KR101594425B1 - Extracts and glabretal-type triterpenoids from the root bark of Dictamnus dasycarpus and its use - Google Patents

Abstract

The present invention relates to four kinds of novel glabeletal-type triterpenoid compounds separated from them and their immunosuppressive compositions containing them as an active ingredient. According to the present invention, it is possible to provide a composition having an excellent immunosuppressive effect with few side effects, thereby providing a composition for prevention and treatment of diseases caused by immune hyperresponsiveness such as organ rejection, autoimmune disease, allergy, It can also be used as an additive.

Description

Triticum aestivum extracts and novel glabertal-type triterpenoids isolated therefrom and uses thereof. Extracts and glabretal-type triterpenoids from the root bark of Dictamnus dasycarpus and its use.

The present invention relates to a white python extract and a novel glabretal-type triterpenoid isolated therefrom and uses thereof. More specifically, the present invention relates to four novel glabertal-type triterpenoid compounds isolated from white line fatigue based on the immunosuppressive activity tracking method and to an immunosuppressive composition comprising the same as an active ingredient.

Currently, the social and economic interest in immune diseases is increasing due to the extension of life span and quality of life in Korea. Immunosuppressants are used for the treatment of autoimmune diseases such as type I diabetes and rheumatoid arthritis, or rejection reactions by immune cells in organ transplantation. The worldwide market is worth $ 10 billion this year, It is expected to expand further.

Cyclosporin A and tacrolimus (FK506), isolated from natural product extracts, are typical immunosuppressants. Cyclosporin A was isolated from Cylindrocarpon lucidum and Tolypocladium inflatum and tacrolimus (FK506) was isolated from Streptomyces tsukubaensis ), which are the most frequently used immunosuppressants in clinical practice today, have been widely used in the prevention and treatment of rejection reactions in organ transplantation (Borel et al., Inflammation Res. 43: 179-186, 1994; Herold et al , J. Immunol., 136: 1315-1321, 1986, Kronke et al., Proc Natl Acad Sci USA 81: 5214-5218, 1984, Mann, Nat.Prod. Rep. 18: 417-430, 2001; Siekierka et al., Nature 341: 755-757).

However, because they are not specific for immune cells, the need for new immunosuppressive agents has been suggested for reasons of renal toxicity, vascular stiffness, lymphoma, skin cancer as well as side effects such as various infectious diseases (Huang et al., Eur. 211: 359-364,1992).

Dictamnus dasycarpus T. is a perennial herb that is native to Asia and Europe. It is a 'gas plant' or 'burning bush' because of the flammable aromatic substances emitted from the leaves. (Gao et al., Chem. Biodiversity, 8: 1234-1244, 2011; Storer et al., Tetrahedron, 29: 1217-1222, 1973). Woo et al., 1996, Planta Med., 1987), have been used since ancient times for the treatment of menstrual irregularities, antifungal, cough, jaundice, rheumatism, 53: 399-401, 1987; Beis et al., Turk. J. Pharm. Sci., 2: 111-124, 2005). This plant is known to contain alkaloids, limonoids, flavonoids, coumarins, sesquiterpene glycosides, essential oils (Storer et al. Tetrahedron, 29: 1217-1222, 1973, Baser et al., Planta Med., 60: 481-482, 1994; Souleles et al., Planta Med., 55: 402, 1989).

The extract of D. albus var. Caucasicus is known to exhibit a blood coagulation effect (Petrov et al., Uch. Zap., Pyatigorskii Farm. Inst. 5: 335-338, 1961). It has also been reported that the furoquinoline alkaloid isolated from D. albus L. inhibits human phosphodiesterase 5, an enzyme involved in smooth muscle relaxation in vitro (Nam et al. , Arch Pharm Res. 28: 675-679, 2005). Furthermore, glycosides isolated from D. dasycarpus T. have been reported to modulate the immune system (Chang et al., J. Nat. Prod. 64: 935-938, 2001; , Planta Med., 68: 425-429, 2002).

As a background of the present invention, Korean Patent Laid-Open Publication No. 10-2012-0124142 (Nov. 13, 2012) discloses a composition for inhibiting multiple drug resistance comprising an extract of white tiger bark as an active ingredient, -1181347 (Sep. 17, 2012) discloses a composition for the prevention and treatment of lipid-related cardiovascular diseases or obesity, which comprises white tiger blood extract as an active ingredient. Korean Patent Registration No. 10-1011671 (Jan. 28, 2011) discloses a composition for preventing or treating a degenerative brain nervous system disease comprising a white line extract or a remonoid compound. However, there is no known immunosuppressive activity of the white line extract and the novel glabretal-type triterpenoid isolated therefrom.

Numerous cited documents and patent documents are referred to and cited throughout this specification. The disclosures of the cited documents and patents are incorporated herein by reference in their entirety to more clearly describe the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 10-2012-0124142 (November 13, 2012) Korean Registered Patent No. 10-1181347 (September 17, 2012) Korean Patent No. 10-1011671 (January 28, 2011)

Disclosure of the Invention The present invention has been conceived to solve the above problems of the prior art. The present inventors have used an extract library for 500 medicinal resources in the domestic market, which is possessed by the inventors in order to isolate a substance having immunosuppressive activity from natural products As a result, strong immunosuppressive activity was observed in white blood. Based on these results, we confirmed the significant activity of the extract and its fractions on the basis of the immunosuppressive activity tracing method. From the result, new compounds were isolated and purified to identify the structure.

