Field of the invention

The invention relates to an extract extracted from an arborescent plant and containing stilbenes and compounds enhancing their effect, as well as a method for producing it. The invention in particular relates to the pharmaceutical and treatment use of the extract. The extract in question suppresses, inhibits and prevents an inflammation reaction of the body.

Background of the invention

As byproducts of mechanical forest industry, many secondary streams are created, which for the time being are mostly used for bioenergy, only. However, the bark, roots, branches, and internal branches of forest trees include a lot of valuable bioactive compounds which could be used much more than nowadays is in products of a higher degree of refining, too.

Internal branches of soft wood species, conifer trees in particular, are not commonly desired material in mechanical or chemical wood processing. A branch creates a point of discontinuity in the wood strength, deteriorating the quality of timber. In the manufacture of pulp and paper, branches are a worse quality raw material due to their shorter fibres and larger amount of extractive agents. In- ternal branches, that is, branch places inside the trunk of the tree, may, however, be separated from other raw wood material and used as raw material for bioactive compounds.

Bioactive compounds may be introduced by extracting raw wood material. The extractive agents of wood comprise particularly much phenolic agents, such as lignans, flavonoids, and stilbenes. They protect wood from microbiological damages and affect tree properties. In addition, the extractive agents include, for example, terpenes, terpenoids, waxes, and fatty acids.

Stilbenoid, pinosylvin, typical for pine trees, is chemically very similar to resveratrol found in grapes and berries, which is reported to have many advan- tageous health effects, such as antidiabetic effect, for example. Both pinosylvin and its monomethylether have been shown to be an effective antibacterial and antifungal substance. Flavonoids are known to have major antioxidant activity.

Because the secondary streams of the wood processing industry are an easily available raw material, there is an evident need to find new usage methods and utilization potential of it. Brief description of the invention

It is an object of the invention to utilize the bioactive compounds obtained from raw wood material. It is a particular object of the invention to develop a method for recovering bioactive compounds. It is a further object to provide new purposes of use for an extract including bioactive agents, obtained by the method. The object of the invention is achieved by an extract, use thereof, and a method for preparing it, which are characterized by what is disclosed in the independent claims. Preferred embodiments of the invention are disclosed in the dependent claims.

The invention is based on an extract which was found to have, in a plurality of in vivo disease models, significant medical, treating, and pharmaceutical characteristics. The invention is further based on a method allowing a selective extraction of desired active ingredient from a material originating from arborescent plants.

The extract of the invention, extracted from arborescent plants and including stilbenes as well as compounds enhancing their effects, prevents inflammatory reaction of the body by preventing inflammation cytokines causing inflammation reactions. The extract also reduces the differentiation of cells into the direction of dendrite cells important in an allergic inflammation. The extract further has a relieving effect of an inflammation. This being the case, the inventive extract is usable in the treatment of any inflammation disease. It is, furthermore, useful in the prevention, alleviating, and/or treating illnesses caused by the inflammation, such as eczema, metabolic syndrome, and type 2 diabetes, as well as rheumatoid arthritis, arthrosis, gout, asthma, and possible also cancer and Alzheimer's disease.

In the light on present-day know-how, there are aspects of an inflammation in many other diseases, too. Inflammation diseases cause, despite treatments, major human suffering and significantly burden the national economy. The inventive extract, or compositions containing the extract, provide a functioning and, from the skin point-of-view, a more advantageous alternative to conventional inflammation-restraining medicines, such as cortisone.

The extract is pharmaceutically effective and non-toxic. The specific issue is that the amounts/concentrations of pinosylvin and monomethylsylvin in the extract are notably lower than the concentrations of absolute agents in the comparison tests, but yet the effects obtained from tests with disease models are at the same level, at least. The active ingredients in the extract thus have a mutually enhancing effect, and a medicinal or treating effect is obtained with very small extract concentrations. The advantage of the extract of the invention is that it does not contain substances, such as resin acids, that cause allergies easily.

The advantage of the method of the invention is that by extracting with water in a pressure it is possible to extract efficiently poorly soluble extractive agents from plant material without the need to use solvents or other chemicals in addition to water. There in not necessarily any need to purify the extract obtained, either. In most extraction known in the field, the extractive agents are first separated from the plant material with an organic solvent, whereby nearly all the small- molecule compounds in the plant will dissolve. After this, the solution thus ob- tained is purified by water-water extractions or chromatography. With the methods according to the invention, the demanding purification steps may be avoided. In the extraction method according to the invention, by changing the temperature and pressure it is possible to affect the solubility of the ingredients extracted, whereby by changing the conditions the desired extractive agents may be recov- ered.

