Extraction and separation method and application of diaryl heptane compound in fructus amomi hainanensis

Abstract

The invention relates to a preparation method for extracting and separating a diaryl heptane compound from fructus amomi hainanensis and application of the diaryl heptane compound. The specific preparation process comprises the following steps: carrying out alcohol extraction, organic solvent extraction, silica gel column chromatography separation, ODS column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatograph purification and the like on fructus amomi hainanensis to obtain one linear diaryl heptane compound which is (3R, 5R)-3, 5-diacetoxy-1, 7-bis (3, 4-dihydroxyphenyl) heptane with a molecular formula of C23H28O8. In-vitro cell research shows that the compound has gastric mucosa protection, anti-enteritis and anti-inflammatory activity. The method for extracting and separating the diaryl heptane compound is clear and controllable, and the obtained compound is few in impurity and high in purity, has potential medicinal development prospects and can be used for preparing gastric mucosal lesion resisting drugs and ulcerative colitis resisting drugs.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a preparation method and application of diarylheptane compounds separated from Amomum villosum. Background Art

[0002] Amomum villosum is pungent and warm in nature, and enters the spleen, stomach, and kidney meridians. It has the effects of removing dampness and appetizing, warming the spleen and stopping diarrhea, regulating qi and calming the fetus. It is used to treat dampness and turbidity blocking the middle part of the body, removing lumps without hunger, spleen and stomach deficiency and cold, vomiting and diarrhea, nausea during pregnancy, and restless fetuses. Amomum villosum has a history of more than 1,300 years of medicinal use and is one of the "Four Major Southern Medicines" in my country. It is also included in the catalog of medicinal materials with the same origin as medicine and food. Modern pharmacological studies have shown that Amomum villosum has the effects of gastrointestinal protection, antibacterial, fetal protection, regulating intestinal flora, and hypoglycemic activity.

[0003] Hainan Amomum is a unique variety of Hainan, and the Amomum produced is a geographical indication product, but the research content of Hainan Amomum is less than that of Yangchun Amomum. In recent years, most of the research on the chemical composition of Hainan Amomum has focused on the analysis of volatile oils, and there are fewer studies on the non-volatile components and efficacy of Hainan Amomum, which cannot reveal the true quality of Hainan Amomum. In addition, due to the unique planting conditions in Hainan Province, the forest economic planting model of Hainan Amomum and other medicinal materials has unique development advantages in Hainan. In recent years, this model has been continuously developing and can bring benefits to Hainan's economy. In addition, the composition and activity research of Hainan Amomum is conducive to the discovery and optimization of its planting conditions. Excellent planting conditions are also helpful to improve its medicinal value, provide a theoretical basis for the development and utilization of tropical medicinal resources, and are conducive to the development of the traditional Chinese medicine industry. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing diarylheptane compounds extracted and separated from Amomum villosum, comprising the following steps:

[0005] (1) Alcohol extraction: Weigh 6 kg of dried fruits of Amomum villosum, crush them, add 70% ethanol and reflux extract for 3 times, with a solid-liquid ratio of 1:5, combine the extracts, recover the solvent, and dry under reduced pressure to obtain an extract;

[0006] (2) organic solvent extraction: dissolving the extract obtained in step (1) in distilled water, and then extracting with an equal volume of dichloromethane for 5 times, combining the dichloromethane extracts, concentrating and drying to obtain a dichloromethane extract;

[0007] (3) Silica gel column chromatography separation: The dichloromethane extract obtained in step (2) is subjected to silica gel column chromatography separation, wet column loading, and gradient elution using a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 10:1, wherein the dichloromethane-methanol mixed solvent with a volume ratio of 50:1 is decompressed and the solvent is recovered to obtain a separated product;

[0008] (4) ODS reverse phase column purification: the separated product obtained in step (3) was eluted with 35% to 50% methanol aqueous solution, wherein the 40% methanol aqueous solution was eluted under reduced pressure to recover the solvent to obtain a crude product 1;

[0009] (5) Sephadex column chromatography separation: the crude product 1 obtained in step (4) is subjected to a Sephadex column for further purification, eluted with pure methanol, the eluate is collected, and the solvent is recovered to obtain a crude product 2;

[0010] (6) Semi-preparative HPLC purification: The crude product 2 obtained in step (5) was purified by semi-preparative HPLC using 60% methanol water as the mobile phase, a volume flow rate of 2 mL / min, and a detection wavelength of 210 nm to separate and obtain a pure diarylheptane compound. The compound is (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane, and its chemical structure is as follows:

[0011]

[0012] The molecular formula is C 23 H 28 O8.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: the extraction and separation method of (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane in Amomum villosum of the present invention is clear and controllable, and the obtained compound has few impurities and high purity; and the compound has gastric mucosal protective activity, anti-enteritis activity and anti-inflammatory activity, providing a source and basis for further developing gastric mucosal protective and anti-enteritis pharmacological substances in Amomum villosum. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane

[0015] Figure 2 (3R,5R)-3,5-Diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane Carbon Spectrum

[0016] Figure 3 Effect Example 1 shows the gastric mucosa protective activity of the compound.

