Diterpenoid component in hyssopus harzianum as well as preparation method and application of diterpenoid component

Abstract

The invention relates to the technical field of separation and purification of hyssopus harzianum, in particular to a diterpenoid compound and a preparation method and application thereof.The diterpenoid compound is obtained by extracting and separating hyssopus harzianum, and the preparation method comprises the steps that hyssopus harzianum extract is extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain dichloromethane extract; and carrying out gradient elution on the dichloromethane extract twice by using silica gel column chromatography, and separating and purifying a fraction obtained after separation by using semi-preparative liquid chromatography to obtain the diterpenoid compound. The invention discloses a diterpenoid compound in hyssop officinalis for the first time, the structure and physicochemical properties of the diterpenoid compound are determined and represented, and experiments prove that the diterpenoid compound has certain anti-inflammatory activity, so that the diterpenoid compound is applied to preparation of medicines for preventing inflammation or / and protecting inflammation and preparation of medicines for preventing or / and resisting skin inflammation.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of Hyssopus sibiricum, and is a diterpenoid component in Hyssopus sibiricum, which is a diterpenoid compound referred to as hyssopusone C, as well as a preparation method and application of the diterpenoid compound. Background Art

[0002] Hyssopus cuspidatus is a plant of the genus Hyssopus in the family Labiatae. As a traditional medicinal material, Hyssopus cuspidatus is widely used by the Uygur people and has anti-inflammatory, antioxidant, and anti-asthmatic effects. Modern literature reports that Hyssopus cuspidatus can treat inflammatory diseases such as colds, fevers, coughs, asthma, and tracheitis.

[0003] Inflammation is a key defense system that the body has gradually built up during the evolution process to resist the invasion of foreign pathogens. However, when this defense mechanism persists or overreacts, it often causes disease and even threatens human life and health. Long-term use of the same drug will gradually expose its shortcomings such as drug resistance and adverse reactions. Therefore, it is particularly important to find highly effective and low-toxic drugs for the effective treatment of diseases.

[0004] The ingredients in Hyssopus officinalis have strong anti-inflammatory activity. Therefore, extracting, separating and purifying the ingredients of Hyssopus officinalis and finding its active ingredients and new pharmacological activities can better utilize the rich plant resources of Hyssopus officinalis, which has far-reaching significance for the comprehensive development and application of Hyssopus officinalis. Summary of the invention

[0005] The present invention provides a diterpene compound and a preparation method thereof, which overcomes the deficiencies of the above-mentioned prior art and discloses for the first time a diterpene component in Hyssopus sclerotium, so that the diterpene component can be used in the preparation of preventive anti-inflammatory drugs and anti-inflammatory drugs.

[0006] One of the technical solutions of the present invention is achieved by the following measures: a diterpenoid compound having a chemical structural formula of: .

[0007] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The above diterpenoid compounds are prepared according to the following steps: The first step is to grind the whole herb of Hyssopus officinalis, extract it with ethanol after reflux, and combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard tip Hyssopus officinalis extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; In the third step, the extract of the dichloromethane part is subjected to the first gradient elution by silica gel column chromatography, and 9 fractions are obtained after separation; Step 4: The fifth fraction of the nine fractions obtained in Step 3 is subjected to a second gradient elution through a silica gel chromatography column to obtain eight sub-fractions in sequence; In the fifth step, the fourth subfraction of the eight subfractions obtained in the fourth step is separated and purified by semi-preparative liquid chromatography to obtain diterpenoid compounds.

[0008] In the first step, the volume fraction of ethanol is 90% to 95%, and the solid-liquid ratio of ethanol reflux extraction is 1:1 to 2.

[0009] In the first step, the reflux extraction is carried out at 50°C to 60°C, and the reflux extraction is performed three times, each time for 2 to 3 hours.

[0010] In the third step, the eluent for the first gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for the gradient elution are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, respectively.

[0011] In the fourth step, the eluent for the second gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for the gradient elution are 1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, respectively.

[0012] In the fifth step, the eluent of the semi-preparative liquid chromatography was a 55% methanol in water solution, the flow rate was 2 mL / min, the chromatographic column was a C18 chromatographic column, and the detection wavelength was 210 nm.