It is a further advantage of the invention that it is economical to implement and extracts of good quality are obtained by it, which may be extensively utilized. In addition to pharmaceutical applications, it may also be utilized as a nutrition additive, as a food products, and in cosmetics. Brief description of the figures

The invention will now be described in greater detail by means of preferred embodiments and with reference to the attached figures, in which:

Figure 1A: Ahma-1 extract prevented an acute allergic paw inflammation. Mice were sensitized to ovalbumin after which they were exposed to the al- lergen in question by injecting ovalbumin to the paw (s.c. injection). The allergic paw swelling was measured after 1.5, 3, and 6 hours from injecting ovalbumin. The minor swelling caused by the vehicle injected in the control paw has been subtracted from the results. Average value + mean error of average value (SEM), n=6- 9,***p<0.001 and ** p<0.01.;

Figure IB. Reference test. Stilbenes effectively prevented an acute allergic paw inflammation. Mice were sensitized to ovalbumin after which they were exposed to the allergen in question by injecting ovalbumin to the paw (s.c. injection). The allergic paw swelling was measured after 3 and 6 hours from injecting ovalbumin. The minor swelling caused by the vehicle injected in the control paw has been subtracted from the results. Average value + mean error of average value (SEM), n=6-9, ** p<0,01 and *p<0,05. Figure 2. Ahma-I extract, stilbenes and dexametason prevented the expression of surface antigen CD54 in the THP-I cells differentiated in the direction of the dendrite cells. The cells were treated with DNC and the compounds being examined. After an incubation of 24 h the cells were recovered and incubated with the antidote of the CD 54 surface antigen for 30 min. The proportion of cells exhibiting CD54 surface antigen was determined by flow cytometry. Average value + mean error of average value (SEM), n=4, *** p<0,001, ** p<0,01 and *p<0,05.

Figure 3: Ahma-I extract, pinosylvin, resveratrol and dexametason prevented the expression of surface antigen CD86 in the THP-I cells differentiated in the direction of the dendrite cells. The cells were treated with DNCB and the compounds being examined. After an incubation of 24 h the cells were recovered and incubated with the antidote of the CD86 surface antigen for 30 min. The proportion of cells exhibiting CD86 surface antigen was determined by flow cytometry. Average value + mean error of average value (SEM), n=4, *** p<0,001, ** p<0,01 and *p<0,05.

Figure 4: The Ahma-1 extract effectively prevented the paw inflammation caused by carrageen, which is a widely-used model of an acute inflammation. The inflammatory swelling was measured after 3 and 6 hours from the injection of carrageen into the paw tissue. The minor swelling caused by the solvent, that is, vehicle injected in the control paw has been subtracted from the results. Average value + mean error of average value (SEM), n=7, ***p<0,001.;

Figure 5: The Ahma-1 extract effectively prevented the number of inflammation factors A) IL-6 and B) MCP-1 produced in the paw tissue in the acute inflammation model. The transmitter levels of the paw tissue were measured after 6 hours from the injection of carrageen. The black column is untreated control and white column is vehicle control (the paw in which only carrageen solvent was injected, that is, the vehicle instead of carrageen solution). Average value + mean error of average value (SEM), n=5, ***p<0.001.

Figure 6: DNCB increased the expression of surface antigens A CD54 and B CD86 on the THP-1 cells, which depicts the differentiation of the cells into the direction of the dendrite cells important to the creation of allergic inflammation. Ahma-1 extract and dexametason prevented the expression of these surface antigens in the THP-1 cells. The cells were treated with DNCB and the compounds being examined. After an incubation of 24 h the cells were recovered and incubated with the antidote of the A CD54 or B CD86 surface antigen for 30 min, and the proportion of cells exhibiting the surface antigen in question was determined by flow cytometry. Average value + mean error of average value (SEM), n=4,***p<0,001 and ** p<0,05.

Figure 7: Ahma-1 extract prevented dosage-dependently A nitrogen ox- ide (its metabolite nitrite was measured), B TNF, C IL-6 and D MCP-1 yield in macrophages. The cells were activated with LPS and the levels of the inflammation transmitters were measured after 24 h incubation. Dexametason was included as a known anti-inflammatory reference compound. Average value + mean error of average value (SEM), n=4, ***p<0,001, *p<0,05.

