Benzyl isopropyl ether and preparation method thereof and application of benzyl isopropyl ether as anti-inflammatory drug

Abstract

The present application relates to the technical field of separation and purification of Schizonepeta tenuifolia, and is a kind of Schizonepeta tenuifolia phenylpropanoid compound, its preparation method and application, Schizonepeta tenuifolia is extracted by heating reflux with ethanol solution and recovered under reduced pressure, and concentrated, the total extract of Schizonepeta tenuifolia is extracted and concentrated with petroleum ether, dichloromethane and ethyl acetate in turn, the extract of ethyl acetate part is separated by gradient elution, the third fraction is separated by gradient elution, the first fraction is separated by gradient elution, the sixth fraction is separated by gradient elution, and phenylpropanoid compound one and phenylpropanoid compound two are obtained at 12.1 minutes and 13.4 minutes, respectively. The present application discloses phenylpropanoid compound one and phenylpropanoid compound two separated from Schizonepeta tenuifolia for the first time, and in vitro anti-inflammatory pharmacodynamic experiments are carried out on the two compounds, which prove that they have a certain inhibitory effect on RAW264.7 cells, and can be applied to the preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology of Nepeta macrocarpa, specifically to a phenylpropanoid component in Nepeta macrocarpa, its preparation method, and its application as an anti-inflammatory drug. Background Technology

[0002] Nepeta cataria is a perennial herb belonging to the genus Nepeta in the family Lamiaceae. Its stem is erect, about 0.3 to 1 meter tall, branched in the upper part, and covered with short, soft hairs. Leaves are opposite, pinnately divided, and covered with short, soft hairs on both sides. Flowers are arranged in verticillasters with pale purple corollas. The above-ground parts, due to their strong medicinal value, possess various pharmacological effects such as detoxification, dispersing lumps, dispelling wind and cold, and relieving cough and phlegm, and have been widely used in the medical field since ancient times. The genus Nepeta contains approximately 250 species, mainly distributed in the Mediterranean, Central Asia, and Africa, extending from North Africa to tropical mountainous regions.

[0003] Phenylpropanoids are a class of natural aromatic compounds found in plants. Their basic skeleton consists of a benzene ring and a three-carbon chain (C6-C3), and they include coumarins, lignans, and flavonoids. Their physiological functions include defending against pathogens and herbivores, participating in cell signaling, providing pigments for flowers and fruits, absorbing ultraviolet radiation, and regulating plant growth. Currently, various anti-inflammatory applications of phenylpropanoids have been disclosed in existing technologies.

[0004] The patent document with publication number CN121108101A discloses a phenylpropanoid component, its preparation method, and its application as an anti-inflammatory drug. It provides for the first time a method for preparing 10 new compounds from the pseudobulb of *Gnaphalium affine*, and systematically evaluates its anti-neuroinflammatory activity, clarifying its application in the development and treatment of drugs for central nervous system diseases.

[0005] Patent document CN108929296B discloses an anti-inflammatory phenylpropanoid component, its extraction method, and its application. This invention extracts a novel phenylpropanoid component from olive fruit, and its physicochemical properties and chemical structure are clarified through physicochemical constants and modern spectroscopic analysis.

[0006] It is evident that phenylpropanoids are common secondary metabolites in plants, possessing various biological activities such as antioxidant, anti-inflammatory, and antibacterial properties. They are widely distributed in plants, including some species of the genus Nepeta.

[0007] However, chemical research on *Nepeta macrocarpa* has primarily focused on terpenoids, with limited research on phenylpropanoids. Furthermore, existing techniques mainly concentrate on phenylpropanoids from specific plant sources, and their anti-inflammatory mechanisms often target single inflammatory pathways, without addressing a systematic study of phenylpropanoids in *Nepeta macrocarpa*. Therefore, research on the structural analysis, optimization of preparation methods, and anti-inflammatory mechanisms of phenylpropanoids in *Nepeta macrocarpa* is of great significance. Summary of the Invention

[0008] This invention provides a phenylpropanoid component from Nepeta macrocephala, its preparation method, and its application as an anti-inflammatory drug, overcoming the shortcomings of the prior art. It discloses for the first time the isolation of phenylpropanoid compound I and phenylpropanoid compound II from Nepeta macrocephala, and conducts in vitro anti-inflammatory pharmacodynamic experiments on these two compounds, proving that they can be used in the preparation of anti-inflammatory drugs.

