A sesquiterpenoid compound, and a preparation method and application thereof

Abstract

The application discloses two novel sesquiterpenes compounds extracted from Hubei Sinodendron, three known sesquiterpenes compounds, application of the sesquiterpenes compounds in preparation of anti-neuroinflammatory drugs and a preparation method of the sesquiterpenes compounds. The sesquiterpenes compounds 1-5 of the application show anti-neuroinflammatory activity, and are characterized by inhibiting expression of IL-1beta and or TNF-alpha. Therefore, the sesquiterpenes compounds can be used for preparation of anti-neuroinflammatory drugs. The application provides a new method for preparation of a novel sesquiterpenes compound with anti-neuroinflammatory activity, and provides an ideal candidate compound for development of an efficient anti-neuroinflammatory drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of compound extraction, and particularly relates to two novel sesquiterpenes and three known sesquiterpenes and application thereof in preparation of anti-neuroinflammatory drugs. BACKGROUND

[0002] Inflammation is a protective response of the body to ensure the removal of harmful stimuli, and a healing process to repair damaged tissues. Oxidative damage and inflammation in the central nervous system have been found to be associated with many nervous system diseases, such as Alzheimer's disease, Parkinson's disease, mental illness, major depression, etc. However, chronic or uncontrolled neuroinflammation associated with cell and tissue damage can accelerate the progression of allergic or autoimmune diseases. At present, most flavonoids and polyphenols drugs have been successfully applied to the clinical treatment of neuroinflammation. However, they are difficult to cross the blood-brain barrier, have low bioavailability, and single target, which limits the clinical application. Therefore, it is urgent to develop new anti-neuroinflammatory drugs with strong liposolubility, high efficiency and small side effects. Natural products have been proved to be a valuable source of anti-neuroinflammatory, which has attracted extensive interest of organic chemists. SUMMARY

[0003] The inventors have found that the ethanol extract of Hubei Chiman has an anti-neuroinflammatory effect, and two novel sesquiterpenes and three known sesquiterpenes are isolated from the effective part, and pharmacodynamic evaluation shows that they have good anti-neuroinflammatory effect.

[0004] The technical problem solved by the present application is to provide two novel sesquiterpenes and three known sesquiterpenes;

[0005] Another technical problem solved by the present application is to provide a preparation method of the two novel sesquiterpenes and the three known sesquiterpenes;

[0006] Still another technical problem solved by the present application is to provide a pharmaceutical composition containing the above-mentioned sesquiterpenes 1-5;

[0007] The technical problem solved by the present application is to provide a pharmaceutical composition containing the above-mentioned sesquiterpenes as an application of anti-neuroinflammatory drugs.

[0008] Specifically, the present application relates to the following compounds:

[0009]

[0010] The present application relates to a preparation method of the two novel sesquiterpenes and the three known sesquiterpenes.

[0011] (1) Dry Hubei gold caladium root powder is extracted with ethanol at room temperature, and the extract is evaporated to obtain an extract.

[0012] (2) The extract in step 1 is adsorbed on diatomite, and is eluted with petroleum ether, ethyl acetate and methanol in sequence to obtain three parts.

[0013] (3) The ethyl acetate part in step 2 is separated by normal phase silica gel column, and is gradient eluted with petroleum ether-ethyl acetate mixed solvent with a volume ratio of 80:1-0:1, and is combined by silica gel thin layer plate to obtain nine components F.A-F.I.

[0014] (4) 11.3 g of component F.B in step 3 is separated by silica gel 580.0 g, and is eluted with dichloromethane / methanol as mobile phase with a volume ratio of 100:1 / 1:1, v / v to obtain eight components F.B1-F.B8.

[0015] (5) 32.4 g of component F.B3 in step 4 is separated by silica gel 100.0 g, and is eluted with petroleum ether / ethyl acetate as mobile phase with a volume ratio of 50:1 / 1:1, v / v to obtain four fractions F.B3a-F.B3e.

[0016] (6) 326.0 mg of component F.B3c in step 5 is separated by Sephadex LH-20 gel, and is eluted with methanol / water as mobile phase with a volume ratio of 60%-90% to obtain eight fractions F.B3c1-F.B3c8.

[0017] (7) 563.6 mg of component F.B3c in step 6 is further purified by semi-preparative YMC-pack ODS-A column, and is eluted with MeCN / H2O as mobile phase with a volume ratio of 70:30 at a flow rate of 7 mL / min by semi-preparative HPLC system to obtain 2, 4 and 5. 829.0 mg of F.B3c8 in step 6 is separated by semi-preparative high performance liquid chromatography, and is eluted with MeCN / H2O as mobile phase with a volume ratio of 60:40 at a flow rate of 7 mL / min to obtain 1 and 3.