Accordingly, it is an object of the present invention to provide an immunosuppressive active composition comprising an extract of tinea cordis, or a novel glabertal-type triterpenoid isolated therefrom.

Another object of the present invention is to provide a novel glabertal-type triterpenoid compound isolated from the white pine bark extract.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an immunosuppressive composition comprising an extract of white radish as an active ingredient.

According to another aspect of the present invention, there is provided a triterpenoid compound selected from Compounds 1 to 4 represented by the following Formulas 1 to 4, respectively.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a triterpenoid compound or a pharmaceutically acceptable salt thereof selected from the compounds 1 to 4 represented by the above Formulas 1 to 4, A composition for immunosuppression is provided.

Preferably, said composition is an organ transplant rejection reaction caused by an immunosensitivity reaction; Autoimmune diseases of lupus or rheumatoid arthritis; Rhinitis, asthma, allergic diseases of atopy.

According to still another aspect of the present invention, there is provided a method of extracting white line extract, comprising extracting white line extract with water, alcohol or a mixed solvent thereof; A second step of suspending the extract extracted from the first step in distilled water, and then sequentially fractionating the extract with hexane and chloroform; And a third step of performing concentration gradient silica gel chromatography on the fraction dissolved in chloroform in the second step using a mixed solvent of chloroform and methanol (CHCl ₃ -MeOH) to obtain a fraction The present invention provides a method for producing a triterpenoid compound according to the present invention.

Hereinafter, the present invention will be described in more detail.

The present inventors confirmed the strong immunosuppressive activity of the tsukuni extract and, based on the immunosuppressive activity tracking method in white blood, the four new glabretal-type triterpenoids, dictabitol A- D (dictabretol A to D, Formulas 1 to 4). The structures of compounds 1 to 4 were confirmed by spectroscopic analysis including 2D NMR. The immunosuppressive activity was measured based on T cells and confirmed that the proliferation of activated T cells was inhibited to an IC _{50 of} 1.48 μM, thereby completing the present invention.

Accordingly, the present invention provides a composition for immunosuppression, which contains an extract of P. tuberculosis as an active ingredient.

As the extraction solvent for preparing the white line extract, which is an active ingredient of the composition of the present invention, there can be used a solvent which can be generally used in the extraction of natural materials, such as water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate , Butyl acetate, 1,3-butylene glycol, and a mixed solvent thereof, and more preferably methanol.

The present invention also provides a triterpenoid compound selected from the compounds 1 to 4 represented by the above Formulas 1 to 4, respectively.

Compounds 1 to 4 represented by the above Formulas 1 to 4 according to the present invention were determined to be Dictabretol A to D (Dictabretol A to D) as described below.

Compound 1 ( Dictabretol  A, Formula 1)

Dictabretol A is separated into a white amorphous solid and has a melting point of 234.4 ° C. The molecular formula was determined to be C ₃₅ H ₅₆ O ₇ by measuring the value of 587.3951 in HRESIMS and the unsaturation of this molecule was found to be 8. The hydroxyl group (3402 cm ^-1 ) and the ester function (1723 cm ^-1 ) were confirmed by IR spectrum. ¹ in the H NMR spectrum (chloroform, Table _{1) δ H 0.88 (H-} 19), 1.31 (H-27), 0.84 (H-28), 0.87 (H-29), 1.04 (H-30), 0.96 ( H-4 '), and 0.95 (H-5'). H-26 from the signals of δ _H 4.65 (H-3), 3.75 (H-7), 5.43 (H-21), 3.94 We confirmed the methine and confirmed the duplicate signal of aliphatic methine and methylene. The characteristic signals of the cyclopropylmethylene group in the higher field region δ _H 0.71 (2H, doublet, J = 4.5 Hz, H-18a) and 0.46 (2H, doublet, J = 5.0 Hz, H-18b) Confirmed. ^In addition to the analysis of ¹ H and ¹³ C NMR spectra (chloroform, Tables 1 and 2), the HMBC spectrum revealed that compound 1 is a glabretal-type triterpenoid (Kamperdick et al., J. Nat. Prod. 66: 675-678, 2003; Mulholland et al., Phytochemistry 34: 579-580, 1993; Su et al., Bioorg. Med. Chem. 14: 960-972, 2006). The ¹ H and ¹³ C NMR spectra of Compound 1 showed a trisubstituted epoxy group signal at C-24 [ δ _H 3.18 (1H, doublet, J = 7.5 Hz) and δ _C 63.38] and C-25 ( δ _C 60.74) . In addition, the presence of hemiacetal groups was deduced from the signals of δ _C 98.26 (C-21) and δ _H 5.43 (1H, overlap, H-21). _{δ H 2.21 (2H, broad doublet} , J = 7.0 Hz, H-2 '), 2.13 (1H, multiplet, H-3'), 0.96 (3H, doublet, J = 2.0 Hz, H-4 '), and The presence of isovaleric acid was confirmed by ¹ H NMR signal of 0.95 (3H, doublet, J = 2.0 Hz, H-5 '). Isovaleric acid exhibited HMBC interrelation between δ _H 0.84 (H-28) and δ _H 0.87 (H-29) and δ _C 77.77 (C-3), 36.16 (C-4), and 41.27 ? _H 4.65 (H-3) and ? _C 33.85 (C-1), 22.88 (C-2), 36.16 (C-4), 41.27 (C-5), 27.79 29), and 172.95 (C-1 ') (Kamperdick et al., J. Nat.