[0009] One of the technical solutions of this invention is achieved through the following measures: a phenylpropanoid component from Nepeta macrocarpa, wherein the phenylpropanoid component includes phenylpropanoid compound one and phenylpropanoid compound two, wherein, The structure of phenylpropanoid compound one is as follows: , The structure of phenylpropanoid compound II is as follows: .

[0010] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: The above-mentioned phenylpropanoid compound one and phenylpropanoid compound two were obtained by the following method: The first step is to pulverize the large-bracted catnip, soak it in ethanol solution, heat and reflux to extract it, combine the reflux extracts, and recover and concentrate them under reduced pressure to obtain the total extract of large-bracted catnip. The second step involves dispersing the total extract of Nepeta macrocarpa in water into a suspension, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. After concentrating the extract, petroleum ether fraction extract, dichloromethane fraction extract, and ethyl acetate fraction extract are obtained. The third step involved separating the ethyl acetate fraction using silica gel column chromatography with gradient elution to obtain eight fractions. The fourth step involves separating the third of the eight fractions using silica gel column chromatography gradient elution to obtain six fractions. Fifth step: Separate the first fraction of the obtained 6 fractions by gradient elution using gel column chromatography to obtain 7 fractions; In the sixth step, the sixth fraction of the seven fractions obtained was purified and separated by semi-preparative liquid chromatography gradient elution, and the eluent was collected. Phenylacetic compound I was obtained at 12.1 minutes, and phenylpropanoid compound II was obtained at 13.4 minutes.

[0011] In the first step above, each 50 kg of Nepeta macrocarpa is extracted with a 95% to 98% ethanol solution by heating to 70°C to 80°C and refluxed two to three times, with each reflux extraction lasting 1.5 to 2.5 hours.

[0012] In the third step above, during gradient elution in silica gel column chromatography, the eluent consists of dichloromethane and methanol in volume ratios of 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0013] In the fourth step above, during gradient elution in silica gel column chromatography, the eluent consists of dichloromethane and methanol in volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 2:1, respectively.

[0014] In the fifth step above, during gradient elution in silica gel column chromatography, the eluent consists of petroleum ether and ethyl acetate in volume ratios of 2:1 and 1:1, and dichloromethane and methanol in volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1, and 5:1, respectively.

[0015] In step six above, during the semi-preparative liquid chromatography gradient elution, the eluent consists of methanol and water in a volume ratio of 52:48.

[0016] The second technical solution of the present invention is achieved through the following measures: a method for preparing phenylpropanoid components from Nepeta macrocarpa, which is carried out according to the following method: The first step is to pulverize the large-bracted catnip, soak it in ethanol solution, heat and reflux to extract it, combine the reflux extracts, and recover and concentrate them under reduced pressure to obtain the total extract of large-bracted catnip. The second step involves dispersing the total extract of Nepeta macrocarpa in water into a suspension, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. After concentrating the extract, petroleum ether fraction extract, dichloromethane fraction extract, and ethyl acetate fraction extract are obtained. The third step involved separating the ethyl acetate fraction using silica gel column chromatography with gradient elution to obtain eight fractions. The fourth step involves separating the third of the eight fractions using silica gel column chromatography gradient elution to obtain six fractions. Fifth step: Separate the first fraction of the obtained 6 fractions by gradient elution using gel column chromatography to obtain 7 fractions; In the sixth step, the sixth fraction of the seven fractions obtained was purified and separated by semi-preparative liquid chromatography gradient elution, and the eluent was collected. Phenylacetic compound I was obtained at 12.1 minutes, and phenylpropanoid compound II was obtained at 13.4 minutes.

