EXTRACT AND A METHOD OF PREPARATION OF THIS EXTRACT

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to the field of interferon (IFN) inducers-a special group of potential antiviral compounds, particularly to Nerium oleander special extract, its ingredients and compositions containing fixed combinations of the Nerium oleander and Glycyrrhiza glabra extracts, as well as to a method of preparation of the extracts using a substance's characteristics information transmission device.

2. Description of the Prior Art The ability of IFN-s to confer an antiviral state on cells is their defining activity as well as the fundamental property. IFNs are essential for the survival of higher vertebrates because they provide an early line of defense against viral infections-hours to days before immune responses.

This vital role has been demonstrated by the exquisite sensitivity to virus infections of mice lacking both IFN a/ (3 and y receptors. Multiple pathways have involved to combat different types of viruses and the various compensatory defense mechanisms that different viruses have involved.

Antiviral mechanisms of interferon action include dsRNA-dependent protein kinase (PKR), the 2- 5A Synthases, and the Mx proteins pathways. PKR pathway mediates signal transduction, inhibition of protein synthesis and transcriptional control, 2-SA system pathway-RNA cleavage, and Mx proteins interfere with viral replication, impairing the growth of influenza and other negative-strand RNA viruses at the level of viral transcription and at other steps.

Any stage in virus replication appears to be inhibited by IFNs, including entry and/or uncoating

(simian virus 40, retroviruses), transcription (influenza virus, vesicular stomatitis virus), RNA stability (picornaviruses), initiation of translation (reoviruses, adenovirus, vaccina), maturation, and assembly and release (retroviruses).

Along with antiviral activity IFNs inhibit cell growth and control apoptosis.

Effects of y-IFN on the immune system includes: - the ability to induce the expression of MHC class II proteins in a wide variety of different cell types, and thereby to promote the development of CD4+ T-cell response.

- generation of activated macrophages, a key effector cell population in innate and adaptive immune responses involved in killing microbial targets.

- Regulation humoral immunity, by regulating the development of specific T helper cell subsets, or directly at the level of B cells and their functions-development of proliferation, immunoglobulin secretion and IG heavy-chain switching.

IFN inducers represent a special group of potential antiviral compounds. The main requirements for them are (1) high IFN-inducing activity, (2) absence od side effects, (3) wide range of antimicrobial activity, (4) broad therapeutic security and, (5) good solubility in water and biologic fluids. IFN inducers may be used against very different infections and conditions. IFN inducers stimulate IFN production in different cells and organs, and that determine the strategy for their application in hepatitis B and C, influenza, rhinoviral and enteroviral infections, encephalitis, rabies, etc.

It has been demonstrated that glycyrrhizin, an active principle of Glycyrrhiza glabra extracts, induces interferon formation. Antiviral activity of glycyrrhizin was observed against vaccinia, herpes simplex I, Newcastle disease, vesicular stomatitis and influenza A viruses.

a substance's characteristics information transmission technique for the purpose of potentiation of the activity of the final product.

Hardly surprising, viruses fight back, not only against host defenses in general, but also against the IFN system in particular, both through novel mechanisms and by subverting host systems through the synthesis of novel proteins and proteins that mimic and thus interfere with host systems. y-IFN is generated mainly by T cells. Many factors are involved in the activation and suppression of T cells. Consequently, there might be multiple modes of INF induction by T cells.

Increasingly research shows that fixed combination of herbs have greater-than-expected medicinal benefit for the treatment of viral diseases, due to the combination of constituents which have synergistic effect and act on different molecular targets. In some cases, the medicinal value of the herb may be entirely due to the combination of substances and can not be reproduced by one or two"active"constituents alone. Complex combination of several plants is a general approach of traditional medicine, particularly of oriental medicine such as Ayurveda and Unani in India, Kampo in China and Japan.

