the Institute of Medical and Biological Problems (Moscow) and Central Skin Venerology Institute (Moscow) by the same methods described above.

EXAMPLE 7 Cosmetic compositions

Various cosmetic compositions were prepared according to the following recipes. These compositions were clinically tested. 1. Prophylactic anti-acne cream. Glycefungin 3.0

Vodexol 3.0

Polyethylenoxιde-400 43.0 Polyethylenoxιde-1500 51.0

2. Nutritive anti-wrmkle cream

Vodexol 0, .5

Esefsol 0, .5

Wax 6. .0

Stearme 5. .0

Stearates 3. .0

Olive oleum 40. .0

Threeethanolamine 1, .0

Nipagm 0, .2

Vitamin E 0, .5

Water till 100. .0

3. Antifungal spray

Glycefungin solution

70% ethyl alcohol 3.0 Twιn-80 1.0 10% solution of Polyvinyl¬ pyrrolidone 3.0 10% solution of sorbitum 2.0 Ethyl alcohol 30% 9.0% Menthae essential oil 2 drops

4. Antifungal pencil

Glycefungin 1.0 Polyethylenoxide-400 1.0 Polyethylenoxide-1500 6.0 Rose oleum 2 drops

5. Anti-acne, anti-inflammatory, antiallergic, antibacterial, antifungal cream for wide application in dermatology

Glycefungin 6.6

Vodexol 2.4 Esefsol 3.0

Emulgator 88.0

Bee wax 25.0

Olive oleum 200.0

Dimethylsulfoxide 10.0 Ethylic alcohol 96% 50.0 Water 630.0

The clinical trials of this cream were performed at the Mycology Dpt . of the Central Skin Venerology Institute (Moscow) . The efficacy of the cream was proved on 46 patients suffered acne vulgaris (12) , acne juveniles (14) , acne rosaceae (2) , atopic dermatitis (11) , contact dermatitis (4) , candidosis of skin folds (3) .

The cream was used twice a day during 1-3 weeks on affected parts of the skin. It was demonstrated after the treatment that 12 patients were completely cured, 11 patients had remarkable improvement of their condition, 21 patients had real improvement, 2 patients had no changes. The best results were found in patients suffering from contact dermatitis, atopic dermatitis and acne vulgaris.

The cream of the invention surpassed some steroid hormone creams in clinical efficacy and showed no side effects. The cosmetic compositions are recommended as potent anti-bacterial, anti-fungal, anti-inflammatory and anti- allergic creams in dermatological practice.

Claims

1. Biologically active composition obtainable by extracting the grass of Glycerrhiza glabra L. or an extract thereof with a suitable extraction agent.

2. Biologically active composition as claimed in claim 1, wherein the extraction agent is selected from the group consisting of methylene chloride, water, a mixture of acetone and water (70%/30% (v/v) ) , acetone, chloroform, glycerin, polyethylene oxide-400, olive oil, diethyl ether, and petroleum ether, or combinations thereof.

3. Biologically active composition as claimed in claim 1 or 2 , wherein the chopped grass without the roots is extracted with methylene chloride under vacuum, after which the extraction agent is evaporated to obtain a substance named "Glycefungin" .

4. Biologically active composition as claimed in claim 1 or 2, wherein the chopped grass without the roots is first extracted with methylene chloride, after which the methylene chloride is evaporated, to yield a dried material which is subsequently extracted with 70% acetone, after which the acetone is evaporated to yield a substance named "Esefsol" .

5. Biologically active composition as claimed in claim 1 or 2, wherein the chopped grass without the roots is first extracted with methylene chloride, after which the methylene chloride is evaporated, to yield a dried material which is subsequently extracted with 70% acetone, after which the acetone is evaporated and the thus obtained material is subsequently extracted with hot water at an elevated temperature, preferably between 80 and 90°C, the extraction is optionally repeated twice, the extraction agent is evaporated of and the material is dried to obtain an aromatic, thick, viscous, dark-brown mass, named "Vodexol".

6. Biologically active composition as claimed in claim, wherein the lipophilic and phenolic components are separated from the chopped grass, the thus obtained material is treated with an alcohol to extract the soluble sugar fraction, the sugars are purified by precipitation with acetic acid and sodium sulphate, the precipitate obtained is subsequently dissoluted in an alcohol and a polysaccharide complex, named "Polysol", is reprecipitated in acetone.

7. Biologically active composition as claimed in claim 1 or 2, wherein the grass is extracted with pure water-free acetone to obtain a composition named "Glycacet"

8. Biologically active composition as claimed in claim 1 or 2, wherein the grass is extracted with chloroform, which extraction leads to a composition named "Glycochlor" .

9. Method for the preparation of Glycefungin, comprising extracting the leaves of the grass Glycerrhiza glabra L with methylene chloride under vacuum, and evaporating the extraction agent

10. Method for the preparation of Vodexol, comprising processing leaves of the grass Glycerrhiza glabra L. with methylene chloride and 70% acetone m water, subsequently extracting the thus obtained material with hot water at an elevated temperature, preferably between 80 and 90°C, optionally repeating the extraction twice, evaporating the extraction agent and drying the material.

11. Method for the preparation of Efesol, comprising extracting leaves of the grass Glycerrhiza glabra L. with methylene chloride, extracting the raw material left after methylene chloride extraction with 70% acetone in water, evaporating the extraction agent and drying.

12. Method for the preparation of Polysol, comprising separating the lipophilic en phenolic components from leaves of the grass Glycerrhiza glabra L. , treating the thus obtained material with an alcohol to extract the soluble sugar fraction, purifying the sugars by precipitation with acetic acid and sodium sulphate, subsequently dissolutmg the precipitate obtained in an alcohol and reprecipitating the Polysol m acetone.

13. Method for the preparation of Glycacet, comprising extracting leaves of the grass Glycerrhiza glabra L. with pure water-free acetone.

14. Method for the preparation of Glycochlor, comprising extracting leaves of the grass Glycerrhiza glabra L. the with chloroform.

15. Use of any of the compositions according to any one of the claims 1 to 8 as a biologically active agent.

16. Use of any one of the compositions of any one of the claims 1 to 8, or combinations thereof, for the preparation of a therapeutical composition for the treatment of fungal, bacterial, viral, inflammatory, tumor, protozoan, allergic diseases.

17. Textile impregnated with any one of the compositions according to any one of the claims 1 to 8, or combinations thereof.

18. Foot inner soles impregnated with any one of the compositions according to any one of the claims 1 to 8, or combinations thereof.

19. Cosmetic composition comprising any one of the compositions according to any one of the claims 1 to 8 , or combinations thereof, and a suitable excipient.

20. Therapeutic compositions comprising any one of the compositions according to any one of the claims 1 to 8, or combinations tehreof, and a suitable excipient.

21. Use of Glycefungin for the prevention of adverse microbial growth in vaccine manufacturing processes.