An alkane compound, its preparation method and application

Abstract

This invention discloses a diphenylheptane compound, a novel chemical component discovered in Alpinia oxyphylla. The invention also identifies the structure of the compound isolated by the above method using physicochemical properties and modern spectroscopic techniques. Furthermore, the invention evaluates the compound's activity using an LPS-induced RAW264.7 cell inflammation model and other activity screening systems, finding that the compound has a certain protective effect on the mouse macrophage line RAW264.7, significantly inhibiting NO release and exhibiting strong anti-inflammatory activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an alkane compound, its preparation method, and its application. Background Technology

[0002] *Alpinia oxyphylla* Miq. is a perennial herb that grows in shady and damp places under forests, mainly distributed in Guangdong and Hainan provinces. The dried, mature fruit of *Alpinia oxyphylla*, a plant in the ginger family, is typically spindle-shaped or elliptical, about 1.2-2 cm long and 1-1.3 cm in diameter. Its surface is brown with 13-20 irregular, intermittent raised lines; the pericarp is thin and hard, tightly adhering to the seed. Inside the seed bulb, a light brown septum divides it into three chambers, each containing 6-11 seeds. The seeds themselves are irregularly polyhedral in shape, 3-4 mm in diameter, and have a pale yellow aril.

[0003] As one of the "Four Great Southern Chinese Herbs," Alpinia oxyphylla has a long history of medicinal use. Its medicinal application was first recorded in *Bencao Shiyi* (Supplement to the Compendium of Materia Medica), where Zang Qi stated that it "stops vomiting and hiccups...and can be held in the mouth to reduce saliva and impurities." *Zheng Lei Ben Cao* (Classified Materia Medica) records its use in treating "spermatorrhea, dribbling urination," highlighting its kidney-warming effect. *Bencao Gangmu* (Compendium of Materia Medica) records its efficacy in treating frequent urination, heart deficiency with urinary incontinence, leukorrhea, abdominal distension, abdominal pain, diarrhea, halitosis, metrorrhagia, and threatened abortion. Classic formulas using Alpinia oxyphylla, such as Suoquan Wan and Yizhi Ren San, have kidney-tonifying and urination-reducing effects. Compound preparations of Alpinia oxyphylla, such as Jiannao Wan, Zhiqi Keli, and Yizhi Xingnao Tang, are clinically used to treat symptoms such as memory decline and neurasthenia. Modern pharmacological research shows that Alpinia oxyphylla possesses a wide range of pharmacological effects, including neuroprotective, nephroprotective, antidiuretic, anti-inflammatory, and antioxidant properties. As a plant resource with high safety that can be used for both medicinal and edible purposes, there is currently very little product development for Alpinia oxyphylla, which limits its application.

[0004] Therefore, further research on the composition of Alpinia oxyphylla in order to obtain an active ingredient with medicinal effects is not only beneficial for understanding the compound composition of Alpinia oxyphylla, but also of great significance for the in-depth development and application of Alpinia oxyphylla. Summary of the Invention

[0005] The present invention aims to conduct a more in-depth study on the anti-inflammatory active ingredients in Alpinia oxyphylla and to discover its active ingredients.

[0006] In view of this, the present invention proposes a diphenylheptane compound or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite, the structure of which is shown in Formula I:

[0007]

[0008] Furthermore, the above-mentioned compound can be,

[0009]

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned compound, characterized by comprising the following steps:

[0011] A) Take Alpinia oxyphylla, reflux extract with 50-70% ethanol, remove the solvent, and obtain the total extract;

[0012] B) The total extract was dissolved in water and separated by macroporous adsorption resin column chromatography, eluting sequentially with water, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol. Each eluent was collected and concentrated under reduced pressure to obtain the water-eluted fraction, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol eluents; each gradient elution was performed using 4 column volumes (the same applies below).

[0013] C) The 65-75% ethanol eluent fraction was separated by silica gel column chromatography, and 10 fractions (3A-3J) were collected by cyclohexane-ethyl acetate gradient elution. Fraction 3F was separated by ODS column chromatography with methanol-water gradient elution to obtain 5 fractions (3F1-3F5). Fraction 3F5 was separated by preparative liquid chromatography to obtain 11 fractions (3F5A-3F5K). 3F5E was separated by preparative liquid chromatography to obtain compound 1. Further, compound 1 can be separated by preparative liquid chromatography to obtain compounds 1a and 1b.

