Diterpenoid anti-inflammatory compounds from hyptis muticus and preparation method and application thereof

Abstract

The present application relates to the technical field of isolation and purification of Hyptis emoryi, and is a diterpenoid compound, a preparation method and application thereof, wherein the diterpenoid compound is extracted and separated from Hyptis emoryi, and the preparation steps comprise: extracting a Hyptis emoryi extract with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain a dichloromethane extract; performing twice gradient elution on the dichloromethane extract by using a silica gel column chromatography; and performing separation and purification on the obtained fractions after separation by using a semi-preparative liquid chromatography to obtain the diterpenoid compound. The present application discloses the diterpenoid compound in Hyptis emoryi for the first time, determines and characterizes the structure and physical and chemical properties of the diterpenoid compound, and proves by experiments that the diterpenoid compound has certain anti-inflammatory activity, so that the diterpenoid compound can be applied to preparation of anti-inflammatory drugs and preparation of anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of isolation and purification of Hyssopus cuspidatus, and is a diterpenoid anti-inflammatory compound in Hyssopus cuspidatus, a preparation method and application thereof, which is referred to as hyssopusone B. BACKGROUND

[0002] Hyssopus cuspidatus is a plant of the genus Hyssopus in the family Labiatae. Hyssopus cuspidatus ) is a plant of the genus Hyssopus ( Labiatae ) in the family Labiatae ( Hyssopus ) and is artificially cultivated as a secondary dry heat. It has the effects of expelling phlegm and wind, inducing perspiration and detoxification, anti-inflammatory and detumescence, and driving intestinal worms.

[0003] Inflammation is a key defense system gradually constructed by the body during evolution to resist the invasion of foreign pathogens. However, when this defense mechanism persists or is overreacted, it often leads to diseases, and even threatens human life and health. Long-term use of the same drug gradually exposes its shortcomings such as drug resistance and adverse reactions, so it is particularly important to find high-efficiency and low-toxicity drugs for effective treatment of diseases.

[0004] The diterpenoid components in Hyssopus cuspidatus have strong anti-inflammatory activity, so it is of great significance to fully develop and utilize the diterpenoid components in Hyssopus cuspidatus, fully explore their potential pharmacological value, and accurately analyze and characterize the structural properties and physicochemical properties of the monomer compounds, for the comprehensive development and application of Hyssopus cuspidatus. SUMMARY

[0005] The present application provides a diterpenoid compound and a preparation method thereof, which overcomes the shortcomings of the prior art. It first discloses the diterpenoid compound in Hyssopus cuspidatus, so that it can be applied in the preparation of anti-inflammatory drugs and anti-inflammatory drugs.

[0006] One of the technical solutions of the present application is achieved by the following measures: a diterpenoid compound, the chemical structural formula of which is:

[0007] .

[0008] The following is a further optimization or / and improvement of the above-mentioned one of the technical solutions:

[0009] The above-mentioned diterpenoid compound is prepared by the following steps:

[0010] Firstly, the whole grass of Hyssopus cuspidatus is crushed, then soaked and refluxed with ethanol to extract, and the extract is combined to obtain a Hyssopus cuspidatus extract;

[0011] The second step is to disperse the extract of *Hedyotis diffusa* in water, extract it sequentially with petroleum ether, dichloromethane, and ethyl acetate, and concentrate the dichloromethane extract to obtain dichloromethane extract.

[0012] The third step involved performing a gradient elution of the dichloromethane extract using silica gel column chromatography to obtain eight fractions after separation.

[0013] The fourth step involves eluting the fourth fraction of the eight fractions obtained in the third step using a silica gel column for a second gradient elution, resulting in five fractions.

[0014] In the fifth step, the second component of the five components obtained from the elution in the fourth step was separated and purified by semi-preparative liquid chromatography to obtain diterpenoid compounds.

[0015] In the first step above, the volume fraction of ethanol is 90% to 95%.

[0016] In the first step above, soaking is performed at room temperature for 3 to 4 hours.

[0017] In the first step above, the reflux extraction is carried out at 50°C to 60°C, with heating and reflux extraction performed three times, each time for 2 to 3 hours.

[0018] In the third step above, the eluent for the first gradient elution is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the gradient elution is 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0019] In the fourth step above, the eluent for the second gradient elution is a mixture of dichloromethane and methanol. The second gradient elution is set with 5 gradients, and the volume ratios of dichloromethane and methanol in the gradient elution are 1:0, 80:1, 50:1, 30:1, and 10:1, respectively.

[0020] In step 5 above, the eluent for the semi-preparative liquid chromatography is an aqueous solution of 35% methanol, and the flow rate is 2 mL / min.