Figure 8. Ahma-1 extract effectively prevented the expression of iNOS in the macrophages. The cells were activated with LPS and the level of the inflammation enzyme was measured after 24 h incubation with the Western blot method.. Dexametason was included as a known anti-inflammatory reference compound and clearly prevented the expression of both inflammation enzymes. Actin is the charging control of the Western blot gel drive. Average value + mean error of average value (SEM), n=4, ***p<0,001.

Detailed description of the invention

The invention relates to an aqueous extract containing stilbene and lignan, extracted from arborescent plants. In particular the invention relates to an extract isolated from arborescent plants, the dry matter of which comprises: stilbenes 0.02-6 % by weight and lignans 0.5-30 % by weight. In addition, the extract may comprise flavonoids approximately 0.1-10 % by weight. The concentrations are determined as percent by weight of the dry matter. In the examples of the invention, the extract is referred to as Ahma-1 extract or ahma-1 extract.

The dry matter of the extract of the invention advantageously comprises approximately 1-6 % by weight, more advantageously 2-5 % by weight, and particularly advantageously 3-4 % by weight of stilbenes, advantageously 1-8 % by weight, more advantageously 3-7 % by weight, and particularly advantageously 4- 5 % by weight of lignans, and potentially approximately 0.1-10 % by weight of fla- vonoids.

In this context, an aqueous extract means that the extract contains water used as a solvent and at most 30 % by volume of alcohol and/or glycerol.

Stilbenes, lignans, and flavonoids are aromatic phenolic compounds. Stilbenes include, for example, pinosylvin and monomethylpinosylvin, lignans in- elude, for example, sekoicolariciresinol and matairesinor, and flavonoids are classified to include, for example, flavonols, flavones, flavanols, isoflavanols, and anto- cyanides.

In the manufacture of the inventive extract, arborescent plants and their parts are used as the raw material. A woody stem is the perennial overground stem of a plant. Arborescent plants are, as to their growth method, usually trees, bushes, twigs, or perennial garland. The following are advantageously used: branch and inner branch of a pine, pine bark, pine root, inner part of a spruce, spruce bark, spruce phloem, spruce root, twigs of forest berries, juniper, blackcurrant and redcurrant bushes. Most advantageously, the inner branch material of a pine and/or spruce is used as the raw material.

In an embodiment, the extract of the invention comprises pinosylvin, pinosylvinmonomethylether, terpin, vanillin, P-hydroxybenzoic acid, 3-hydroxy- 4metoxybenzoic alcohol, vanillin acid, azelaic acid, diconiferyl alcohol, caffeic acid, sekoicolariciresinol, nortrakelogenin, matairesinol, and pinoresinol.

In an embodiment, the extract is made from the bark of arborescent plants. In the bark extract, out of the dry matter content, the proportion of stilbenes is advantageously 0.020-0.1 % by weight, more advantageously 0.035-0.065 % by weight, and particularly advantageously 0.045-0.055 % by weight, proanthocya- nidins advantageously approximately 2.5-10 % by weight, more advantageously 4.4-8.4 % by weight and particularly advantageously 5.7-7.1 % by weight. The bark extract has an insignificant amount of lignans.

The present invention relates to the user of the inventive extract as a medicine or an active ingredient. The extract may be part of a medicine composi- tion or it may be used as the medicine is prepared. A medicine or active ingredient refers to a substance used to deter, prevent, alleviate, and/or treat an illness.

In particular, the invention relates to the use of the aqueous extract of the invention, isolated from arborescent plants, as a substance preventing and restraining an inflammation. An inflammation reaction is normally an essential de- fence mechanism of the organs, which aims at eliminating a bacterium or another disease causing element, and to restore the normal state of the organs. A failure of the regulation mechanism of an inflammation reaction may cause the reaction to turn against the organs and consequently to an emergence of diseases.

In in vivo test carried out, the extract of the invention was found to no- tably and statistically significantly prevent swelling and inflammation reactions and reduce the transmitters typical for an allergic contact eczema. The extract was further found to alleviate an inflammation. The alleviating effect of an inflammation by the extract was detected with a plurality of different meters. It is particularly interesting that the inventive extract affects both the symptoms of an inflammation, such as pain or swelling, and the mechanisms strengthening the inflammation, such as the activation of inflammation genes and gathering of inflammation cells on the inflammation area. In cell culture tests, the extract showed preventive effects on the differentiation of dendrite cells, expression of sensibilization cells and production of inflammation factors, so it may have a favourable effect on the mechanisms of allergies.