[0017] Figure 4 The anti-enteritis activity of the compound in Effect Example 2.

[0018] Figure 5 Effect Example 3 shows the anti-inflammatory activity of the compound. DETAILED DESCRIPTION

[0019] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all should be included in the protection scope of the present invention.

[0020] Example 1: Preparation of (3R, 5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane:

[0021] (1) Alcohol extraction: Weigh 6 kg of dried Amomum villosum fruit, crush it, add 70% ethanol and reflux extract for 3 times, with a solid-liquid ratio of 1:5, 1 h / time, combine the extracts, recover the solvent, and dry it under reduced pressure by rotary evaporation to obtain 1461.5 g of extract;

[0022] (2) Organic solvent extraction: The extract obtained in step (1) was dissolved in distilled water, and then extracted with an equal volume of dichloromethane. The extraction was repeated 5 times, and the dichloromethane extracts were combined, concentrated and dried to obtain 233.4 g of dichloromethane extract;

[0023] (3) Silica gel column chromatography separation: The dichloromethane extract obtained in step (2) was subjected to silica gel column chromatography separation, wet column loading, and eluted with dichloromethane:methanol (100:0, 50:1, 20:1, 10:1) to obtain four fractions: FrA, Fr B, Fr C, and Fr D. Different fractions were detected by silica gel thin layer plate (TCL), and those with the same main components were combined. FrA obtained 9 fractions (FrA1-9), Fr B obtained 18 fractions (Fr B1-18), Fr C obtained 10 fractions (Fr C1-10), and Fr D obtained 8 fractions (Fr D1-8). The purity of the compound in Fr B13 was 70.62%;

[0024] (4) ODS reverse phase column purification: Fr B13 was purified by ODS reverse phase column, eluted with methanol water (35%, 40%, 45%, 50%), and different fractions were detected by silica gel thin layer plate (TCL). The main components were the same and combined to obtain Fr B13-40% component with a purity of 82.05%;

[0025] (5) Sephadex column chromatography separation: Fr B13-40% was purified again by Sephadex LH-20 Sephadex column and eluted with pure methanol to obtain Fr B13-40%-N12 fraction with a purity of 90.78%;

[0026] (6) Semi-preparative HPLC purification: FrB13-40%-N12 was purified by semi-preparative HPLC with 60% methanol in water, a volume flow rate of 2 mL / min, and a detection wavelength of 210 nm to obtain (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane with a purity of 94.24%.

[0027] Example 2: The structure of (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane was determined. Its hydrogen and carbon spectra are shown in the attached Figure 1 and 2 , its mass spectrum and spectrum data are as follows:

[0028] 1 H-NMR (CD3OD, 400MHz) δ: 6.57 (2H, d, J = 2.1Hz, H-2', 2”), 6.64 (2H, d, J = 8.0H z, H-5', 5"), 6.46 (2H, dd, J = 8.0Hz, 2.1Hz, H-6', 6"), 2.42 (4H, t, J = 7.9Hz, H- 1,7), 1.75 (4H, m, H-2, 6), 4.89 (2H, m, H-3, 5), 1.81 (2H, m, H-4), 1.94 (6H, m, CH3); 13C-NMR (CD3OD, 100MHz) δ: 30.52 (C-1), 36.24 (C-2), 70.09 (C-3), 38.0 6(C-4), 70.09(C-5), 36.24(C-6), 30.52(C-7), 132.89(C-1'), 114.90(C-2' ), 144.65(C-3'), 142.86(C-4'), 115.06(C-5'), 119.20(C-6'), 132.89(C-1” ), 114.90 (C-2"), 144.65 (C-3"), 142.86 (C-4"), 119.20 (C-5"), 119.20 (C-6"), 20.16 (-CH3), 171.47 (C=O) identified as (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane.

[0029] Effect Example 1 Study on the gastric mucosal protective activity of (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane

[0030] 1. Cell culture and passaging

[0031] Resuscitated human gastric mucosal epithelial cells (GES-1 cells) were cultured in complete culture medium (containing 10% FBS + 1% P / S double antibody + 89% RPMI-1640 culture medium). After 24 hours, new culture medium was replaced to allow GES-1 cells to adhere to the wall and grow. After the GES-1 cells grew to a suitable density (about 80%), they were subcultured. The old culture medium was discarded, and the GES-1 cells were slowly rinsed with PBS to rinse the residual serum on the cell surface. About 1 mL of 0.25% trypsin was added to each T25 culture flask to digest the cells. After 2 minutes, 3 mL of complete culture medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and an appropriate amount of complete culture medium was added to resuspend the cells, and subculture was performed at an appropriate cell concentration.

