Agent eliciting immunostimulating effect

FIELD: medicine, immunology.

SUBSTANCE: invention is designated for treatment and prophylaxis of diseases associated with the immune system insufficiency in chronic relapsing inflammatory diseases, in cases of insufficient effectiveness of antibacterial and anti-inflammatory therapy. Agent eliciting the immunostimulating effect comprises dropwort (Filipendula ulmaria L.) dried, milled, above-ground part, dropwort (Filipendula ulmaria L. Maxim.) dry an aqueous or dry alcoholic extract prepared in the definite ratio of raw : extractant.

EFFECT: valuable medicinal properties of agent.

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The invention relates to medicine, more specifically to immunology and AIDS, having an immunostimulating action, used for the treatment and prevention of diseases associated with deficiency of the immune system in chronic relapsing diseases of an inflammatory nature, in cases of lack of effectiveness of antibacterial and anti-inflammatory therapy.

Known means of synthetic and natural origin used to stimulate the immune system. These include substances of microbial (bacterial and fungal) origin (prodigiozan, pyrogenes, ribomunyl, nukleinat sodium); substances of vegetable origin (tonsillar, immunal, different preparations of Echinacea). Immunoregulatory peptides of natural origin: drugs from the thymus gland (thymalin, taktivin, timoptic, vilonen); preparations of spleen (splenin); preparations of bone marrow: mielopid. Cytokines (natural and recombinant). Synthetic and chemically pure immunomodulators: analogs of drugs of endogenous origin (timogen, immunofan, Likopid); drugs (dalargin, levamisole, Dibazol, ditiocarb, polyoxidonium) [2, 3, 5, 7, 8, 9].

The presence of side effects in the majority of the listed drugs in the form of chills, fever headache, dyspeptic disorders, pain at the site of parenteral administration of the drug, adverse effects during pregnancy, asthma, allergic diseases greatly restrict the use of many drugs with immune stimulating effects.

Closest to the claimed tool is of plant origin, having an immunostimulating action, tincture of Echinacea purpurea (Echinacea purpurea (L) Moench (Asteraceae)) (prototype) [1].

Development of new natural immunomodulators with low side effects that are relevant.

The problem solved by this invention is an expanding Arsenal of low means, having an immunostimulating action, for prophylaxis and treatment of acute and chronic inflammatory processes, immune disorders of different origin and other conditions associated with low activity of the immune system without side effects and with more prolonged stimulant.

This object is achieved in that as a means of having an immunostimulating action, use dried powdered aerial part of meadowsweet Filipendula ulmaria (L) Maxim (LV 1); a dry aqueous extract of dried powdered aerial parts meadowsweet is atlantaga Filipendula ulmaria (L) Maxim when the ratio of raw material : solvent 1:9 (LV 2); a dry alcoholic extract 40 percent ethyl alcohol dried powdered aerial parts of meadowsweet Filipendula ulmaria (L) Maxim when the ratio of raw material : solvent 1:9 (LV 3); dry alcoholic extract 70 percent ethyl alcohol dried powdered aerial parts of meadowsweet Filipendula ulmaria (L) Maxim when the ratio of raw material : solvent 1:9 (LV 4).

Meadowsweet (Filipendula vyazolistny) - Filipendula ulmaria (L) Maxim. The family Rosaceae. A perennial herb with a creeping rhizome and high solid ribbed stems, up to 150, see the Leaves are thick, broken-pinnate, green top, white bottom. Flowers yellowish-white, gathered in a dense panicle. Blooms in June-July.

Common in forest and forest-steppe and mountain-forest belt everywhere. Grows in damp woods and groves, stands on the banks of rivers and marshes, wet meadows [4, 6].

The plant contains an essential oil consisting of salicylic aldehyde and heliotropine, ascorbic acid, carotene, the glycoside serein, vanilla, terpenes, free salicylic acid, anthocyanin, flavone, tanning matters and pyrogallol groups, volatile[1, 4, 6].

New in the present invention is that identified a new property aerial parts of meadowsweet (Filipendula ulmaria (L) Maim), namely its immunostimulating effect. He has a longer-lasting stimulatory effect on the performance of specific humoral immune response and nonspecific resistance of the organism in comparison with widely used in practical medicine Echinacea purple.

From literature data it is known the use of drugs of meadowsweet (Filipendula ulmaria (L.) Maxim) in the form of a decoction made from the roots and flowers as an antiseptic, anti-inflammatory, diuretic, astringent remedies for eczema, atopic dermatitis, gout, rheumatism, edema, pleurisy [1, 3], in the form of lotions and mouthwashes with stomatitis, gingivitis, gum disease [3]. The decoction is used for gynecological disorders, in the treatment of purulent wounds, ulcers, [1, 4]. In clinical medicine is used as an integral part of medicine, Zdenko assigned when the bladder papillomatosis and anatsidnyh gastritis, tachycardia [I]. Shows high antioxidant activity of the decoction of the flowers of meadowsweet (Fihpendula ulmdria (L.) Maxim) [Melpomenia, 2000]. Immunostimulatory properties of meadowsweet not described.