The invention relates to the use of the extract to deter, prevent, alleviate, and/or treat any illness caused by an inflammation reaction. It is particularly usable in the prevention and/or treating illnesses caused by eczema, metabolic syndrome, and type 2 diabetes, as well as rheumatoid arthritis, arthrosis, gout, asthma, and possible also cancer or Alzheimer's disease. The invention additionally relates to a method or treating an illness caused by an inflammation reaction, whereby the extract is administered to a patient as seen fit.

In addition to an aqueous extract, the inventive extract may be in the form of a solid matter. As a medicine, the extract of the invention may be in any form of dosage, such as a tablet, injection, lotion, or powder in an inhalator. The suitable pharmaceutical dosage form is determined on the basis of the purpose of use, administration point and method, and the formulation of the medicine.

The present invention also related to a pharmaceutical composition which is a mixture of the extract acting as the active ingredient and potential other active ingredients and pharmaceutically approved carriers or excipients. An excip- ient refers to a pharmacologically inactive substance which is formulated as a combination with the pharmaceutically active ingredient of the pharmaceutical composition.

Advantageously, the extract may be in the form of an ointment (paste), wax, cream, moisturising cream, gel, or milk-like cream (lotion), which is spread or smoothed on the skin inflammation area, or in the form of a liquid, such as a water- oil emulsion, oil-water emulsion, suspension or solution sprayed on the skin of the inflammation area (e.g. aerosol sprays). Nonwoven swabs or similar containing the active ingredient may also be suitable. Particularly usable are oil-carrying sprays, ointments (paste), waxes, creams, moisturising creams and gels.

The inventive extract may also be used as a dietary supplement or food product or its supplement, such as: powder, liquid, spread, tablets, and capsules. It may be a functional food product, nutritive substance, supplement, health food or drink. The inventive extract may also be used in cosmetics.

The inventive extract is safe to use. Based on toxicity tests, it is nontoxic. The dosage of the extract depends on the proof and use and method of use. An effective amount of the extract is a dosage that is enough to at least alleviate the symptoms caused by the inflammation.

The invention further relates to a method for extracting arborescent plant or their parts. In particular, the invention relates to extracting arborescent plants by subcritical hydrophilic solvent extraction. Subcritical extraction refers to extraction taking place in a pressure vessel, above the boiling point determined for the solvent in question at a normal pressure, but not in the supercritical area. By adjusting the temperature, the polarity of the solvent may be affected to so that as the temperature rises the dielectricity factor of the solvent decreases. By adjusting the temperature, it is also possible to affect the extract agent profile obtained from the extracted material. In the present invention, extraction advantageously takes place at 80 - 180 °C, more advantageously 100 - 160 °C and most advantageously 120 - 140 °C.

Hydrophilic solvent extraction refers to extraction where hydrophilic solvent is used, as a result of which non-soluble extraction agents may be left out of the extract by filtering. The solvent may be, for example, water, water-ethanol mixture, or water-glycerol mixture. Advantageously the mixture is water. The mixture may have ethanol at most 30 % and/or glycerol at most 30 %. By adding an organic solvent, such as ethanol, extraction is enhanced. Ethanol changes the polarity of the solution as acts as a chaotropic agent and thus improves the solubility of hydrophobic substances and facilitates the degradation of solid matter particles. Most of the extraction agents become easily oxidized. Consequently, oxygen may be removed from the solvent being used by, for example, boiling or bubbling by nitrogen gas or ultrasound.

In hydrophilic extraction, by varying the extraction conditions in rela- tion to the solvent, temperature and pH, the extractive agent profile dissolving in the extract. In such a case, it is possible to selectively extract different compound groups based on their polarity and properties of the functional groups. pH adjustment may be performed on the solvent prior to extraction or during extraction. The idea of pH adjustment is that the solubility of the extractive agents is based on the pH value of the solution. This is due to the fact the solubility of a molecule is affected by its partial charges, the charge of the pendant groups of faint acids and bases is in turn dependant on the pH of the solution. Adjusting the extraction solvent to the base direction enhances extraction, the pH may be, for example, pH 8.25-11.