[0017] The third technical solution of the present invention is achieved through the following measures: the application of phenylpropanoid components in Nepeta macrocephala in the preparation of anti-inflammatory drugs.

[0018] This invention discloses for the first time the isolation of phenylpropanoid compound I and phenylpropanoid compound II from Nepeta macrocarpa, and conducts in vitro anti-inflammatory pharmacodynamic experiments on these two compounds, demonstrating that both phenylpropanoid compound I and phenylpropanoid compound II have a certain inhibitory effect on RAW264.7 cells, and can be applied to the preparation of anti-inflammatory drugs. Attached Figure Description

[0019] Appendix Figure 1 The phenylpropanoid compound one in Example 11 of this invention 1 H-NMR spectrum.

[0020] Appendix Figure 2 The phenylpropanoid compound one in Example 11 of this invention 13 C-APT spectrum.

[0021] Appendix Figure 3 The image shows the HH COSY spectrum of phenylpropanoid compound 1 in Example 11 of this invention.

[0022] Appendix Figure 4 This is the HSQC spectrum of phenylpropanoid compound 1 in Example 11 of the present invention.

[0023] Appendix Figure 5 This is the HMBC spectrum of phenylpropanoid compound 1 in Example 11 of the present invention.

[0024] Appendix Figure 6 This is the NOESY spectrum of phenylpropanoid compound 1 in Example 11 of the present invention.

[0025] Appendix Figure 7 The phenylpropanol compound II in Example 11 of this invention 1 H-NMR spectrum.

[0026] Appendix Figure 8 The phenylpropanol compound II in Example 11 of this invention 13 C-APT spectrum.

[0027] Appendix Figure 9 The image shows the HH COSY spectrum of phenylpropanoid compound II in Example 11 of this invention.

[0028] Appendix Figure 10 This is the HSQC spectrum of phenylpropanoid compound II in Example 11 of the present invention.

[0029] Appendix Figure 11 This is the HMBC spectrum of phenylpropanoid compound II in Example 11 of the present invention.

[0030] Appendix Figure 12 The NOESY spectrum of phenylpropanoid compound II in Example 11 of this invention is shown. Detailed Implementation

[0031] 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.

[0032] The present invention will be further described below with reference to embodiments: Example 1: Phenylpropanoid components in *Nepeta macrocarpa*, wherein the phenylpropanoid components include phenylpropanoid compound one and phenylpropanoid compound two, wherein, The structure of phenylpropanoid compound one is as follows: , The structure of phenylpropanoid compound II is as follows: .

[0033] Example 2: As an optimization of the above examples, phenylpropanoid compound one and phenylpropanoid compound two were obtained according to the following method: The first step is to pulverize the large-bracted catnip, soak it in ethanol solution, heat and reflux to extract it, combine the reflux extracts, and recover and concentrate them under reduced pressure to obtain the total extract of large-bracted catnip. The second step involves dispersing the total extract of Nepeta macrocarpa in water into a suspension, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. After concentrating the extract, petroleum ether fraction extract, dichloromethane fraction extract, and ethyl acetate fraction extract are obtained. The third step involved separating the ethyl acetate fraction using silica gel column chromatography with gradient elution to obtain eight fractions. The fourth step involves separating the third of the eight fractions using silica gel column chromatography gradient elution to obtain six fractions. Fifth step: Separate the first fraction of the obtained 6 fractions by gradient elution using gel column chromatography to obtain 7 fractions; In the sixth step, the sixth fraction of the seven fractions obtained was purified and separated by semi-preparative liquid chromatography gradient elution, and the eluent was collected. Phenylacetic compound I was obtained at 12.1 minutes, and phenylpropanoid compound II was obtained at 13.4 minutes.