[0014] Specifically, the Alpinia oxyphylla kernel can be the dried, mature fruit of Alpinia oxyphylla.

[0015] Further, step A) includes: taking dried Alpinia oxyphylla kernels, refluxing them with 3-5 times the amount of 50-70% ethanol 1-3 times, each time for 1-3 hours, combining the extracts, removing the solvent under reduced pressure, and obtaining the total extract.

[0016] Preferably, step B) includes: eluting sequentially with water, 50% ethanol, 70% ethanol, and 95% ethanol, collecting each eluent separately, concentrating under reduced pressure until no alcohol odor is detected, and obtaining water-eluted fractions, 50% ethanol-eluted fractions, 70% ethanol-eluted fractions, and 95% ethanol-eluted fractions.

[0017] The cyclohexane-ethyl acetate gradient elution in step C) is performed at a volume ratio of 100-90:0-10 to 0:100; the methanol-water gradient elution is performed at a volume ratio of 30-50:70-50 to 100:0.

[0018] Specifically, the macroporous adsorption resin includes one or more of the following: D101 type macroporous adsorption resin, HP-20 type macroporous adsorption resin, HPD-100 type macroporous adsorption resin, HPD-100A type macroporous adsorption resin, or HPD-300 type macroporous adsorption resin.

[0019] Further, the cyclohexane-ethyl acetate gradient elution in step C) is performed with volume ratios of 98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, and 0:1; the methanol-water gradient elution is performed with volume ratios of 40:60, 45:55, 50:40, 55:45, 65:35, 80:20, and 100:0.

[0020] Further, step A) involves reflux extraction with 60% ethanol twice, each time for 2 hours.

[0021] Specifically, the conditions for preparing the liquid chromatography include: a specification of C 18 The mobile phase for separating fraction 3F5 was 5μm and 10×250mm (Phenomenaex Gemini); the mobile phase for separating fraction 3F5 was methanol-water (volume ratio 55:45); the mobile phase for separating fraction 3F5E was acetonitrile-water (volume ratio 35:65); the detection wavelength was 223nm and the flow rate was 3mL / min.

[0022] Furthermore, the method further includes: performing preparative liquid-phase separation on the compound 1 using an EnantinPak Y3, 5μm, C18, 250×4.60mm column; wherein the mobile phase volume ratio is 40:60 acetonitrile-water, the detection wavelength is 223nm, and the flow rate is 1mL / min.

[0023] The purpose of this invention is to provide the use of the above-mentioned compounds or their pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules, and metabolites in the preparation of anti-inflammatory drugs.

[0024] The present invention also proposes a drug comprising, as described above, a diphenylheptane compound or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite.

[0025] Furthermore, the drug contains a therapeutically effective amount of the compound of formula (I) above, or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite, and one or more pharmaceutically acceptable carriers.

[0026] Specifically, the drug can be any dosage form as defined in pharmaceutics, including tablets, capsules, soft capsules, gels, oral preparations, suspensions, granules, patches, ointments, pills, powders, injections, infusions, lyophilized injections, intravenous emulsions, liposome injections, suppositories, sustained-release preparations, or controlled-release preparations.

[0027] Furthermore, the pharmaceutically acceptable carrier refers to conventional pharmaceutical carriers, such as diluents, excipients, and water; fillers such as starch, sucrose, lactose, and microcrystalline cellulose; binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; wetting agents such as glycerin; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as hexadecyl alcohol and sodium dodecyl sulfate; adsorbents such as kaolin and soap clay; and lubricants such as talc, calcium and magnesium stearate, micronized silica gel, and polyethylene glycol. Other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0028] The diphenylheptane compound described in this invention is a novel chemical component discovered by researchers in Alpinia oxyphylla, and it was found to be stable in all batches of Alpinia oxyphylla. The inventors further investigated its properties using physicochemical methods and modern spectroscopic techniques (MS, etc.). 1 H-NMR, 13 The compounds isolated by the above methods (such as C-NMR) were structurally identified, confirming that they are novel compounds with the structure shown in formula (I). This invention also used an LPS-induced RAW 264.7 cell inflammation model and other activity screening systems for activity evaluation, finding that the compound has a certain protective effect on the mouse macrophage line RAW 264.7, can significantly inhibit NO release, and shows strong anti-inflammatory activity. It has good research and development prospects. Attached Figure Description

[0029] Figure 1 The HR-ESI-Q-TOF-MS spectrum of compound 1 of the present invention is shown below.