Extraction may be performed as a single extraction or series extraction. Extraction may be enhanced by mixing during the extraction process. This may be implemented with a circulation pump or any mixing technology suitable for the extraction process. Extraction may further be boosted by ultrasound led to the extraction vessel during extraction.

The raw extract acquired from the extraction may be first filtered by a coarse filter to remove big solid matter, after which by a ceramic filter whose pore size is advantageously between 0.4 and 1.2 μηι, without restricting thereto. With an appropriate filter, a significant amount, advantageously more than 90 %, of allergenic resin acids may be removed from a pine inner branch extract, for example. Because the extractive agents become easily oxidised, filtering may also be carried out in an oxygen-free atmosphere. This may be implemented by displacing oxygen from the filter line by an oxygen-free, inert gas in relation to the desired extractive agents, such as nitrogen gas.

The permeate obtained from the extraction process may thereafter be concentrated. Concentration is most advantageously performed by reverse osmosis, nanofilms, freezing or evaporating, or combinations of the above. Once the de- sired concentration degree has been reached, the liquid concentrated extract is recovered and packed.

The extract obtained from the extraction process may be further processed into dry extract by lyophilisation or evaporation, for example.

If needed, the plant material to be extracted is preprocessed by grading and/or cleaning, as well as crushing or cutting into a suitable granular size. The extracted plant material may be fractioned before extraction by sedimenting in water. Inner branches, for example, may be separated from the other wood material by grinding the wood material into a small granular size, such as approximately 2 mm. The raw material may also be fermented.

To implement the invention, an extraction apparatus is needed, by means of which compound groups may be selectively extracted from a biomass, by using as solvent, for example, water, mixture of water and ethanol or water and glycerol, and by varying the extraction conditions in relation to the pressure, temperature and extraction time. The extraction apparatus needed in the invention comprises a preprocessing part, extraction part, separation part, and concentration part of the raw material. The preprocessing part may also be associated with a separate raw material fermentation apparatus with its storage tanks. The extraction part is a pressure vessel. The extraction part further comprises temperature and pressure gauges, by means of which the conditions are monitored. The pressure vessel has a gas regulator valve. The extraction part may further comprise a pH adjustment/feed assembly and ultrasound probe. The separation part comprises a condenser of gas exhausts, a drainage pump, coarse filter; process vessels; micro- filter; ultrafilter; nitrogenous gas apparatus, and circulation pump. The concentration part comprises a cooling concentrator with its cooling aggregate or a nano or reverse osmosis concentration apparatus or evaporation apparatus or a combination of the above.

Examples

Example 1. Acute allergic skin inflammation model

The paw inflammation model caused by ovalbumin has been used to de- scribe an allergic skin inflammation. In this model, mice are first sensitised to ovalbumin by administering ovalbumin, aluminium hydroxide, and whooping cough toxin to the abdominal cavity (intraperitoneally, i.p.] After five days, a booster dosage of ovalbumin and aluminium hydroxide is administered i.p. After two weeks from the sensibilization, the mice are put to sleep and then under the skin (subcu- taneously s.c.) of the animals to the rear paw, ovalbumin or a vehicle (solution to which ovalbumin has been dissolved, in this test PBS) is injected. The swelling response caused by inflammation is estimated by measuring the paw thickness by a plethysmometer at the beginning of the test and during the test up until six hours from the injection of ovalbumin. The compounds examined are administered to the animals orally or to the abdominal cavity 1-2 h before the ovalbumin exposure. The animals are under anaesthesia during the entire paw swelling test; they are monitored and more anaesthetic is administered, if needed. At the end of the test, the animals are put down and the paw tissue is recovered for immunological and molecule biological definitions.

The C57BL/6 mice were bred to the target and bought from a commercial actor (Scanbur A/S, Denmark). Male mice of the age of 10 -12 weeks were used in the study. The animal tests were conducted at the test animal unit of the University of Tampere and the mice were kept in standard conditions (12:12 h light: dark cycle, temperature +22 ± 1 °C, humidity 50-60 %), water and food available ad libi- turn. The animal tests were conducted in compliance with test animal legislation and appropriate project permissions (ESAVI/2139/04.10.03/2012 ja ESAVI/3280/04.10.07/2015).

500 μΐ of the extract (ahma-1) was administered to the animals (weight appr. 25 g) intraperitoneally 1 h before the ovalbumin exposure. With this dosage, the ahma-1 extract clearly and statistically significantly prevented an acute allergic swelling reaction (Figure LA).