[0034] Example 3: As an optimization of the above example, in the first step, every 50 kg of Nepeta macrocarpa is extracted with a 95% to 98% ethanol solution by volume, heated to 70°C to 80°C and refluxed two to three times, with each reflux extraction lasting 1.5 to 2.5 hours.

[0035] Example 4: As an optimization of the above example, in the second step, the volume ratio of the total extract of Nepeta macrocephala to petroleum ether, dichloromethane and ethyl acetate is 1:1.

[0036] Example 5: As an optimization of the above example, in the third step, during gradient elution of silica gel column chromatography, the eluent is composed of dichloromethane and methanol in volume ratios of 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively.

[0037] Example 6: As an optimization of the above example, in the fourth step, during gradient elution of silica gel column chromatography, the eluent is composed of dichloromethane and methanol in volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 2:1, respectively.

[0038] Example 7: As an optimization of the above example, in the fifth step, during gradient elution of silica gel column chromatography, the eluent consists of petroleum ether and ethyl acetate in volume ratios of 2:1 and 1:1, and dichloromethane and methanol in volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1, and 5:1, respectively.

[0039] Example 8: As an optimization of the above example, in step 6, during the semi-preparative liquid chromatography gradient elution, the eluent consists of methanol and water in a volume ratio of 52:48.

[0040] Example 9: The preparation method of phenylpropanoid components in this Nepeta macrocarpa is carried out according to the following method: The first step is to pulverize the large-bracted catnip, soak it in ethanol solution, heat and reflux to extract it, combine the reflux extracts, and recover and concentrate them under reduced pressure to obtain the total extract of large-bracted catnip. The second step involves dispersing the total extract of Nepeta macrocarpa in water into a suspension, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. After concentrating the extract, petroleum ether fraction extract, dichloromethane fraction extract, and ethyl acetate fraction extract are obtained. The third step involved separating the ethyl acetate fraction using silica gel column chromatography with gradient elution to obtain eight fractions. The fourth step involves separating the third of the eight fractions using silica gel column chromatography gradient elution to obtain six fractions. Fifth step: Separate the first fraction of the obtained 6 fractions by gradient elution using gel column chromatography to obtain 7 fractions; In the sixth step, the sixth fraction of the seven fractions obtained was purified and separated by semi-preparative liquid chromatography gradient elution, and the eluent was collected. Phenylacetic compound I was obtained at 12.1 minutes, and phenylpropanoid compound II was obtained at 13.4 minutes.

[0041] Example 10: Application of phenylpropanoid components in the preparation of anti-inflammatory drugs from *Nepeta macrocarpa*.

[0042] Example 11: The phenylpropanoid components in this Nepeta macrocarpa were obtained according to the following method: The first step involved crushing 50 kg of Nepeta macrocarpa into powder, soaking it in a 95% ethanol solution, and then refluxing it three times at 80°C for 2 hours each time. The reflux extracts were then combined, recovered under reduced pressure, and concentrated to obtain the total extract of Nepeta macrocarpa. The second step involves dispersing the total extract of Nepeta macrocarpa in water into a suspension, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate in a volume ratio of 1:1. After concentrating the extract, petroleum ether extract, dichloromethane extract, and ethyl acetate extract are obtained. The third step involved separating the ethyl acetate extract using silica gel column chromatography with gradient elution (the eluent consisted of dichloromethane and methanol in volume ratios of 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, yielding eight fractions). In the fourth step, the third fraction of the obtained 8 fractions was separated by gradient elution using silica gel column chromatography (the eluent consisted of dichloromethane and methanol in volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 2:1, respectively), resulting in 6 fractions. In the fifth step, the first fraction of the obtained 6 fractions was separated by gradient elution using gel column chromatography (the eluent consisted of petroleum ether and ethyl acetate in volume ratios of 2:1 and 1:1, and dichloromethane and methanol in volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1, and 5:1, respectively), to obtain 7 fractions; Step 6: The 6th fraction of the 7 fractions obtained was purified and separated by semi-preparative liquid chromatography gradient elution (the eluent consisted of methanol and water in a volume ratio of 52:48), and the eluent was collected. Phenylacetic compound 1 (Chinese name: (2E)-3-(4-hydroxyphenyl)prop-2-enoic acid-3-oxo-2,5-dioxaheptan-7-yl(2E)-3-(4-hydroxyphenyl)prop-2-enoate) was obtained at 12.1 minutes, and phenyllacetic compound 2 (Chinese name: 3-(4-hydroxyphenyl)propionic acid-(2S)-2-hydroxy-3-methoxypropyl ester, i.e. (2S)-2-hydroxy-3-methoxypropyl3-(4-hydroxyphenyl)propanoate(2)) was obtained at 13.4 minutes.

