RU2166325C2 - Vegetable immunostimulating agent - Google Patents

Abstract

FIELD: medicine, phyto- -therapy. SUBSTANCE: invention proposes to use (+)-R-licorici- dine (I) as a component of Ural licorice roots and also dried powder and treated Ural licorive roots extract containing 12-38 wt.-% of substance as the source of immunostimulating agents of vegetable origin. Proposed immunostimulating agents are not toxic, they can be isolated from available vegetable raw and do not show adverse effects that are characteristic for the known plant immunostimulating agents of terpenoid nature. EFFECT: improved properties of agent. 2 cl, 3 tbl, 4 ex

Description

 The invention relates to medicine, specifically to means for increasing the body's defenses. The effectiveness of vaccines that cause persistent and intense immunity, to a large extent, depends on drugs that can enhance the effect of immunization. For this purpose, adjuvants and immunostimulants are used. Currently, an active search and study of such drugs from plant materials is underway.

 It is known to use the QuilA fraction, isolated from the South American tree Quillaia saponaria and representing a mixture of triterpene glycosides, to activate the immune response of a saponin fraction [1]. Based on this extract, a drug is being developed - ISCOMs - an immunostimulating complex consisting of antigen, cholesterol, phospholipids and natural saponins [2]. The immunostimulating activity of cycloarthran glycosides is known — curculigosaponins C and F from Curculigo orchioides (content 0.13–0.20%) [3].

 The disadvantage of these drugs is the inaccessibility of plant materials, the manifestation of the indicated drugs immunosuppressive properties when used in high doses, as well as low efficiency when administered orally.

 The immunostimulating effect of β glycyrrhizic acid, the main component of licorice root, is also known. The main disadvantage of this drug in terms of its widespread use is the mineralocorticoid effect [4].

 The objective of the invention is to expand the arsenal of funds from domestic raw materials that increase the body's defenses.

 The problem is solved by using a well-known substance of the flavonoid class, a group of isoflavans, specifically (+) - (R) -licoricidin, formula (I) [5], isolated from ethanol extract of Ural licorice Glycyrrhiza uralensis F.

Аналогами по строению соединения (I) являются природные изофлаваны - (-)-дауртин (II), (+) И (-)-мукронулатолы (III), (+) и (-)-веститолы (IV), унаниизофлавон (V) [6], выделенные из бразильских видов лекарственных растений, а также (+) 7-O-метилликорицидин (VI), найденный в солодке уральской [7] . Описан также полный синтез соединения (VI) [8]. Соединение (VI) и содержащие его экстракты корня солодки обладают транквилизирующей и антидотной активностью [7].

The analogs in the structure of compound (I) are natural isoflavans - (-) - daurtin (II), (+) And (-) - mucronulatols (III), (+) and (-) - westitols (IV), unaniisoflavon (V) [6], isolated from Brazilian species of medicinal plants, as well as (+) 7-O-methyllicoricidin (VI), found in Ural licorice [7]. The complete synthesis of compound (VI) is also described [8]. Compound (VI) and licorice root extracts containing it possess tranquilizing and antidote activity [7].

II (R¹=R⁴=OH; R²=R³=R⁵=R⁶OMe; R⁷=R⁸=H);
III (R¹=R⁴=OH; R²=R⁶=R⁷=R⁸=H; R³=R⁵=OMe);
IV (R¹=R³=OH; R²=R⁴=R⁶=R⁷=R⁸=H; R⁵=OMe;
V (R¹=R⁴=OH; R²=R⁷=H; R³=R⁵=R⁸=H; R⁶=Me₂CH);
VI (R¹=R⁷=OMe; R²=R⁶=H; R³=R⁵=OH; R⁴=R⁸=CH₂CH C(Me)₂).

II (R ¹ = R ⁴ = OH; R ² = R ³ = R ⁵ = R ⁶ OMe; R ⁷ = R ⁸ = H);
III (R ¹ = R ⁴ = OH; R ² = R ⁶ = R ⁷ = R ⁸ = H; R ³ = R ⁵ = OMe);
IV (R ¹ = R ³ = OH; R ² = R ⁴ = R ⁶ = R ⁷ = R ⁸ = H; R ⁵ = OMe;
V (R ¹ = R ⁴ = OH; R ² = R ⁷ = H; R ³ = R ⁵ = R ⁸ = H; R ⁶ = Me ₂ CH);
VI (R ¹ = R ⁷ = OMe; R ² = R ⁶ = H; R ³ = R ⁵ = OH; R ⁴ = R ⁸ = CH ₂ CH C (Me) ₂ ).

 Licorice preparations are known - liquiquiriton - the sum of flavonoids (content of at least 55% in terms of lucurazide) and flacarbin used in the treatment of gastric ulcer [9]. The immunostimulating properties of these extracts, as well as individual phenolic compounds from licorice, have not been studied [10, 11].

