Diterpenoid components in hyssopus officinalis and separation and extraction method and application thereof

Abstract

The present application relates to the technical field of hard tip hyssopus extract and separation and purification, and is a diterpenoid component in hyssopus and a separation and extraction method and application thereof, the diterpenoid component in the hyssopus is obtained by the following method: total extract of hard tip hyssopus is extracted, extracted by petroleum ether, separated by silica gel column chromatography multiple gradient elution, and finally purified and separated by high performance liquid chromatography gradient elution, so that the diterpenoid component in the hyssopus, that is, hyssopusone D, can be obtained.The present application discloses the compound hyssopusone D for the first time, and performs in-vitro anti-inflammatory pharmacodynamic experiment on the compound.According to the experimental results, it can be seen that the compound has a strong inhibitory effect on RAW 264.7 cells, so that the compound can be used for preparing an anti-inflammatory drug.

Description

Technical Field

[0001] This invention relates to the field of extract and purification technology of *Hedyotis diffusa*, specifically a diterpenoid component from *Hedyotis diffusa* and its extraction method, and also relates to the application of the diterpenoid component from *Hedyotis diffusa* in the preparation of drugs for preventing and / or fighting inflammation. Background Technology

[0002] *Hyssopus cuspidatus* is a plant belonging to the genus *Hyssopus* in the family Lamiaceae. The whole plant is used medicinally. It has a slightly fragrant aroma, a bland taste, and is slightly moist in nature. It enters the lung and liver meridians and has antitussive, antiasthmatic, heat-clearing, and dampness-removing effects. Clinically, it is used to treat symptoms such as bronchitis, cough, asthma, colds, fever, and difficulty urinating. Furthermore, *Hyssopus cuspidatus* is readily available, inexpensive, clinically effective, and has few toxic side effects. To date, there is limited research on the focus and basic theories of *Hyssopus cuspidatus*. Modern pharmacology indicates that its extracts have significant anti-inflammatory and antibacterial activities, but research on its chemical components is still lacking. *Hyssopus cuspidatus* may be a valuable species for treating respiratory diseases.

[0003] Therefore, it has received much attention in recent years.

[0004] Chinese patent application CN114044734A discloses a rosinane diterpenoid, its preparation method, and its application. It discloses the compound (nepetabrate D) for the first time and conducts in vitro anti-inflammatory pharmacodynamic experiments on the compound. The experiments clearly show that the compound has a strong inhibitory effect on RAW 264.7 cells, Staphylococcus aureus, and Escherichia coli, thus enabling the compound to be used in the preparation of drugs for preventing inflammation and inhibiting bacteria, or / and in the preparation of anti-inflammatory and antibacterial drugs, or / and in the preparation of health products for preventing and treating inflammation and inhibiting bacteria.

[0005] Chinese patent application CN115708840A discloses the application of *Hedyotis diffusa* extract in the preparation of anti-lung cancer drugs. The provided *Hedyotis diffusa* extract significantly inhibits the proliferation, migration, and invasion of human lung cancer cells A549 and NCI-H1975, affecting cell morphology and the release of inflammatory factors TNF-α and IL-6 in the cell supernatant in a dose-dependent manner. It also effectively inhibits the secretion of matrix metalloenzymes MMP-2 and MMP-9, which are related to invasion. Pathological observation using orthotopic Lewis lung cancer models in mice showed that *Hedyotis diffusa* extract can reduce the number of tumor cells in Lewis lung cancer mice, significantly increase the concentration levels of IL-2 and IFN-γ in mouse serum, and effectively enhance the proliferation capacity of lymphocytes and the number of NK cells in the spleen, thereby regulating the immune function of Lewis lung cancer mice. Furthermore, its combination with cisplatin can reduce the immunosuppressive effect of cisplatin on lung cancer mice.

