Alkane compound as well as preparation method and application thereof

Abstract

The invention discloses a diphenyl heptane compound which is a new chemical component found in fructus alpiniae oxyphyllae. Structural identification is carried out on the compound separated by the method through physicochemical properties and modern spectroscopy means. According to the invention, activity evaluation is carried out by using an LPS-induced RAW264.7 cell inflammation model and other activity screening systems, and it is found that the compound has a certain protection effect on a mouse macrophage line RAW264.7, can significantly inhibit release of NO, and shows a strong anti-inflammatory effect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an alkane compound and a preparation method and application thereof. Background Art

[0002] Alpinia oxyphylla Miq. is a perennial herb that grows in the shade and wetness of forests. It is mainly distributed in Guangdong, Hainan and other provinces. Alpinia oxyphylla is the dried mature fruit of the ginger plant Alpinia oxyphylla. It is usually spindle-shaped or oval in shape, 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, close to the seeds. There is a light brown septum in the seed ball that divides it into three chambers, each of which contains 6-11 seeds. The seeds themselves are irregular polyhedral in shape, 3-4 mm in diameter, with a light yellow aril.

[0003] As one of the "four major southern medicines", Yiqi has a long history of use. Its use as medicine was first recorded in "Compendium of Materia Medica". Zang Qi said: "It can stop vomiting and hiccups,...it can be used to absorb saliva and dirt". "Zhenglei Bencao" records that Yiqi is used to treat "spermatorrhea, urine retention", highlighting the kidney warming effect of Yiqi. "Compendium of Materia Medica" records that Yiqi has the effects of treating frequent urination, heart deficiency, slippery urine, red and white turbidity, white turbidity and abdominal distension, abdominal pain, diarrhea, bad breath, metrorrhagia, and fetal leakage. The classic prescriptions of Yiqi, Suoquan Pills and Yiqiren Powder, have the effects of tonifying the kidney and reducing urine. The compound preparations of Yiqi, Jiannao Pills, Zhiqi Granules, and Yiqixingnao Decoction are often used in the clinic to treat symptoms such as memory loss and neurasthenia. Modern pharmacological studies have shown that Yiqi has a wide range of pharmacological effects such as neuroprotection, renal protection, anti-diuresis, anti-inflammatory, and antioxidant. As a highly safe plant resource that can be used as both medicine and food, there are currently very few product developments on Alpinia oxyphylla, which limits its application.

[0004] Therefore, further research on the composition of Alpinia oxyphylla kernel in order to obtain an active ingredient of Alpinia oxyphylla kernel with medicinal effects is not only helpful for understanding the composition of compounds in Alpinia oxyphylla kernel, but also has important significance for the in-depth development and application of Alpinia oxyphylla kernel. Summary of the invention

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

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

[0007]

[0008] Furthermore, the above compound may be,

[0009]

[0010] Another object of the present invention is to provide a method for preparing the above compound, characterized in that it comprises the following steps:

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

[0012] B) the total extract is dissolved in water, separated by macroporous adsorption resin column chromatography, eluted with water, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol in sequence, and the eluates are collected and concentrated under reduced pressure to obtain water elution fraction, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol elution fraction; each gradient elution is 4 column volumes (the same below);

[0013] C) The 65-75% ethanol elution portion was separated by silica gel column chromatography, and cyclohexane-ethyl acetate gradient elution was used to collect 10 fractions 3A-3J, 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 phase separation to obtain compound 1; further, compound 1 can be separated by preparative liquid phase to obtain compounds 1a and 1b.

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

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

[0016] Preferably, the step B) comprises: eluting with water, 50% ethanol, 70% ethanol and 95% ethanol in sequence, collecting each eluate respectively, and concentrating under reduced pressure until there is no alcohol taste, thereby obtaining a water elution fraction, a 50% ethanol elution fraction, a 70% ethanol elution fraction and a 95% ethanol elution fraction.

[0017] The cyclohexane-ethyl acetate gradient elution in the 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 D101 macroporous adsorption resin, HP-20 macroporous adsorption resin, HPD-100 macroporous adsorption resin, HPD-100A macroporous adsorption resin or HPD-300 macroporous adsorption resin.

[0019] Furthermore, the cyclohexane-ethyl acetate gradient elution in the step C) is performed with a volume ratio 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 a volume ratio of 40:60, 45:55, 50:40, 55:45, 65:35, 80:20, and 100:0.

[0020] Furthermore, the step A) is to extract by reflux with 60% ethanol twice, each time for 2 hours.