Following to this concept we showed in this study - the efficacy of a fixed combination of two plants extracts standardized for its active ingredients: Glycyrrhizin (Glycyrrhiza glabra L) and polysaccharides (Nerium oleander L.), - potentiating effect of a substance's characteristics information transmission technique [Gotovski Yu et al.., Patents of Russia 2070405,2070406,2065297,1996] on the IFN- inducing activity of the extracts, in vitro experiments on whole blood cells culture (IFN-inducing activity) and on human carcinoma squamous HEp-2 cells infected by the mice encephala-myocarditis virus (virucide effect).

It has been shown in several publications that extracts of Nerium oleander leaves, well known as a very poisonous plant due to presence of cardiac glycosides, have antitumor, immunomodulating and antibacterial activity. The results of these studies are very controversial both about efficacy and the main constituents responsible for these effects.

As is described in US Patent 5,135.745"Extracts of Nerium species, methods of preparation, and use therefore", the water extract of Nerium oleander is useful in ameliorating cell-proliferative disease in animals. As a result, the inventor disclosed in this patent: - a polysaccharide enriched extract of Nerium species containing an immunologically active polysaccharide useful in treating call-proliferative disease in mammals, wherein the active polysaccharide comprises acidic homo-poly-galacturonans or arabino-galacturonans,

- a method of preparation of Nerium species containing an immunologically active polysaccharide useful in ameliorating call-proliferative disease in mammals, by boiling of plant material in inorganic solvent for several hours to obtain density about 1010, - a method of ameliorating cell-proliferative diseases (malignancy, adenocarcinoma,, psoriasis) by administrating (parenterally, subcutaneously, intramuscularly, intraperitoneally, intracavity, intravenously, transdermal, nasopharyngeally, or mucosal absorption) of a polysaccharide enriched extract of Nerium species extracts.

However, - it has never been scientifically demonstrated that extracts of Nerium oleander and its components could have IFN-inducing and/or antiviral activity, - Nerium species have never been used in combinations with other herbal drugs, particularly with Glycyrrhiza extract for treatment any diseases, - described method of preparation of the Nerium extracts does not include treatment with

SUMMARY OF THE INVENTION It is the object of the present invention to provide plant special extract of Nerium oleander leaves that exhibits IFN-inducing activity in whole blood cell culture.

It is another object of the present invention to found an active principle of that extract.

It is another object of the present invention to evaluate the IFN inducing and antiviral activity of the extract prepared from fixed combination of Nerium Oleander L. leaves and Glycyrrhiza glabra L roots in vitro experiments on whole blood cells culture (IFN-inducing activity) and on human carcinoma squamous HEp-2 cells infected by the mice encephalo- myocarditis virus (virucide effect). As a result inventors found that this combination is more active then each ingredient alone.

Another object of the present invention is to provide the method of preparation of the IFN- stimulating extracts, wherein these extracts are obtained by the treatment of a substance's characteristics with information transmission device. As a result inventors found that this technique statistically significant potentiates the activity of final products.

Detailed description of the invention The present invention relates to new y-Interferon-inducing agents, more particularly to the Nerium oleander extract, its active ingredients and its fixed combinations with Glycyrrhiza glabra extracts, and to the method their preparation using a substance's characteristics information transmission technique for potentiation of the activity of extracts and uses therefore as potential antiviral drugs.

In general, to obtain Nerium species extracts, water extraction of the air dried leaves, flowers and stems at high temperature for several hours (enough for decomposition of toxic

cardiac glycosides) is used in accordance with US Patent No 5,135,745, which disclosed the extract itself, as well the use of this extract in ameliorating cell-proliferative diseases (malignancy, adenocarcinoma, psoriasis) by administrating (parenterally, subcutaneously, intramuscularly, intraperitoneally, intracavity, intravenously, transdermal, nasopharyngeally, or mucosal absorption).

Our invention concerns a new method of preparation of a special extract of Nerium oleander leaves, which exhibits quit new property, particularly IFN inducing activity. This special extract is significantly different from mentioned in US patent extract prepared without any treatment with the substance's information transmission technique, particularly in vitro experiments where IFN-inducing activity is evaluated.

Active ingredients of these extracts are shown to be 50 to 500 KDa polysaccharides containing trihydroxybutiric acid, ribose, arabinose, xylose, lyxose, mannose, talose, glucitol, glucose and galacturonic acid.