[0021] The second technical solution of the present invention is achieved through the following measures: a method for preparing a diterpenoid compound, comprising the following steps:

[0022] The first step is to crush the whole herb of *Hedyotis diffusa*, soak it in ethanol and reflux extract it, and then combine the extracts to obtain *Hedyotis diffusa* extract.

[0023] The second step is to disperse the extract of *Hedyotis diffusa* in water, extract it sequentially with petroleum ether, dichloromethane, and ethyl acetate, and concentrate the dichloromethane extract to obtain dichloromethane extract.

[0024] The third step involved performing a gradient elution of the dichloromethane extract using silica gel column chromatography to obtain eight fractions after separation.

[0025] The fourth step involves eluting the fourth fraction of the eight fractions obtained in the third step using a silica gel column for a second gradient elution, resulting in five fractions.

[0026] In the fifth step, the second component of the five components obtained from the elution in the fourth step was separated and purified by semi-preparative liquid chromatography to obtain diterpenoid compounds.

[0027] The third technical solution of the present invention is achieved through the following measures: the application of a diterpenoid compound (hyssopusone B) in the preparation of an anti-inflammatory drug.

[0028] The fourth technical solution of the present invention is achieved through the following measures: the application of a diterpenoid compound (hyssopusone B) in the preparation of anti-inflammatory drugs.

[0029] This invention discloses for the first time a diterpenoid compound (hyssopusone B) from *Hedyotis diffusa*, determines and characterizes its structure and physicochemical properties, and demonstrates through experiments that it has certain anti-inflammatory activity, thus enabling its application in the preparation of anti-inflammatory drugs and preventive anti-inflammatory drugs. Attached Figure Description

[0030] Appendix Figure 1 The diterpenoid compound (hyssopusone B) of this invention 1 H-NMR spectrum.

[0031] Appendix Figure 2 The diterpenoid compound (hyssopusone B) of this invention 13 C-APT spectrum.

[0032] Appendix Figure 3 This is the COSY spectrum of the diterpenoid compound (hyssopusone B) of the present invention.

[0033] Appendix Figure 4 This is the QC spectrum of the diterpenoid compound (hyssopusone B) of the present invention.

[0034] Appendix Figure 5 This is the BC spectrum of the diterpenoid compound (hyssopusone B) of the present invention.

[0035] Appendix Figure 6 This is the NOESY spectrum of the diterpenoid compound (hyssopusone B) of the present invention. Detailed Implementation

[0036] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solution of this invention and the actual situation. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; room temperature in this invention generally refers to a temperature between 15°C and 25°C, and is generally defined as 25°C.

[0037] The present invention will be further described below with reference to embodiments:

[0038] Example 1: The chemical structural formula of this diterpenoid compound (denoted as hyssopusone B) is as follows:

[0039] .

[0040] The systematic name of the diterpenoid compound (hyssopusone B) of this invention is: (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; the 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]hept-2,2'-epoxy]-3-one.

[0041] Example 2: As an optimization of the above example, the diterpenoid compound (hyssopusone B) was prepared according to the following steps:

[0042] The first step is to crush the whole herb of *Hedyotis diffusa*, soak it in ethanol and reflux extract it, and then combine the extracts to obtain *Hedyotis diffusa* extract.

[0043] The second step is to disperse the extract of *Hedyotis diffusa* in water, extract it sequentially with petroleum ether, dichloromethane, and ethyl acetate, and concentrate the dichloromethane extract to obtain dichloromethane extract.

[0044] The third step involved performing a gradient elution of the dichloromethane extract using silica gel column chromatography to obtain eight fractions after separation.

[0045] The fourth step involves eluting the fourth fraction of the eight fractions obtained in the third step using a silica gel column for a second gradient elution, resulting in five fractions.

[0046] In the fifth step, the second of the five components eluted in the fourth step was separated and purified by semi-preparative liquid chromatography to obtain a diterpenoid compound (hyssopusone B).

[0047] Example 3: As an optimization of the above example, in the first step, the volume fraction of ethanol is 90% to 95%.

[0048] Example 4: As an optimization of the above example, in the first step, the soaking is carried out at room temperature for 3 to 4 hours.

[0049] 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 extraction is carried out three times, each time for 2 to 3 hours.

[0050] 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 ratio of dichloromethane to methanol in the gradient elution is 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0051] 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. The second gradient elution is set with 5 gradients, and the volume ratio of dichloromethane to methanol in the gradient elution is 1:0, 80:1, 50:1, 30:1, and 10:1, respectively.

[0052] Example 8: As an optimization of the above example, in the fifth step, the eluent for the semi-preparative liquid chromatography is an aqueous solution of 35% methanol, and the flow rate is 2 mL / min.

[0053] Example 9: The application of this diterpenoid compound (hyssopusone B) in the preparation of anti-inflammatory drugs.

[0054] Example 10: The application of this diterpenoid compound (hyssopusone B) in the preparation of anti-inflammatory drugs.