[0006] The medicinal effects of *Hedyotis diffusa* mainly come from its terpenoid compounds, including monoterpenoids and diterpenoids. Among them, the dominant diterpenoid components have good anti-inflammatory and antibacterial activities. Therefore, developing and utilizing the diterpenoid monomeric compounds of *Hedyotis diffusa*, further exploring its potential medicinal value, and determining and characterizing the structure and physicochemical properties of its monomeric compounds are of great significance for the development and utilization of *Hedyotis diffusa*. Summary of the Invention

[0007] This invention provides a diterpenoid component from hyssopus, its separation and extraction method, and its application. This invention discloses for the first time the diterpenoid component (i.e., hyssopusone D) from hyssopus and for the first time the application of hyssopusone D in the preparation of drugs for preventing inflammation and / or anti-inflammatory or antibacterial purposes.

[0008] One of the technical solutions of this invention is achieved through the following measures: a diterpenoid component (i.e., hyssopusone D) from hyssopus, with the following chemical structural formula:

[0009]

[0010] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions:

[0011] The chemical name of the above-mentioned hyssopusone D is (1S,2R,5S)-2-hydroxy-2-(2-(1R,2S,5R)-2-hydroxy-6,6-dimethyl-3-oxobicyclo[3.1.1]heptane-2-yl)ethyl)-6,6-dimethylbicyclo[3.1.1]hept-3-one.

[0012] The English name of hyssopusone D is:

[0013] (1S,2R,5S)-2-hydroxy-2-(2-((1R,2S,5R)-2-hydroxy-6,6-dimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)ethyl)-6,6-dimethylbicyclo[3.1.1]heptan-3-one.

[0014] The diterpenoid components in the above-mentioned thyme were prepared according to the following steps:

[0015] The first step is to crush the herb *Hedyotis diffusa* and add ethanol, soak it at room temperature, and then heat and reflux it at 40°C to 60°C at least twice, with each extraction time being 1 to 3 hours. Combine the reflux extracts from each extraction and recover and concentrate them under reduced pressure to obtain the total extract of *Hedyotis diffusa*.

[0016] The second step is to dissolve the total extract of *Hedyotis diffusa* in water and extract it with petroleum ether to obtain the petroleum ether fraction extract.

[0017] The third step involves separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 1:0, 100:1, 50:1, 25:1, 8:1, 5:1, 1:1, and 1:0, respectively.

[0018] The fourth step involves further separating the sixth fraction from the eight fractions by silica gel column chromatography gradient elution to obtain five fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 20:1, 10:1, 3:1, 1:1, and 0:1, respectively.

[0019] The fifth step involves further separating the third of the five fractions by silica gel column chromatography gradient elution to obtain three more fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of petroleum ether and ethyl acetate of 10:1, 5:1, and 3:1, respectively.

[0020] Step 6: The third fraction of the three fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluent is collected. The eluent collected at 27.0 minutes is the diterpenoid component of senna.

[0021] In the first step above, 8 ml to 10 ml of ethanol is added for every 1 g of hard-tipped angelica.

[0022] In the first step above, the soaking time is 3 to 4 hours.

[0023] In the first step above, the heating and reflux extraction is performed three times.

[0024] In step six above, the eluent for gradient elution in high performance liquid chromatography is a mixture of methanol and water, wherein the volume ratio of methanol to water is 85:15.

[0025] The second technical solution of the present invention is achieved through the following measures: a method for separating and extracting diterpenoid components from thyme, comprising the following steps:

[0026] The first step is to crush the herb *Hedyotis diffusa* and add ethanol, soak it at room temperature, and then heat and reflux it at 40°C to 60°C at least twice, with each extraction time being 1 to 3 hours. Combine the reflux extracts from each extraction and recover and concentrate them under reduced pressure to obtain the total extract of *Hedyotis diffusa*.

[0027] The second step is to dissolve the total extract of *Hedyotis diffusa* in water and extract it with petroleum ether to obtain the petroleum ether fraction extract.

[0028] The third step involves separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 1:0, 100:1, 50:1, 25:1, 8:1, 5:1, 1:1, and 1:0, respectively.

[0029] The fourth step involves further separating the sixth fraction from the eight fractions by silica gel column chromatography gradient elution to obtain five fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 20:1, 10:1, 3:1, 1:1, and 0:1, respectively.

[0030] The fifth step involves further separating the third of the five fractions by silica gel column chromatography gradient elution to obtain three more fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of petroleum ether and ethyl acetate of 10:1, 5:1, and 3:1, respectively.

[0031] Step 6: The third fraction of the three fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluent is collected. The eluent collected at 27.0 minutes is the diterpenoid component of senna.