[0030] Figure 2 Compound 1 of the present invention 1 H-NMR spectrum

[0031] Figure 3 Compound 1 of the present invention 13 C-NMR spectrum;

[0032] Figure 4 Compound 1 of the present invention 13 C-NMR and DEPT-135 spectra;

[0033] Figure 5 H is the compound 1 of the present invention.1 -H 1 COSY spectrum;

[0034] Figure 6 The HSQC spectrum of compound 1 of this invention is shown below.

[0035] Figure 7 The HMBC spectrum of compound 1 of the present invention is shown below.

[0036] Figure 8 The NOESY spectrum of compound 1 of this invention;

[0037] Figure 9 The UV spectrum of compound 1 of this invention is shown below.

[0038] Figure 10 The IR spectrum of compound 1 of the present invention is shown below.

[0039] Figure 11 The HPLC chromatograms of compounds 1a / 1b separated by chiral column for compound 1 of the present invention;

[0040] Figure 12 The X-ray spectra of compounds 1a and 1b of this invention are shown.

[0041] Figure 13 IC50 inhibitors of NO release from compounds 1a and 1b of the present invention 50 curve. Detailed Implementation

[0042] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings and examples, so as to better understand the solution of the present invention and its advantages in various aspects. However, the specific embodiments and examples described below are for illustrative purposes only and are not intended to limit the present invention.

[0043] The following will provide a detailed description based on the experimental examples.

[0044] It is particularly important to note that similar substitutions and modifications made to this invention are obvious to those skilled in the art, and they are all considered to be included in this invention. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0045] Unless otherwise specified, all conditions in this invention are performed under conventional conditions or conditions recommended by the manufacturer. Raw materials, excipients, reagents, and instruments used, unless otherwise specified, are all commercially available products. Alpiniae Oxyphallae Fruit was purchased from Anguo Shiyuan Trading Co., Ltd. in 2020; its place of origin is Hainan Province.

[0046] The present invention will be further illustrated below with reference to the embodiments:

[0047] Example 1: Preparation of the compound of the present invention

[0048] Step (1): Take the dried, mature fruit of Alpinia oxyphylla, and extract it twice with 50% ethanol for 2 hours each time. Combine the extracts, remove the solvent under reduced pressure, and obtain the total extract. Dissolve the total extract in water and separate it by HP-20 macroporous adsorption resin column chromatography. Elute sequentially with water, 50% ethanol, 70% ethanol, and 95% ethanol, with 4 column volumes for each gradient (the same below). Collect each eluent and concentrate it under reduced pressure until there is no alcohol odor to obtain the water eluent, 50% ethanol eluent, 70% ethanol eluent, and 95% ethanol eluent.

[0049] Step (2): Take the 70% ethanol eluent from step (1), separate it by silica gel column chromatography, and perform gradient elution with cyclohexane-ethyl acetate at volume ratios of 98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, and 0:1 to collect 10 fractions (3A-3J). Fraction 3F is eluted by ODS column chromatography with a methanol-water gradient (40:60, 45:55, 50:40, 55:45, 65:35, 80:20, and 100:0, v / v) to obtain 5 fractions 3F1-3F5. Fraction 3F5 is separated by preparative liquid chromatography I to obtain 11 fractions 3F5A-3F5K. 3F5E is separated by preparative liquid chromatography II to obtain compound 1. Compound 1 is separated by preparative liquid chromatography III to obtain compounds 1a and 1b.

