Diterpenoid anti-inflammatory compound in hyssopus harzianum as well as preparation method and application of diterpenoid anti-inflammatory compound

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 the 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 inflammation prevention drugs and preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of Hyssopus sibiricum, and discloses a diterpenoid anti-inflammatory compound in Hyssopus sibiricum and a preparation method and application thereof. The diterpenoid anti-inflammatory compound is referred to as hyssopusone B. Background Art

[0002] Hyssopus cuspidatus is a plant of the genus Hyssopus in the family Labiatae. Artificially cultivated ones have a second-level dry heat, aromatic aroma and bitter taste. They have the effects of removing phlegm and dispersing wind, inducing sweating and detoxification, resisting inflammation and swelling, and expelling intestinal worms.

[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 diterpenoid components in Hyssopus sibiricus have strong anti-inflammatory activity. Therefore, in-depth development and utilization of the diterpenoid components in Hyssopus sibiricus, full exploration of its potential pharmacological value, and accurate analysis and characterization of the structural characteristics and physicochemical properties of its monomer compounds have far-reaching significance for the comprehensive development and application of Hyssopus sibiricus. 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 the diterpene compound in Hyssopus sclerotium, so that the diterpene compound can be used in the preparation of 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, soak it in ethanol and extract it by reflux, and then combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard-pointed hyssop extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; The third step is to perform the first gradient elution on the dichloromethane extract by silica gel column chromatography to obtain 8 fractions after separation; Step 4: The fourth fraction of the eight fractions obtained in the third step is subjected to a second gradient elution through a silica gel chromatography column to sequentially obtain five components; In the fifth step, the second component of the five components eluted 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%.

[0009] In the first step, the soaking is performed at room temperature for 3 to 4 hours.

[0010] 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.

[0011] 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, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0012] In the fourth step, the eluent for the second gradient elution was a mixture of dichloromethane and methanol. Five gradients were set for the second gradient elution. The volume ratios of dichloromethane and methanol for the gradient elution were 1:0, 80:1, 50:1, 30:1, and 10:1, respectively.

[0013] In the fifth step, the eluent of the semi-preparative liquid chromatography was a 35% methanol in water solution, and the flow rate was 2 mL / min.

[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, soak it in ethanol and extract it by reflux, and then combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard-pointed hyssop extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; The third step is to perform the first gradient elution on the dichloromethane extract by silica gel column chromatography to obtain 8 fractions after separation; Step 4: The fourth fraction of the eight fractions obtained in the third step is subjected to a second gradient elution through a silica gel chromatography column to sequentially obtain five components; In the fifth step, the second component of the five components eluted 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 (hyssopusone B) in the preparation of a drug for preventing inflammation.

[0016] The fourth technical solution of the present invention is achieved by the following measures: use of a diterpenoid compound (hyssopusone B) in the preparation of an anti-inflammatory drug.

[0017] The present invention discloses for the first time a diterpenoid compound (hyssopusone B) in Hyssopus sclerotium, determines and characterizes its structure and physicochemical properties, and proves through experiments that it has certain anti-inflammatory activity, thereby enabling it to be used in the preparation of anti-inflammatory drugs and anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0023] Attached Figure 6 It is the NOESY spectrum of the diterpenoid compound (hyssopusone B) 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 (referred to as: hyssopusone B) has the chemical structural formula: .

[0026] The diterpenoid compound (hyssopusone B) of the present invention has a systematic name of (1R,5R)-3'-(((1S,5S)-2-hydroxy-6,6-dimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)methyl)-6,6-dimethylspiro[bicyclo[3.1.1]heptane-2,2'-oxiran]-3-one; its Chinese name is (1R,5R)-3'-((1S,5S)-2-hydroxy-6,6-dimethyl-3-oxobicyclo[3.1.1]heptane-2-yl)methyl)-6,6-dimethylspiro[bicyclo[3.1.1]heptane-2,2'-oxiran]-3-one.

[0027] Example 2: As an optimization of the above example, the diterpenoid compound (hyssopusone B) was prepared according to the following steps: The first step is to grind the whole herb of Hyssopus officinalis, soak it in ethanol and extract it by reflux, and then combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard-pointed hyssop extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; The third step is to perform the first gradient elution on the dichloromethane extract by silica gel column chromatography to obtain 8 fractions after separation; Step 4: The fourth fraction of the eight fractions obtained in the third step is subjected to a second gradient elution through a silica gel chromatography column to sequentially obtain five components; In the fifth step, the second component of the five components eluted in the fourth step is separated and purified by semi-preparative liquid chromatography to obtain a diterpenoid compound (hyssopusone B).

[0028] Example 3: As an optimization of the above example, in the first step, the volume fraction of ethanol is 90% to 95%.

[0029] Embodiment 4: As an optimization of the above embodiment, in the first step, the soaking is carried out at room temperature for 3 to 4 hours.

[0030] Example 5: 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.

[0031] Example 6: 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 ratios of dichloromethane and methanol for the gradient elution are 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0032] Example 7: 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 second gradient elution is set with 5 gradients. The volume ratios of dichloromethane and methanol in the gradient elution are 1:0, 80:1, 50:1, 30:1, and 10:1, respectively.