A broad single signal of δ _H 4.65 (1H, broad singlet, H-3) and 3.75 (1H, broad singlet, H-7) indicates that isovaleric and hydroxyl groups are in the alpha direction (Su et al., Bioorg. Med. Chem. 14: 960-972, 2006, Nalli et al., Molecules 15: 5866-5877, 2010). Further, the relative arrangement of compound 1 to the steric structure was confirmed by comparison of the NOE interrelation between H-3 / H-19 and H-7 / H-30 and the literature value of the glabretal-type triterpenoid (Kamperdick et al. , J. Nat. Prod. 66: 675-678, 2003, Mulholland et al., Phytochemistry 34: 579-580, 1993, Su et al., Bioorg. Med. Chem. 14: 960-972, 2006, al., Molecules 15: 5866-5877, 2010; Ochi et al., Bull. Chem. Soc. Jpn. 61: 3225-3229, 1988). In addition, this compound has a 21C carbon atom that is isolated from a closely matched signal of H-21 (δH 5.43 overlap) and C-21 (δC 98.26 and 102.17) and ¹³ C NMR spectra identified from the HSQC spectrum (Kamperdick et al., J. Nat. Prod. 66: 675-678, 2003; Su et al., Bioorg Med. Chem. 14: 960-972, 2006; , ≪ / RTI > Molecules 15: 5866-5877, 2010). Based on the above results, the structure of dictabretol A, a new glabretal-type triterpenoid, was determined.

[Chemical Formula 1]

Compound 2 ( Dictabretol  B, formula (2)

Dictabretol B was isolated as a white amorphous solid with a melting point of 229.9 ° C and a HRESIMS value of 589.4103 to confirm that the molecular formula was C ₃₅ H ₅₈ O ₇ and an unsaturation of 7. ^{The 1} H and ¹³ C NMR spectra were very similar to those of compound 1 (Tables 1 and 2), indicating that this compound is also a glabretal-type triterpenoid. In addition, the single signal methyl group [δH 1.28 (3H, singlet) and δC 26.93] of 26 carbon replaced the methylene signal [δH 3.65 (2H, doublet, J = 12.5 Hz) and δC 64.95] Indicating that the hydroxyl group of the hydroxyl group has disappeared. Chem. 14: 960-972, 2006), the authorship of C-24 (δC 75.27) and C-25 (δC 73.72) of compound No. 2 specify the opening of the epoxide ring (Su et al., Bioorg. , Which proved to reduce the degree of unsaturation by one compared to compound 1. [ Thus, the structure of compound 2 clearly determined that the 26th carbon had a single methyl group and had a 24,25-dihydroxy group. Similarly, Compound 2 was also isolated as an epimeric mixture. However, only the ¹ H and ¹³ C NMR results of the main isomer were included in the table, as the 1D and 2D NMR signals of the isomers are not clear enough to complete the structural analysis (Tables 1 and 2).

(2)

Compound 3 ( Dictabretol  C, Formula 3)

Dictabretol C was isolated as a colorless oil. The molecular formula was found to be C ₄₀ H ₆₆ O ₇ and an unsaturation degree of 8 according to the value of 657.4731 in HRESIMS. The ¹ H and ¹³ C NMR spectra of compound 3 (Tables 1 and 2) were very similar to Compound 1 except that there was no signal corresponding to isovaleric acid, indicating that it was a glabretal-type triterpenoid. ^{In the 1} H NMR spectrum, the presence of decanoic acid was confirmed by methyl group signals of δ _H 1.24-1.61 and δ _H 0.87 corresponding to the C-4'-C-7 'long aliphatic chain, which was confirmed by mass spectra . Further, in the HMBC analysis, δ _H 4.63 (H-3) and δ _C 33.85 (C-1), 36.20 (C-4), 41.25 (C-5), 27.71 , And 173.65 (C-1 '), and δ _H 2.32 (H-2') and δ _C 77.78 (C-3), 173.65 (C-1 '), 25.19 (C-4 '), the decanoic acid was located at carbon number 3. Thus, through the 1D and 2D NMR spectra, it was determined that compound 3 was a glabretal triterpene having a decanoic acid substituent at carbon number 3.

(3)

Compound 4 ( Dictabretol  D, Formula 4)

Dictabretol D was isolated in yellow oil. The molecular formula was found to be C ₄₁ H ₆₈ O ₇ , with an unsaturation of 8 according to the value of 671.4887 in HRESIMS. In the ¹ H and ¹³ C NMR spectra of the compound 4 (Tables 1 and 2), the compound 3 almost coincided with the exception of the typical signal of the methyl side chain of the anteoxy form at the 8 'carbon. The presence of acid is a methyl group attached to the 8-position is a _{δ H 0.86 (H-10 '} ) and _{δ C 34.36 (C-8'} ) , and _{δ C 29.71 (C-9 '} ) correlating and δ _H from HMBC analysis The correlation was determined based on the correlation of 0.84 (H-11 ') with δ _C 36.58 (C-7'), δ _C 34.36 (C-8 '), and δ _C 29.71 (C-9'). Furthermore, high-field transfer of the terminal methyl group located at 10 '( δ _C 11.41) was confirmed due to the gamma effect due to the methyl substitution of the carbon atom of the 8' carbon, which is attributable to the fact that the methyl side chain of the aniso- (Kim et al., J. Antibiot. 59: 797-800, 2006; Wang et al., J. Nat. Prod. 66: 51-56, 2003). Thus, the structure of compound 4 was clearly determined to have a methyl substituent at the 8 ' carbon in the decanoic acid moiety of compound 3. [

[Chemical Formula 4]

The triterpenoid compounds represented by formulas (1) to (4), respectively, can be used in the form of pharmaceutically acceptable salts, and include salts, hydrates and solvates prepared by conventional methods.