(+) - R-Licoricidin (I) as an immunostimulant can be used both in pure form or in the form of a dried extract of licorice root containing from 12 to 38% of substance (I). In addition to the indicated flavan, the extract contains flavans, flavonoids and isoflavonoids containing prenyl substituents at the positions of C ⁵ , C ⁷ , C ⁸ , C ^{2 '} , C ^3' , C ^{4 '} atoms, as well as glycoside-containing (iso) -flavonoids.

 A method for producing extracts consists in processing the ground roots of Glycyrrhiza uralensis F. with ethanol at room temperature, followed by extraction of the active components from the evaporated and dried alcohol extract with methyl tert.-butyl ether. The yield of evaporated and dried extract is 8.5 - 10% for air-dry raw materials. The content of substance (1) in the studied extracts was 6–55%. The output of the sum of extractive substances of phenolic nature, as well as the ratio of components depends on the place of collection and the time of collection of raw materials.

 Compound (I) is isolated from an evaporated alcoholic extract of licorice root by treatment with diethyl ether followed by column chromatography of an ether solution on silica gel. To obtain an individual crystalline substance, the enriched fractions were recrystallized from ether-hexane. The yield of the compound is 0.31% on air-dry raw materials.

 An advantage of the invention is the possibility of the integrated use of plant materials of Ural licorice by processing the phenolic components of licorice that accumulate upon receipt of glycyrrhizic acid drugs - glycyrrhizum and glycyrrhizin [9].

 The immunostimulating properties of the patented compound and its extracts were studied on outbred mice weighing 18-20 g when immunized with a weak antigen [bovine serum albumin (BSA)]. The immunization schedule was as follows. The primary immune response in animals was induced by subcutaneous administration of 500 μg BSA in 0.5 ml of sterile saline. The secondary immune response was elicited by reintroduction of the antigen after 28 days (at a dose of 100 μg).

 The test substances were administered in doses of 5 mg / kg or 100 mg / kg in 0.5 ml of sterile saline solution intraperitoneally (ip) or subcutaneously (s / c). The control group of animals was injected with antigen without drug (negative control) or with complete Freund's adjuvant (positive control).

 Blood for obtaining the test serum was taken on days 14 and 36 after the first immunization individually from each animal, the level of specific antibodies in the sera was evaluated by enzyme immunoassay (ELISA) by a standard method.

 For comparison, the immunostimulating properties of glycyrrhizic acid (VII), the main triterpene component of licorice root, and also liquiquirithin (VIII), the main phenolic component of licorice extracts, were studied [10, 11].

Результаты испытаний приведены в табл. 1, 2 и 3.

The test results are given in table. 1, 2 and 3.

 From the data table. 1 shows that licorice root extract 1, containing 20% of substance (I) according to HPLC, has pronounced immunostimulating properties, since antibody titers in serum obtained from immunization with albumin with this drug are similar to antibody titers in serum when immunized with full Freund’s adjuvant and 100 times higher than with albumin without the drug. It should be noted that extracts 2 (according to HPLC the content of licoricidin is 10%) and 3 (according to HPLC the content of licoricidin is 40%), as well as glycyrrhizic acid (VII) are significantly lower in activity than Freund's complete adjuvant. Extract 1 is the most effective immunostimulant also with subcutaneous administration. The extract also does not cause immunosuppressive properties when administered in a large dose.

 In the table. 2 shows data on the immunostimulating effect of the individual components of the extract - licoricidin (I), liquiquirithin (VIII), as well as extracts containing 11% (extract 4) and 45-50% (extracts 5, 6) of substance (I).

 From the data table. 2 shows that all extracts have immunostimulating activity. The greatest immunostimulating activity both with subcutaneous and oral administration is possessed by compound (I). It should be noted that the compound (VIII) - has less activity than any of the extracts.

 In the table. 3 shows data on the immunostimulating effect of extracts containing various amounts of compound (I).

 From the data table. Figure 3 shows that all extracts in terms of ELISA titer exceed the effect of the control group. The highest activity, comparable with the effect of Freund's adjuvant, has extract 9, and with subcutaneous administration - extract 10.

 Thus, from the data table. 1-3, extracts containing compound (I) in a concentration of 12-38 wt.% Have the highest immunostimulating activity.

Compound (I) and its extracts belong to the 3rd class of moderately hazardous substances. LD ₅₀ for (I) - 700 mg / kg, for extracts 1, 9 and 10 -> 5000 mg / kg.

 The invention is illustrated by examples.

Example 1. Obtaining extracts 1-12. 100 g of air-dry crushed roots of Glycyrrhiza uralensis F. pour 250 ml of 95% ethanol, insist 12 hours at 20 ^o C, and then 8 hours at 50-60 ^o C with stirring. The alcohol tincture is filtered off, evaporated, dried in vacuo, and a dry loose amorphous powder is obtained (weight 11.2 - 12.3 g, depending on the time and place of collection of raw materials). 10 g of the obtained dry extract was treated with 20 ml of methyl tert.-butyl ether under reflux, filtered off by cooling, the filtrate was evaporated, the residue was triturated with hexane. The precipitate formed (8.2–9.5 g) (8.5–10.0%) was filtered off and dried in vacuo. The content of compound (I) in the extract was determined by HPLC [Chromatograph - Milichrom A-02, column ⌀ 2x75 mm, Nucleosil 5-C 18, eluent A: EtOH: 0.03% CF ₂ COOH, 10:90; eluent B: EtOH: H ₂ O, 90:10, detector: 230 and 260 nm].