[0018] The present application establishes a BV-2 cell microglial neuroinflammation model induced by LPS. The mRNA levels of IL-1β and TNF-α are detected by RT-qPCR, which are two key immune response mediators in a wide inflammatory response. Compounds 2, 4 and 5 have inhibitory effects on the expression of IL-1β and TNF-α, and compounds 1 and 3 only have inhibitory effects on the expression of IL-1β.

[0019] Another aspect of the present application provides a pharmaceutical composition which employs one or more selected from the above-mentioned sesquiterpenes as a raw material, and comprises a therapeutically effective amount of one or more selected from the above-mentioned sesquiterpenes as an active ingredient. The composition can further comprise pharmaceutically acceptable pharmaceutical adjuvants such as carriers, excipients, adjuvants and / or diluents, etc. The pharmaceutical composition is used for treating neuroinflammation, etc.

[0020] Still another aspect of the present application provides a method for treating neuroinflammation, which comprises administering to a subject in need of such treatment a therapeutically effective amount of one or more selected from the above-mentioned sesquiterpenes or the above-mentioned pharmaceutical composition.

[0021] Advantages

[0022] Based on the advantages of such sesquiterpenes in terms of novel chemical structure and significant biological activity, they have good development prospects and are expected to develop into novel drugs for treating neuroinflammation, etc. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the key to compounds 1-2 1 H– 1 H COSY and HMBC correlation, where the bold black solid line represents 1 H– 1 HCOSY two-dimensional nuclear magnetic correlation, and single arrow represents HMBC two-dimensional nuclear magnetic correlation. Further determine the structure of novel sesquiterpenes 1 and 2.

[0024] Figure 2 is the anti-neuroinflammatory activity determination of compounds 2, 4 and 5 at different concentrations and the research results of their action mechanisms. (A-F) RT-qPCR detection of the effects of compounds 2, 4 and 5 on IL-1β and TNF-α mRNA expression. (G) Immunoblotting and densitometric analysis of p-IKBα / IKBα (H) and p-p65 / p65 (I), respectively. *p<0.05, **p<0.01 compared with the Con group; #p<0.05 and ##p<0.01 compared with the Mod group.

[0025] Figure 3 is the in vitro anti-neuroinflammatory effect of compounds 1 and 3. Respectively, RT-qPCR detection of the effects of compounds 1 and 3 on IL-1β and TNF-α mRNA expression. DETAILED DESCRIPTION

[0026] The following implementation and pharmacological activity experiments further illustrate the present application, but do not mean any limitation on the present application.

[0027] The dried root powder of Lycoris chinensis was extracted with ethanol at room temperature. The extract was evaporated to dryness to give a residue. The residue was adsorbed on diatomite and eluted with petroleum ether, ethyl acetate and methanol successively to give three fractions. The ethyl acetate fraction was separated by normal phase silica gel column and eluted with petroleum ether-ethyl acetate (80:1-0:1, v / v) to give nine fractions F.A-F.I. Fraction F.B (11.3 g) was separated by silica gel (580.0 g) and eluted with dichloromethane / methanol (100:1 / 1:1, v / v) to give eight fractions F.B1-F.B8. Fraction F.B3 (32.4 g) was separated by silica gel (100.0 g) and eluted with petroleum ether / ethyl acetate (50:1 / 1:1, v / v) to give four fractions F.B3a-F.B3e. Fraction F.B3c (326.0 mg) was separated by Sephadex LH-20 gel and eluted with methanol / water (60%-90%) to give eight fractions F.B3c1-F.B3c8. Fraction F.B3c5 (563.6 mg) was further purified by semi-preparative HPLC system (YMC-pack ODS-A column, MeCN / H2O, 70:30, 7 mL / min) to give 2, 4 and 5. Fraction F.B3c8 (829.0 mg) was separated by semi-preparative HPLC system (MeCN / H2O, 60:40, 7 mL / min) to give 1 and 3.

[0028] The above compounds were separated by HPLC (YMC-pack ODS-A, 250 x 20 mm, S-5 μm, 12 nm, 7 mL / min).

[0029] Structure identification of compounds 1-5

[0030] The novel compounds 1 and 2 were tested for structural analysis and the following physicochemical property data were obtained:

[0031] Compound 1: Colorless crystals (MeOH); m.p. 169-170 °C; [α] D 25 = +56.4 (c = 0.05 in MeOH); 1 H and 13 C NMR data, see Tables 1 and 2; IR: v max = 1683 cm -1 (C=0); UV (MeOH): λ max (log ε) = 245 (0.86) nm; (+)-HRESIMS: m / z 279.1222 [M+H] +(calcd for C 15 H 19 O5,279.1227).