The compounds of formulas (1) to (4) according to the present invention can be obtained by extracting and purifying from white ring fat, and can be obtained through organic synthesis.

A method for separating and purifying a novel triterpenoid according to the present invention is as follows.

According to still another aspect of the present invention, there is provided a method of extracting white line extract, comprising extracting white line extract with water, alcohol or a mixed solvent thereof; A second step of suspending the extract extracted from the first step in distilled water, and then sequentially fractionating the extract with hexane and chloroform; And a third step of performing concentration gradient silica gel chromatography on the fraction dissolved in chloroform in the second step using a mixed solvent of chloroform and methanol (CHCl ₃ -MeOH) to obtain a fraction do.

Further, the fraction obtained in the third step was purified by using Sephadex LH-20 as a mobile phase with chloroform and methanol (CHCl ₃ -MeOH), and the purified fraction was purified by silica gel column chromatography using hexane-acetone as a mobile phase To obtain a fraction; The fraction obtained in the fourth step is subjected to a fifth step of obtaining a fraction using a reverse phase (RP) column using acetonitrile and water (MeCN-H ₂ O), and a fifth step of obtaining the fraction obtained in the fifth step Is subjected to chromatography using a mixed solvent of dichloromethane and methanol (CH ₂ Cl ₂ -MeOH) to obtain compound 1 of formula (1).

Further, the fraction obtained in the fourth step is subjected to a fifth step of obtaining a fraction using a reverse phase (RP) column using acetonitrile and water (MeCN-H ₂ O) The separated fractions were chromatographed using a mixed solvent of hexane and acetone, a mixed solvent of dichloromethane and methanol (CH ₂ Cl ₂ -MeOH) or a mixed solvent of chloroform and methanol (CHCl ₃ -MeOH) To obtain Compound 2 of Formula 2, Compound 3 of Formula 3, or Compound 4 of Formula 4, respectively.

Further, the present invention relates to a pharmaceutical composition for immunosuppression or a food composition containing the novel triterpenoid or a pharmaceutically acceptable salt thereof as an active ingredient of the above-mentioned tsunami extract or the compounds 1 to 4 isolated therefrom .

Wherein said composition is an organ transplant rejection reaction caused by an immune hypersensitivity reaction; Autoimmune diseases of lupus or rheumatoid arthritis; Rhinitis, asthma, allergic diseases of atopy, and the like.

The immunosuppressive activity against the compounds 1 to 4 isolated according to the present invention was measured by proliferation in T cells and secretion of cytokines. To this end, transgenic mice with OT-II T cell receptors 8-10 weeks old Were used. Cell viability was measured by propidium iodide staining and showed no toxicity to T cells at concentrations that were active in cell proliferation and cytokine secretion. As a result of cell proliferation measurement, IC ₅₀ values of compounds 1, 3 and 4 were 1.49, 1.78, and 1.48 μM [using cyclosporin A (IC _{50 of} 0.05 μM) as a positive control] (Table 3). The degree of secretion of cytokines IL-2 and IFN-y was measured in further activated T cells, but no compound showed any specific effect on cytokine secretion. These results indicate that compounds 1, 3, and 4 do not affect cytokine secretion and inhibit the proliferation of T cells.

The pharmaceutically acceptable salts of the compounds 1 to 4 of the present invention include a salt of a hydroxy group which may exist in the compound of the formula unless otherwise indicated, and examples of the salt of the hydroxy group include alkali metals such as lithium, sodium, And alkaline earth metal salts such as salts, magnesium and calcium. Preferably, physiologically acceptable salts with sodium, potassium, calcium and the like can be cited, and these salts can be produced through the production method or the production process of salts known in the art.

In addition, the compounds 1 to 4 of the present invention can be produced as pharmaceutically acceptable non-toxic salts and solvates, according to methods conventional in the art. Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

The composition of the present invention can be formulated by methods well known in the pharmaceutical or food science field and can be formulated into pharmaceutical or food-grade preparations in admixture with itself or a pharmaceutically acceptable carrier, excipient or the like, For example, it may be formulated into a liquid, a syrup, a capsule or the like. Can further be suitably formulated according to each disease or ingredient, using appropriate methods in the art or as disclosed in Remington ' s Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the intended method.

The term "health supplementary food" used in the present invention refers to a nutritional supplement which is obtained by using a raw material or ingredient having a useful function in the human body, Refers to a functional food such as a health food or beverage, yogurt, or cheese prepared and processed in the form of a capsule, a powder, a granule, a liquid agent, or a pill.

The composition of the present invention is appropriately selected depending on the degree of absorption of the active ingredient in the body, the excretion rate, the age and weight of the patient, the sex and condition of the patient, the severity of the disease to be treated and the like, but is generally 0.01 to 500 mg / Preferably 0.1 to 200 mg / kg. The unit dosage formulations thus formulated may be administered several times at predetermined time intervals as necessary. The dose is not intended to limit the scope of the invention in any way.

The white roots extract of the present invention and the compounds 1 to 4 isolated and purified therefrom can be added to the raw material in food production or mixed with the cooked food appropriately to produce the above health promoting food or drink, The content of each of the compounds 1 to 4 isolated and purified from the white line bark extract in the prepared food or beverage ranges from 0.01 to 30% by weight.