[0032] 2. Toxicity of the compound to GES-1 cells

[0033] GES-1 cells were cultured at 1×10 4 100 μL of complete medium was added to the normal group (NC), and the monomer was dissolved with DMSO to prepare a mother solution with a concentration of 100 mM. The drug treatment groups were added with drug solutions diluted with complete medium to concentrations of 500.000, 250.000, 125.000, 62.500, 31.250, 15.625, and 7.812 μM, 100 μL per well, and 4 replicate wells were set. After 24 hours of culture in an incubator at 37°C and 5% CO2, the supernatant was discarded and 10% CCK-8 solution was added, 100 μL per well. After incubation for 1 hour, the OD value of each well was detected on an ELISA reader (450 nm), and the cell TC was calculated. 50 value.

[0034] 3. Study on the protective effect of this compound on ethanol-induced GES-1 cell damage

[0035] GES-1 cells were cultured at 1×10 4 100 μL of complete medium was added to each well of the normal group (NC) and the model group (EC) based on the cytotoxicity results. The drug treatment concentrations were set up. The drug treatment groups were added with 200.000, 100.000, 50.000, 25.000, 12.500, 6.250, and 3.125 μM of drug-containing medium, 100 μL per well. After 24 hours of drug intervention, the old medium was discarded. Except for the normal group, the other groups were added with 9% ethanol to model and cultured for 3 hours. The gastric mucosal protective activity was evaluated by CCK-8 method with cell survival rate as an indicator.

[0036] The half toxic concentration (TC) of the compound on GES-1 cells 50) is 220.412±23.430μM. Figure 1 As shown, the compound exhibited significant protective activity against ethanol-induced GES-1 cell damage at 25.000 μM to 100.000 μM (P<0.05), indicating that the compound has gastric mucosal protective activity.

[0037] Effect Example 2: Study on the Anti-Enteritis Activity of (3R,5R)-3,5-Diacetoxy-1,7-Bis(3,4-dihydroxyphenyl)heptane

[0038] 1. Cell culture and passaging

[0039] The revived human cloned colon adenocarcinoma cell line (Caco-2 cells) was cultured in complete culture medium (containing 10% FBS + 1% P / S double antibody + 89% DMEM culture medium). After 24 hours, the new culture medium was replaced to allow the Caco-2 cells to adhere to the wall and grow. After Caco-2 cells have grown to a suitable density (about 80%), they were subcultured. The old culture medium was discarded, and the Caco-2 cells were slowly rinsed with PBS to rinse the residual serum on the cell surface. About 1 mL of 0.25% trypsin was added to each T25 culture flask to digest the cells. After 2 minutes, 3 mL of complete culture medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and an appropriate amount of complete culture medium was added to resuspend the cells. The cells were subcultured at an appropriate cell concentration.

[0040] 2. Toxicity of the compound to Caco-2 cells

[0041] Caco-2 cells were cultured at 1×10 4 100 μL of complete medium was added to the normal group (NC), and the monomer was dissolved with DMSO to prepare a mother solution with a concentration of 100 mM. The drug treatment groups were added with drug solutions diluted with complete medium to concentrations of 500.000, 250.000, 125.000, 62.500, 31.250, 15.625, and 7.812 μM, 100 μL per well, and 4 replicate wells were set. After 24 hours of culture in an incubator at 37°C and 5% CO2, the supernatant was discarded and 10% CCK-8 solution was added, 100 μL per well. After incubation for 1 hour, the OD value of each well was detected on an ELISA reader (450 nm), and the cell TC was calculated. 50 value.

[0042] 3. Study on the protective effect of the compound on LPS-induced Caco-2 cell damage

[0043] Caco-2 cells were cultured at 1×10 4Each well was inoculated in a 96-well plate. After 24 hours of adherent growth, the old culture medium was discarded. 100 μL of complete culture medium was added to each well of the normal group (NC) and the model group (EC). Based on the cytotoxicity results, the drug treatment concentration of the drug treatment group was established. The drug treatment group was added with 125.000, 62.500, 31.250, 15.625, and 7.815 μM drug-containing culture medium, 100 μL per well. After 24 hours of drug intervention, the old culture medium was discarded. Except for the normal group, the other groups were added with 2.5 μg / mL of LPS for modeling and cultured for 12 hours. The anti-enteritis activity was evaluated by CCK-8 method with cell survival rate as an indicator.

[0044] The half toxic concentration (TC) of the compound on Caco-2 cells 50 ) is 235.065±15.322μM. Figure 2 As shown, the compound exhibited significant protective activity against LPS-induced Caco-2 cell injury at 62.500 μM to 125.000 μM (P<0.01), indicating that the monomer compound has anti-enteritis activity.