[0043] The phenylpropanol compound I and phenylpropanol compound II obtained in Example 11 of this invention were subjected to 1H NMR spectroscopy. 1 H-NMR and carbon nuclear magnetic resonance (NMR) 13 C-APT analysis and a comprehensive analysis of a series of two-dimensional spectra (HSQC, HMBC, COSY, NOESY).

[0044] Phenylacetic compound one 1 H-NMR spectrum as shown Figure 1 As shown, phenylpropanoid compound one 13 C-APT spectrum as shown Figure 2 As shown, phenylpropanoid compound II 1 H-NMR spectrum as shown Figure 7 As shown, phenylpropanoid compound II 13 C-APT spectrum as shown Figure 8 As shown, for Figure 1 and Figure 2 Perform spectrum analysis, and analyze spectrum 1 and... Figure 2 Each peak is assigned a location. Figure 1 and Figure 2 The peak assignments are shown in Table 1. Figure 7 and Figure 8 Perform spectrum analysis, and analyze spectrum 7 and... Figure 8 Each peak is assigned a location. Figure 7 and Figure 8 The peak assignments are shown in Table 1. The HSQC spectrum of phenylpropanoid compound one is shown in... Figure 3 As shown, the HMBC spectrum of phenylpropanoid compound one is as follows. Figure 4 As shown, the COSY spectrum of phenylpropanoid compound one is as follows: Figure 5 As shown, the NOESY spectrum of phenylpropanoid compound one is as follows: Figure 6 As shown; the HSQC spectrum of phenylpropanoid compound II is as follows. Figure 9 As shown, the HMBC spectrum of phenylpropanol compound II is as follows. Figure 10 As shown, the COSY spectrum of phenylpropanoid compound II is as follows. Figure 11 As shown, the NOESY spectrum of phenylpropanoid compound II is as follows. Figure 12 As shown in Table 1, the chemical structural formula of phenylpropanoid compound one is as follows: The chemical structural formula of phenylpropanoid compound II is shown below: Both phenylpropanoid compound I and phenylpropanoid compound II are readily soluble in chloroform and methanol.

