Diterpenoid components in ethanol extract of hyptis emoryi and extraction method and application thereof

Abstract

The present application relates to the technical field of hard tip godfrey herb extract, and discloses a diterpene component in hard tip godfrey herb ethanol extract, an extraction method and application thereof, wherein the aboveground part of hard tip godfrey herb is extracted by adding ethanol, and then extracted and concentrated; the petroleum ether part is extracted, washed and eluted, and separated; and the third fraction is purified and separated, and the compound (1R, 5R, 6'R, 7a'R)-6, 6, 7', 7'-tetramethyl tetrahydrospiro [bicyclo [3.1.1] heptane-2, 2'-[6, 7a] methyl benzofuran]-3, 4' (5'H)-dione is obtained at the 23rd minute. The present application discloses the diterpene component in hard tip godfrey herb ethanol extract for the first time, and through in-vitro antitumor pharmacodynamics experiments, it is found that the diterpene component has a strong inhibitory effect on HepG-2 cells and HeLa cells, so that it can be used for preparing a drug for preventing liver cancer or / and cervical cancer and an anti-liver cancer or / and cervical cancer drug.

Description

Technical Field

[0001] This invention relates to the field of extract technology of *Hedyotis diffusa*, specifically to a diterpenoid component in an ethanol extract of *Hedyotis diffusa*, its extraction method, and its application. It also relates to the application of the diterpenoid component in the ethanol extract of *Hedyotis diffusa* in the preparation of drugs for preventing and / or fighting tumors. The diterpenoid component in the ethanol extract of *Hedyotis diffusa* is the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione. Background Technology

[0002] Terpenes are a large class of natural products, and one of the most structurally complex and bioactively diverse categories. Currently, terpenes can be isolated from plants of the genus *Hymenoplastrus*.

[0003] Hard-tipped sage is a plant belonging to the genus *Sage* in the family Lamiaceae. The *Zhu Yi Dian* states: "It stops cough and resolves phlegm, reduces inflammation and relieves asthma, brightens the skin, eliminates wounds, softens hardened masses and reduces swelling, eliminates tinnitus, invigorates blood and removes blood stasis, and expels intestinal parasites. It is mainly used to treat pleurisy, pneumonia, asthma, whooping cough, pale complexion, facial wounds, indurated swelling, tinnitus, blood stasis in the whites of the eyes, and intestinal parasites." The *Baidi Yi Yao Shu* states: "It reduces inflammation and relieves asthma, stops cough and resolves phlegm, dispels cold and relieves pain, clears the liver and softens the spleen, promotes urination and reduces swelling, eliminates the toxicity of poisonous insects, and expels intestinal parasites. It is mainly used to treat pneumonia, shortness of breath and asthma, persistent cough, colds, toothache, sciatica, back pain, liver obstruction and spleen swelling, edema, and poisonous insect bites." Therefore, the whole plant of *Hedyotis diffusa* can be used medicinally, possessing heat-clearing, detoxifying, and anti-inflammatory effects. Furthermore, modern pharmacological studies have shown that its terpenoid compounds have a wide range of pharmacological activities, including effects on the central nervous system, cardiovascular system, anti-tumor, and antiviral activity.

[0004] Chinese patent application CN115010589B discloses a macrophage diterpenoid component in senna extract, its preparation method, and its application. The macrophage diterpenoid component in senna extract is an abbreviation for NepetabrateE. However, it only relates to the strong anti-inflammatory effect of NepetabrateE on lipopolysaccharide (LPS)-stimulated RAW264.7 cells, thus enabling NepetabrateE to be used in the preparation of anti-inflammatory and preventative anti-inflammatory drugs.

[0005] Chinese patent application CN115010604A discloses a diterpenoid component of *Hedyotis diffusa*, its preparation method, and its application. The diterpenoid component in *Hedyotis diffusa* is an abbreviation for Nepetabrate I. Animal experiments have shown that Nepetabrate I has significant anti-inflammatory and anti-inflammatory effects, can play a synergistic anti-inflammatory role, has definite efficacy, high safety, and is readily available. It can be used in the preparation of drugs with anti-inflammatory effects. However, it only involves the application of Nepetabrate I in the preparation of anti-inflammatory drugs.