[0050] The conditions for preparing liquid chromatography I and II in step (2) above are as follows: preparative column: Phenomenex Gemini, C 18The preparative high-performance liquid chromatograph (HPLC) was a Shimadzu (Japan) with a 5μm, 10×250mm column, an LC-6AD (SHIMADZU, LIQUID CHROMATOGRAPH) pump, an SPD-20A (prominence UV / VIS DETECTOR) detector, and an LC solution workstation. The preparative HPLC III conditions were as follows: Separation column: Enantin Pak Y3 (C18, 5μm, 4.60×250mm) column; preparative HPLC was a Shimadzu (Japan) with a LC-6AD (SHIMADZU, LIQUID CHROMATOGRAPH) pump; an SPD-20A (prominence UV / VIS DETECTOR) detector; and an LC solution workstation. The mobile phase for fraction 3F5 was methanol-water (55:45 v / v), with a detection wavelength of 223 nm and a flow rate of 3 mL / min. The mobile phase for compound 1 was acetonitrile-water (35:65 v / v), with a detection wavelength of 223 nm and a flow rate of 3 mL / min. The mobile phases for compounds 1a and 1b were acetonitrile-water (40:60 v / v), with a detection wavelength of 223 nm and a flow rate of 1 mL / min.

[0051] Example 2: Preparation of the compound of the present invention

[0052] (1) Take the dried, mature fruit of Alpinia oxyphylla and extract it twice with 60% ethanol for 2 hours each time. Combine the extracts and remove the solvent under reduced pressure to obtain the total extract. Dissolve the total extract in water and separate it by HP-20 macroporous adsorption resin column chromatography. Elute with water, 45% ethanol, 65% ethanol and 90% ethanol in sequence. Collect each eluent and concentrate it under reduced pressure until there is no alcohol odor to obtain the water eluent, 45% ethanol eluent, 65% ethanol eluent and 90% ethanol eluent.

[0053] (2) Take the 65% ethanol eluent from step (1), separate it by silica gel column chromatography, and elute with cyclohexane-ethyl acetate gradient (98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 0:1 v / v) to collect 10 fractions (3A-3J). Fraction 3F is eluted by ODS column chromatography with methanol-water gradient (40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0, v / v) to obtain 5 fractions 3F1-3F5. Fraction 3F5 is separated by preparative liquid chromatography to obtain fractions 3F5A-3F5K, a total of 11 fractions. 3F5E is separated by preparative liquid chromatography to obtain compound 1. Compound 1 is separated by preparative liquid chromatography to obtain compounds 1a and 1b.

[0054] The conditions for preparing liquid chromatography I and II in step (2) above are as follows: preparative column: Phenomenex Gemini, C 18 The preparative high-performance liquid chromatograph (HPLC) was a Shimadzu (Japan) with a 5μm, 10×250mm column, an LC-6AD (SHIMADZU, LIQUID CHROMATOGRAPH) pump, an SPD-20A (prominence UV / VIS DETECTOR) detector, and an LC solution workstation. The preparative HPLC III conditions were as follows: Separation column: Enantin Pak Y3 (C18, 5μm, 4.60×250mm) column; preparative HPLC was a Shimadzu (Japan) with a LC-6AD (SHIMADZU, LIQUID CHROMATOGRAPH) pump; an SPD-20A (prominence UV / VIS DETECTOR) detector; and an LC solution workstation. The mobile phase for fraction 3F5 was methanol-water (55:45 v / v), with a detection wavelength of 223 nm and a flow rate of 3 mL / min. The mobile phase for compound 1 was acetonitrile-water (35:65 v / v), with a detection wavelength of 223 nm and a flow rate of 3 mL / min. The mobile phases for compounds 1a and 1b were acetonitrile-water (40:60 v / v), with a detection wavelength of 223 nm and a flow rate of 1 mL / min.

[0055] Example 3: Preparation of the compound of the present invention

[0056] (1) Take dried, ripe Alpinia oxyphylla fruit and use... 70% ethanol The extract was refluxed twice, 2 hours each time. The extracts were combined, and the solvent was removed under reduced pressure to obtain the total extract. The total extract was dissolved in water and separated by HP-20 macroporous adsorption resin column chromatography. The extract was eluted sequentially with water, 55% ethanol, 75% ethanol, and 100% ethanol. The eluents were collected separately and concentrated under reduced pressure until no alcohol odor was detected, yielding the water-eluted fraction, the 55% ethanol-eluted fraction, the 75% ethanol-eluted fraction, and the 100% ethanol-eluted fraction.