[0045] The structure of phenylpropanoid compound 1 was identified as follows: 1The 1H-NMR spectrum shows two aromatic protons with δH: 7.59 (2H,d,J=8.52Hz, H-2,6) and δH: 6.81 (2H,d,J=8.46Hz, H-3,5), indicating the presence of a benzene ring; two double bond protons with δH: 7.63 (1H,d,J=15.9Hz, H-7) and δH: 6.48 (1H,d,J=15.9Hz, H-8), indicating the presence of one double bond; and three methylene protons with δH: 2.55 (1H,s,H-10a), 2.53 (1H,s,H-10b), and 3.41 (1H,t,J=6Hz,H-11a), 3 0.39 (1H,t,J=6Hz,H-11b), δH:4.76 (2H,s,H-12); 3.69 (3H,s,13-OCH3) indicates the presence of a methoxy group in the structure, consistent with the δC:51.913 CNMR spectrum; in the APT spectrum, the resonance signals at δC:160.3, 130.6, 124.7, and 115.8 are aromatic carbons; the signals at δC:146.1 and 112.9 are tertiary carbons attached to double bonds; the signals at δC:168.5, 166.1, and 160.3 are oxygen-bound quaternary carbons, and the signal at 160.3 is also in the aromatic carbons, indicating that a hydroxyl group is attached to the carbon at 160.3. In the HMBC spectrum, the correlation between δH: 7.63 (1H, d, J = 15.9 Hz, H-7) and δC: 124.7 (C-1), 130.6 (C-2, 6), 112.9 (C-8) indicates that the double bond is attached to C-1; the correlation between δH: 3.69 (3H, s, 13-OCH3) and δC: 168.5 (C-13) indicates that the methoxy group is attached to C-13; δH: 4.76 (2H, s) The correlation between δH: 168.5 (C-13) and δC: 168.5 indicates that the methylene group is also connected to C-13; in the 1H-1HCOSY spectrum, the correlation between δH: 3.41 (1H,t,J=6Hz,H-11a), 3.39 (1H,t,J=6Hz,H-11b) and δH: 2.55 (1H,s,H-10a), 2.53 (1H,s,H-10b) indicates that the two methylene groups are linked together.

[0046] Therefore, the compound of p-phenylpropanoid is named as: (2E)-3-(4-hydroxyphenyl)prop-2-enoic acid-3-oxo-2,5-dioxaheptan-7-yl(2E)-3-(4-hydroxyphenyl)prop-2-enoate.

[0047] The structure of compound 2 phenylpropanoid was identified as follows: 1The 1H-NMR spectrum shows two aromatic protons with δH: 6.96 (2H,d,J=8.4Hz, H-2,6), δH: 7.17 (1H,d,J=8.4Hz, H-12), and δH: 6.65 (2H,d,J=8.46Hz, H-3,5), indicating the presence of a benzene ring; and four methylene protons with δH: 3.39 (1H,m,H-12a), 2.55 (1H,m,H-7a), 2.52 (1H,m,H-10a), 2.44 (2H,m,H-7b,H-12b), 2.43 (1H,m,H-10b), and 2.42 (1H,m,H-8a). 2.31 (1H, m, H-8b); δH: 3.56 (3H, s, 12-OCH3) and 3.80 (1H, s, H-11) are protons connected to oxygen, and the peak area at δH: 3.56 shows three protons, indicating the presence of a methoxy group in the structure; consistent with the δC: 51.213 CNMR spectrum; in the APT spectrum, the resonance signals at δC: 155.2, 132.0, 129.1, and 115.0 are aromatic carbons; the signals at δC: 171.8 and 155.2 are oxygen-connected quaternary carbons, and the signal at δC: 155.2 is also in the aromatic carbons, indicating that a hydroxyl group is attached to the carbon at 155.2.The signal at δC:171.8 is stronger than that at δC:155.2, indicating that the carbon signal at δC:171.8 is an ester carbonyl signal; the signal at δC:66.5 is an oxygen-bonded tertiary carbon, corresponding to an oxygen-bonded proton at δH:3.80 (1H,s,H-11) in the 1H-NMR spectrum, indicating that a hydroxyl group is attached to the carbon at δC:66.5; the signal at δC:57.1 is an oxygen-bonded secondary carbon, indicating that the methylene group here is bonded to an oxygen atom, and that this is also present in HMBC. In the HMBC spectrum, the proton signal δH: 2.43 of a methylene group is remotely correlated with the carbon signal δC: 171.8 at the ester carbonyl group, indicating that this methylene group is attached to the oxygen atom of the ester carbonyl group. In the HMBC spectrum, the correlation between δH: 2.55 (1H,m,H-7a) and 2.44 (2H,m,H-7b) and δC: 132.0 (C-1) and 129.1 (C-2,6) indicates that the methylene group is attached to C-1. The correlation between δH: 2.52 (1H,m,H-10a) and 2.43 (…) indicates that the methylene group is attached to C-1. The correlation between δH: 1H, m, H-10b, 3.39 (1H, m, H-12a), 2.44 (2H, m, H-12b) and δC: 66.5 (C-11) indicates that the methylene group attached to the carbonyl oxygen of the ester is bonded to the carbon of the hydroxyl group, and there is another methylene group also bonded to the carbon of the hydroxyl group; the correlation between δH: 3.39 (1H, m, H-12a), 2.44 (2H, m, H-12b) and δC: 51.2 (12-OCH3) indicates that the methoxy group is bonded to one... The methylene groups are linked together; the correlation between δH: 2.42 (1H,m,H-8a), 2.31 (1H,m,H-8b) and δC: 171.8 indicates that one methylene group is linked to the carbonyl carbon of the ester; in the 1H-1HCOSY spectrum, the correlation between δH: 2.55 (1H,m,H-7a), 2.44 (2H,m,H-7b) and δH: 2.42 (1H,m,H-8a), 2.31 (1H,m,H-8b) indicates that these two methylene groups are linked together.

