European verbena herb extract as well as extraction method and application thereof

Abstract

The invention relates to a verbena extract as well as an extraction method and application thereof. The extraction method comprises the following steps: (1) obtaining stems and leaves of lumpy European verbena, adding ethanol for reflux extraction to obtain an ethanol extracting solution, and then concentrating under reduced pressure to obtain an ethanol extract; the volume concentration of the ethanol is 85%-95%; the mass ratio of the ethanol to the massive stems and leaves of the verbena is 1: (5-7); (2) dissolving the ethanol extract in water to obtain a mixed solution, extracting with petroleum ether to obtain a petroleum ether phase and a residual mixture, concentrating the petroleum ether phase under reduced pressure to obtain a petroleum ether extract, extracting the residual mixture with ethyl acetate, and concentrating under reduced pressure to obtain an ethyl acetate extract; mixing the petroleum ether extract and the ethyl acetate extract to obtain a crude verbena extract; and (3) carrying out normal-phase silica gel column chromatography on the crude verbena extract, and then eluting to obtain the verbena extract. The verbena extract disclosed by the invention can relieve inflammation, and can be used for preparing medicines for treating inflammatory diseases or used as a raw material of anti-inflammatory, soothing and repairing cosmetics.

Description

Technical Field

[0001] This invention relates to the field of verbena extraction technology, specifically to a verbena extract, its extraction method, and its application. Background Technology

[0002] Inflammation is an immune response initiated by the body in response to pathogen invasion, tissue damage, or harmful stimuli. Moderate inflammation is beneficial for pathogen invasion and promoting tissue repair. However, when the inflammatory response is excessive or out of control, it leads to abnormal function of the human immune system, damage to its own tissues and cells, manifested as redness, swelling, fever, pain, and dysfunction. Inflammation is also an important aspect of skin problems.

[0003] Traditional Chinese medicine (TCM) and natural drugs have been an important source of drug discovery and development since ancient times. An increasing number of drugs derived from TCM have attracted the attention of medicinal chemists. Abundant natural drug resources, a long history of TCM, and profound traditional TCM theories give us a unique advantage in discovering new anti-inflammatory drugs from TCM. Furthermore, the active ingredients of TCM, due to their anti-inflammatory activity, have been widely used in the cosmetics industry. With social progress and consumption upgrades, the cosmetics market increasingly embraces the concept of "returning to nature," and all-natural skincare products are gradually becoming the focus of consumer attention. Among these, cosmetics containing natural TCM active ingredients are increasingly favored due to their safety, wide applicability, and unique advantages that traditional chemically synthesized cosmetics cannot match.

[0004] Verbena has a long history of medicinal use, having been practiced clinically in my country for thousands of years. It has few toxic side effects and exhibits different pharmacological effects in the traditional medicine of various countries. Numerous preclinical and clinical studies have confirmed its antibacterial, anti-inflammatory, anticonvulsant, neuroprotective, neuroprotective, cardioprotective, and antitumor activities. Research has found that verbena decoction has a positive impact on the clinical condition of patients with chronic systemic gingivitis. Verbena water extract improves the periodontal microenvironment through a multi-mechanism synergistic effect (inhibiting the release of inflammatory factors + regulating pathogenic bacteria), demonstrating its potential as a natural adjunctive treatment for chronic gingivitis.

[0005] However, there is still limited research on the pharmacological effects and material basis of verbena. Therefore, in-depth research and development to apply it to the preparation of drugs for treating inflammatory diseases or whitening, anti-aging, soothing and repairing cosmetics is urgently needed. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a verbena extract, its extraction method, and its application. The verbena extract prepared by the present invention has a clear relieving effect on inflammation and can be used to prepare anti-inflammatory drugs and cosmetics.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] The first objective of this invention is to provide a method for extracting verbena extract, comprising the following steps:

[0009] (1) Obtain the tuberous verbena stems and leaves, add ethanol solution for reflux extraction to obtain ethanol extract, and then concentrate the ethanol extract under reduced pressure to obtain ethanol extract.

[0010] (2) The ethanol extract is dissolved in water to obtain a mixed solution, and extracted with petroleum ether to obtain a petroleum ether phase and a remaining mixture. The petroleum ether phase is concentrated under reduced pressure to obtain a petroleum ether extract. The remaining mixture is extracted with ethyl acetate and concentrated under reduced pressure to obtain an ethyl acetate extract. The petroleum ether extract and the ethyl acetate extract are mixed to obtain a crude extract of verbena.

[0011] (3) The crude extract of verbena was subjected to normal phase silica gel column chromatography and then eluted to obtain verbena extract.

[0012] The beneficial effects of this invention are: the present invention first crushes the stems and leaves of verbena and then dissolves them in ethanol for processing, so that the components of verbena are dissolved in ethanol first, which makes the subsequent extraction steps more effective. In the process of preparing verbena extract, multiple extractions are carried out using petroleum ether and ethyl acetate, which can fully extract the anti-inflammatory active ingredients of verbena.