[0057] (2) Take the 75% ethanol eluent from step (1), separate it by silica gel column chromatography, and elute with cyclohexane-ethyl acetate gradient (98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 0:1 v / v) to collect 10 fractions (3A-3J). Fraction 3F is eluted by ODS column chromatography with methanol-water gradient (40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0, v / v) to obtain 5 fractions 3F1-3F5. Fraction 3F5 is separated by preparative liquid chromatography to obtain 11 fractions 3F5A-3F5K. 3F5E is separated by preparative liquid chromatography to obtain compound 1. Compound 1 is separated by preparative liquid chromatography to obtain compounds 1a and 1b.

[0058] The conditions for preparing liquid chromatography I and II in step (2) above are as follows: preparative column: Phenomenex Gemini, C 18 The preparative high-performance liquid chromatograph (HPLC) was a Shimadzu (Japan) with a 5μm, 10×250mm column, an LC-6AD (SHIMADZU, LIQUID CHROMATOGRAPH) pump, an SPD-20A (prominence UV / VIS DETECTOR) detector, and an LC solution workstation. The preparative HPLC III conditions were as follows: Separation column: Enantin Pak Y3 (C18, 5μm, 4.60×250mm) column; preparative HPLC was a Shimadzu (Japan) with a LC-6AD (SHIMADZU, LIQUID CHROMATOGRAPH) pump; an SPD-20A (prominence UV / VIS DETECTOR) detector; and an LC solution workstation. The mobile phase for fraction 3F5 was methanol-water (55:45 v / v), with a detection wavelength of 223 nm and a flow rate of 3 mL / min. The mobile phase for compound 1 was acetonitrile-water (35:65 v / v), with a detection wavelength of 223 nm and a flow rate of 3 mL / min. The mobile phases for compounds 1a and 1b were acetonitrile-water (40:60 v / v), with a detection wavelength of 223 nm and a flow rate of 1 mL / min.

[0059] Example 4: Structural identification of the compounds of the present invention

[0060] Yellow oily substance. HR-ESI-MS gives m / z 329.1758 [M+H] + (Calculated value is 329.1753), the molecular formula of the compound is determined to be C. 20 H 24 O4, the calculated degree of unsaturation is 9.

[0061] As shown in Table 1 and Figures 1-12 As shown, compound 1 1 In the H-NMR (600MHz, in CDCl3) spectrum, a monosubstituted benzene ring hydrogen signal [δ] was observed. H 7.28(2H,m,H-3″,5″),7.19(3H,m,H-2″,4″,6″)], 1,2,4-substituted benzene ring hydrogen signals [δ H 6.95 (1H,brs,H-2′), 6.88 (2H,d,J=7.7Hz,H-5′,6′)], 3 SPs 3 Hybridized hydroxymethyl hydrogen signal [δ] H 4.77 (1H,dd,J=11.8,2.0Hz,H⁻¹),4.35 (1H,m,H⁻³),3.96 (1H,m,H⁻⁵)], 1-methoxyhydrogen signal [δ] H 3.91(3H,s,3′-OCH3)], and several SPs 3 Hybridized methylene hydrogen signal. 13 The C-NMR (150MHz, in CDCl3) spectrum combined with the DETP 135 spectrum showed a total of 20 carbon signals, including one methoxy signal (δ). C 56.0), 4 methylene signals (δ C 40.6, 38.6, 38.0, 31.8), 11 methine signals (δ C 128.6×2, 128.5×2, 125.8, 118.9, 114.2, 108.9, 73.4, 71.3, 65.2), 4 quaternary carbon signals (δ C (146.5, 144.9, 142.5, 135.2). Based on 1D-NMR analysis, compound 1 is inferred to be a diphenylheptane compound.

[0062] In the HMBC spectrum, H-1 was observed to be correlated with C-2, 3, 5, 1′, 2′, 6′, and H-7 was observed to be correlated with C-5, 6, 1″, 2″, 6″; (in summary...) 1 H- 1 The correlation between H-1 / H2-2 / H-3 / H2-4 / H-5 in the HCl COSY spectrum indicates that the two benzene rings are connected through a pyran ring.