Main registered drugs meadowsweet are: meadowsweet flowers (haemostatic, protivogemorroidalny, diuretic), registration number 88/229/7; meadowsweet castelpetroso to revise and root (an integral part of medicine, Zdrenka, hemostatic, protivogemorroidalny, diuretic), registration number 74/270/6.

The authors are not found in the analyzed literature, this combination of essential features for use as an immunostimulating agent of above-ground dried crushed part of meadowsweet, dry, aqueous and alcoholic extract.

A comparison of the claimed funds: LV 1, LV 2, LV 3, LV 4 with known shows that first proposed the use of extracts from dried powdered aerial parts of meadowsweet as an immunostimulating agent.

Thus, the claimed means of having immunostimulant effect, meets the criteria of the invention of "Novelty" and "Inventive step"because it is not obvious to a person skilled in the art. The proposed solution meets the criteria of the invention "Industrially applicable"because it can be successfully used in practical health care.

Immunotropic activity funds: LV 1, LV 2, LV 3, LV 4, was studied in experiments on animals, and shows its immunostimulating effect in comparison with the immunostimulating action of tinctures of Echinacea purpurea. We studied the activity of extracts from the aerial parts of meadowsweet, polucen is x in the following ways:

Example 1. the aboveground part of meadowsweet was dried and crushed to fine powder (LV 1);

Example 2. dried powdered aerial part of meadowsweet was boiled in distilled water at the ratio of raw material and extractant 1:9 on a water bath for 15 min, then was cooled for 45 min, filtered through 4 layers of gauze, and then was evaporated to dryness (LV 2);

Example 3. dried powdered aerial part of meadowsweet was filled with 40 percent ethyl alcohol in the ratio of raw material and extractant 1:9, insisted 14 days, the extract obtained was filtered through 4 layers of gauze, and then was evaporated to dryness (LV 3);

Example 4. dried powdered aerial part of meadowsweet filled in 70 percent ethyl alcohol in the ratio of raw materials and extravanga 1:9, insisted 14 days, the extract obtained was filtered through 4 layers of gauze, and then was evaporated to dryness (LV 4).

Material and methods.

The experiments were performed on 180 mice of both sexes CBA/CaLac 1 category (conventional linear mouse) (certificate available)obtained from the nursery of the Institute of pharmacology of RAMS Siberian branch. The animals were conducted in accordance with rules adopted by the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes Strasbourg, 1986). Before and during the experiment, the mouse was in standard plastic cages (10-12 individuals) with small wood chips on a standard diet. All experiments were conducted in the autumn-winter period.

Experimental model of cytotoxic immunosuppression was created using standard drug cyclophosphamide (production of Saransk plant "Biochemist"), which was dissolved in sterile saline and were injected into mice intraperitoneally once at the maximum tolerated dose (250 mg/kg). Model experimental stress reproduced by 16-hour immobilization in a hollow closed cylinder.

In this work, we used a tincture of Echinacea purpurea 40% ethyl alcohol (NEP) "Echinacea-Galeno Pharm" (JSC "Pharmaceutical factory of St. Petersburg") (prototype), as the comparison drug. Before the introduction of the experimental animals, the extract was diluted with distilled water 2 times and evaporated to the original volume to remove the alcohol. Tincture of Echinacea purpurea and the proposed means of LV 1, LV 2, LV 3, LV 4, obtained in accordance with examples 1, 2, 3 and 4, animals were injected through a tube into the stomach within 5 days from the experimental exposure dose of 50 mg/kg as a suspension in distilled water to a volume of 0.2 ml of the Control animals were injected with the appropriate volume of the distilled water.

In the study of humoral immune response of mice were immunized minimum dose of sheep erythrocytes (DL) - 5× 106/mouse (once intraperitoneally in a volume of 0.2 ml) at the end of the 5-day course introducing the NEP or LV 1, 2, 3 and 4. On day 7 after immunization were determined absolute (× 106/organ) number of antibody productive cells (AFC) in the spleen of mice according to the method of Cuningham [10] and the tiger (Log2) antibodies (AT) in the serum using standard reaction of haemagglutination (DSA).