[0021] Specifically, the conditions of the preparative liquid chromatography include: specifications of C 18 ,5μm,10×250mm PhenomenexGemini; the mobile phase for separation fraction 3F5 was methanol-water with a volume ratio of 55:45; the mobile phase for separation 3F5E was acetonitrile-water with a volume ratio of 35:65; the detection wavelength was 223nm, and the flow rate was 3mL / min. .

[0022] Furthermore, the method also includes: performing preparative liquid separation on the compound 1, with a specification of 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 object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite in the preparation of anti-inflammatory drugs.

[0024] The present invention also provides a drug comprising the above-mentioned diphenylheptane compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof.

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

[0026] Specifically, the drug can be any dosage form mentioned in pharmacy, 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 drug carriers in the pharmaceutical field, such as diluents, excipients and water, fillers such as starch, sucrose, lactose, microcrystalline cellulose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol and sodium lauryl sulfate; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, micropowdered silica gel and polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0028] The diphenylheptane compounds described in the present invention are new chemical components discovered by researchers in Alpinia oxyphylla seeds, and it is found that the compounds are stably present in each batch of Alpinia oxyphylla seeds. The inventors have studied the physical and chemical properties and modern spectroscopy methods (MS, 1 H-NMR, 13 C-NMR, etc.), the compound separated by the above method was structurally identified, and it was confirmed that it was a new compound with a structure as shown in formula (I). The present invention also uses an activity screening system such as an LPS-induced RAW 264.7 cell inflammation model to evaluate the activity, and it is found that the compound has a certain protective effect on the mouse macrophage cell line RAW 264.7, can significantly inhibit the release of NO, and shows a strong anti-inflammatory effect. It has a good research and development prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the HR-ESI-Q-TOF-MS spectrum of compound 1 of the present invention;

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

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

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

[0033] Figure 5 is H of the compound 1 of the present invention1 -H 1 COSY spectrum;

[0034] Figure 6 is the HSQC spectrum of compound 1 of the present invention;

[0035] Figure 7 is the HMBC spectrum of compound 1 of the present invention;

[0036] Figure 8 is the NOESY spectrum of compound 1 of the present invention;

[0037] Fig. 9 is the UV spectrum of compound 1 of the present invention;

[0038] Fig.10 is the IR spectrum of compound 1 of the present invention;

[0039] Fig.11 HPLC spectrum of chiral column separation of compound 1a / 1b of compound 1 of the present invention;

[0040] Fig.12 X-ray spectra of compounds 1a and 1b of the present invention;

[0041] Fig.13 The IC values ​​of the compounds 1a and 1b of the present invention for inhibiting NO release are 50 curve. DETAILED DESCRIPTION

[0042] The following is a more detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings and examples, so that the scheme of the present invention and its advantages in various aspects can be better understood. However, the specific embodiments and examples described below are only for the purpose of illustration, rather than for limiting the present invention.

[0043] The following is a detailed description based on the contents of the experimental examples.

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

[0045] If the specific conditions are not specified in the present invention, they are carried out under conventional conditions or conditions recommended by the manufacturer. The raw materials or excipients used, as well as the reagents or instruments used without indicating the manufacturer, are all conventional products that can be obtained commercially. Alpiniae oxyphyllae fructus was purchased from Anguo Shiyuan Trading Co., Ltd. in 2020, and its origin is Hainan Province.

[0046] The present invention will be further described below in conjunction with embodiments:

[0047] Example 1 Preparation of the compounds of the present invention

[0048] Step (1): Take the dried mature fruit of Alpinia oxyphylla, extract it with 50% ethanol under reflux twice, each time for 2 hours, combine the extracts, remove the solvent under reduced pressure, and obtain the total extract. The total extract is dissolved in water, separated by HP-20 macroporous adsorption resin column chromatography, eluted with water, 50% ethanol, 70% ethanol, and 95% ethanol in sequence, and each gradient elution is 4 column volumes (the same below), and each eluate is collected respectively, and concentrated under reduced pressure until there is no alcohol taste, to obtain a water elution fraction, a 50% ethanol elution fraction, a 70% ethanol elution fraction, and a 95% ethanol elution fraction;