[0063] In the NOESY spectrum, H-1 and H-5 were observed to be correlated, indicating that they are located on the same side. Compound 1 was separated into a pair of enantiomers, 1a and 1b, by chiral HPLC. Combined with CuKα diffraction data obtained from X-ray single-crystal diffraction, the absolute configurations of 1a and 1b were determined to be 1S,3S,5S and 1R,3R,5R, respectively, and identified as (1S,3S,5S)-3-hydroxy-1,5-epoxy-1-(3-hydroxy-4-methoxyphenyl)-7-phenylhept an-3-ol and (1R,3R,5R)-3-hydroxy-1,5-epoxy-1-(3-hydroxy-4-methoxyphenyl)-7-phenylhept an-3-ol, with the following structures:

[0064]

[0065] Table 1 Compound 1 1 H and 13 C NMR data

[0066]

[0067] Multiplets and or overlapped signals are reported without designatingmultiplicity

[0068] The compounds in each example were identified using the same method, and the results were consistent. Example 5: In vitro anti-PGE2 test of the compounds of the present invention.

[0069] 1. Materials

[0070] 1.1 Drug compounds 1a and 1b;

[0071] 1.2 Cell Model: Mouse macrophage line RAW 264.7, obtained from the Chinese Academy of Traditional Medicine; culture conditions: DMEM + 10% fetal bovine serum (FBS), 37℃, 5% CO2.

[0072] 2. Principles and Methods

[0073] 2.1 Experimental Principle

[0074] Lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria (Sigma-Aldrich, lot number: 114M4009) is one of the most important pathogenic molecules mediating infectious inflammatory damage, and many diseases are closely associated with LPS-induced persistent subclinical inflammation. LPS has been widely used to induce inflammation in animal and cell experiments.

[0075] Macrophages play a crucial role in the inflammatory response. When stimulated, macrophages produce inflammatory factors and mediators, which is a key process of inflammation. The inhibition of these factors is often used as an important indicator for evaluating the anti-inflammatory activity of drugs.

[0076] 2.2 Drug inhibition test on NO secretion

[0077] 1. Experimental Procedure

[0078] Cells were pipetted off and the cell density was adjusted to 2 × 10⁶ cells / mL in DMEM medium containing 10% FBS. 5 The sample was inoculated at a density of 100 μL / mL into each well of a 96-well plate. After inoculation, the plate was placed in an incubator for 24 hours. Subsequently, the 96-well plate was removed, the supernatant was aspirated, and drug-containing medium prepared with serum-free DMEM was added. The specific experimental setup is as follows:

[0079] (1) Solvent control group (group 1): 100 μL of serum-free DMEM medium containing 1 / 1000 DMSO was added to each well;

[0080] (2) Model group (+ group): 100 μL of serum-free DMEM medium with LPS at a final concentration of 0.5 μg / ml was added to each well;

[0081] (3) Drug administration sample group (including compounds 1a and 1b, positive control hydrocortisone): 100 μL of culture medium containing the corresponding concentration of sample and 0.5 μg / ml LPS was added to each well;

[0082] After the drug was added, the 96-well plate was placed in a CO2 cell culture incubator and cultured for 24 hours.

[0083] 2. Experimental Results

[0084] (1) The concentration of nitrite in the culture medium, an indicator of NO production, was measured according to the Griess method (Beyotime Biotechnology, S0021). Cell-free supernatant and Griess reagent (nitrate reduction assay reagent) were thoroughly mixed in equal 50 μL volumes; the absorbance of the final product was measured at 540 nm using a microplate reader. The nitrite concentration and inhibition rate were calculated based on the standard calibration curve. Details of the nitrite concentration results for each experimental group and the control group are provided below. Figure 13 ;

[0085] (2) The inhibitory effect of the tested compound on LPS-induced NO production is described as IC50. 50 The values ​​are detailed in Table 2.

[0086] Table 2. Inhibitory effects of compounds on LPS-induced NO production in RAW264.7 cells.