[0006] Therefore, developing and utilizing the terpenoid compounds of *Hedyotis diffusa*, further exploring its potential medicinal value, determining and characterizing the structure and physicochemical properties of its monomeric compounds, and systematically evaluating the antitumor ability of terpenoid compounds are of great significance for the development and utilization of *Hedyotis diffusa*. Summary of the Invention

[0007] This invention provides a diterpenoid component in the ethanol extract of *Hedyotis diffusa*, its extraction method, and its application, overcoming the shortcomings of the prior art. It discloses for the first time the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione, which exhibits strong inhibitory effects on HepG-2 and HeLa cells, thus enabling it to be used as a drug for the prevention and / or treatment of liver cancer and / or cervical cancer.

[0008] One of the technical solutions of this invention is achieved through the following measures: a compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione, 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 above was extracted using the following method:

[0012] The first step is to crush and sieve the aerial parts of *Hedyotis diffusa* and add ethanol. After soaking at room temperature, the mixture is heated and refluxed for extraction. The reflux extracts are combined and recovered under reduced pressure and concentrated to obtain the total extract of *Hedyotis diffusa*.

[0013] The second step involves dispersing the total extract of *Hedyotis diffusa* in water into a suspension, then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated to obtain extracts from each part.

[0014] The third step involved separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions.

[0015] In the fourth step, the third fraction of the eight fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 23 minutes, the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione was obtained.

[0016] In the first step above, add 8 to 12 mL of ethanol to every 1 g of the aerial parts of *Hedyotis diffusa*.

[0017] In the first step above, the soaking time is 4.5 to 5.5 hours, the heating temperature is 40°C to 60°C, and the reflux extraction is performed 3 times, with each reflux extraction lasting 2 to 3 hours.

[0018] In the third step above, the silica gel column chromatography gradient eluent includes chloroform and methanol, with volume ratios of chloroform and methanol of 1:0, 100:1, 40:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0019] In the fourth step above, 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 75:25.

[0020] The second technical solution of the present invention is achieved through the following measures: an extraction method for a compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione, which is carried out according to the following method:

[0021] The first step is to crush and sieve the aerial parts of *Hedyotis diffusa* and add ethanol. After soaking at room temperature, the mixture is heated and refluxed for extraction. The reflux extracts are combined and recovered under reduced pressure and concentrated to obtain the total extract of *Hedyotis diffusa*.

[0022] The second step involves dispersing the total extract of *Hedyotis diffusa* in water into a suspension, then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated to obtain extracts from each part.

[0023] The third step involved separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions.

[0024] In the fourth step, the third fraction of the eight fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 23 minutes, the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione was obtained.

[0025] The following are further optimizations and / or improvements to the second technical solution of the above invention:

[0026] In the first step above, add 8 to 12 mL of ethanol to every 1 g of the aerial parts of *Hedyotis diffusa*.

[0027] In the first step above, the soaking time is 4.5 to 5.5 hours, the heating temperature is 40°C to 60°C, and the reflux extraction is performed 3 times, with each reflux extraction lasting 2 to 3 hours.

[0028] In the third step above, the silica gel column chromatography gradient eluent includes chloroform and methanol, with volume ratios of chloroform and methanol of 1:0, 100:1, 40:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0029] In the fourth step above, 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 75:25.

[0030] The third technical solution of the present invention is achieved through the following measures: the use of a compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione in the preparation of a drug for the prevention of liver cancer and / or cervical cancer.

[0031] The fourth technical solution of the present invention is achieved by the following measures: the use of a compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione in the preparation of drugs for treating liver cancer and / or cervical cancer.

[0032] This invention discloses the compound for the first time, and through in vitro antitumor pharmacodynamic experiments, it was found that it has a strong inhibitory effect on HepG-2 cells and HeLa cells, thus enabling it to be used as a drug for the prevention and / or treatment of liver cancer and / or cervical cancer. Attached Figure Description

[0033] Appendix Figure 1The present invention is for Hyssopusone E 1 H-NMR spectrum.

[0034] Appendix Figure 2 The present invention is for Hyssopusone E 13 C-APT spectrum.

[0035] Appendix Figure 3 The present invention is for Hyssopusone E 1 H- 1 HCOSY spectrum.

[0036] Appendix Figure 4 The image shows the HSQC spectrum of Hyssopusone E according to this invention.