The pharmaceutical composition or health promotion food or drink of the present invention may further comprise other herbal medicines or extracts thereof that are pharmaceutically acceptable for elevating or supplementing the desired effect.

As described above, the white line extract and the novel compound according to the present invention are highly effective in suppressing immune function in animal models of immune cells and rheumatoid arthritis, and can be usefully used as competitive immunosuppressants.

In addition, since the extract of the present invention, which is an effective ingredient of the present invention, is a natural substance with little toxicity and side effects, it can be safely used even when taken for a long time.

Brief Description of the Drawings Fig. 1 is a graph showing the effect of the white line extract of the present invention on T lymphocyte activity. Fig.
FIG. 2 is a graph showing the effect of a novel triterpenoid compound separated from white blood cells on cell survival of immune cells according to the present invention. FIG.
FIGS. 3A to 3D are graphs showing the results of analyzing the effect of the novel triterpenoid compound isolated from the white blood line according to the present invention on the cell proliferation inhibitory effect of immune cells. FIG.
FIG. 4 is a graph showing the results of analyzing the effect of the novel triterpenoid compound isolated from the white blood line according to the present invention to inhibit the cell cycle activity of immune cells.
FIGS. 5A and 5B are graphs showing the results of analysis of cell cycle-related regulators of immune cells by novel triterpenoid compounds isolated from white blood line according to the present invention. FIG.
FIG. 6 is a graph showing the therapeutic effect on a rheumatoid arthritis model animal by a novel triterpenoid compound isolated from white tiger blood according to the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples illustrate the present invention, and the scope of the present invention is not limited by the following examples.

Materials and methods

plant material

The white pine bloom was purchased at the Kyungdong market in Korea in January 2008 and was recognized by Professor Emeritus Lee Kyung Soon of the College of Pharmacy, Chungbuk National University, Republic of Korea. Herbarium specimens were kept at the Korea University College of Life Sciences (SS45-01142008).

Efficacy analysis for cell proliferation and cytokine secretion

(1 × 10 ⁶ cells / well) collected from transgenic mice (H-2b haplotype; The Jackson Laboratory, Bar Harbor, ME, USA) well plate. The cells were then stimulated with OVA323-339 peptide (323 ISQAVHAAHAEINEAGR339; AnaSpec, Fremont, CA, USA) and cultured in RPMI-10 medium containing 5% CO ₂ and 37 ° C , And DMSO (0.1%) were used as controls. The next day propidium iodide staining was measured by running the cargo cell viability, and the results were analyzed by flow cytometry using a ^{FACSCalibur TM system with CellQuestsoftware TM (BD} Biosciences, San Jose, CA). Relative cell growth rates were measured by MTT assay kit (Promega, Madison, WI, USA) three days after each compound treatment. The level of secretion of IL-2 and IFN-γ in the supernatant was measured by sandwich ELISA kit (Invitrogen, Carlsbad, CA, USA).

Example  One. White line blood  Extract and from this Glavlevall -type Triterpenoid  detach

In order to separate the active substance from the white ring blood, 5 kg of the dried white ring was extracted three times with methanol (MeOH) at room temperature, and the solvent was removed in vacuo through filtration. The methanol extract was suspended in distilled water, and then fractionated by sequential use of hexane ( n- Hexane), chloroform (CHCl ₃ ), and ethyl acetate (EtOAc).

Depending on the immunosuppressive activity results, CHCl ₃ Extract (95 g) by column chromatography on silica gel _{(CHCl 3 -MeOH, 100: 0} to 50:50) were run to give the five fractions (SS45-47-1-SS45-47-5). The detailed fraction SS45-47-4 (12.8 g) was purified with 9 fractions (SS45-51-1-SS45-51-9) using CHCl ₃ -MeOH (1: 1) as mobile phase with Sephadex LH-20 . Among the obtained fractions, SS45-51-3 (4.4 g) was separated by silica gel column chromatography using hexane-acetone (10: 1 to 1: 1, pure acetone) as mobile phase, and 11 fractions (SS45-56- 1-SS45-56-11). Among them, SS45-56-5 (900 mg) was eluted with acetonitrile-water (40-100% acetonitrile and acetonitrile-acetone 1: 1) through vacuum liquid chromatography using RP-18, Eluting with CH ₂ Cl ₂ -MeOH (1: 0, 90: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1) (dictabretol A, 65 mg). Fractions SS45-56-4 (203 mg) were purified by column chromatography using RP-18 using acetonitrile-water (3: 7, 5: 5, 7: 3, 8: (SS45-57-1-SS45-57-9) as the mobile phase. Of the obtained fractions, SS45-57-2 (44 mg) was isolated by silica gel column chromatography using hexane-acetone (10: 1 to 1: 1) as a mobile phase to give Compound 2 (dictabretol B, 33 mg) . The fraction SS45-57-6 (36 mg) was purified by silica gel column chromatography using a mobile phase of CH ₂ Cl ₂ -MeOH (1: 0, 80: 1, 50: 1, 30: 1, 20: And the compound 3 (dictabretol C, 23 mg) was obtained. Fraction SS45-57-8 (9 mg) was purified by silica gel column chromatography using mobile phase CHCl ₃ -MeOH (1: 0, 80: 1, 50: 1, 30: 1, 20: 1, 10: 1) , And Compound 4 (dictabretol D, 5 mg) was obtained.