[0033] Example 8: As an optimization of the above example, in the fifth step, the eluent of the semi-preparative liquid chromatography is a 35% methanol in water solution, and the flow rate is 2 mL / min.

[0034] Example 9: Use of the diterpenoid compound (hyssopusone B) in the preparation of a drug for preventing inflammation.

[0035] Example 10: Use of the diterpenoid compound (hyssopusone B) in the preparation of anti-inflammatory drugs.

[0036] Example 11: The diterpenoid compound (hyssopusone B) was prepared according to the following steps: In the first step, the whole herb of Hyssopus officinalis is crushed and 90% ethanol is added. After soaking at room temperature for 3 hours, it is heated to reflux extraction at 50°C for 3 times, each time for 2 hours. The reflux extracts are combined and recovered and concentrated under reduced pressure to obtain Hyssopus officinalis extract.

[0037] In the second step, the hard-pointed Hyssopus officinalis extract is dispersed with water, extracted with petroleum ether, dichloromethane and ethyl acetate in sequence, and the extract is concentrated under reduced pressure to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract.

[0038] In the third step, the dichloromethane extract was separated by gradient elution on a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography was a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol were 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0039] In the fourth step, the fourth fraction obtained in the third step is passed through a silica gel chromatography column and gradient eluted with a volume ratio of dichloromethane to methanol ranging from 1:0 to 0:1, and five components Fr.4-1, Fr.4-2, Fr.4-3, Fr.4-4, and Fr.4-5 are separated in sequence.

[0040] In the fifth step, the second component Fr.4-2 was further separated and purified by semi-preparative liquid chromatography. The eluent was 35% methanol in water. The flow rate was 2 mL / min. The eluted product was collected to obtain a diterpene compound (hyssopusone B).

[0041] Example 12: The diterpenoid compound (hyssopusone B) was prepared according to the following steps: In the first step, the whole herb of Hyssopus officinalis is crushed and 95% ethanol is added. After soaking at room temperature for 3.5 hours, it is heated to reflux extraction at 60°C for 3 times, each time for 3 hours. The reflux extracts of each time are combined and recovered and concentrated under reduced pressure to obtain Hyssopus officinalis extract.

[0042] In the second step, the hard-pointed Hyssopus officinalis extract is dispersed with water, extracted with petroleum ether, dichloromethane and ethyl acetate in sequence, and the extract is concentrated under reduced pressure to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract.

[0043] In the third step, the dichloromethane extract was separated by gradient elution on a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography was a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol were 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0044] In the fourth step, the fourth fraction obtained in the third step is passed through a silica gel chromatography column and gradient eluted with a volume ratio of dichloromethane to methanol ranging from 1:0 to 0:1, and five components Fr.4-1, Fr.4-2, Fr.4-3, Fr.4-4, and Fr.4-5 are separated in sequence.

[0045] In the fifth step, the second component Fr.4-2 was further separated and purified by semi-preparative liquid chromatography. The eluent was 35% methanol in water. The flow rate was 2 mL / min. The eluted product was collected to obtain a diterpene compound (hyssopusone B).

[0046] Example 13: The diterpenoid compound (hyssopusone B) was prepared according to the following steps: In the first step, the whole herb of Hyssopus officinalis is crushed and 90% ethanol is added. After soaking at room temperature for 4 hours, it is heated to reflux extraction at 55°C for 3 times, each time for 3 hours. The reflux extracts of each time are combined and recovered and concentrated under reduced pressure to obtain Hyssopus officinalis extract.

[0047] In the second step, the hard-pointed Hyssopus officinalis extract is dispersed with water, extracted with petroleum ether, dichloromethane and ethyl acetate in sequence, and the extract is concentrated under reduced pressure to obtain petroleum ether extract, dichloromethane extract and ethyl acetate extract.

[0048] In the third step, the dichloromethane extract was separated by gradient elution on a silica gel column chromatography to obtain 8 fractions, wherein the gradient eluent of the silica gel column chromatography was a mixture of dichloromethane and methanol, and the volume ratios of dichloromethane and methanol were 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0049] In the fourth step, the fourth fraction obtained in the third step is passed through a silica gel chromatography column and gradient eluted with a volume ratio of dichloromethane to methanol ranging from 1:0 to 0:1, and five components Fr.4-1, Fr.4-2, Fr.4-3, Fr.4-4, and Fr.4-5 are separated in sequence.

[0050] In the fifth step, the second component Fr.4-2 was further separated and purified by semi-preparative liquid chromatography. The eluent was 35% methanol in water. The flow rate was 2 mL / min. The eluted product was collected to obtain a diterpene compound (hyssopusone B).

[0051] Embodiment 14: The diterpenoid compound (hyssopusone B) obtained in Examples 11 to 13 above was subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR) and carbon nuclear magnetic resonance spectroscopy ( 13 C-APT) analysis. 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 ; and the obtained compound was analyzed by chromatogram, and the peak attribution is shown in Table 1. The chemical structure formula of the compound can be determined by the data in Table 1, and the compound is easily soluble in methanol.