In the reference test (Figure 1 B), pinosylvin, monomethylpinosylvin and resveratrol were used in 30 mg/kg dexametason in 2 mg/kg dosages. All the compounds being examined, pinosylvin, monomethyloinosylvin and resveratrol, as well as the dexametason used as the control compound clearly and statistically significantly prevented the inflammation reaction.

Example 2. Measuring surface antigens by flow cytometry

THP-1 cells are used in the so-called extended h-CLAT model: when cells are cultivated with compounds causing allergic contact dermatitis, they dif- ferentiate in the direction of dendrite cells and start to produce certain transmitters typical for allergic contact eczema. Of the compounds causing allergic contact dermatitis, the model employed 2,4-dinitrochlorobenze (DNCB), and the expression of CD54 and CD86 surface antigens was measured with flow cytometry. In flow cytometry, cells marked with fluorescent substance are radiated with a laser beam and the light the cells scatter and the light the excited tracer emits are measured. The cell populations may be distinguished from each other on the basis of the structure, size or, like in this study, the surface antigens identified by the tracer.

THP-I cells were divided on a 24 microwell plate at cell density 1 million cells/well and DNCB (5 ug/ml) and the examined compounds were added immedi- ately after the cell division. The cells were incubated for 24 h, after which the cell suspension was recovered, cells were centrifuged down (400xg, 5 min) and purified by resuspending then twice with 1 % BSA-PBS. After the purifications, the cell samples were blocked (20 min, human Fc receptor binding inhibitor, eBioscience, USA) and incubated with antidotes or isotype controls of surface antigens CD54 (Anti-Human [lCAM-1] FITC, eBioscience, USA) and CD86 (Anti-Human [B7-2] PerCP-eFluor 710, eBIOscience, USA) for 30 minutes. The cell samples were purified twice 1 % BSA-PBS, 400xg, 5 min) and resuspended to 0.4 ml of 0.1 % of BSA- PBS solution and transferred to 12 x 75 mm tubes. The proportion of cells expressing CD54 and CD86 surface antigens was determined with flow cytometry (FACSCanto 11 Flow Cytometer, BD Biosciences, USA). DNCB 5 (pg/ml) significantly added the proportion of CD54 and CD86 positive cells compared to vehicle control. Ahma-I extract, pinosylvin and resvera- trol prevented statistically significantly the expression of surface antigens CD 54 and CD86, and thus the differentiation of the THP-I cells in the direction of the den- drite cells under the conditions examined. Ahma-I extract had a minor, but statistically significant effect on the expression of both surface antigens (Figures 2 and 3).

Example 3. Acute inflammation model (paw swelling caused by carrageen)

As the model of an acute inflammation, a paw inflammation caused by carrageen, is widely used. The compounds examined are administered to the ani- mals orally or to the abdominal cavity 1-2 h before putting the animal to sleep. The sizes of the rear paws of the animal put to sleep are measured by plethysmometer, after which carrageen, starting an acute inflammation reaction is injected to the right paw, and vehicle to the left paw (solution to which the carrageen has been dissolved, in this test PBS). The swelling of both paws is measured during the test up until 6 h from the carrageen injection. The animals are under anaesthesia during the entire paw swelling test; they are monitored and more anaesthetic is administered, if needed. At the end of the test, the animals are put down and the paw tissue is recovered for immunological and molecule biological definitions.

The C57BL/6 mice were bred to the target and bought from a coramer- cial actor (Scanbur A/S, Denmark). Male mice of the age of 10 -12 weeks were used in the study. The animal tests were conducted at the test animal unit of the University of Tampere and the mice were kept in standard conditions (12:12 h light: dark cycle, temperature +22 ± 1 °C, humidity 50-60 %), water and food available ad libitum. The animal tests were conducted in compliance with test animal legislation and project permission ESAVI/5019/04.10.03/2012.

Ahma-1 extract was administered to the animals (average weight 25 g) 500 μΐ intraperitoneally 1 h before the carrageen injection. The pinosylvin and monomethylpinosylvin concentrations of the extract were 50 and 90 μg/ml (Joonas Kortelainen /Ahma insinoorit Oy) whereby the computational pinosylvin dosage was 1 mg/kg and monomethylpinosylvin 1.8 mg/kg.