Example  2. Structural analysis of compounds

In order to analyze the structure of the compound obtained in Example 1, the following analysis was carried out. Melting points and optical intensity were measured using Stanford Research System MPA100 and JASCO P-2000 luminometer, respectively. IR spectra were recorded on a Varian 640-IR. ^{The 1} H, ¹³ C, and 2D NMR spectra were recorded using Varian 500 MHz NMR and CDCl ₃ with internal standard TMS. The chemical shifts were expressed as δ values. The high-resolution electrospray ionization (HRESI) mass spectra were measured using a Waters Q-TOF mass spectrometer. Column chromatography was performed on silica gel (Kieselgel 60, 70-230 and 230-400 mesh, Merck, Darmstadt, Germany), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), and YMC-gel ODS- , YMC, Kyoto, Japan) were used. Thin-film chromatography was performed on pre-coated silica gel 60 F ₂₅₄ plates (0.25 mm, Merck, Darmstadt, Germany).

Compound 1 (Formula 1)

Dictabretol A (1): White, amorphous solid, melting at 234.4 ° C. As a result of the measurement of roundness, values of [?] ²⁷ _D -22.6 (c 0.3, CHCl ₃ ) were obtained. IR spectra were measured at 3402, 2967, 1723, 1216 and 1054 cm ^-1 . ¹ H and ¹³ C NMR data (500 MHz, CHCl ₃ ) are shown in Tables 1 and 2. In the voice ^{ESIMS 587 [MH] -, 633} [M + HCOO] - in this, the positive ESIMS was observed value 611 [M + Na], the value of ⁺ were observed, in the audio resolution ^{ESIMS 587.3951 [MH] - (C} 35 H ₅₅ O ₇ , calculated as 587.3948).

Compound 2 (Formula 2)

Dictabretol B (2): white amorphous solid form with melting point of 229.9 ° C. As a result of the measurement of roundness, values of [?] ²⁷ _D -20.2 (c 0.3, CHCl ₃ ) were obtained. IR spectra were measured at 3444, 2930, 1727, 1387 and 1095 cm ^-1 . ¹ H and ¹³ C NMR data (500 MHz, CDCl ₃ ) are shown in Tables 1 and 2. In the voice ^{ESIMS 589 [MH] -, 635} [M + HCOO] - was the value of the observation, 613 [M + Na], the value of ⁺ were observed in the positive ESIMS, in voice resolution ^{ESIMS 589.4103 [MH] - (C} 35 H ₅₇ O ₇ , calculated as 589.4104).

Compound 3 (Formula 3)

Dictabretol C (3): Colorless oil, measured by optical rotation [α] ²⁷ _D -33.2 (c 0.3, CHCl ₃ ). IR spectra were measured at 3421, 2931, 1721, 1215 and 1025 cm ^-1 . ¹ H and ¹³ C NMR data (500 MHz, CDCl ₃ ) are shown in Tables 1 and 2. In the voice ^{ESIMS 657 [MH] -, 703} [M + HCOO] - was the value of the observation, in the positive ESIMS 681 [M + Na], the value of ⁺ were observed, in the audio resolution ^{ESIMS 657.4731 [MH] - (C} 40 H ₆₅ O ₇ , calculated as 657.4730).

Compound 4 (Formula 4)

Dictabretol D (4): Yellow oil type, and its optical density was measured. The value of [α] ²⁷ _D -17.9 (c 0.3, CHCl ₃ ) was obtained. IR spectra were measured at 3392, 2931, 1718, 1216 and 1023 cm ^-1 . ¹ H and ¹³ C NMR data (500 MHz, CDCl ₃ ) are shown in Tables 1 and 2. In the voice ^{ESIMS 671 [MH] -, 717} [M + HCOO] - was the value of the observation, which was the 695 [M + Na] value of ⁺ observed in the positive ESIMS, in voice resolution ^{ESIMS 671.4887 [MH] - (C} 41 H ₆₇ O ₇ , calculated as 671.4887).

Table 3 shows the inhibitory effect of the isolated compounds 1-4 on T cell proliferation using splenocytes isolated from transgenic mice with 8-10 week OT-II T cell receptors.

Example  3. Analysis of efficacy against immunosuppression

One) White line blood  Effect of extract treatment on T lymphocyte activity

(H-2b haplotype; The Jackson Laboratory, Bar Harbor, ME, USA) with 8-10 week OT-II T cell receptor to investigate the effect of the extract on the immune system. Cells (1 x 10 ⁶ cells / well) were used. Immunocytes were isolated from splenocytes and seeded at 3 × 10 ³ cells / well in a 96-well plate. After the stimulation of the T lymphocyte activity by the addition of OVA peptide (323 ISQAVHAAHAEINEAGR339; AnaSpec, Fremont, CA, USA), the white blood cell extract was incubated at a concentration of 10 μg / ml, 2 μg / ml, 0.4 μg / ml And cultured for 3 days.

On the third day, MTS assay was used to measure the proliferation of immune cells. As a result, when compared with PC (positive control) and NC (negative control, non-stimulated state), the proliferation of immune cells was markedly suppressed even at 0.4 μg / ml of white blood.

2) From white ring blood  Measurement of cell viability of immune cells by separate compound treatment

In order to investigate the cell viability of immune cells treated with the compounds 1, 3 and 4 isolated from white blood, PI (Propidium Iodide) and Annexin V staining were performed and analyzed by flow cytometry (FACSCalibur ^TM system with Cell viability was measured via CellQuestsoftware ^TM (BD Biosciences, San Jose, Calif.).