[0087]

[0088] Experimental data show that the diphenylheptane compounds 1a and 1b isolated from Alpinia oxyphylla in this study both inhibited LPS-induced NO production in RAW264.7 cells (see details). Figure 13 The IC50 of some of the compounds was determined by examining their inhibitory effects on LPS-induced NO in RAW264.7 cells. 50 The results showed that compound 1a had an IC50 value for inhibiting NO in LPS-induced RAW264.7 cells. 50 The value was 31.84 μM, and its anti-inflammatory effect was stronger than that of compound 1b. The IC50 value of compound 1b for inhibiting NO in LPS-induced RAW264.7 cells was 31.84 μM. 50 The value is 44.63 μM.

[0089] The above descriptions are merely embodiments of the present invention. Commonly known technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims. The specific embodiments described in the specification can be used to interpret the content of the claims. The above descriptions are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A diphenylheptane compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in Formula I:

2. The compound according to claim 1, wherein the compound has the following structure:

3. A method for preparing the compound as described in claim 1, characterized in that, Includes the following steps: A) Take Alpinia oxyphylla, reflux extract with 50-70% ethanol, remove solvent, and obtain total extract; B) The total extract was dissolved in water and separated by macroporous adsorption resin column chromatography. It was eluted sequentially with water, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol. The eluents were collected separately and concentrated under reduced pressure to obtain the water-eluted fraction, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol eluents. C) The 65-75% ethanol eluent was separated by silica gel column chromatography, and 10 fractions (3A-3J) were collected by gradient elution with cyclohexane-ethyl acetate. Fraction 3F was separated by ODS column chromatography with methanol-water gradient elution to obtain 5 fractions (3F1-3F5). Fraction 3F5 was separated by preparative liquid chromatography to obtain 11 fractions (3F5A-3F5K). 3F5E was separated by preparative liquid chromatography to obtain compound 1.

4. The preparation method according to claim 3, characterized in that, Step A) includes: taking Alpinia oxyphylla fruit, refluxing it with 3-5 times the amount of 60% ethanol 1-3 times, each time for 1-3 hours, combining the extracts, removing the solvent under reduced pressure, and obtaining the total extract. Step B) includes: eluting sequentially with water, 50% ethanol, 70% ethanol, and 95% ethanol, collecting each eluent separately, concentrating under reduced pressure until no alcohol odor is detected, and obtaining water-eluted fractions, 50% ethanol-eluted fractions, 70% ethanol-eluted fractions, and 95% ethanol-eluted fractions. The cyclohexane-ethyl acetate gradient elution in step C) is performed at a volume ratio of 100:0 to 0:100; the methanol-water gradient elution is performed at a volume ratio of 40:60 to 100:

0.

5. The preparation method according to claim 3, characterized in that, The macroporous adsorption resin includes one or more of the following: D101 type macroporous adsorption resin, HP-20 type macroporous adsorption resin, HPD-100 type macroporous adsorption resin, HPD-100A type macroporous adsorption resin, or HPD-300 type macroporous adsorption resin.

6. The preparation method according to claim 3, characterized in that, The cyclohexane-ethyl acetate gradient elution in step C) is performed with volume ratios of 98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, and 0:1; the methanol-water gradient elution is performed with volume ratios of 40:60, 45:55, 50:40, 55:45, 65:35, 80:20, and 100:

0.

7. The preparation method according to claim 3, characterized in that, Step A) involves reflux extraction with 60% ethanol twice, each time for 2 hours; The conditions for the preparative liquid chromatography of fraction 3F5 include: a specification of C 18 The mobile phase for separating fraction 3F5 was 5μm and 10×250mm Phenomenex Gemini; the mobile phase for separating fraction 3F5 was methanol-water with a volume ratio of 55:45; the mobile phase for separating fraction 3F5E was acetonitrile-water with a volume ratio of 35:65; the detection wavelength was 223nm and the flow rate was 3mL / min.

8. The preparation method according to claim 7, characterized in that, The method further includes: preparative liquid phase separation of the compound 1, wherein the specification is EnantinPak Y3, 5μm, C18, 250×4.60mm column, the mobile phase volume ratio is 40:60 acetonitrile-water, the detection wavelength is 223nm, and the flow rate is 1mL / min.

9. The use of the diphenylheptane compound as described in claim 1 or its pharmaceutically acceptable salt in the preparation of anti-inflammatory drugs.

10. A medicine comprising the diphenylheptane compound of claim 1 or a pharmaceutically acceptable salt thereof.

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