The phagocytic activity of peritoneal macrophages was determined by [11]. After decapitate mice were injected intraperitoneally with 5 ml of physiological solution and produced the fence contents. The concentration of living cells brought to 0.75× 106cells/ml of the resulting suspension was poured into plastic Petri dishes with a diameter of 40 mm, incubated 1 hour at 37° C, then poured adosados and was added a solution of neutral red, made of 1 part saturated solution of neutral red and 99 parts of Hanks solution. After incubation for 1 hour at 37° adosados poured in each Cup was added to 3 ml lyse solution (8 ml of 0.2 M solution of PA2NRA4·N2About connected with 12 ml of 0.1 M citric acid solution, using 1 N NaOH solution was made pH to 4.2; 24 parts received nitrate buffer is added 76 parts 96% ethanol) and stirred. The solution was poured into the cell width of 0.5 cm and measured the optical density on the photocolorimeter CPK-2MP at a wavelength of 540 nm, expressing the activity of peritoneal macrophages in units of optical density x1000.

Statistical processing of the results was carried out using the statistical software package "Statistica" with a preliminary estimate of the normal distribution and the t-student test.

The results of the study.

In the study of the absolute amount of antibody productive cells in the spleen of mice treated with LV 1, LV 2, LV 3, LV 4 in norm and after the extreme effects (immobilization stress, cyclophosphamide), it was revealed that LV 1, LV 2, LV 3, LV 4 on day 7 observations led to the normalization of the absolute number of KLA in animals immunized with EB on the background exposure to stress or administration of cyclophosphane, in contrast to the experimental group who did not receive the proposed means (Table 1).

Administration of the extract NEP (prototype) led to the increase of the studied index on day 7 observations, but the absolute amount of the KLA in these experimental groups was statistically lower than in the introduction of the proposed tools (Table 1).

The content of serum hemagglutinins class M with the introduction of the proposed tools on the 7 day of observations was reduced in the group animal is, immunized DL after immobilizing stress, in comparison with the corresponding group not treated with the drug (Table 2). This fact indicates the offset switching synthesis of IgM to IgG in the earlier period, which is indirectly confirmed by the increase on day 7 research hemagglutinin titer of class G to the corresponding values in the control group (Table 3). After the introduction of the proposed means to mice given cyclophosphamide, there was an increase tiger IgM antibodies in comparison with the corresponding control group (Table 2). The titer of serum hemagglutinin class M with the introduction of the NEP group of animals after application of cyclophosphamide was on day 7 of the studies was significantly lower than in the groups of animals treated with the proposed tool: LV 1, LV 2, LV 3, LV 4.

Dynamics of the content of immunoglobulines class G in the serum of animals treated with the NEP, was similar to the dynamics of this indicator in the groups of animals after the course the introduction of the proposed tools.

When comparing the phagocytic activity of peritoneal macrophages in the introduction as the NEP (prototype), LV 1, LV 2, LV 3 and LV 4 (claimed vehicle) on day 7 observations revealed a stimulating effect of all the drugs in the target rate in the group who underwent immobilization stress, or the introduction of qi is lafontana compared to the respective control groups without the injection.

Thus, the proposed means of LV 1, LV 2, LV 3, LV 4 resulted in a more pronounced and prolonged stimulation of the humoral immune response than the introduction of the NEP (prototype). This LV 1, LV 2, LV 3 and LV 4 almost similar level of impact on the specific humoral response. The influence of nonspecific resistance of the organism (the phagocytic activity of peritoneal macrophages) LV 1, LV 2, LV 3 and LV 4 (rated medium) was comparable with the effect of the NEP (prototype).

Literature.

1. Savrasov VI, Kitaev R.I., Khmelev CYP Medicinal plants: Therapeutic and prophylactic use. Voronezh: Publishing house of Voronezh state University, 1993. - 480 S.

2. Leskov VP, Chiradeep A.N., Gorlina NICHOLAS, Novozhenov VG Clinical immunology for clinicians. M., 1997. - 124 S.

3. Mashkovsky PPM Medicines. In 2 parts.- 12th ed., revised and expanded. M.: Medicine, 1993.

4. Minaev VG Medicinal plants of Siberia. Novosibirsk: Nauka, 1991. - 431 S.

5. D.K. Novikov, Derkach YU.N., Novikov P.D. Immunotropic drugs // In the world of drugs. - 1999. No. 2. - P.48-57.

6. Plant resources of the USSR: Flowering plants, their chemical composition and use; Family Hydrangeaceae - Haloragaceae, Leningrad: Nauka, 1987. - 326 S.

7. Xed B.C., Sergeev, A.S., Korostelev, S.A. the Analysis of contemporary trends in the creation of immunotropic of the Experimental and clinically. Pharmacol. - 1995. No. 3. - P.3-7.

