JP4895552B2 - Anti-inflammatory compounds - Google Patents

Description

  The present invention relates to a compound having an anti-inflammatory effect, and more particularly, to an anti-inflammatory compound isolated from shaved spines.

  Shaphi (Hippophae rhamnoides L.) is a deciduous shrub belonging to the family Gumiaceae, and is distributed mainly in Europe, the former Soviet Union, West Asia, the Middle East, etc. In China, shaved fruits have long been used for asthma, indigestion and inflammation.

  In recent years, shaved spines have been applied to arteriosclerosis, angina pectoris, ulcers, radiation injury and the like. It has been reported that chemical components of this plant include flavonoid glycosides, sterols, essential oils, and triterpene derivatives contained in fruits, seeds, stems and leaves. As the pharmacological action, blood pressure reduction, anti-arteriosclerosis, antitussive, expectorant, flat asthma, antibacterial action are known.

For example, Japanese Patent Application Laid-Open No. 2004-217545 discloses a novel flavonoid glycoside having a protein non-enzymatic glycation-inhibiting activity, an aldose reductase inhibitory activity, and a free radical scavenging activity, which is derived from shaba spine and has diabetes or diabetic complications. It is described as being effective for the prevention and treatment of diseases, diseases such as aging, cancer, arteriosclerosis and cerebral infarction.
JP 2004-217545 A

  Traditionally, it has been known that the spinach has an anti-inflammatory effect, but the active ingredient was unknown. It is an object of the present invention to clarify a compound having anti-inflammatory effect derived from shaved spines and to develop a new use of the compound.

  As a result of intensive studies on the active ingredient derived from shaved spine, the present inventors have clarified a compound having an anti-inflammatory effect. The present invention has been made based on such findings, and provides a triterpene derivative represented by the following general formula (1). This triterpene derivative and a pharmacologically acceptable salt have an anti-inflammatory action (a nitric oxide production inhibitory action) and a radical scavenging action.

（上記式中、Ｒ₁、Ｒ₂及びＲ₃は同時に又は各々独立して水酸基、水素原子、Ｃ_1-6アルキル基又はアルキルエーテル基を意味する。）

(In the above formulas, R ₁ , R ₂ and R ₃ simultaneously or independently represent a hydroxyl group, a hydrogen atom, a C _1-6 alkyl group or an alkyl ether group.)

  The present invention also provides a nitric oxide inhibitor containing a benzopyran derivative represented by the following general formula (2) or a pharmacologically acceptable salt as an active ingredient. This benzopyran derivative or a pharmacologically acceptable salt also has a radical scavenging action.

（上記式中、Ｒ_４は水素原子、アルキル基、アシル基、糖類を意味する。）

(In the above formula, R ₄ represents a hydrogen atom, an alkyl group, an acyl group, or a saccharide.)

  The triterpene derivative represented by the above general formula (1), the benzopyran derivative represented by the above general formula (2), and pharmacologically acceptable salts thereof have an anti-inflammatory action (a nitric oxide production inhibitory action) and a radical scavenging action. Have. Therefore, it is considered effective for treating various inflammatory diseases, various allergic diseases, diabetes and the like.

  In order to use the novel triterpene derivative and benzopyran derivative and their pharmacologically acceptable salts for therapeutic purposes, each compound and its non-toxic salt are used as active ingredients and are administered orally or parenterally. The dose varies depending on symptoms, age, sex, body weight, dosage form, etc., but for example, when administered orally to an adult, the daily dose is usually 0.1-1000 mg.

  There are no limitations on the dosage form for formulating novel triterpene derivatives and benzopyran derivatives and their pharmacologically acceptable salts, and solid agents such as tablets, pills, capsules, powders, granules, solutions, suspensions Liquid preparations such as liquids and emulsions can be used orally, and intravenous, intramuscular and subcutaneous injections, suppositories, patches and the like can be used parenterally.

  When used as a solid agent, excipients such as starch, lactose, glucose, calcium phosphate, magnesium stearate, carboxymethyl cellulose can be used, and if necessary, lubricants, disintegrants, coating agents, coloring agents, etc. Can be used. In the case of injections and liquid preparations, stabilizers, solution aids, suspending agents, emulsifiers, buffers, preservatives and the like can be contained.