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

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

[0051] Example 2 Preparation of the compounds of the present invention

[0052] (1) Take the dried mature fruit of Alpinia oxyphylla, extract it with 60% ethanol under reflux twice, each time for 2 hours, combine the extracts, remove the solvent under reduced pressure, and obtain the total extract. The total extract is dissolved in water, separated by HP-20 macroporous adsorption resin column chromatography, and eluted with water, 45% ethanol, 65% ethanol, and 90% ethanol in sequence, and the eluents are collected respectively, and concentrated under reduced pressure until there is no alcohol taste, to obtain the water elution fraction, the 45% ethanol elution fraction, the 65% ethanol elution fraction, and the 90% ethanol elution fraction;

[0053] (2) The 65% ethanol elution portion of step (1) was separated by silica gel column chromatography and gradient eluted with cyclohexane-ethyl acetate (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 was subjected to ODS column chromatography with methanol-water (40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0, v / v) to obtain 5 fractions from 3F1 to 3F5. Fraction 3F5 was separated by preparative liquid chromatography to obtain 11 fractions from 3F5A to 3F5K. 3F5E was separated by preparative liquid phase to obtain compound 1. Compound 1 was separated by preparative liquid phase to obtain compounds 1a and 1b.

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

[0055] Example 3 Preparation of the compounds of the present invention

[0056] (1) Take dried ripe fruit of Alpinia oxyphylla and use 70% ethanol Reflux extraction was performed twice, each time for 2 hours, the extracts were combined, and the solvent was removed under reduced pressure to obtain the total extract. The total extract was dissolved in water, separated by HP-20 macroporous adsorption resin column chromatography, and eluted with water, 55% ethanol, 75% ethanol, and 100% ethanol in sequence. The eluates were collected respectively, and concentrated under reduced pressure until there was no alcohol taste, and the water elution fraction, the 55% ethanol elution fraction, the 75% ethanol elution fraction, and the 100% ethanol elution fraction were obtained;

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

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

[0059] Example 4 Structural Identification of Compounds of the Invention

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

[0061] As shown in Table 1 and Figure 1-Figure 12 As shown, compound 1 1 In the H-NMR (600MHz, inCDCl3) spectrum, a monosubstituted benzene ring hydrogen signal [δ H 7.28 (2H, m, H-3″, 5″), 7.19 (3H, m, H-2″, 4″, 6″)], 1,2,4-substituted benzene ring hydrogen signal [δ H 6.95 (1H, brs, H-2′), 6.88 (2H, d, J=7.7 Hz, H-5′, 6′)], 3 SP 3 Hybridized oxygen-linked methylene hydrogen signal [δ H 4.77 (1H, dd, J = 11.8, 2.0 Hz, H-1), 4.35 (1H, m, H-3), 3.96 (1H, m, H-5)], 1 methoxy hydrogen signal [δ H 3.91(3H,s,3′-OCH3)], and several SP 3 Hybridized methylene hydrogen signals. 13 The C-NMR (150 MHz, inCDCl3) spectrum combined with the DETP 135 spectrum showed a total of 20 carbon signals, including: 1 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, it was inferred that compound 1 was a diphenylheptane compound.

[0062] In the HMBC spectrum, it was observed that H-1 correlated with C-2, 3, 5, 1′, 2′, 6′, and H-7 correlated with C-5, 6, 1″, 2″, 6″; 1 H- 1 The H-1 / H2-2 / H-3 / H2-4 / H-5 correlations in the H COSY spectrum confirmed that the two benzene rings were connected by a pyran ring.

[0063] In the NOESY spectrum, H-1 was observed to be correlated with H-5, indicating that the two were located on the same side. Compound 1 was separated into a pair of enantiomers 1a and 1b by chiral HPLC. Combined with the CuKα diffraction data collected in the X-ray single crystal diffraction test, the absolute configurations of 1a and 1b were determined to be 1S, 3S, 5S and 1R, 3R, 5R, respectively, and they were 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, respectively, with the following structures:

[0064]

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

[0066]

[0067] Multiplets andoroverlappedsignals arereportedwithoutdesignatingmultiplicity

[0068] The same method was used to identify the compounds in each example, and the results were the same. 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 The mouse macrophage cell line RAW 264.7 was obtained from the Chinese Academy of Traditional Chinese Medicine; culture conditions: DMEM + 10% fetal bovine serum (FBS), 37°C, 5% CO2.

[0072] 2. Principles and methods

[0073] 2.1 Experimental Principle

[0074] Lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria (Sigma, USA, batch number: 114M4009) is one of the most important pathogenic molecules that mediate infectious inflammatory damage. Many diseases are closely related to persistent subclinical inflammation induced by LPS. In animal and cell experiments, LPS is widely used to induce inflammation.