8. Khaitov R.M P.M., Pinegin B.V. Basic concepts of immunotropic drugs // Immunology. - 1996. No. 6. - P.4-9.

9. Shirinsky B.C. Secondary immunodeficiencies problems of diagnosis and treatment. Novosibirsk, 1997. - 111

10. Cuningham, A.I. // Nature. - 1965. - V.207. - R.

11. Vrav B., J. Hoebeke, Saint-Girillain M. e.a. // Scand. J. Immunol. - 1980. - Vol.11 - P.147-153.

Table 1
GroupAOK
Intact control2,28± 0,18
Immunization DLof 11.69± 0,29* * *
Immobilization + immunizationa 3.87± 0,24* # # #
Cyclophosphamide + immunization4,54± 0,53* # # #
NEP + immunization11,98± 0,13* * *
NEP + immobilization + immunization6,54± 0,52* * * # # $
NEP + cyclophosphamide + immunization9,24± 0,98* * * # $$
LV + immunizationwas 9.33± 0,67* * *
LV + immobilization + immunizatioof 10.58± 0,78* *
LV + cyclophosphamide + immunizationa 9.09± 0,75* * *
LV + immunization
LV + immobilization + immunizationwas 9.33± 0,67* * *
LV + cyclophosphamide + immunization8,63± 1,01* * * # #
LV + immunization9,14± 0,17* * *
LV + immobilization + immunization10,24± 0,58* *
LV + cyclophosphamide + immunizationwas 12.75± 0,81* * * $$$
LV + immunization13,12± 0,97* * *
LV + immobilization + immunization10,89± 0,84* *
LV + cyclophosphamide + immunization9,98± 0,81* * * $
Note (hereinafter in tables 2, 3). * - reliability of differences with the background group; # - with the control group EB $ - with an appropriate control group (group immunized EB on the background of injection).

Table 2
GrouplgM
Immunization DL2,75± 0,92
Immobilization + immunization3,99± 0,52# # #
Cyclophosphamide + immunization2,32± 0,32#
NEP + immunization 3,05± 0,22
NEP + immobilization + immunization3,63± 0,92# $
NEP + cyclophosphamide + immunization2,92± 0,57$
LV +immunization2,95± 0,97
LV +immobilization + immunization3,00± 0,78# $
LV +cyclophosphamide + immunization2,250,75#
LV + immunization2,65± 0,65
LV + immobilization + immunization2,95± 0,47# $$
LV + cyclophosphamide + immunace2,50± 0,41
LV + immunization2,25± 0,22
LV + immobilization + immunization3,20± 0,16# $
LV + cyclophosphamide + immunization3,40± 0,38# # $$
LV + immunization2,00± 0,35# #
LV + immobilization + immunization3,00± 0,74# # $$
LV + cyclophosphamide + immunization2,65± 0,68$

Table 3
GroupIgG
Immunization DL11,57± 0,92
Immobilization + immunization7,65± 0,28# #
Cyclophosphamide + immunization 6,52± 0,37# #
NEP + immunization10,52± 1,07
NEP + immobilization + immunization9,12± 0,80$
NEP + cyclophosphamide + immunization7,92± 0,49$
LV 1 + immunization10,02± 1,00
LV 1 + immobilization + immunization6,65± 0,88# # $
LV 1 + cyclophosphamide + immunization6,82± 0,65# #
LV + immunization9,02± 0,69#
LV + immobilization + immunization7,12± 0,57# #
LV + cyclophosphamide + immunization7,02± 1,01# #
LV + immunization11,82± 0,26
LV + immobilization + immunization11,12± 1,16$$
LV + cyclophosphamide + immunization9,92±, and 0.40$
LV + immunizationof 10.72± 1,27
LV + immobilization + immunization9,92± 0,89$$
LV + cyclophosphamide + immunization7,92± 0,81# $

Table 4
GroupFAPM
Intact control91,2± 7,20
Immobilization80,9± 8,20* *
Cyclophosphamide51,2± 6,60* * *
NEP125,3± 3,10*
NEP + immobilization114,3± 6,54$$
NEP + cyclophosphamide103,2± 3,69$$$
LVof 120.5± at 8.36* *
LV + immobilization105,2± 9,6$$
LV + cyclophosphamide95,2± 5,77$$
LV96,2± 6,60
LV + immobilization115,6± 13,65* $
LV + cyclophosphamide90,3± 5,56* $
LV150,8± 7,60* *
LV + immobilization138,1± 15,60
LV + cyclophosphamide128,8± 6,78* $$
LV145,6± 8,65* *
LV + immobilization130,2± to 10.62
LV + cyclophosphamide11b,3± 9,78* $$
Note. * - reliability of differences with the background group; # - with the control group EB $ - with an appropriate control group (receiving only medication).

1. Means, having an immunostimulating action, including the extraction of plant material, characterized in that it contains su is th water or dry alcoholic extract of meadowsweet Filipendula ulmaria (L.) Maxim, obtained when the ratio of raw material:solvent 1:9.

2. The tool, according to claim 1, characterized in that it consists of the dried powdered aerial part of meadowsweet.

3. The tool, according to claim 1, characterized in that it contains 40%alcohol extract.

4. The tool, according to claim 1, characterized in that it contains 70%alcohol extract.



 

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