[0055] Example 11: The diterpenoid compound (hyssopusone B) was prepared according to the following steps:

[0056] The first step is to crush the whole herb of *Hedyotis diffusa* and add 90% ethanol. After soaking at room temperature for 3 hours, the herb is extracted by reflux at 50°C three times for 2 hours each time. The reflux extracts are combined and concentrated under reduced pressure to obtain *Hedyotis diffusa* extract.

[0057] The second step involves dispersing the extract of *Hedyotis diffusa* in water, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated under reduced pressure to obtain petroleum ether extract, dichloromethane extract, and ethyl acetate extract.

[0058] The third step involves separating the dichloromethane extract using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient eluent is a mixture of dichloromethane and methanol, with volume ratios of 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0059] In the fourth step, the fourth fraction obtained in the third step was subjected to gradient elution using a silica gel column with a volume ratio of dichloromethane to methanol ranging from 1:0 to 10:1, to sequentially separate five components: Fr.4-1, Fr.4-2, Fr.4-3, Fr.4-4, and Fr.4-5.

[0060] In the fifth step, the second component Fr.4-2 was further separated and purified by semi-preparative liquid chromatography. The eluent was an aqueous solution of 35% methanol, the flow rate was 2 mL / min, and the elution product was collected to obtain the diterpenoid compound (hyssopusone B).

[0061] Example 12: The diterpenoid compound (hyssopusone B) was prepared according to the following steps:

[0062] The first step is to crush the whole herb of *Hedyotis diffusa* and add 95% ethanol. After soaking at room temperature for 3.5 hours, the herb is extracted by reflux at 60°C three times for 3 hours each time. The reflux extracts are combined and concentrated under reduced pressure to obtain *Hedyotis diffusa* extract.

[0063] The second step involves dispersing the extract of *Hedyotis diffusa* in water, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated under reduced pressure to obtain petroleum ether extract, dichloromethane extract, and ethyl acetate extract.

[0064] The third step involves separating the dichloromethane extract using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient eluent is a mixture of dichloromethane and methanol, with volume ratios of 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0065] In the fourth step, the fourth fraction obtained in the third step was subjected to gradient elution using a silica gel column with a volume ratio of dichloromethane to methanol ranging from 1:0 to 10:1, to sequentially separate five components: Fr.4-1, Fr.4-2, Fr.4-3, Fr.4-4, and Fr.4-5.

[0066] In the fifth step, the second component Fr.4-2 was further separated and purified by semi-preparative liquid chromatography. The eluent was an aqueous solution of 35% methanol, the flow rate was 2 mL / min, and the elution product was collected to obtain the diterpenoid compound (hyssopusone B).

[0067] Example 13: The diterpenoid compound (hyssopusone B) was prepared according to the following steps:

[0068] The first step is to crush the whole herb of *Hedyotis diffusa* and add 90% ethanol. After soaking at room temperature for 4 hours, the herb is heated and refluxed at 55°C for 3 hours each time. The reflux extracts are combined and concentrated under reduced pressure to obtain *Hedyotis diffusa* extract.

[0069] The second step involves dispersing the extract of *Hedyotis diffusa* in water, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated under reduced pressure to obtain petroleum ether extract, dichloromethane extract, and ethyl acetate extract.

[0070] The third step involves separating the dichloromethane extract using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient eluent is a mixture of dichloromethane and methanol, with volume ratios of 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0071] In the fourth step, the fourth fraction obtained in the third step was subjected to gradient elution using a silica gel column with a volume ratio of dichloromethane to methanol ranging from 1:0 to 10:1, to sequentially separate five components: Fr.4-1, Fr.4-2, Fr.4-3, Fr.4-4, and Fr.4-5.

[0072] In the fifth step, the second component Fr.4-2 was further separated and purified by semi-preparative liquid chromatography. The eluent was an aqueous solution of 35% methanol, the flow rate was 2 mL / min, and the elution product was collected to obtain the diterpenoid compound (hyssopusone B).

[0073] Example 14:

[0074] The diterpenoid compounds (hyssopusone B) obtained in Examples 11 to 13 above were analyzed by 1H-NMR and 13C-APT nuclear magnetic resonance spectroscopy. 1 H-NMR spectrum as shown Figure 1 As shown, 13 C-APT spectrum as shown Figure 2 As shown, the two-dimensional maps are respectively Figures 3 to 6The obtained compounds were subjected to spectral analysis, and the peak assignments are shown in Table 1. The chemical structural formula of the compounds can be determined from the data in Table 1, and the compounds are readily soluble in methanol.