[0052] Embodiment 15: The diterpenoid compound (hyssopusone B) of the present invention was subjected to an in vitro anti-inflammatory pharmacodynamic experiment. The in vitro anti-inflammatory pharmacodynamic experiment adopted the MTT method: a cell suspension consisting of a certain number of RAW 264.7 cells was placed in a culture bottle, 2 mL to 3 mL of DMEM culture medium containing 10% FBS was added, and the cells were cultured in a 37°C, 5% CO2 incubator, and the cell growth was observed to be good; the cells could be passaged when the density was 80% to 90%, and the cells with good growth were taken, washed twice with 0.1% mol / L phosphate buffer, and about 0.1 mL / cm 2 TPS was incubated at 37℃ for 1min, then taken out, the digestion solution was washed off, and an appropriate amount of culture medium was added. The culture medium was aspirated with a pipette, and the cells attached to the wall were blown gently to make them fall off. The cell density was adjusted to 5×10 4 / mL, replace the culture bottle and culture medium to maintain normal cell metabolism; grouping is hydrocortisone control group and experimental group i.e. compound; take RAW264.7 cells in logarithmic growth period, dilute and mix with blank culture medium to make cell suspension, inoculate 200uL / well in 96-well plate, culture at 37℃, put each well in 5% CO2 incubator for 24h, add phosphate buffer to each group, adjust the concentration to 1ug / mL, continue to culture for 24h, centrifuge, take supernatant and place in 96-well plate for use; standard is diluted with DMEM culture medium containing 10% FBS, and the sample is added to 96-well plate at 50uL / well; add 50uL of Griess Reagent I and II at room temperature to each well, mix well, place in dark at room temperature for 10min, measure absorbance at 540nm with microplate reader, and calculate NO content; NO content = 1-(A administration-A control) / A control. Each step of the experiment strictly followed the method provided by the MTT kit for experimental and data processing. The results of the inhibitory effects of diterpenoids (hyssopusone B) and positive drugs on NO production in LPS-stimulated RAW264.7 cells are shown in Table 2.

[0053] The diterpenoid compound (hyssopusone B) of the present invention has a certain inhibitory effect on the release of NO, indicating that the diterpenoid compound (hyssopusone B) has broad application prospects as a potential anti-inflammatory drug.

[0054] In summary, the present invention provides a diterpenoid anti-inflammatory compound (hyssopusone B) in Hyssopus sclerotium and its preparation method and application. The compound is obtained from the whole plant of Hyssopus sclerotium through chromatographic separation and purification. The diterpenoid compound (hyssopusone B) of the present invention has a certain inhibitory effect on the release of NO in RAW264.7 macrophages induced by lipopolysaccharide, laying a foundation for the development and application of such compounds in Hyssopus sclerotium in potential anti-inflammatory drugs.

[0055] 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.

[0056]

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 the hard-pointed hyssop, soak it in 8 times the amount of ethanol, reflux extract it, and combine the extracts to obtain the hard-pointed hyssop extract; The second step is to disperse the hard-pointed hyssop extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; The third step is to perform the first gradient elution on the dichloromethane extract by silica gel column chromatography to obtain 8 fractions after separation; Step 4: The fourth fraction of the eight fractions obtained in the third step is subjected to a second gradient elution through a silica gel chromatography column to sequentially obtain five components; In the fifth step, the second component of the five components eluted 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%, and the soaking is for 3 to 4 hours at room temperature.

4. The diterpenoid compound according to claim 2 or 3, characterized in that 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.

5. The diterpenoid compound according to any one of claims 2 to 4, 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, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

6. The diterpenoid compound according to any one of claims 2 to 5, characterized in that In the fourth step, the eluent for the second gradient elution was a mixture of dichloromethane and methanol. Five gradients were set for the second gradient elution. The volume ratios of dichloromethane and methanol for the gradient elution were 1:0, 80:1, 50:1, 30:1, and 10:1, respectively.

7. The diterpenoid compound according to any one of claims 2 to 6, characterized in that In the fifth step, a semi-preparative C18 chromatographic column was used, and the eluent for liquid chromatography was 35% methanol in water at a flow rate of 2 mL / min.

8. A method for preparing a diterpenoid compound according to any one of claims 1 to 7, characterized in that Follow these steps: The first step is to grind the whole herb of Hyssopus officinalis, soak it in ethanol and extract it by reflux, and then combine the extracts to obtain Hyssopus officinalis extract; The second step is to disperse the hard-pointed hyssop extract with water, extract it with petroleum ether, dichloromethane and ethyl acetate in sequence, and concentrate the dichloromethane extract to obtain the dichloromethane extract; The third step is to perform the first gradient elution on the dichloromethane extract by silica gel column chromatography to obtain 8 fractions after separation; Step 4: The fourth fraction of the eight fractions obtained in the third step is subjected to a second gradient elution through a silica gel chromatography column to sequentially obtain five components; In the fifth step, the second component of the five components eluted 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 a drug for preventing inflammation.

10. Use of the diterpenoid compound according to any one of claims 1 to 7 in the preparation of anti-inflammatory drugs.

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