Another object of our invention is the fixed combination of Nerium oleander leaves and Glycyrrhiza glabra roots, potentially useful in treating viral disease in mammals, which exhibits y-IFN-inducing and virucide activity in vitro experiments on whole blood cells culture (IFN- inducing activity) and on human carcinoma squamous HEp-2 cells infected by the mice encephala-myocarditis virus (virucide effect). These fixed combination of Nerium oleander and Glycyrrhiza glabra extracts (FC-NO: GG) are more active then each component separately (NOE and GGE). Fixed combination of special extracts (FC-NO: GG-SE) obtained by the treatment of substance's characteristics information transmission device is significantly more active than samples FC-NO: GG (without such a treatment).

The present invention will be described in detail hereinafter:

Example 1. Preparation of Nerium oleander L. extract (NOE).

Add about 10 g of air dried, sliced into pieces leaves, branches and flowers of Nerium oleander to 100 ml of distilled water in 200 ml of round-bottom flask, heat it up to 100°C and keep boiling for 3 h. During boiling, add distilled water to maintain a constant water level. After boiling, bring the temperature of the mixture to 20-25°C, filter using a coarse filter in order to separate from plant material and remove from particulate matters. Bring filtrate to the volume of 100 ml with distilled water and subject it to sterile filtration into sterile flask using Millipore filters with pore size 0.45 u. M and 0.22 LM consequently.

Specification : Total saccharides content-3.9-4.1 mg/ml, total polysaccharides content-1.4- 1.6 mg/ml, dry residue-10-12 mg/ml.

Example 2. Preparation of Nerium oleander L. special extract (NOSE).

Add about 10 g of air dried, sliced into pieces leaves, branches and flowers of Nerium oleander to 100 ml of distilled water in 200 ml of round-bottom flask, heat it up to 100°C and keep boiling for 3 h. During boiling, add distilled water to maintain a constant water level. After boiling, bring the temperature of the mixture to 20-25°C, filter using a coarse filter in order to separate from plant material and remove from particulate matters. Bring filtrate to the volume of 100 ml with distilled water and subject it to sterile filtration into sterile flask using Millipore filters with pore size 0.45 uM and 0.22 u. M consequently. On the filling line after sterile filtration fix active electrodes of apparatus for the substance's characteristics information transmission"Transfer-P" (Imedis-BRT Ltd., Moscow). The apparatus is set up on the straight transmission ofy-IFN information characteristics and transmission of information characteristics of human organism specific response on certain groups of viruses (Hepatitis A, B, C, D, measles) by"Imedis

Bioresonance"software equipped with Pauly & Shmidt's"Substances'Bio-resonance Database" (Imedis-BRT Ltd., Moscow). Store solution at 4°C for one year.

Specification : Total saccharides content-3. 9-4.1 mg/ml, total polysaccharides content-1.4- 1.6 mg/ml, dry residue-10-12 mg/ml.

Example 3. Isolation of Nerium oleander polysaccharides (NOPS) Apply 20 p1 oiNOE to Shodex Suger Ionpak K-804 ion-exchange resin gel of sulfonated styrene- divinylbenzene copolymer HPLC column (Showa Denko K. K., Japan) and elute the column with water to obtain PS fractions I-III, figure 1. These fractions were further characterized for their monosaccharides composition by GC-MS of their TMS-derivatives, figure 3.

Example 4. Preparation of Glycyrrhiza glabra L. extract (GGE).

Glycyrrhiza glabra L. extract (GGE) is prepared as described above for NOE (example 1).

Specification : Glycyrrhizin content-2.0-2.5 mg/ml, total saccharides content-4.3-4.5 mg/ml, total polysaccharides content-2.8-3.0 mg/ml, dry residue-12-14 mg/ml.

Example 5. Preparation of the fixed combination of the extracts of Nerium oleander and Glycyrrhiza glabra (FC-NO: GG-SE).