[0013] In the third step, the silica gel chromatographic column for the first gradient elution uses silica gel of 100 to 200 meshes, and in the fourth step, the silica gel chromatographic column for the second gradient elution uses silica gel of 200 to 300 meshes.

[0014] The second technical solution of the present invention is achieved by the following measures: A method for preparing a diterpenoid compound is carried out according to the following steps: The first step is to grind the whole herb of Hyssopus officinalis, extract it with ethanol after reflux, and combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard tip Hyssopus officinalis extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; In the third step, the extract of the dichloromethane part is subjected to the first gradient elution by silica gel column chromatography, and 9 fractions are obtained after separation; Step 4: The fifth fraction of the nine fractions obtained in Step 3 is subjected to a second gradient elution through a silica gel chromatography column to obtain eight sub-fractions in sequence; In the fifth step, the fourth subfraction of the eight subfractions obtained in the fourth step is separated and purified by semi-preparative liquid chromatography to obtain diterpenoid compounds.

[0015] The third technical solution of the present invention is achieved through the following measures: use of a diterpenoid compound in the preparation of drugs for preventing inflammation and / or protecting inflammation.

[0016] The fourth technical solution of the present invention is achieved through the following measures: use of a diterpenoid compound in the preparation of a drug for preventing and / or resisting skin inflammation.

[0017] The present invention discloses for the first time the diterpenoid compounds in Hyssopus sclerotium, determines and characterizes the structure and physicochemical properties thereof, and proves through experiments that the diterpenoid compounds have certain anti-inflammatory activity, thereby enabling the compounds to be used in the preparation of drugs for preventing inflammation and / or protecting against inflammation and in the preparation of drugs for preventing or / and resisting skin inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Attached Figure 1 The diterpenoid compound (hyssopusone C) of the present invention 1 H-NMR spectrum.

[0019] Attached Figure 2 The diterpenoid compound (hyssopusone C) of the present invention 13 C-APT spectrum.

[0020] Attached Figure 3 This is the COSY spectrum of the diterpenoid compound (hyssopusone C) of the present invention.

[0021] Attached Figure 4 It is the QC spectrum of the diterpenoid compound (hyssopusone C) of the present invention.

[0022] Attached Figure 5 It is the BC spectrum of the diterpenoid compound (hyssopusone C) of the present invention.

[0023] Attached Figure 6 It is the NOESY spectrum of the diterpenoid compound (hyssopusone C) of the present invention. DETAILED DESCRIPTION

[0024] The present invention is not limited by the following embodiments, and the specific implementation method can be determined according to the technical scheme of the present invention and the actual situation. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all known and commonly used chemical reagents and chemicals in the prior art; unless otherwise specified, the percentages in the present invention are all mass percentages; the normal temperature and room temperature in the present invention generally refer to the temperature of 15°C to 25°C, and are generally defined as 25°C.

[0025] The present invention will be further described below in conjunction with embodiments: Example 1: The diterpenoid compound (denoted as hyssopusone C) has the chemical structural formula: .

[0026] The diterpenoid component in the hard-pointed hyssopus of the present invention is a diterpenoid compound (hyssopusone C), and its systematic name is: (1R, 2S, 5R)-2-(2-((1R, 5S)-6,6-dimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)ethyl)-2-hydroxy-6,6-dimethylbicyclo[3.1.1]heptan-3-one. The Chinese systematic name is: (1R, 2S, 5R)-2-(2-((1R, 5S)-6,6-dimethyl-3-oxobicyclo[3.1.1]heptane-2-yl)ethyl)-2-hydroxy-6,6-dimethylbicyclo[3.1.1]heptan-3-one.

[0027] Example 2: As an optimization of the above example, the diterpenoid compound (hyssopusone C) was prepared according to the following steps: The first step is to grind the whole herb of Hyssopus officinalis, extract it with ethanol after reflux, and combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard tip Hyssopus officinalis extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; In the third step, the extract of the dichloromethane part is subjected to the first gradient elution by silica gel column chromatography, and 9 fractions are obtained after separation; Step 4: The fifth fraction of the nine fractions obtained in Step 3 is subjected to a second gradient elution through a silica gel chromatography column to obtain eight sub-fractions in sequence; In the fifth step, the fourth subfraction of the eight subfractions obtained in the fourth step is separated and purified by semi-preparative liquid chromatography to obtain a diterpenoid compound (hyssopusone C).