[0032] The following are further optimizations and / or improvements to the second technical solution of the above invention:

[0033] In the first step above, 8 ml to 10 ml of ethanol is added for every 1 g of hard-tipped angelica.

[0034] In the first step above, the soaking time is 3 to 4 hours.

[0035] In the first step above, the heating and reflux extraction is performed three times.

[0036] In step six above, the eluent for gradient elution in high performance liquid chromatography is a mixture of methanol and water, wherein the volume ratio of methanol to water is 85:15.

[0037] The third technical solution of the present invention is achieved through the following measures: the application of a diterpenoid component of thyme in the preparation of a drug for preventing inflammation and / or anti-inflammatory purposes.

[0038] The following are further optimizations and / or improvements to the third technical solution of the above invention:

[0039] The aforementioned anti-inflammatory drugs refer to drugs that prevent inflammation of RAW 264.7 cells.

[0040] The aforementioned anti-inflammatory drugs refer to anti-inflammatory drugs targeting RAW 264.7 cell inflammation.

[0041] This invention discloses the compound hyssopusone D for the first time and conducts in vitro anti-inflammatory pharmacodynamic experiments on the compound. The experimental results clearly show that the compound has a strong inhibitory effect on RAW 264.7 cells, thus enabling the compound to be used in the preparation of drugs for the prevention of inflammation and / or anti-inflammatory purposes. Attached Figure Description

[0042] Appendix Figure 1 This is a chemical structure diagram of hyssopusone D as described in this invention.

[0043] Appendix Figure 2 The hyssopusone D described in this invention 1 H-NMR spectrum.

[0044] Appendix Figure 3 The hyssopusone D described in this invention 13 C-APT spectrum. Detailed Implementation

[0045] The present invention is not limited to the following embodiments, and the specific implementation can be determined according to the technical solution of the present invention and the actual situation.

[0046] Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are known and commonly used chemical reagents and chemical products in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; unless otherwise specified, all solutions in this invention are aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous solution of hydrochloric acid; room temperature in this invention generally refers to a temperature between 15°C and 25°C, and is generally defined as 25°C.

[0047] The present invention will be further described below with reference to embodiments:

[0048] Example 1: A diterpenoid component from hyssopusone, namely hyssopusone D, has the following chemical structural formula:

[0049]

[0050] The chemical name of hyssopusone D is (1S,2R,5S)-2-hydroxy-2-(2-(1R,2S,5R)-2-hydroxy-6,6-dimethyl-3-oxobicyclo[3.1.1]heptane-2-yl)ethyl)-6,6-dimethylbicyclo[3.1.1]hept-3-one.

[0051] The English name of hyssopusone D is:

[0052] (1S,2R,5S)-2-hydroxy-2-(2-((1R,2S,5R)-2-hydroxy-6,6-dimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)ethyl)-6,6-dimethylbicyclo[3.1.1]heptan-3-one.

[0053] Example 2, as an optimization of the above examples, the diterpenoid components in the herb were prepared according to the following steps:

[0054] The first step is to crush the herb *Hedyotis diffusa* and add ethanol, soak it at room temperature, and then heat and reflux it at 40°C to 60°C at least twice, with each extraction time being 1 to 3 hours. Combine the reflux extracts from each extraction and recover and concentrate them under reduced pressure to obtain the total extract of *Hedyotis diffusa*.

[0055] The second step is to dissolve the total extract of *Hedyotis diffusa* in water and extract it with petroleum ether to obtain the petroleum ether fraction extract.

[0056] The third step involves separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 1:0, 100:1, 50:1, 25:1, 8:1, 5:1, 1:1, and 1:0, respectively.

[0057] The fourth step involves further separating the sixth fraction from the eight fractions by silica gel column chromatography gradient elution to obtain five fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 20:1, 10:1, 3:1, 1:1, and 0:1, respectively.

[0058] The fifth step involves further separating the third of the five fractions by silica gel column chromatography gradient elution to obtain three more fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of petroleum ether and ethyl acetate of 10:1, 5:1, and 3:1, respectively.

[0059] Step 6: The third fraction of the three fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluent is collected. The eluent collected at 27.0 minutes is the diterpenoid component of senna.