Cells were transfected with 5 × 10 ⁵ / well of RMA cell line and 10% of fetal bovine serum of A20 cell line in RTA cell line of mouse T lymphocyte, A20 cell of mouse B lymphocyte, and J774 cell line of mouse macrophage cell line in 6 well tissue culture plate (FBS), RPMI-1640 (Invitrogen) medium containing 1% penicillin / streptomycin and 25 mM HEPES and the J774 cell line was cultured in DMEM containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin in vitro culture medium.

At this time, the compounds 1, 3, and 4 isolated from the white blood line were dissolved in PBS containing 10% DMSO (Sigma), and the cells were treated by taking 1/100 of the total culture volume. , 5 uM, 2 uM concentration, and the cell viability was measured after 24 hours. The results are shown in Fig. At this time, the measurement of cell viability was considered to be the survival of PI ^low and Annexin V ^low cells.

3) From white ring blood  Analysis of inhibitory effect on cell proliferative capacity of immune cells by treatment with isolated compounds

Cell proliferation was measured using the CFSE [Carboxyfluorescein succinimidyl ester] proliferation assay to measure the effect of the compounds 1 to 4 on the cell proliferation of immune cells.

For cell line experiments, 1 x 10 ⁷ of the same cell lines as in Example 3-2 were mixed with 1 ml of PBS containing 0.1% fetal bovine serum in 1.5 μM CFSE at room temperature for 8 minutes, mixed with 1 ml of 100% fetal bovine serum Lt; RTI ID = 0.0 > 37 C < / RTI > for 10 minutes. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded. Cells were washed 3 times with PBS containing 2% fetal bovine serum and CFSE staining was performed. Then, the cells were counted and counted in a ^6- well tissue culture plate at 1 × 10 ⁵ / well Cells were cultured in the same conditions as in Example 3-2.

At this time, the compounds 1, 2, 3, and 4 isolated from the white blood line were treated to a concentration of 2 uM in a final concentration of 0.1% DMSO, and after one day, three days, and five days, And the degree of cell proliferation was measured. The results are shown in FIGS. 3A to 3C.

In order to measure the effect on cell proliferation in primary cells, spleen of OT II mice transgenic to 10-week old transgenic mice (trnasgenic mice) was isolated and 5 x 10 ⁷ cells were scaled up in the same manner as above After CFSE staining, cells were cultured in RPMI-1640 (Invitrogen) medium containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin and 25 mM HEPES at a concentration of 5 x 10 ⁵ / well in 96-well round tissue culture plates. To stimulate splenocytes in vitro stimulation signals were treated with 1 uM / ml of OVA _{323 -339} peptide ( ³²³ ISQAVHAAHAEINEAGR ³²⁹ ; AnaSpec, Fremont, CA, USA).

At this time, the compounds 1, 2, 3, and 4 isolated from the white blood line were treated to a concentration of 0.5 uM in a final concentration of 0.1% DMSO, and after one day, three days, and five days, And the degree of cell proliferation was measured. The results are shown in Fig.

4) From white ring blood  Analysis of inhibitory effect on cell cycle activity of immune cells by treatment with isolated compounds

In order to investigate the cell cycle activity of immune cells treated with the compounds 1 to 4 isolated from white blood, PI (Propidium iodide) cell cycle analysis was carried out using a flow cytometry analyzer.

RMA cell line and J774 cell line were used to culture cells at a concentration of 5 x 10 ⁵ / well in a 6-well tissue culture plate in RPMI-1640 containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin and 25 mM HEPES, 1640 (Invitrogen) medium. The J774 cell line was cultured in DMEM (Invitrogen) medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

At this time, the compounds 1, 2, 3, and 4 isolated from the white blood line were dissolved in PBS containing 10% DMSO (Sigma), and the cells were treated with 1/100 of the total volume of the culture, . After 24 hours, the cells were harvested, washed with PBS, and fixed with 70% ethanol at -20 ° C for 12 hours. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded and the cells were eluted with 500 μl of PBS containing 50 μg / ml of PI (Propidium Iodide), 0.1 mg / ml of RNaseA and 0.05% Triton X-100, After incubation for a few minutes, the cells were washed with PBS and analyzed using a flow cytometer. The results are shown in FIG. As shown in Fig. 4, the S group was observed to decrease in the experimental group treated with the compounds 1, 3 and 4 in the RMA cell line.

5) From white ring blood  Immune cell cell cycle-related Adjuvant analysis by separate compound treatment

It was confirmed that the treatment of compounds 1 to 4 isolated from white blood cells inhibited the transition from the RMA cell line to the S phase in the cell cycle. Therefore, it was confirmed that the white blood cell compound inhibited cyclin D1 and cyclin E, which are cell cycle positive regulators, and p27 ^KIP1 , p21 ^CIP1 And to investigate the effect on the expression of c-Myc to inhibit the p27 ^KIP1 and p21 ^CIP1 in upper, anti-cyclin D1 (Santa- cruz), anti-cyclin E (Santa-cruz), anti-p27KIP1 (Santa Western blotting was performed using anti-p21 CIP1 (Santa-cruz) and anti-c-Myc (Santa-cruz) antibodies.