[0028] Example 3: As an optimization of the above example, in the first step, the volume fraction of ethanol is 90% to 95%, and the solid-liquid ratio of ethanol reflux extraction is 1:1 to 2.

[0029] Example 4: As an optimization of the above example, in the first step, the reflux extraction is carried out at 50°C to 60°C, and the reflux extraction is performed three times, each time for 2 to 3 hours.

[0030] Example 5: As an optimization of the above example, in the third step, the eluent for the first gradient elution is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol for the gradient elution is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1. After the first gradient elution, 9 fractions were obtained, and the fraction with a volume ratio of dichloromethane to methanol of 10:1 (i.e., the 5th fraction among the 9 fractions) was collected for the next step.

[0031] Example 6: As an optimization of the above example, in the fourth step, the eluent for the second gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for the gradient elution are 1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1. After the second gradient elution, 8 fractions are obtained, and the fraction with a volume ratio of dichloromethane to methanol of 10:1 (i.e., the 4th sub-fraction among the 8 sub-fractions) is collected for the next step.

[0032] Example 7: As an optimization of the above example, in the fifth step, the eluent of the semi-preparative liquid chromatography is a 55% methanol in water solution, the flow rate is 2 mL / min, the chromatographic column is a C18 chromatographic column, and the detection wavelength is 210 nm.

[0033] Example 8: As an optimization of the above example, in the third step, the silica gel chromatography column for the first gradient elution uses silica gel with a mesh size of 100 to 200, and in the fourth step, the silica gel chromatography column for the second gradient elution uses silica gel with a mesh size of 200 to 300.

[0034] Example 9: Use of the diterpenoid compound (hyssopusone C) in the preparation of drugs for preventing inflammation and / or protecting against inflammation.

[0035] Example 10: Use of the diterpenoid compound (hyssopusone C) in the preparation of a drug for preventing and / or resisting skin inflammation.

[0036] Example 11: The diterpenoid compound (hyssopusone C) was prepared according to the following steps: In the first step, the dried whole herb of Hyssopus officinalis was crushed and subjected to heating reflux extraction with 95vt% ethanol for 3 times at a reflux extraction temperature of 55°C for 2.5 hours each time, and the combined filtrates were concentrated under reduced pressure until there was no alcohol taste, thereby obtaining Hyssopus officinalis extract.

[0037] In the second step, the hard-pointed Hyssopus officinalis extract is dispersed with water, and then extracted with 1 to 1.3 times the volume of petroleum ether, dichloromethane and ethyl acetate solutions in turn until it becomes colorless, and concentrated under reduced pressure to obtain the petroleum ether extract, dichloromethane extract and ethyl acetate extract respectively.

[0038] In the third step, the extract of the dichloromethane fraction was applied to a silica gel chromatography column (100 to 200 meshes) and gradient elution was performed using dichloromethane-methanol as the mobile phase (the volume ratio of dichloromethane:methanol was 1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1, respectively) to obtain 9 subfractions.

[0039] In the fourth step, the fifth subfraction obtained in the third step was applied to a silica gel chromatography column (200 to 300 meshes) and gradient eluted with dichloromethane-methanol as the mobile phase (the volume ratio of dichloromethane:methanol was 1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1), to obtain 8 subfractions.

[0040] In the fifth step, the fourth subfraction (10:1 fraction) of the eight subfractions obtained in the fourth step was applied to a semi-preparative liquid phase using a C18 column, a mobile phase of 55vt% methanol, a flow rate of 2mL / min, and a detection wavelength of 210nm to obtain a diterpenoid compound (hyssopusone C).