[0075] Example 15:

[0076] In vitro anti-inflammatory pharmacodynamic experiments were conducted on the diterpenoid compound (hyssopusone B) of this invention. The in vitro anti-inflammatory pharmacodynamic experiments used the MTT assay: a certain number of RAW 264.7 cells were placed in a culture flask, and 2 to 3 mL of DMEM medium containing 10% FBS was added. The flask was incubated at 37°C in a 5% CO2 incubator, and the cell growth was observed. When the cell density reached 80% to 90%, the cells were passaged. Cells with good growth were taken, washed twice with 0.1% mol / L phosphate buffer, and approximately 0.1 mL / cm³ of DMEM medium was added. 2 After incubating the TPS at 37°C for 1 minute, remove the cells, wash away the digestion solution, add an appropriate amount of culture medium, aspirate the culture medium with a pipette, gently blow away the adherent cells to detach them, and adjust the cell density to 5 × 10⁻⁶. 4 Cells were cultured at a concentration of 10 μL / mL, and the culture flasks and medium were changed to maintain normal cell metabolism. The cells were divided into a control group (hydrocortisone) and an experimental group (the compound). Logarithmically growing RAW264.7 cells were diluted with blank medium to form a cell suspension. 200 μL / well was seeded into 96-well plates and cultured at 37°C. Each well was placed in a 5% CO2 incubator for 24 hours to allow the cells to adhere. Phosphate buffer was added to each group to adjust the concentration to 1 μg / mL. After culturing for another 24 hours, the cells were centrifuged, and the supernatant was collected and placed in 96-well plates for later use. Standards were diluted with DMEM medium containing 10% FBS, and samples were added to 96-well plates at a concentration of 50 μL / well. 50 μL of room temperature Griess Reagent I and II were added to each well, mixed thoroughly, and incubated at room temperature in the dark for 10 minutes. The absorbance was measured at 540 nm using a microplate reader, and the NO content was calculated. NO content = 1 - (A-drug-A-control) / A-control. Each step of the experiment was strictly performed in accordance with the methods provided by the MTT kit. The results of the inhibitory effects of diterpenoids (hyssopusone B) and positive control on NO production in LPS-stimulated RAW264.7 cells are shown in Table 2.

[0077] The diterpenoid compound (hyssopusone B) of this 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.

[0078] In summary, this invention provides a diterpenoid anti-inflammatory compound (hyssopusone B) from *Hedyotis diffusa*, its preparation method, and its application. This compound is obtained from the whole herb of *Hedyotis diffusa* through chromatographic separation and purification. The diterpenoid compound (hyssopusone B) of this invention exhibits a certain inhibitory effect on lipopolysaccharide-induced NO release from RAW264.7 macrophages, laying the foundation for the development and application of such compounds from *Hedyotis diffusa* in potential anti-inflammatory drugs.

[0079] The above technical features constitute various embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

[0080]

[0081]

Claims

1. A diterpenoid compound, characterized in that... The chemical structural formula is: 。 2. A method for preparing a diterpenoid compound according to claim 1, characterized in that... It is prepared according to the following steps: The first step is to crush the whole herb of *Hedyotis diffusa*, soak it in 8 times the amount of ethanol and reflux it for extraction, and then combine the extracts to obtain *Hedyotis diffusa* extract. The second step is to disperse the extract of *Hedyotis diffusa* in water, extract it sequentially with petroleum ether, dichloromethane, and ethyl acetate, and concentrate the dichloromethane extract to obtain dichloromethane extract. The third step involved performing a gradient elution of the dichloromethane extract using silica gel column chromatography to obtain eight fractions after separation. The fourth step involves eluting the fourth fraction of the eight fractions obtained in the third step using a silica gel column for a second gradient elution, resulting in five fractions. Fifth step: The second component of the five components obtained from the elution in the fourth step is separated and purified by semi-preparative liquid chromatography to obtain diterpenoid compounds; In the first step, the volume fraction of ethanol is 90% to 95%, and the soaking is carried out at room temperature for 3 to 4 hours. In the third step, the eluent for the first gradient elution is a mixture of dichloromethane and methanol. The volume ratios of dichloromethane and methanol in the gradient elution are 1:0, 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively. In the fourth step, the eluent for the second gradient elution is a mixture of dichloromethane and methanol. The second gradient elution is set with 5 gradients, and the volume ratio of dichloromethane to methanol in the gradient elution is 1:0, 80:1, 50:1, 30:1, and 10:1, respectively. In the fifth step, a semi-preparative C18 column was used, and the eluent for liquid chromatography was an aqueous solution of 35% methanol at a flow rate of 2 mL / min.

3. The method for preparing diterpenoid compounds according to claim 2, characterized in that... In the first step, the reflux extraction is carried out at 50°C to 60°C, with heating and reflux extraction performed three times, each time for 2 to 3 hours.

4. The use of a diterpenoid compound according to claim 1 in the preparation of an anti-inflammatory drug.

5. The use of a diterpenoid compound according to claim 1 in the preparation of an anti-inflammatory drug.

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