[0048] Therefore, the name of compound 2 of phenylpropanoid is: 3-(4-hydroxyphenyl)propanoic acid-(2S)-2-hydroxy-3-methoxypropyl ester ((2S)-2-hydroxy-3-methoxypropyl3-(4-hydroxyphenyl)propanoate (2).

[0049] In vitro anti-inflammatory pharmacodynamic experiments were conducted on phenylpropanoid compound one and phenylpropanoid compound two obtained in Example 11 of the present invention. The in vitro anti-inflammatory pharmacodynamic experiments were performed using the MTT colorimetric method.

[0050] Phenylacetic compound I and phenylpropanoid compound II were used as experimental groups, indomethacin as the control group, and a blank group was also set up. RAW 264.7 (mouse macrophages) cells were used as experimental subjects in the experimental, control, and blank groups. After dilution of the culture medium, the cells were inoculated at 4 × 10⁻⁶. 5 The culture medium was seeded at a density of 100 μL per well in 96-well plates. After normal incubation for 24 hours, the corresponding drugs were added to each group to achieve final drug concentrations of 6.25 μm / mL (Group 1), 12.5 μm / mL (Group 2), 25 μm / mL (Group 3), 50 μm / mL (Group 4), 100 μm / mL (Group 5), and 200 μm / mL (Group 6), for a total of 6 concentrations, with 3 replicates per 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 min 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 (μg / mL), IC50 for optical density value calculation 50 The calculation method is a known existing technique. The IC50 values ​​of RAW264.7 cells in the experimental and control groups were compared. 50 As shown in Table 2, the data in Table 2 indicate that both phenylpropanoid compound I and phenylpropanoid compound II of this invention have a certain inhibitory effect on RAW264.7 cells.

[0051] In summary, this invention discloses for the first time the isolation of phenylpropanoid compound I and phenylpropanoid compound II from Nepeta macrocarpa, and conducts in vitro anti-inflammatory pharmacodynamic experiments on these two compounds, demonstrating that both phenylpropanoid compound I and phenylpropanoid compound II have a certain inhibitory effect on RAW264.7 cells and can be applied to the preparation of anti-inflammatory drugs.

[0052] 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.

[0053]