[0032] Compound 2: Colorless crystals (MeOH); m.p. 170-171 °C; [α] D 25 = +51.20 (c = 0.05 in MeOH); 1 Hand 13 C NMR data, see Tables 1 and 2; IR: v max = 3434 cm -1 (O-H), 1762 cm -1 (C=0); UV (MeOH): λ max (log ε) = 206 (0.26), 275 (0.67) nm; (+)-HRESIMS: m / z 285.1089 [M + Na] + (calcd for C 15 H 18 O4Na, 285.1097).

[0033] Table 1

[0034] 1 H NMR spectroscopic data of compounds 1-2 in CDCl3(δ H in ppm, J in Hz, Measured at 600MHz).

[0035]

[0036] Table 2

[0037] 13 C NMR spectroscopic data of compounds 1-2 in CDCl3(δ C in ppm, Measured at 150MHz).

[0038]

[0039] Anti-neuroinflammatory activity assay of compounds 1, 2, 3, 4 and 5

[0040] Research has found that sesquiterpenes have a wide range of pharmacological activities, and this class of chemical components has novel structure and wide distribution, and has always been a hot spot in the research of natural medicinal chemistry. The following is the determination of the anti-neurogenic effect of sesquiterpenes.

[0041] 1. Cell culture, stimulation and treatment

[0042] BV-2 mouse microglial cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics, and stored in a cell incubator providing 5% CO2 at 37°C. Cells (1 x 10 5 cells / well) were inoculated in a 6-well plate, and then subsequent experiments were performed.

[0043] 2. Quantitative reverse transcription polymerase chain reaction

[0044] Total RNA samples of cultured BV-2 cells were extracted with TRIzol TM reagent. RNA was quantified by a microplate reader, and then converted to cDNA using Evo M-MLV reverse transcriptase premix. Then, according to the manufacturer's instructions, RT-qPCR detection was performed using SYBR Green Pro Taq HS qPCR kit to detect mRNA levels.

[0045] 3. Western blot analysis

[0046] All protein samples were extracted with RIPA buffer containing protease and phosphatase inhibitors. The protein concentration was determined by BCA kit, then about 60 μg of protein was separated by 10% polyacrylamide gel and transferred to PVDF membrane, after blocking in 5% non-fat milk, incubated with primary antibody reactive to IKBα, p-IKBα, NF-κB p65, p-NF-κB p65, β-actin, followed by secondary antibody incubation, finally, the membrane was visualized by ECL kit and analyzed by ImageJ software.

[0047] 4. Statistical analysis

[0048] Statistical analysis was performed using GraphPad 9.0 software. All data were expressed as mean ± standard deviation (SD) of at least 3 biological replicates, and analyzed by one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.

Claims

1. A sesquiterpene compound, characterized by having the structure shown below: 。 2. The method for preparing the sesquiterpene compound of claim 1, comprising the following steps: (1) dry Hubei Chrysomyrtus root powder is extracted with ethanol at room temperature, and the extract is evaporated to obtain an extract; (2) the extract in step 1 is adsorbed on diatomite, and is eluted with petroleum ether, ethyl acetate and methanol in sequence to obtain three parts; (3) the ethyl acetate part in step 2 is separated by normal phase silica gel column, gradient eluted with petroleum ether-ethyl acetate mixed solvent with a volume ratio of 80:1-0:1, and combined with silica gel thin layer plate to obtain nine components F.A-F.I; (4) 11.3 g of component F.B in step 3 is separated by silica gel 580.0 g, using dichloromethane-methanol as mobile phase with a volume ratio of 100:1-1:1, to obtain eight components F.B1-F.B8; (5) 2.4 g of component F.B3 in step 4 is separated by silica gel 100.0 g, using petroleum ether-ethyl acetate as mobile phase with a volume ratio of 50:1-1:1, to obtain four fractions F.B3a-F.B3e; (6) 326.0 mg of component F.B3c in step 5 is separated by Sephadex LH-20 gel, using methanol / water as mobile phase with a volume ratio of 60%-90%, to obtain eight fractions F.B3c1-F.B3c8; (7) Separation of F.B3c8 29.0 mg from step 6 by semi-preparative HPLC with MeCN / H2O 60:40 as mobile phase at a flow rate of 7 mL / min gave 1 .

3. The sesquiterpene compound of claim 1 for use in the preparation of an anti-neuritis drug.

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