Example 2. Isolation of compound (I). 10 g of the alcoholic extract obtained under the conditions of Example 1 are treated with 45 ml of diethyl ether at room temperature, the precipitate formed (flavonoids, glycosides) is filtered off, the filtrate is evaporated, the residue (0.8-0.9 g) is dried in a vacuum and subjected to column chromatography on silica gel (eluent - benzene - ethyl acetate). Fraction 2 (benzene eluent) (weight 0.35 g) was recrystallized from 3: 1 hexane: ether. Obtain 0.31 g of substance (1), so pl. 157-159 ^o C, [α] ₅₈₀ +23.2 ^o (s 0.5, MeOH), which according to the NMR and mass spectra corresponds to licoricidin [4]. The absolute configuration of the compound is established as (3R) from the analysis of optical circular dichroism (CD) [θ] ₃₂₈ + 1.2 • 10 ³ (positive maximum), [θ] ₂₈₂ + 2.2 • 10 ³ (positive maximum).

Example 3. Isolation of compound (VIII). 10 g of the alcoholic extract obtained under the conditions of Example 1 were added to a mixture of 15 ml of water and 15 ml of ethyl acetate. The colored layers are separated on a separatory funnel, the aqueous layer is cooled to 10 ^° C. After 12 hours, 0.7 g of compound (VIII) is filtered off, which, according to the NMR and mass spectra, corresponds to lycirithin [11].

 Example 4. The immunostimulating properties of the claimed substances were studied in 288 animals. Outbred mice were injected subcutaneously with 500 μg of bovine albumin in 0.5 ml of sterile saline. The test compound was administered at doses of 5 mg / kg or 100 mg / kg intraperitoneally or subcutaneously. Bovine albumin was administered to control animals without preparation (negative control) or with Freund's complete adjuvant (positive control). Re-administration of albumin at a dose of 100 mg per mouse was performed on day 28. Serum was obtained on days 14 and 36 and was examined by enzyme-linked immunosorbent assay individually from each animal. The experiments were repeated twice, 6 mice were taken for each dose and route of administration. The results are shown in table. 1-3.

Thus, the compound (I), as well as extracts containing it, have the following advantages:
- they are available, as they stand out from available plant materials (Ural licorice);
- the method of obtaining simple technological design and does not require expensive reagents and equipment;
- the compound (I) and its extracts have a pronounced immunostimulating activity, without showing immunosuppressive properties inherent in the known immunostimulants of a terpenoid nature.

Sources of information
1. Bomford R., Stapleton M., Winsor S., Beesley JE, Jessup EA Vaccine. 1992. V. 10. N 9. P.572-577.

 2. Kersten G. F. A., Crommelin D.J.A. Biochim. Biophys. Acta. 1995. V. 1241. N 2. P. 117-138.

 3. Xu J.-P., Xu R., Li X.-Y. Phytochemistry. 1992. V. 31. N 1. P. 233-236.

 4. Hikino H. in Economic and Medicinal Plant Research. // Ed. by Wagner H., Hikino H., Farnsworth N.R., v. I, p. 55-60, Academic Press, 1985.

 5. Fukai T., Toyono M., Nomura T. On the Structure of Licoricidin. // Heterocycles. 1988. V. 27. N 10. P. 2309-2313.

 6. Bandyukova V.A., Kazakov A.L. Advances in the chemistry of natural isoflavonoids. HPS, 1978. N 6. S. 670-689.

 7. Pat. USA 4.639.466 MKI A 61 K 031/35. S.A., 1987.106. 189006 p.

 8. T. L. Shin, M.J. Wyvratt, H. Morzik, J. Org. Chem. 1987. V. 52. N 10. P. 2029-2013.

 9. Mashkovsky M. D. Medicines, v. 1, S.361-362, Medicine, Moscow, 10th ed.), 1987.

 10. Tolstikov G.A., Schulz E.E., Baltina L.A., Tolstikova T.G. Licorice. Unused opportunities of healthcare of Russia, Chemistry in the interests of sustainable development, 1997. N 5. P.57-73.

 11. Okuda, T., Yoshida, T., Hatano, T. In Food Phytochemicals for Cancer Prevention II // ACS Symp. Ser. 547. Ch. 15. P. 133-143, 1996.

Claims (2)

1. The use of (+) - R-licoricidin of the formula I

as an immunostimulating agent.  2. Plant immunostimulant based on an extract of Ural licorice root, characterized in that it contains 12 - 38 wt.% (+) - r-licoricidin of the formula I.

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