[0045] Effect Example 3: Study on the anti-inflammatory activity of (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane

[0046] 1. Cell culture and passaging

[0047] The revived mouse mononuclear macrophage leukemia cells (RAW 264.7 cells) were cultured in complete culture medium (containing 10% FBS + 1% P / S double antibody + 89% DMEM culture medium). After 24 hours, the new culture medium was replaced to allow the RAW 264.7 cells to grow on the wall. After the RAW 264.7 cells have grown to a suitable density (about 80%), they were subcultured. The old culture medium was discarded, and the RAW264.7 cells were slowly rinsed with PBS to rinse the residual serum on the cell surface. About 1 mL of 0.25% trypsin was added to each T25 culture flask to digest the cells. After 2 minutes, 3 mL of complete culture medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and an appropriate amount of complete culture medium was added to resuspend the cells. The cells were subcultured at an appropriate cell concentration.

[0048] 2. Toxicity of the compound to RAW 264.7 cells

[0049] RAW 264.7 cells were cultured at 1 × 10 4100 μL of complete medium was added to the normal group (NC), and the monomer was dissolved with DMSO to prepare a mother solution with a concentration of 100 mM. The drug treatment group was added with a drug solution diluted with complete medium to a concentration of 400.000, 200.000, 100.000, 50.000, 25.000, 12.500, and 6.250 μM, 100 μL per well, and 4 replicate wells were set. After 24 hours of culture in an incubator at 37°C and 5% CO2, the supernatant was discarded and 10% CCK-8 solution was added, 100 μL per well. After incubation for 1 hour, the OD value of each well was detected on an ELISA reader (450 nm), and the cell TC was calculated. 50 value.

[0050] 3. Study on the protective effect of the compound on LPS-induced RAW 264.7 cell damage

[0051] RAW 264.7 cells were cultured at 1 × 10 4 Each well was inoculated in a 96-well plate. After 24 hours of adherent growth, the old culture medium was discarded. 100 μL of complete culture medium was added to each well of the normal group (NC) and the model group (EC). Based on the cytotoxicity results, the drug treatment concentration was established. The drug treatment groups were added with 50.000, 25.000, 12.500, 6.250, and 3.125 μM drug-containing culture medium, 100 μL per well. After 24 hours of drug intervention, the old culture medium was discarded. Except for the normal group, 0.5 μg / mL of LPS was added to the other groups for modeling. The culture was cultured for 12 hours. The CCK-8 method was used to evaluate the anti-inflammatory activity with the cell survival rate as an indicator.

[0052] The half toxic concentration (TC) of the compound on RAW 264.7 cells 50 ) is 120.215±13.320μM. Figure 3 As shown, the compound exhibited significant protective activity against LPS-induced RAW 264.7 cell injury at 12.500 μM to 50.000 μM (P<0.01), indicating that the monomer compound has anti-inflammatory activity.

Claims

1. A method for preparing diarylheptane compounds extracted and separated from Amomum villosum, comprising the following steps: (1) Alcohol extraction: Weigh 6 kg of dried fruits of Amomum villosum, crush them, add 70% ethanol and reflux extract for 3 times, with a solid-liquid ratio of 1:5, combine the extracts, recover the solvent, and dry under reduced pressure to obtain an extract; (2) organic solvent extraction: dissolving the extract obtained in step (1) in distilled water, and then extracting with an equal volume of dichloromethane for 5 times, combining the dichloromethane extracts, concentrating and drying to obtain a dichloromethane extract; (3) Silica gel column chromatography separation: The dichloromethane extract obtained in step (2) is subjected to silica gel column chromatography separation, wet column loading, and gradient elution using a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 10:1, wherein the dichloromethane-methanol mixed solvent with a volume ratio of 50:1 is decompressed and the solvent is recovered to obtain a separated product; (4) ODS reverse phase column purification: the separated product obtained in step (3) was eluted with 35% to 50% methanol aqueous solution, wherein the 40% methanol aqueous solution was eluted under reduced pressure to recover the solvent to obtain a crude product 1; (5) Sephadex column chromatography separation: the crude product 1 obtained in step (4) is subjected to a Sephadex column for further purification, eluted with pure methanol, the eluate is collected, and the solvent is recovered to obtain a crude product 2; (6) Semi-preparative HPLC purification: The crude product 2 obtained in step (5) was purified by semi-preparative HPLC using 60% methanol water as the mobile phase, a volume flow rate of 2 mL / min, and a detection wavelength of 210 nm to separate and obtain a pure diarylheptane compound, which is (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane.

2. Use of the diarylheptane compounds obtained according to the preparation method of claim 1 in the preparation of drugs for resisting gastric mucosal injury.

3. Use of the diarylheptane compounds obtained according to the preparation method of claim 1 in the preparation of anti-ulcerative colitis drugs.

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