The ahma-1 extract clearly and statistically significantly prevented an acute inflammation reaction in a paw swelling model caused by carrageen (see Figure 4). The result is in line with a previously published test in which it was noted thatstilbenes (100 mg/kg) and the control compound dexametason effectively pre- vent an acute inflammation reaction in a similar model (Laavola et al, J Agric Food Chem 2015, 63, 3445-3453). After the last test point, the animals were put down and the paw tissue was recovered in a buffer solution [Tris (50 mM, pH 7.4), NaCl (150 mM), 0.5 % Triton X ja protease and phosphatase inhibitors phenylmethylsulphonylfluoride (0,5 mM), sodiumorthovanadate (2 mM), leupeptin (0.10 μ /ηι1), aprotinin (0.25 μ / ι1) and NaF (1.25 mM)]. Inflammation cytokine IL-6 and MCP-1 concentrations were measured from the tissue by ELISA (DuoSet, R&D Systems).

Ahma-1 extract prevented effectively and statistically significantly the production of both the studied inflammation cytokines IL-6 and MCP-1 in the paw tissue (Figure 5).

In Ahma-1 extract, the pinosylvin and monomethylpinosylvin concentrations are 50 and 90 μg/ml, whereby in the in vivo models the computational stil- bene dosages administered to the animals were 1 mg/kg (pinosylvin) and 1.8 mg/kg monomethylpinosylvin). Thus, the stilbene dosages obtained from the extract were significantly smaller than the stilbene amounts (30 and 100 mg/kg) ad- ministered as absolute agents in the reference tests, but the efficiency of the extract was in the same neighbourhood.

Example 4. Effect of Ahma-1 extract on dendrite cells - extended h-CLAT model

THP-1 cells are used in the so-called extended h-CLAT model: when cells are cultivated with compounds causing allergic contact dermatitis, they differentiate in an allergic inflammation in the direction of dendrite cells and start to produce transmitters typical for allergic contact eczema. The model employed type compound 2,4-dinitrochlorobenzene (DNCB) causing allergic contact dermatitis, and the expression of CD54 and CD86 surface antigens was measured with flow cytometry. In a further study, the mechanisms were studied by measuring p38, JNK and ERK1/2 MAP kinase phosphorylation (activation) by the Western Blot method.

In this extended h-CLAT model, the effect of the ahma-1 extract on the expression of the surface antigens was studied. Dexametason was included as a known anti-inflammatory control compound.

The differentiation of the THP-1 cells into the direction of the dendrite cells important to the creation of allergic inflammation was measured by determining the expression of the cell antigens CD54 and CD86 by flow cytometry. In flow cytometry, cells marked with fluorescent substance are radiated with a laser beam and the light the cells scatter and the light the excited tracer emits are measured. The cell populations may be distinguished from each other on the basis of the structure, size or, like in this study, the surface antigens identified by the tracer. THP-1 cells were divided on a 24 microwell plate at cell density 1 million cells/well and DNCB (5 μ /ηι1) and the examined compounds were added immediately after the cell division. The cells were incubated 24 h, after which the cell suspension was recovered, cells were centrifuged down (400xg, 5 min) and puri- fied by 1 % BSA-PBS twice. After the purifications, the cell samples were blocked (20 min, human Fc receptor binding inhibitor, eBioscience, USA) and incubated with antidotes or isotype controls of surface antigens CD54 (Anti-Human [lCAM-1] FITC, eBioscience, USA) and CD86 (Anti-Human [B7-2] PerCP-eFluor 710, eBIOsci- ence, USA) for 30 minutes. The cell samples were purified twice (1 % BSA-PBS, 400xg, 5 min) and resuspended to 0.4 ml of 0.1 % of BSA-PBS solution and transferred to 12 x 75 mm tubes. The proportion of cells expressing CD54 and CD86 surface antigens was determined with flow cytometry (FACSCanto 11 Flow Cytom- eter, BD Biosciences, USA).

DNCB 5 ^g/ml) significantly added the proportion of CD54 and CD86 positive cells compared to vehicle control. The control compound dexametason (1 uM) an to a lesser extent ahma-1 extract (1:500 dilution) prevented statistically significantly the expression of surface antigens CD54 and CD86, and thus the differentiation of the THP-1 cells in the direction of the dendrite cells under the conditions examined (Figure 6). Example 5. Effect of Ahma-1 extract on J774 macrophages

The J774 macrophages of a mouse are a widely-used cell line in inflammation studies. When cells are exposed to a bacterial lipopolysaccharide (LPS), they activate to produced enzymes typical for a inflammation, such as inducible nitrogen oxide synthase (iNOS) and cyklooxygenase 2 (COX-2) and nitrogen oxide and inflammation cytokines.