[0041] The diterpenoid compound (hyssopusone C) obtained by the present invention is a light yellow solid, easily soluble in methanol and chloroform, and has a specific rotation of = +1.0 ( c = 0.1, MeOH ). HR-ESI-MS m / z : 341.2137 [M+Na] + (calculated value 341.2093), molecular formula is C 20 H 30 O3, named (hyssopusone C). Diterpenoid compound (hyssopusone C) 1 H-NMR spectrum Figure 1 As shown, 13 C-APT spectrum Figure 2 As shown, the two-dimensional maps are Figures 3 to 6 , 1The H NMR (600 MHz, CDCl3) and APT NMR (600 MHz, CDCl3) data are shown in Table 1. Through the above tests, it was finally determined that the compound was a new diterpenoid compound.

[0042] Example 12: Study on the anti-inflammatory activity of diterpenoid compound (hyssopusone C) (1) Cell culture Take out the cryovial of RAW264.7 cells from liquid nitrogen or a low-temperature refrigerator and quickly thaw it in a 37°C water bath. After thawing, disinfect the outside of the cryovial with 75% alcohol, and then transfer the cell suspension to a centrifuge tube containing an appropriate amount of high-glucose DMEM medium (preheated to 37°C) under sterile conditions. Centrifuge the cell suspension, remove the supernatant, and then resuspend the cells in fresh high-glucose DMEM medium (containing 10% fetal bovine serum, preheated to 37°C). Transfer the cell suspension to a cell culture dish or culture flask and culture it in a constant temperature incubator at 37°C and 5% CO2. Observe the cell growth status regularly and replace the culture medium to maintain healthy cell growth. When the cells reach the logarithmic growth phase, use a hemocytometer or an automatic cell counter to count the cells. According to the counting results, dilute the cell suspension to the required cell density, i.e. 8×10 5 cells / μL. Use a pipette to add the diluted cell suspension to a sterile 96-well plate, adding 200 μL to each well. Place the inoculated 96-well plate in a constant temperature incubator at 37°C and 5% CO2, and collect the cell culture supernatant after a certain period of incubation.

[0043] (2) MTT assay to detect the effect of drugs on cell viability The cells that are growing vigorously and vigorously in the logarithmic growth phase were controlled to have a cell concentration of 8×10 5 / μL, inoculated in a 96-well plate, 200μL per well, and divided into positive control group and experimental group. After culturing the cells with drugs for 24h, 20μL MTT solution was added to each well (MTT can be reduced to purple formazan crystals by dehydrogenase in the mitochondria of living cells, and its production is proportional to the number of living cells.) Continue to culture for 2h, remove the supernatant and add 200μL DMSO, shake it in the dark for 10min, use a full-wavelength microplate reader to detect the optical density (OD) value of each well at a wavelength of 570nm, and calculate the cell survival rate. Cell survival rate = (OD value of experimental group or positive control group / OD value of blank control group) × 100%.

[0044] (3) Detection of the effect of diterpenoids on the release of nitric oxide (NO) from cells Take RAW264.7 cells in the logarithmic growth phase with vigorous mitosis and dilute the cell concentration to 2×10 5 / mL, placed in a 96-well plate, 200μL per well, cultured at 37℃, 5% CO2 for 24h, removed the supernatant, and divided the cells into a control group, lipopolysaccharide group, and compounds of different mass concentrations (1.0μg / mL lipopolysaccharide + 100, 50, 25, 12.5, 6.25, 3.125μg / ml). After adding drugs or blank culture medium to each group of cells, continue to culture for 24h. Take 100μL of the supernatant, add 100μL of Griess reagent, place at room temperature for 10min, set the wavelength to 570nm, measure the absorbance on the microplate reader, substitute it into the standard curve, and calculate the NO release. NO content = 1-(A drug-A control) / A control.

[0045] (4) Experimental results The results of testing cell viability using the MTT method showed that compared with the blank control group, the concentration of diterpenoids in the range of 0.1μg / ml to 100μg / ml was non-toxic to cells, and the cell activity was above 87%. The cell NO release rate was tested using the Griess reagent, and the results showed that diterpenoids had certain anti-inflammatory activity at 0.5μg / ml to 100μg / ml, and the amount of NO released decreased with increasing concentration. The anti-inflammatory activity showed that the compound had strong anti-inflammatory activity. When the concentration of diterpenoids was greater than 0.25μg / ml, it could significantly inhibit the NO release in RAW264.7 cells induced by lipopolysaccharide, thereby inhibiting the NO-mediated inflammatory response.