  Next, production examples of the triterpene derivative represented by the general formula (1) and the benzopyran derivative represented by the general formula (2) will be described.

  A spinach (Hippophae rhamnoides L.) branch bark (3 kg) was immersed in 80% acetone (10 L) for 48 hours to obtain an extract. This was repeated three times, and the resulting extract was concentrated and dried to obtain 929.84 g of extract.

  929.84 g of this extract was dissolved in water, and partitioned and extracted sequentially with n-hexane, chloroform, ethyl acetate and n-butanol, and concentrated under reduced pressure to give an n-hexane fraction (21.14 g) and a chloroform fraction ( 29.34 g), an ethyl acetate fraction (37.12 g), an n-butanol fraction (180.23 g), and a water fraction (631.73 g) were obtained.

  Each fraction was subjected to a nitric oxide (NO) production inhibition test described later at a sample concentration of 100 μg / mL. As a result, an n-hexane fraction (89.5%) and a chloroform fraction (90.3%) were obtained. %), Ethyl acetate fraction (67.2%), butanol fraction (24.6%), and water fraction (0.8%) showed NO production inhibitory activity.

  Subsequently, MTT assay was performed for each fraction, and 28.1 g of the chloroform fraction having the strongest activity was applied to a silica gel column (wako gel C-300, 6.5 × 18 cm), and then to n−. Elution sequentially with xylene, ethyl acetate: n-hexane (2:98, 4:96, 8:92, 15:85, 20:80, 40:60, 80:20), ethyl acetate and methanol, Fraction fr. 1 (1.3 g), fr. 2 (1.0 g), fr. 3 (2.0 g), fr. 4 (1.5 g), fr. 5 (2.8 g), fr. 6 (12.2 g) was obtained.

  Next, fr. Normal fraction HPLC for 3 fractions, column is Shiseido Silica SG 80A 10 × 250 mm, mobile phase is eluted with hexane: ethyl acetate 70:30, flow rate 4.0 mL / min (room temperature), retention time 8 minutes 20 seconds The peak eluting at 1 was fractionated using a Shodex RI-72 (differential refractometer). This fraction (230 mg) was again eluted by reverse phase HPLC with column Capcell PAK C18 10 × 250 mm, mobile phase with methanol: water 95: 5, flow rate 4.0 mL / min (room temperature), retention time 10 min 40 The peak eluting at 1 second was fractionated using UV (210 nm) to obtain Compound 1 (24.9 mg). The structural formula (3) of Compound 1 is shown below.

  fr. The 4 fractions were applied to an ODS column (Chromatorex ODS100-200 mesh, 5.5 × 15 cm) and eluted with water: methanol (50: 50 → 0: 100), 9 (fr.4-1 to fr.4). A fraction of -9) was obtained. Of these, fr. 4-1 (0.12 g), reverse phase HPLC, column Capcell PAK C18 10 × 250 mm, mobile phase eluting with methanol: water 35:65, flow rate 4.0 mL / min (room temperature), retention time 13 minutes 40 The peak eluting at 1 second was fractionated using UV (254 nm) to obtain Compound 3 (12.8 mg). The structural formula (4) of Compound 2 is shown below.

The ¹ H- and ¹³ C-NMR data, MS, IR, and UV data of the obtained compound are shown below. Compounds 3 and 4 were measured by VSRIAN Mercury 300, ¹ H-NMR was measured at 300 MHz, and ¹³ C-NMR was measured at 75 MHz.

Table 2 shows ¹ H-NMR and ¹³ C-NMR data (300 MHz, in Chloroform-d) of Compound 2.

  fr. Normal phase HPLC for 4-6 (0.07 g), column Shiseido Silica SG 80A 10 × 250 mm, mobile phase eluted with hexane: ethyl acetate: acetone 60: 35: 5, flow rate 4.0 mL / min (room temperature) The peak eluting at a retention time of 11 minutes and 13 seconds was fractionated using UV (254 nm) to obtain Compound 3 (6.0 mg). The structural formula (5) of Compound 3 is shown below.