J774 cells (American Type Culture Collection, USA) were cultivated at 37 °C and in 5 % C02 DMEM cultivation medium (Lonza Group Ltd, Switzerland), containing 10 % serum (Lonza Group Ltd, Switzerland) and 100 U/ml penicillin, 100 ug/ml streptomycin and 250 ng/ml amphotericin B (Invitrogen Co., USA). The cells were divided on a 24 microwell plate at cell density 0.2 million cells/well and cultivated for 72 hours before the beginning of the test.

At the beginning of the test the cells were treated by LPS and the examined compounds and after 24 h incubation the cell cultivation medium was recovered for determining nitrite (metabolite of nitrogen oxide in cell cultivation me- dium) and inflammation cytokines TNF, IL-6 and MCP-1, and proteins were isolated from the cells to measure iNOS expression. Nitrite was measured with the Griess method (Green et al, Anal Biochem 1982, 126, 131-138), TNF, IL-6 and MCP- 1 production with the ELISA methods, and iNOS protein expression with the Western Blot method.

Ahma-1 extract prevented dosage-dependently the production of in- flammation factors nitrogen oxide, TNF, IL-6 and MCP-1 in activated J774 macrophage cells (Figure 7).

To measure the expression levels of iNOS enzymes, important in an inflammation, the cells were broken and cell proteins isolated to an extraction solution (10 mM Tris, pH 7.4, 5 mM EDTA, 50 mM NaCl, 1 % Triton X-100, 0.5 mM PMSF, 1 mM sodiumorthovanadite, 20 μg/ml leupeptin, 50 μg/ml aprotinin, 5 mM sodium fluoride, 2 mM sodiumpyrophosphate and 10 μΜ n-octyl-E-D-glucopyra- noside). The samples were centrifuged (13 400xg, 4 °C, 10 min), the supernatants were recovered and frozen (-20 °C) in and SDS buffer. The protein content of the supernatant was determined with the Coomassie sini method.

Protein samples were analyzed with the Western blot method. The cell sample (20 μg of protein) was pipetted to (10 % SDS polyacrylamidegel and after the gel run was transferred on a nitrocellulose membrane (iBlot®, Thermo Fisher Scientific, USA). After the transfer, the membrane was incubated in the blocking solution (5 % milk TBS/T 20 mM Tris pH 7.6, 150 mM NaCl, 0.1 % Tween-20) 1 h at room temperature and then with the primary antidote (5 % milk-TBS/T) at +4 °C overnight. The next day the membrane was washed with TBS/T, incubated with the secondary antidote (5 % milt TBS/T) 1 h at room temperature and washed with TBS/T. The combined antidote was determined by means of chemilumines- cence (Super Signal® West Dura, Pierce, Thermo Fisher Scientific, USA) Image Quant LAS 4000 mini imaging equipment (GE Healthcare Bio-Sciences AB, Sweden).

The quantization of the chemiluminescence was done with the Image Quant TL program (GE Healthcare Bio-Sciences AB, Sweden). The antidotes used are shown in appendix 3.

Even as a low concentration (1: 500 dilution), Ahma-1 effectively prevented, and as higher concentrations entirely, the expression of iNOS protein (Figure 8).

Based on the results, ahma-1 extract has an obvious inflammation-alleviating effect measured at the cell level. Example 6. Cytotoxicity measurements

The cytotoxicity of the Ahma-1 extract in the cell lines used was measured with the XTT test (Cell Proliferation Kitt II; Roche Diagnostics, Germany). The test measures the effect of the examined compound on the activity of the mitochon- drional dehydrogenase of the cells. The toxicity of Ahma-1 extract at different concentrations was dependent on the cell line as per Table 1.

Table 1. Cytotoxicity of Ahma-1 extract

X = toxic

0 = non-toxic

With J774 cells and HaCaT cells an 1: 10 dilution from the original ahma-1 extract was toxic on the cells measured after 24 h incubation and concentrations weaker than this were not toxic. With THP-1 cells, dilutions up to 1:200 were toxic on the cells. A person skilled in the art will find it obvious that, as technology advances, the basic idea of the invention may be implemented in many different ways. The invention and its embodiments are thus not restricted to the above-described examples but may vary within the scope of the claims.