[0046] The above results show that the diterpenoid compound (hyssopusone C) has no toxicity to cells, and can effectively inhibit the release of NO induced by lipopolysaccharide when the concentration is greater than 0.25 μg / ml, thereby inhibiting the inflammatory response. Therefore, it has the potential to be developed into an anti-inflammatory drug.

[0047] In summary, the present invention selects Hyssopus officinalis as the research object. This raw material is widely used by the Uyghurs and has high medicinal value. For the first time, a new diterpene component (hyssopusone C) was obtained from Hyssopus officinalis. The extraction and separation methods are quick, simple and have good purity. The diterpene compound exhibits significant anti-inflammatory activity, which is of great significance in the field of research and development of anti-inflammatory drugs.

[0048] The above technical features respectively constitute various embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.

[0049]

Claims

1. A diterpenoid compound, characterized in that The chemical structure is: 。 2. The diterpenoid compound according to claim 1, characterized in that Prepared according to the following steps: The first step is to grind the whole herb of Hyssopus officinalis, extract it with ethanol after reflux, and combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard tip Hyssopus officinalis extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; In the third step, the extract of the dichloromethane part is subjected to the first gradient elution by silica gel column chromatography, and 9 fractions are obtained after separation; Step 4: The fifth fraction of the nine fractions obtained in Step 3 is subjected to a second gradient elution through a silica gel chromatography column to obtain eight sub-fractions in sequence; In the fifth step, the fourth subfraction of the eight subfractions obtained in the fourth step is separated and purified by semi-preparative liquid chromatography to obtain diterpenoid compounds.

3. The diterpenoid compound according to claim 2, characterized in that In the first step, the volume fraction of ethanol is 90% to 95%, the solid-liquid ratio of ethanol reflux extraction is 1:1 to 2, the reflux extraction is carried out at 50°C to 60°C, and the reflux extraction is heated for 3 times, each time for 2 to 3 hours.

4. The diterpenoid compound according to claim 2 or 3, characterized in that In the third step, the eluent for the first gradient elution was a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for the gradient elution were 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, respectively.

5. The diterpenoid compound according to any one of claims 2 to 4, characterized in that In the fourth step, the eluent for the second gradient elution is a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol for the gradient elution are 1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, respectively.

6. The diterpenoid compound according to any one of claims 2 to 5, characterized in that In the fifth step, the eluent of the semi-preparative liquid chromatography was a 55% methanol in water solution, the flow rate was 2 mL / min, the chromatographic column was a C18 chromatographic column, and the detection wavelength was 210 nm.

7. The diterpenoid compound according to any one of claims 2 to 6, characterized in that In the third step, the silica gel chromatographic column for the first gradient elution uses silica gel of 100 to 200 meshes, and in the fourth step, the silica gel chromatographic column for the second gradient elution uses silica gel of 200 to 300 meshes.

8. A method for preparing a diterpenoid compound according to any one of claims 1 or 3 or 4 or 5 or 6 or 7, characterized in that Follow these steps: The first step is to grind the whole herb of Hyssopus officinalis, extract it with ethanol after reflux, and combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard tip Hyssopus officinalis extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; In the third step, the extract of the dichloromethane part is subjected to the first gradient elution by silica gel column chromatography, and 9 fractions are obtained after separation; Step 4: The fifth fraction of the nine fractions obtained in Step 3 is subjected to a second gradient elution through a silica gel chromatography column to obtain eight sub-fractions in sequence; In the fifth step, the fourth subfraction of the eight subfractions obtained in the fourth step is separated and purified by semi-preparative liquid chromatography to obtain diterpenoid compounds.

9. Use of the diterpenoid compound according to any one of claims 1 to 7 in the preparation of drugs for preventing inflammation and / or protecting against inflammation.

10. Use of the diterpenoid compound according to any one of claims 1 to 7 in the preparation of a drug for preventing or / and resisting skin inflammation.

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