[0037] Appendix Figure 5 The image shows the HMBC spectrum of Hyssopusone E according to this invention.

[0038] Appendix Figure 6 The NOESY spectrum of Hyssopusone E of this invention is shown. Detailed Implementation

[0039] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemicals mentioned in this invention are well-known and commonly used chemical reagents and chemicals 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, generally defined as 25°C.

[0040] The present invention will be further described below with reference to embodiments:

[0041] Example 1: The compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione has the following chemical structural formula:

[0042] .

[0043] This invention discloses for the first time the diterpenoid component in the ethanol extract of *Hyssopus spp.*, namely the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methanobenzofuran]-3,4'(5'H)-dione, systematically named (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methanobenzofuran]-3,4'(5'H)-dione (abbreviated as hyssopusone E). Experiments demonstrate that it has a strong inhibitory effect on HepG-2 and HeLa cells, thus making Hyssopusone... E can be used to prepare drugs for the prevention of tumors (liver cancer and / or cervical cancer) and anti-tumor drugs (liver cancer and / or cervical cancer).

[0044] Example 2: As an optimization of the above examples, the following method was used to extract the following:

[0045] The first step is to crush and sieve the aerial parts of *Hedyotis diffusa* and add ethanol. After soaking at room temperature, the mixture is heated and refluxed for extraction. The reflux extracts are combined and recovered under reduced pressure and concentrated to obtain the total extract of *Hedyotis diffusa*.

[0046] The second step involves dispersing the total extract of *Hedyotis diffusa* in water into a suspension, then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated to obtain extracts from each part.

[0047] The third step involved separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions.

[0048] In the fourth step, the third fraction of the eight fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 23 minutes, the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione was obtained.

[0049] Example 3: As an optimization of the above example, in the first step, 8 mL to 12 mL of ethanol is added to every 1 g of the aerial parts of *Hedyotis diffusa*.

[0050] Example 4: As an optimization of the above example, in the first step, the soaking time is 4.5 hours to 5.5 hours, the heating temperature is 40°C to 60°C, the reflux extraction is performed 3 times, and the reflux extraction time is 2 hours to 3 hours each time.

[0051] Example 5: As an optimization of the above example, in the third step, the silica gel column chromatography gradient eluent includes chloroform and methanol, and the volume ratio of chloroform to methanol is 1:0, 100:1, 40:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0052] Example 6: As an optimization of the above example, in the fourth 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 75:25.

[0053] Example 7: The extraction method of the compound 1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione was carried out according to the following method:

[0054] The first step is to crush and sieve the aerial parts of *Hedyotis diffusa* and add ethanol. After soaking at room temperature, the mixture is heated and refluxed for extraction. The reflux extracts are combined and recovered under reduced pressure and concentrated to obtain the total extract of *Hedyotis diffusa*.

[0055] The second step involves dispersing the total extract of *Hedyotis diffusa* in water into a suspension, then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated to obtain extracts from each part.

[0056] The third step involved separating the petroleum ether fraction using silica gel column chromatography gradient elution to obtain eight fractions.

[0057] In the fourth step, the third fraction of the eight fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 23 minutes, the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione was obtained.

[0058] Example 8: The use of the compound 1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione in the preparation of drugs for the prevention of liver cancer and / or cervical cancer.

[0059] Example 9: The use of the compound 1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione in the preparation of drugs for treating liver cancer and / or cervical cancer.

[0060] Example 10: The compound 1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione was extracted according to the following method:

[0061] The first step is to crush and sieve 6 kg of the aerial parts of *Hedyotis diffusa* and add 40 L of ethanol. After soaking at room temperature for 3 hours, heat to 50 °C and reflux extract 3 times, 2 hours each time. Combine the reflux extracts and recover and concentrate under reduced pressure to obtain the total extract of *Hedyotis diffusa*.

[0062] The second step involves dispersing 437.0g of total extract of *Hedyotis diffusa* in water into a suspension, then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. The extract is then concentrated to obtain extracts from each part.

[0063] The third step involved taking 134.8g of petroleum ether extract and separating it using silica gel column chromatography gradient elution to obtain 8 fractions. The volume ratios of chloroform and methanol in the silica gel column chromatography gradient elution were 1:0, 100:1, 40:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0064] In the fourth step, the third fraction of the eight fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 23 minutes, the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione was obtained.