[0060] Example 3, as an optimization of Example 2 above, in the first step, 8 ml to 10 ml of ethanol is added for every 1 g of hard-tipped vanilla.

[0061] Example 4, as an optimization of Example 2 above, in the first step, the soaking time is 3 to 4 hours.

[0062] Example 5, as an optimization of Example 2 above, involves heating and reflux extraction three times in the first step.

[0063] Example 6, as an optimization of Example 2 above, in the sixth step, the eluent for the high performance liquid chromatography gradient elution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 85:15.

[0064] Example 7: Application of diterpenoid components in this herb in the preparation of anti-inflammatory and / or anti-inflammatory drugs.

[0065] Example 8, as an optimization of Example 7 above, refers to a drug for preventing inflammation of RAW 264.7 cells.

[0066] Example 9, as an optimization of Example 7 above, refers to an anti-inflammatory drug that targets RAW 264.7 cell inflammation.

[0067] Example 10: The diterpenoid components of this herb were prepared according to the following steps:

[0068] The first step is to crush the herb *Hedyotis diffusa* and add ethanol. Add 8 to 10 ml of ethanol to every 1 g of herb *Hedyotis diffusa*. Soak at room temperature for 3 to 4 hours. Then, heat and reflux at 40 to 60°C for 3 extractions, each extraction lasting 1 to 3 hours. Combine the reflux extracts and recover and concentrate under reduced pressure to obtain the total extract of *Hedyotis diffusa*.

[0069] The second step is to dissolve the total extract of *Hedyotis diffusa* in water and extract it with petroleum ether to obtain the petroleum ether fraction extract.

[0070] The third step involves separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 1:0, 100:1, 50:1, 25:1, 8:1, 5:1, 1:1, and 1:0, respectively.

[0071] The fourth step involves further separating the sixth fraction from the eight fractions by silica gel column chromatography gradient elution to obtain five fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 20:1, 10:1, 3:1, 1:1, and 0:1, respectively.

[0072] The fifth step involves further separating the third of the five fractions by silica gel column chromatography gradient elution to obtain three more fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of petroleum ether and ethyl acetate of 10:1, 5:1, and 3:1, respectively.

[0073] Step 6: The third fraction of the three fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluent is collected. The eluent collected at 27.0 minutes is the diterpenoid component of senna. The eluent for high performance liquid chromatography gradient elution is a mixture of methanol and water with a volume ratio of 85:15.

[0074] The diterpenoid component (hyssopusone D) in the hyssopus obtained in Example 10 was analyzed by 1H-NMR and 13C-APT nuclear magnetic resonance spectra.

[0075] The 1H-NMR spectrum of hyssopusone D described in Example 10 is as follows: Figure 2 As shown.

[0076] The 13C-APT spectrum of hyssopusone D described in Example 10 is as follows: Figure 3 As shown.

[0077] right Figure 2 and Figure 3 Perform spectrum analysis and... Figure 2 and Figure 3 The peaks were assigned to determine the chemical shifts and peak types of carbon and hydrogen in the structure of compound hyssopusoneD. Its chemical structural formula is shown below. Figure 1 As shown. Among them, Figure 2 and Figure 3 The peak assignments are shown in Table 1.

[0078] In vitro anti-inflammatory pharmacodynamic experiments of the diterpenoid component (hyssopusone D) in the herb described in this invention:

[0079] Hyssopusone D obtained in Example 10 of this invention was subjected to in vitro anti-inflammatory pharmacodynamic experiments using the MTT colorimetric method.