[0075] Macrophages play a vital role in inflammatory responses. After being stimulated, macrophages produce inflammatory factors and mediator activation, which is a key process of inflammation. Their inhibition 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 steps

[0078] The cells were pipetted off and the cell density was adjusted to 2 × 10 5 / mL; evenly inoculate into 96-well plates, 100 μL per well, and place the plates in an incubator for 24 hours. Then, take out the 96-well plates, remove the supernatant, and add the drug-containing medium prepared with serum-free DMEM medium. The experimental settings are as follows:

[0079] (1) Solvent control group (-group): 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 containing LPS at a final concentration of 0.5 μg / ml was added to each well;

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

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

[0083] 2. Experimental results

[0084] (1) The nitrite concentration in the culture medium as an indicator of NO production was measured according to the Griess method (Beyotime Biotechnology, S0021). The cell-free supernatant and Griess reagent (nitrate reduction test reagent) were completely mixed in equal amounts of 50 μL; the absorbance of the final product was measured on a microplate reader at 540 nm. The nitrite concentration and inhibition rate were calculated based on the standard calibration curve. The results of nitrite concentration in each experimental group and the control group are detailed in Fig.13 ;

[0085] (2) The inhibitory effect of the test compound on LPS-induced NO production is described as IC 50 The results are shown in Table 2.

[0086] Table 2 Inhibitory effect of compounds on LPS-induced NO production in BV-2 cells

[0087]

[0088] Experimental data showed that the diphenylheptane compounds 1a and 1b isolated from Alpinia oxyphylla had an inhibitory effect on the production of NO in RAW264.7 cells induced by LPS (see Fig.13 ). The IC values ​​of some of the compounds for their inhibitory effects on LPS-induced NO in RAW264.7 cells were determined. 50 The IC values ​​of compound 1a for the inhibition of LPS-induced NO in RAW264.7 cells were 50 The value was 31.84 μM, which showed that its anti-inflammatory effect was stronger than that of 1b. The IC value of compound 1b on the inhibition of LPS-induced NO in RAW264.7 cells was 50 The value is 44.63 μM.

[0089] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical schemes and / or characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical scheme of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims. The above is only the preferred implementation method of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A diphenylheptane compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof, wherein the compound structure is shown in Formula I:

2. According to the compound of claim 1, the compound has the following structure:

3. A method for preparing the compound according to claim 1, characterized in that: The following steps are involved: A) extracting Alpinia oxyphylla seeds with 50-70% ethanol under reflux, removing the solvent, and obtaining a total extract; B) the total extract is dissolved in water, separated by macroporous adsorption resin column chromatography, eluted with water, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol in sequence, and the eluates are collected and concentrated under reduced pressure to obtain water elution fraction, 45-55% ethanol, 65-75% ethanol, and 90-100% ethanol elution fraction; C) The 65-75% ethanol elution portion was separated by silica gel column chromatography, and cyclohexane-ethyl acetate gradient elution was used to collect 10 fractions 3A-3J. 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: The step A) comprises: taking the fruit of Alpinia oxyphylla, refluxing and extracting it with 3-5 times the amount of 60% ethanol for 1-3 times, each time for 1-3 hours, combining the extracts, and removing the solvent under reduced pressure to obtain the total extract; The step B) comprises: eluting with water, 50% ethanol, 70% ethanol, and 95% ethanol in sequence, collecting each eluate, and concentrating under reduced pressure until there is no alcohol smell, thereby obtaining a water elution portion, a 50% ethanol elution portion, a 70% ethanol elution portion, and a 95% ethanol elution portion; 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 D101 macroporous adsorption resin, HP-20 macroporous adsorption resin, HPD-100 macroporous adsorption resin, HPD-100A macroporous adsorption resin or HPD-300 macroporous adsorption resin.

6. The preparation method according to claim 3, characterized in that: The cyclohexane-ethyl acetate gradient elution in the step C) is performed with a volume ratio 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 a volume ratio 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: The step A) is to extract by reflux with 60% ethanol twice, each time for 2 hours; The conditions for the preparative liquid chromatography of the fraction 3F5 include: specifications of C 18 ,5μm,10×250mmPhenomenex Gemini; the mobile phase for separation fraction 3F5 was methanol-water with a volume ratio of 55:45; the mobile phase for separation 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: performing preparative liquid separation on 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. Use of the diphenylheptane compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule or metabolite thereof as claimed in claim 1 in the preparation of an anti-inflammatory drug.

10. A drug comprising the diphenylheptane compound of claim 1 or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, or metabolite thereof.

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