Cell culture was carried out under the same conditions as in Example 3-4. One day after the culture, western blot was harvested and cultured in RIPA lysis buffer containing proteinase inhibitor cocktail (50 mM Tris, 150 mM NaCl, 1.0% (GE Healthcare, Buckinghamshire, UK) by electrophoresis on 10% SDS polyacrylamide gels, and then lysed with 0.5% deoxycholate, 0.1% SDS. After that, immunoblot was performed using the antibody. The immunoreactive band was visualized with a Biomolecular Imaging System (ImageQuantTM LAS4000mini Bio-molecular Imager, GE Healthcare, Buckinghamshire, UK) using an ECL system and the results are shown in FIG.

In order to investigate the effect of white blood cells on the cell cycle in primary cells, cyclin E, a cell cycle positive regulator, and p27 ^KIP1 , a negative regulator, were subjected to western blotting using the same antibody. The cells were cultured in the same manner as in Example 3-3 except that OT II mouse spleen cells were cultured in a 96-well round tissue culture plate at a concentration of 1 × 10 ⁶ / well containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin and 25 mM HEPES 0.0 > RPMI-1640 < / RTI > (invitrogen). To stimulate splenocytes in vitro stimulation signals were treated with 1 uM / ml of OVA _{323 -339} peptide ( ³²³ ISQAVHAAHAEINEAGR ³²⁹ ; AnaSpec, Fremont, CA, USA). At this time, one sample of the compound isolated from the white blood line was treated to a concentration of 0.5 uM in a final concentration of 0.1% DMSO, and after 3 days, the cells were harvested and subjected to Western blotting under the same conditions as above. .

6) From white ring blood  By separated compound Rheumatism  Therapeutic effect on arthritis model animals

To investigate the therapeutic effect of the collagen-induced arthritis model induced by collagen-induced compounds by the compounds isolated from tinea bloom, a type II collagen solution (2.5 mg / ml) was added at 0.05 M acetic acid and stored at 4 ° C. Immediately prior to immunization, the same volume of Complete Freund's Adjuvant (CFA) was mixed using a three-way stopcock. This mixed solution was subcutaneously injected into the tail bottom portion of the mouse at a dose of 140 μl. After 3 weeks, the collagen solution was mixed with the same volume of IFA (Incomplete Freund's Adjuvant) and subcutaneously injected into the tail at 100 μl. From 10 days after the second inoculation, the treatment was continued for 3 weeks once every 2 days. Negative controls were treated with vehicle (0.5% DMSO) and positive controls treated with Enbrel (8 mg / kg). One sample of the white line compound was tested at 10 mg / kg.

The results are shown in FIG. 6, wherein clinical evaluation of arthritis ranked four arthritis edema and redness as arthritis scores. 0 - no symptoms; 1 - mild edema and fever limited to one toe and joint; 2-normal flare and edema of two or more types of joints; 3 - severe redness and swelling of the whole foot, including the toes; 4 - maximal inflammation of the limbs including multiple joints, redness and edema (the largest cumulative clinical arthritis score per animal 16)

Composition Example

Preparation Example 1. Preparation of powder

2g of white pine bark extract

Lactose 1g

The above components were mixed and packed in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

100mg of white radish extract

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Formulation Example 3. Preparation of capsules

100mg of white radish extract

Corn starch 100 mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Formulation Example 4. Preparation of capsules

200 mg of the chloroform fraction of Example 1

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate qs

The above components were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules.

Formulation Example 5. Preparation of Injection

100 mg of white radish extract

180 mg mannitol,

Na ₂ HPO _{4 12} H ₂ O 26 mg

2974 mg of distilled water

After mixing the above components, an injection was prepared according to the usual production method.

Formulation Example 6. Preparation of Healthy Foods

100 mg of white radish extract

Vitamin mixture quantity

 Vitamin A acetate 70 μg

 Vitamin E 1.0 mg

 Vitamin B1 0.13 mg

 0.15 mg of vitamin B2

 Vitamin B6 0.5 mg

 Vitamin B12 0.2 μg

 Vitamin C 10 mg

 Biotin 10 μg

 Nicotinic acid amide 1.7 mg

 Folic acid 50 μg

 Calcium pantothenate 0.5 mg

Mineral mixture quantity

 1.75 mg of ferrous sulfate

 0.82 mg of zinc oxide

 Magnesium carbonate 25.3 mg

 Potassium monophosphate 15 mg

 Secondary calcium phosphate 55 mg

 Potassium citrate 90 mg

 Calcium carbonate 100 mg

 Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

Claims (6)

delete 1. A triterpenoid compound selected from the group consisting of compounds represented by the following formulas (1), (3) and (4), respectively.
[Chemical Formula 1]

(3)

[Chemical Formula 4]

3. The triterpenoid compound according to claim 2, wherein the triterpenoid compound is separated from the white blood line. 1. A composition for immunosuppression comprising a triterpenoid compound or a pharmaceutically acceptable salt thereof selected from the group consisting of compounds represented by the following Formulas (1) to (4) as an active ingredient.
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

5. The method of claim 4, wherein the composition is an organ transplant rejection reaction caused by an immunological hypersensitivity reaction; Autoimmune diseases of lupus or rheumatoid arthritis; Rhinitis, asthma, allergic diseases of atopy, and the like. Extracting the white line extract with water, alcohol or a mixed solvent thereof;
A second step of suspending the extract extracted from the first step in distilled water, and then sequentially fractionating the extract with hexane and chloroform; And
In the second step to perform the density gradient silica gel chromatography (gel chromatography) using a mixed solvent of chloroform and methanol fractions (CHCl ₃ -MeOH) it was dissolved in the chloroform to a third step of obtaining a fraction (1) to (4), wherein the compound represented by the general formula (1) is a compound represented by the following general formula (1).
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

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