Add about 5 g of air dried, sliced into pieces leaves, branches and flowers of Nerium oleander and 5 g of air dried, sliced into pieces roots of Glycyrrhiza glabra to 100 ml of distilled water in 200 ml of round-bottom flask, heat it up to 100°C and keep boiling for 3 h. During boiling, add distilled water to maintain a constant water level. After boiling, bring the temperature of the mixture to 20-25°C, filter using a coarse filter in order to separate from plant material and remove from particulate matters. Bring filtrate to the volume of 100 ml with distilled water and subject it to sterile filtration into sterile flask using Millipore filters with pore size 0.45 ZM and 0.22 AM consequently. On the filling line after sterile filtration fix active electrodes of apparatus for the

substance's characteristics information transmission"Transfer-P" (Imedis-BRT Ltd., Moscow).

The apparatus is set up on the straight transmission of y-IFN information characteristics and transmission of information characteristics of human organism specific response on certain groups of viruses (Hepatitis A, B, C, D, measles)"Imedis Bioresonance"software equipped with Pauly & Shmidt's"Substances'Bio-resonance Database" (Imedis-BRT Ltd., Moscow).

Specification : Total saccharides content-5.0-5.4 mg/ml, total polysaccharides content-1.4- 1.6 mg/ml, Glycyrrhizin-1.15 mg/ml, dry residue-13-15 mg/ml. Free and bound monosaccharides were characterized by GC-MS of their TMS-derivatives, figure2.

Example 6. IFN-y inducing activity of the extracts Test samples : Concentrations of the extracts and their ingredients in stock solution used for further dilutions of test samples were: (a)-10.4 mg/ml of Nerium oleander extract (NOE) with 1.5 mg/mi of Nerium oleander polysaccharides (NOPS) (b)-13.9 mg/ml of Glycyrrhiza glabra extract (GGE) with 2.9 mg/ml of Glycyrrhiza glabra PS and 2.3 mg/ml of Glycyrrhizin (c)-13. 9 mg/ml of fixed combination (FC) of NOE : GGE with 1.2 mg/ml of PS and 1.15 mg/ml of Glycyrrhizin (d)-13.9 mg/ml of fixed combination Nerium oleander and Glycyrrhiza glabra special extract FC-NO: GG-SE with 1.2 mg/ml of PS and 1.15 mg/ml of Glycyrrhizin (e)-10.4 mg/ml of Nerium oleander special extract (NOSE) with 1. 5 mg/ml of NOPS (f)-0. 2 mg/ml of NO-PS-I (g)-0.2 mg/ml of NO-PS-II

(h)-1. 0 mg/ml of NO-PS-III Then solutions (a), (b), (e), (f) and (g) were diluted with water in ten times (1 : 9), while solutions (c), (d) in five times (1: 4), to obtain dilution (1: 10) with concentrations of Glycyrrhizin and PS equal to that in all other samples (a)- (g). From these solutions other dilutions (1: 100,1: 1000 and 1: 10000) of test samples were prepared.

Final concentrations of the extracts and their ingredients in incubation media were ten times less than in test samples since one volume of test sample is added to nine volumes of incubation media.

Incubation procedure Effect of extracts and their components on INF-y production in whole blood cell culture was studied as described by Wang et al., 2000.

Whole blood of healthy donors (n=6) was collected into 4 ml sterile heparinized (lithium heparin) Vacutainer tubes (Vacuete, Greiner). 100 pl of blood samples were added to each well of 12-well tissue culture plate (Falcon), containing 800 u. I cultivation media (RPMI-1640 medium containing 2 ml L-glutamine, 1 mM sodium pyruvate, 100 IU penicillin and streptomycin and 10 % fetal calf serum) and 100 pLI test solutions with known concentrations of active ingredients, glycyrrhizin and polysaccharides (PS). Cells were cultivated for 72 hours in a humidifier atmosphere, containing 6 % CO2. and centrifuged in JOUAN BR4 centrifuge rotor with 12-well plate adapters at 3000 rpm for 15min. Cell-free culture supernatants were collected and stored at-80°C until use.