[0065] The compound (1R, 5R, 6'R, 7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione obtained in Example 10 of this invention was subjected to 1H NMR spectroscopy. 1 H-NMR and carbon nuclear magnetic resonance (NMR) 13 C-APT analysis. 1 H-NMR spectrum, 13 C-APT spectrum, 1 H- 1 HCOSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, etc. Figures 1 to 6 As shown.

[0066] Combination Figures 1 to 6 Perform spectral analysis, and Figure 1 and Figure 2 Each peak is assigned a location. Figure 1 and Figure 2The peak assignments are shown in Table 1.

[0067]

[0068] pass Figure 3 , Figure 4 As shown in Table 1, the chemical structural formula of the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione of the present invention is as follows:

[0069] .

[0070] The compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione in this invention is an amorphous powder that is readily soluble in chloroform and methanol.

[0071] The compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione described in this invention was subjected to in vitro antitumor pharmacodynamic experiments using the MTT assay.

[0072] The experimental group used compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione, and the control group used Paclitaxel (a paclitaxel antitumor drug). HepG-2 (human liver cancer cells) and HeLa (human cervical cancer cells) cells were used as experimental subjects in both groups. After dilution of the culture medium, the cells were incubated at 6 × 10⁻⁶. 4 100 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 value. 50(μg / mL), IC50 for optical density value calculation 50 The calculation method is a known existing technique. The IC50 values ​​of the experimental and control groups for HepG-2 and HeLa cells were compared. 50 As shown in Table 2.

[0073]

[0074] As shown in Table 2, the compound (1R,5R,6'R,7a'R)-6,6,7',7'-tetramethyltetrahydrospiro[bicyclo[3.1.1]heptane-2,2'-[6,7a]methylbenzofuran]-3,4'(5'H)-dione described in this invention has a certain inhibitory effect on both HepG-2 cells and HeLa cells.

[0075] In summary, this invention discloses the compound for the first time, and through in vitro antitumor pharmacodynamic experiments, it was found that it has a strong inhibitory effect on HepG-2 cells and HeLa cells, thus enabling it to be used as a drug for the prevention and / or treatment of liver cancer and / or cervical cancer.

[0076] The above technical features constitute the 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.

Claims

1. A compound, characterized in that, The chemical structural formula is 。 2. A process for the preparation of a compound according to claim 1, characterized in that, The following methods are used: In the first step, the aerial part of the hard-pointed godfrey herb is crushed and sieved, and then added with ethanol. After soaking at room temperature, the reflux extraction is heated. The reflux extraction liquids are combined and recovered and concentrated under reduced pressure to obtain the total extract of the hard-pointed godfrey herb; In the second step, the total extract of the hard-pointed godfrey herb is dispersed into a suspension with water, and then extracted with petroleum ether, dichloromethane and ethyl acetate in sequence. The extraction liquids are concentrated to obtain the extract of each part; In the third step, the extract of the petroleum ether part is separated by silica gel column chromatography with gradient elution to obtain 8 fractions; In the fourth step, the third fraction of the 8 fractions is purified and separated by high performance liquid chromatography with gradient elution, and the eluate is collected. The compound of claim 1 is obtained at the 23rd minute, In the first step, the soaking time is 4.5 hours to 5.5 hours, the heating reflux extraction temperature is 40°C to 60°C, the reflux extraction times are 3 times, and the reflux extraction time of each time is 2 hours to 3 hours, In the third step, the gradient eluent of the silica gel column chromatography is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol is 1:0, 100:1, 40:1, 20:1, 10:1, 5:1, 2:1 and 1:1 in sequence, In the fourth step, the gradient eluent of the high performance liquid chromatography is a mixture of methanol and water, and the volume ratio of methanol to water is 75:

25.

3. The process for the preparation of a compound according to claim 2, characterized in that, In the first step, 8 mL to 12 mL of ethanol is added to 1 g of the aerial part of the hard-pointed godfrey herb.

4. Use of the compound of claim 1 in the preparation of a drug for preventing cervical cancer.

5. Use of the compound of claim 1 in the preparation of an anti-cervical cancer drug.

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