Data relating to the properties of Compound 3 are shown in Table 3. HR-FAB-MS (m / z) is measured using JOEL GC mate, UV λ _max nm (logε) is measured using Shimadzu UV-160, and IR ν ^cm-1 _max is measured. This was done using JASCO IR A-2.

Table 4 shows ¹ H-NMR and ¹³ C-NMR data (600 MHz, in Methanol-d ₄ ) of Compound 4.

[Test Example 1] NO production inhibitory activity test Compounds 1 to 3 were used as samples to perform a nitric oxide production inhibitory activity test in the following manner. Each sample was dissolved in DMSO. Separately, a medium in which 5 mL of L-glutamine (200 mM) and 50 mL of FBS were added to 500 mL of F-12HAM medium was prepared so that DMSO in the medium was 0.2 wt%. Next, 50 mL of confused RAW264.7 cells were added to the prepared medium to obtain a cell suspension. This cell suspension was placed in a Falcon tube.

RAW264.7 cells were spun down with a centrifuge (1000 rpm, 3 min, 4 ° C.), and the supernatant was removed using an aspirator. Then, 20 mL of fresh medium was added to the Falcon tube from which the supernatant was removed, and suspended to obtain a suspension adjusted to a concentration of 1.5 × 10 ⁵ cells / mL. Then, 200 μL of the suspension was dispensed into a 96-well plate (8096R manufactured by Sumitomo Bakelite Co., Ltd.), and the cells were brought into close contact with a CO ₂ incubator for 1 hour.

After incubation, 2 μL of LPS (10 μg / mL, O55: B5 manufactured by Sigma), 2 μL of mouse INF-γ (33 ng / mL, manufactured by Genzyme) and 0.4 μL of the compound solution were added to a 96-well plate. This was cultured for 16 hours in a CO ₂ incubator, and 100 μL of the culture supernatant was collected.

To this was added 50 μL of a 0.1% naphthyldiamine solution and 50 μL of a sulfanilamide solution. D. Measurements were taken at 570 nm (control 655 nm). The cell viability was determined by microscopic observation and the MTT method.
Inhibition rate (%) = {1- (X−Y) / (Z−Y)} × 100
X: NO ₂ induced by IFN-gamma and LPS in the presence of test compound ^- amount of Y: test compound, IFN-gamma and NO ₂ LPS-induced in the absence ^- of the amount Z: and IFN-gamma The amount of NO ₂ − induced by LPS Further, from the calculated value, the concentration at which the NO production inhibitory activity by the sample compound was inhibited by 50% (IC ₅₀ ) was determined. The results are shown in Table 5 together with the results of cell viability. In the table, “A” means NO production inhibition rate (%), and “B” means cell survival rate (%).
Evaluation criteria

[Test Example 2] Radical scavenging activity test Using compounds 1 to 3 as samples, a radical scavenging activity test was performed based on the method of Okada et al. To a 96-well plate (Sumitomo Bakelite, # 8096R) was added 0.2 M acetate buffer (pH 5.5) 40 μL, 12% aqueous ethanol solution 120 μL, and 0.4 μL of a test compound dissolved in dimethyl sulfoxide. 40 μL of 5 mM DPPH solution was added and left in the dark for 30 minutes. Thereafter, the absorbance at 520 nm was measured with a plate reader (Bio-Rad 3550 plate reader). And about the obtained measured value, the elimination rate of DPPH radical was computed using the following formula. Further, from the calculated value, the concentration at which radical scavenging activity by the sample compound was inhibited by 50% (IC ₅₀ ) was determined. The results are shown in Table 6.
Erase rate (%) = {1− (X−Z) / (Y−Z)} × 100
X: Absorbance when test compound is added Y: Absorbance when no test compound is added Z: Absorbance of only dimethyl sulfoxide and 12% ethanol solution

Claims (3)

A triterpene derivative represented by the following general formula (1).

(In the above formulas, R ₁ , R ₂ and R ₃ simultaneously or independently represent a hydroxyl group, a hydrogen atom, a C _1-6 alkyl group or an alkyl ether group.)   A nitric oxide production inhibitor comprising the triterpene derivative according to claim 1 or a pharmacologically acceptable salt as an active ingredient.   A radical scavenger comprising the triterpene derivative according to claim 1 or a pharmacologically acceptable salt as an active ingredient.