If assay IFN-y content in cell culture supernatants was determined using commercially available human Interferon-y TiterZyme@2 EIA kits (Assay Designs, Inc. MI, USA) as described in instruction sheet.

IFN-y Standards calibration curve was prepared and concentrations of IFN-y were automatically calculated using optical density data at 450 nm obtained by multichannel microplate-reader PR-2100 (Sanofi, Pasteur). All measurements were performed in duplicate, means and standard deviations were recorded.

Results All tested samples were active in dilutions 1: 100 (actually in final dilutions 1: 1000), figure 3. That corresponds to concentrations of extracts from 10 to 30 ug/ml, polysaccharides-from 1.5 to 3 llg/ml, and glycyrrhizin-2.2 u. g/ml (2.7 I1M). At lower concentrations they were inactive, and in higher-cytotoxic.

Figure 3 shows that Nerium oleander extracts (NOE and NOSE) stimulates y-IFN production, and polysaccharides I-III are active ingredients of these extracts.

Figure 3 shows that fixed combination of Nerium oleander and Glycyrrhiza glabra extracts (FC- NO: GG) is more active then each component separately (NOE and GGE).

Figure 3 shows that usage of substance's characteristics information transmission technique significantly potentates the IFN induction in whole blood in vitro test. Test samples of NOSE and FC-NO: GG-SE obtained by the treatment of substance's characteristics information transmission device are significantly more active then samples NOE and FC-NO: GG (without such a treatment); all these samples have the same concentrations of active ingredients.

Example 4. Antiviral activity of FC-NO : GG extract Materials and Methods.

Various methods are available for in vitro and in vivo antiviral screening of plant materials [Vlietinck and Vanden Berghe, 1991]. In our study we used methods earlier described for encephalitis virus [Fokina et al., 1991]

The following materials were used: Eagle's growth medium with Earl's solution containing 10% fetal bovine serum (FBS) purchased from the. Chumakov's Institute of Poliomyelitis and Viral Encephalitis, Moscow, Russia.

0. 25% Trypsin solution. Chumakov's Institute of Poliomyelitis and Viral Encephalitis, Moscow, Russia.

0. 02% Versenate Solution. Chumakov's Institute of Poliomyelitis and Viral Encephalitis, Moscow, Russia Fetal bovine serum (FBS). Chumakov's Institute of Poliomyelitis and Viral Encephalitis, Moscow, Russia. Each batch of the FBS is inactivated for 30 min at 56° C before use.

Maintaining medium-Eagle's minimal medium containing 2% fetal serum.

Goriaew's hemocytometer (Krasnogvardeec Company, Sankt-Petersburg, Russia).

50 ml and 250 ml vials (Costar Co.) 0. 02-1 ml adjustable automatic single channel ant multi-channel samplers,. (Labsystems, Finland).

96 well MaxiSorp Nunc microplates-COSTAR.

0. 025 ml multi-channel sampler with dosing tips.

0. 025 ml and 0.1 ml sampler.

Thermostat with CO2 supply.

Sterile containers and pipets for dilution.

Virus suspension with known concentration Methods:

a) Passage and maintenance of human carcinoma squamous cell HEp-2 (Caucasian, larynx) and mouse embryo cell NIH 3T3 (NIH Swiss), cultures.

Re-sowing of the cells Pour out the growth media from the vial containing the cell culture; 'Add 0.25% Trypsin solution (or equal volumes of 0.25% Trypsin solution and 1: 5000 Solutio Versenate) into the vial. 0.5 ml of the solution is enough for the vial with 25 cm2 surface. Distribute it equally on the surface of cells gently shaking of the vial; Keep the vial was stored at 36° C, unless all cells are separated from the growth surface (control by microscopic examination) Re-suspended the cells in the growth medium (4.5 ml per vial with 25 cm2 surface), which is neglecting the trypsin action. Suck in the suspension several times through a thin sampler tip to disintegrate the cells aggregations; Dilute the cell suspension in the growth medium to required concentration, or dilute it in ratio 1: 2 v/v.; Sow the cells suspension in vials, close tightly and place in thermostat at 36° C.