Claims

1. A phenylpropanoid component from Nepeta macrocarpa, characterized in that... The phenylpropanoid components include phenylpropanoid compound one and phenylpropanoid compound two, and the structure of phenylpropanoid compound one is as follows: ， The structure of phenylpropanoid compound II is as follows: 。 2. The phenylpropanoid components in Nepeta macrocarpa according to claim 1, characterized in that... Phenylacetic compound I and phenylpropanoid compound II were obtained by the following method: The first step is to pulverize the large-bracted catnip, soak it in ethanol solution, heat and reflux to extract it, combine the reflux extracts, and recover and concentrate them under reduced pressure to obtain the total extract of large-bracted catnip. The second step involves dispersing the total extract of Nepeta macrocarpa in water into a suspension, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. After concentrating the extract, petroleum ether fraction extract, dichloromethane fraction extract, and ethyl acetate fraction extract are obtained. The third step involved separating the ethyl acetate fraction using silica gel column chromatography with gradient elution to obtain eight fractions. The fourth step involves separating the third of the eight fractions using silica gel column chromatography gradient elution to obtain six fractions. Fifth step: Separate the first fraction of the obtained 6 fractions by gradient elution using gel column chromatography to obtain 7 fractions; In the sixth step, the sixth fraction of the seven fractions obtained was purified and separated by semi-preparative liquid chromatography gradient elution, and the eluent was collected. Phenylacetic compound I was obtained at 12.1 minutes, and phenylpropanoid compound II was obtained at 13.4 minutes.

3. The phenylpropanoid components in Nepeta macrocarpa according to claim 2, characterized in that... In the first step, each 50 kg of Nepeta macrocarpa is extracted with a 95% to 98% ethanol solution by volume, heated to 70°C to 80°C and refluxed two to three times, with each reflux extraction lasting 1.5 to 2.5 hours.

4. The phenylpropanoid components in Nepeta macrocarpa according to claim 2 or 3, characterized in that... In the second step, the total extract of Nepeta macrocephala was mixed with petroleum ether, dichloromethane, and ethyl acetate in a volume ratio of 1:

1.

5. The phenylpropanoid components in Nepeta macrocarpa according to any one of claims 2 to 4, characterized in that... In the third step, during gradient elution in silica gel column chromatography, the eluent consists of dichloromethane and methanol in volume ratios of 100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, respectively.

6. The phenylpropanoid components in Nepeta macrocarpa according to any one of claims 2 to 5, characterized in that... In the fourth step, during gradient elution in silica gel column chromatography, the eluent consists of dichloromethane and methanol in volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 2:1, respectively.

7. The phenylpropanoid components in Nepeta macrocarpa according to any one of claims 2 to 6, characterized in that... In the fifth step, during gradient elution in silica gel column chromatography, the eluent consists of petroleum ether and ethyl acetate in volume ratios of 2:1 and 1:1, and dichloromethane and methanol in volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1, and 5:1, respectively.

8. The phenylpropanoid components in Nepeta macrocarpa according to any one of claims 2 to 7, characterized in that... In step six, during the semi-preparative liquid chromatography gradient elution, the eluent consists of methanol and water in a volume ratio of 52:

48.

9. A method for preparing phenylpropanoid components from Nepeta macrocarpa according to any one of claims 3 to 8, characterized in that... Perform the following steps: The first step is to pulverize the large-bracted catnip, soak it in ethanol solution, heat and reflux to extract it, combine the reflux extracts, and recover and concentrate them under reduced pressure to obtain the total extract of large-bracted catnip. The second step involves dispersing the total extract of Nepeta macrocarpa in water into a suspension, and then extracting it sequentially with petroleum ether, dichloromethane, and ethyl acetate. After concentrating the extract, petroleum ether fraction extract, dichloromethane fraction extract, and ethyl acetate fraction extract are obtained. The third step involved separating the ethyl acetate fraction using silica gel column chromatography with gradient elution to obtain eight fractions. The fourth step involves separating the third of the eight fractions using silica gel column chromatography gradient elution to obtain six fractions. Fifth step: Separate the first fraction of the obtained 6 fractions by gradient elution using gel column chromatography to obtain 7 fractions; In the sixth step, the sixth fraction of the seven fractions obtained was purified and separated by semi-preparative liquid chromatography gradient elution, and the eluent was collected. Phenylacetic compound I was obtained at 12.1 minutes, and phenylpropanoid compound II was obtained at 13.4 minutes.

10. The use of phenylpropanoid components from Nepeta macrocephala according to any one of claims 1 to 8 in the preparation of anti-inflammatory drugs.

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