[0080] Hyssopusone D was used as the experimental group, and Aspinin (aspirin, an anti-inflammatory drug) was used as the control group. A blank control group was also set up. RAW 264.7 cells were used as experimental subjects in the experimental, control, and blank control groups. After dilution of the culture medium, the cells were injected at a concentration of 6 × 10⁻⁶. 4100 μl of the drug was seeded at a density of 1 / ml in 96-well plates. After normal incubation for 24 hours, the corresponding drugs were added to each group to achieve final drug concentrations of 2.5 μg / ml (Group 1), 5 μg / ml (Group 2), 10 μg / ml (Group 3), 20 μg / ml (Group 4), and 40 μg / ml (Group 5), for a total of 5 concentrations, with 3 replicates for each concentration. After 48 hours of incubation, 10 μl of MTT was added to each well for staining. After another 4 hours of incubation, the original culture medium was discarded, and 150 μl of DMSO was added to each well. The plates were shaken at low speed for 10 minutes to fully dissolve the crystals. The optical density was measured at 570 nm using an ELISA reader, and the 50% inhibitory concentration (IC50) was calculated based on the optical density. 50 IC is calculated using optical density values ​​(μM). 50 The calculation method is a known existing technique. The IC50 values ​​of the experimental and control groups for RAW 264.7 cells were compared. 50 As shown in Table 2.

[0081] As can be seen from the data in Table 2, the hyssopusone D of the present invention has a certain inhibitory effect on RAW 264.7 cells.

[0082] In summary, this invention discloses the compound hyssopusone D for the first time and conducts in vitro anti-inflammatory pharmacodynamic experiments on the compound. The experiments clearly show that the compound has a strong inhibitory effect on RAW 264.7 cells, thus enabling the compound to be used in the preparation of drugs for the prevention of inflammation and / or anti-inflammatory and antibacterial purposes.

[0083] 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.

[0084] Table 1

[0085]

[0086]

[0087] Table 2

[0088] compound <![CDATA[IC 50 (μM)]]> Aspinin 0.36±1.22 hyssopusoneD 25.7±0.1

Claims

1. A diterpenoid component from a fragrant herb, characterized in that, Its chemical structural formula is: ； Its chemical name is (1S,2R,5S)-2-hydroxy-2-(2-(1R,2S,5R)-2-hydroxy-6,6-dimethyl-3-oxobicyclo[ 3.1.1]Heptane-2-yl)ethyl)-6,6-dimethylbicyclo[3.1.1]heptan-3-one.

2. A method for separating and extracting diterpenoid components from *Hedyotis diffusa* as described in claim 1, characterized in that, Follow these steps: The first step is to crush the herb *Hedyotis diffusa* and add ethanol. Add 8 to 10 ml of ethanol to every 1 g of herb *Hedyotis diffusa*. After soaking at room temperature, heat and reflux extract at 40°C to 60°C at least twice, with each extraction time being 1 to 3 hours. Combine the reflux extracts from each extraction and recover and concentrate under reduced pressure to obtain the total extract of *Hedyotis diffusa*. The second step is to dissolve the total extract of *Hedyotis diffusa* in water and extract it with petroleum ether to obtain the petroleum ether fraction extract. The third step involves separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 1:0, 100:1, 50:1, 25:1, 8:1, 5:1, 1:1, and 1:0, respectively. The fourth step involves further separating the sixth fraction from the eight fractions by silica gel column chromatography gradient elution to obtain five fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of 20:1, 10:1, 3:1, 1:1, and 0:1, respectively. The fifth step involves further separating the third of the five fractions by silica gel column chromatography gradient elution to obtain three more fractions. The silica gel column chromatography gradient elution solution includes petroleum ether and ethyl acetate, with volume ratios of petroleum ether and ethyl acetate of 10:1, 5:1, and 3:1, respectively. Step 6: The third fraction of the three fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluent is collected. The eluent collected at 27.0 minutes is the diterpenoid component of senna. The eluent for high performance liquid chromatography gradient elution is a mixture of methanol and water, wherein the volume ratio of methanol to water is 85:

15.

3. The method for separating and extracting diterpenoid components from *Hedyotis diffusa* according to claim 2, characterized in that, In the first step, the soaking time is 3 to 4 hours; or / and, in the first step, the extraction is performed by heating and reflux 3 times.

4. The use of diterpenoid components from the herb *Hedyotis diffusa* according to claim 1 in the preparation of anti-inflammatory and / or anti-inflammatory drugs.

5. The application of the diterpenoid components in *Hedyotis diffusa* according to claim 4 in the preparation of drugs for preventing and / or fighting inflammation, characterized in that... Anti-inflammatory drugs refer to drugs that prevent inflammation of RAW 264.7 cells; and / or anti-inflammatory drugs refer to anti-inflammatory drugs that inhibit inflammation of RAW 264.7 cells.

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