Incubate cell culture for 48-72 hours to obtain monolayer, then change the growth medium for maintenance media. Interweave the cell culture every 5-7 days. b) Cell count

The cell count can be exactly counted in the suspension by a hemocytometer.

Thoroughly disperse cells by recurrent suction through a thin dropper before the counting. Dilute 0.1 ml of cell suspension with 0.9 ml of 0.1% Trypan Blue in the Eagle's medium. The dead cells have are colored blue.

Mix suspension thoroughly by Paster pipet and apply in the chambers of hemocytometer.

Count the cells in 10 small squares of the both nets of the chamber were counted, excluding the cells, placed on lines.

If cell aggregates are detected the result was not taken into consideration, and the count has to be repeated with a re-suspended cell suspension.

Calculate the mean cell count in a square by the results obtained for three samples.

Calculate the concentration of cells in 1 ml by formula: C = M x 25000 Where: C = initial concentration of cells in 1 ml M = mean value of cells in 10 small squares (3 samples).

The amount of cells applied into the well of the microplate usually has to be 2-3x104 cells per 0.1 ml of growth medium.

Results To evaluate virulicidal activity of FC-NO: GG its effect on replication of the encephalo- myocarditis virus (EMCV) in the HEp-2 cells was studied. The mixture of two equal volumes of NG extract and EMCV was incubated for 2-3 hours, and then was added to 48 hours old HEp-2 cells.

Before to start the experiments, sterile filtration of all extracts was performed by micro-filters with pore size 0.8 and 0.2 llm (Gelman Sciences).

Test was performed as follows:

0.2 ml of the tested drug solutions was applied into the each microplate's wells coated with Hep-2 and NIH 3T3 cells monolayers, except the wells of last (H) row (control). Three microplates for each cell culture were prepared 0.2 ml of the tested drug solutions were applied into the each well of the last row H (control- no cells) of the microplate, diluted solutions of the EMCV were prepared in doses of 50 and 100 TCDso (tissue cytotoxic dose) Calculation of the virus'titer was performed by Koerber's formula: log TCD50= L-d (S-0.5) Where: L-logarithm of starting test concentration d-a difference between log subsequent dilutions S-sum of the proportions of the test-objects which gave a positive result (cytopathic effect) The mixture of two equal volumes of NG extract and EMCV was incubated for 2-3 hours.

0.2ml of incubation mixture was applied in each microplate's well coated with Hep-2 cells monolayer, except the wells in the last H row (control). 3 plates were prepared.

After incubation for 48 hours the results were evaluated under a microscope, and then cells were colored with crystal-violet (0.2 ml was added in each well) Results of the tests were evaluated by the quantity of wells, in which the extract protected the cells monolayer from cytopathic action of the virus in doses of 50 and 100 TCDso (Table 1).

Tablel. Virucide action of the NS-extract on EMCV (mean of 3 series of test) Dilution Level of protective effect of NG Control Control + NG of NG 50 TCDSO 100 TCDso Qty % Qty % Qty % Qty % 1/500 8 from 66. 6 2 from 8 25 12 100 12 100 12 from from 12 12 1/1000 0 from 6 0 0 from 6 0 6 from 100 6 Control 0 from 8 0 0 from 8 0 EMCV Thus, in dilution 1/500 and a low viral load (50 TCDso) the NG protects the cell monolayer on 66.6%, and in higher dose of virus (100 TCDSo)-on 25%. In dilution 1/1000 not any virucide action was observed.

Table 2. Data of statistical analysis X2for 1/500 dilution of NG (mean of 3 series of tests) Dose of virus Freedom level x2 Significance (p) SO TCDSO 1 6. 238* <0. 05 100 TCD5o 1 0. 633 Not significant * Significant to control Conclusions.

In conclusion, fixed combination of Nerium oleander and glycyrrhiza glabra special extracts (FC- NO: GG) in dilution 1/500 significantly exhibits virucide effect on the mice encephala-myocarditis virus in vitro.