[0013] Verbena is bitter, pungent, and slightly cold in nature. It enters the liver and spleen meridians. Its functions include clearing heat and detoxifying, promoting blood circulation and regulating menstruation, promoting diuresis and reducing swelling, and treating erysipelas. The main effects of verbena are cooling the blood, breaking up blood stasis, clearing heat and detoxifying, promoting blood circulation and regulating menstruation, promoting diuresis and reducing swelling, and treating erysipelas. It also has effects such as treating dysmenorrhea, anti-tumor, anti-inflammatory and analgesic, neuroprotective, immune-regulating, antioxidant, and anti-early pregnancy effects. This invention does not involve mixing verbena with other drugs for synergistic treatment; it only extracts the effective active ingredients of verbena for anti-inflammatory purposes. The extraction steps of this invention are simple, and the anti-inflammatory activity is good. The verbena extract preparation method of this invention is simple, has low equipment cost, is inexpensive, and is easy to promote and industrialize.

[0014] Based on the above technical solution, the present invention can be further improved as follows.

[0015] Furthermore, the above-ground part of the verbena is crushed to obtain blocky verbena stems and leaves, wherein the length of the crushed verbena stems and leaves is 0.5cm to 2cm.

[0016] Furthermore, in step (1), before performing the reflux extraction, the tuberous verbena stems and leaves need to be soaked in the ethanol solution for 1 to 2 hours; the reflux extraction is performed 3 times, with temperatures of 78℃ to 82℃, 83℃ to 87℃, and 83℃ to 87℃ respectively; and the times are 2 hours to 3 hours, 1 hour to 2 hours, and 1 hour to 2 hours respectively.

[0017] The volume concentration of the ethanol solution is 85% to 95%; the mass ratio of the ethanol solution to the stems and leaves of the verbena is 1:5 to 7.

[0018] Furthermore, the temperature for vacuum concentration in step (1) is 45℃~50℃; the vacuum degree is 30mbar~60mbar.

[0019] Further, in step (2), the volume concentration of ethanol extract in the mixed solution is 10% to 20%; the mass ratio of petroleum ether to the mixed solution is 1:100 to 1:50; and the mass ratio of ethyl acetate to the mixed solution is 1:50 to 1:40.

[0020] Furthermore, the normal-phase silica gel column in step (3) is 200-300 mesh; the elution solution is petroleum ether-acetone elution solution.

[0021] A second objective of this invention is to provide a verbena extract obtained by the aforementioned extraction method.

[0022] Furthermore, the verbena extract comprises the following compounds by mass fraction: 0.1%–0.3% luteolin, 0.2%–0.4% baicalin, 0.1%–0.3% cyanidin, and 0.3%–0.5% eupatorium flavonoids.

[0023] The beneficial effects of adopting the above-mentioned further scheme are: This invention provides a systematic study of the anti-inflammatory active components of Verbena officinalis, which is of great significance for the development of low-toxicity and highly effective anti-inflammatory drugs. Flavonoids are important secondary metabolites in Verbena species, characterized by a C6-C3-C6 skeleton, including two benzene rings and one oxygen-containing heterocycle, exhibiting various biological activities such as antioxidant, anti-inflammatory, and anti-tumor effects. Flavonoids isolated from Verbena mainly exist in the forms of flavones, flavonols, dihydroflavones, and their glycosides. Reported flavonoid glycosides include apigenin glycoside, luteolin glycoside, and quercetin glycoside, but the variety and activity studies are limited. Therefore, the discovery of new flavonoids and the study of their anti-inflammatory activities have great potential in the development of new anti-inflammatory drugs.

[0024] A third objective of this invention is to provide an application of verbena extract, wherein the verbena extract is used in the preparation of medicaments for treating inflammatory diseases and / or anti-inflammatory and soothing cosmetics.

[0025] Furthermore, the inflammatory diseases include at least one of acute and chronic respiratory infections, rhinitis, pharyngitis, prostatitis, atopic dermatitis, inflammatory bowel disease, arthritis, and autoimmune diseases.

[0026] Furthermore, verbena extract is formulated into pharmaceutically permissible dosage forms, such as tablets, hard capsules, soft capsules, powders, tinctures, oral liquids, syrups, granules, pills, or injections.

[0027] The beneficial effects of this invention are: the verbena extract prepared by the method of extracting the effective components from verbena can be used in the preparation of anti-inflammatory drugs, and can prepare drugs of various dosage forms, making the drugs suitable for different occasions and populations.

[0028] A fourth objective of this invention is to provide a verbena medicine for treating inflammatory diseases, wherein the active ingredient of the medicine is the verbena extract, and the dosage form of the medicine includes at least one of tablets, capsules, oral liquids, lozenges, granules, suspensions, suppositories, injections, powder injections, pellets, sustained-release agents, and controlled-release agents.

[0029] The fifth objective of this invention is to provide an anti-inflammatory and soothing cosmetic product, wherein the effective ingredient of the anti-inflammatory and soothing cosmetic product is the verbena extract mentioned above, and the anti-inflammatory and soothing cosmetic product includes at least one of the following: serum, face cream, face mask, lotion, cleansing water, facial cleanser, and shampoo and conditioner.

[0030] Furthermore, the preparation steps of the anti-inflammatory and soothing cosmetic are as follows:

[0031] (1) Preparation of phase A: Deionized water, glycerol, panthenol and 1% sodium hyaluronate solution were added to a beaker and stirred at room temperature until completely mixed to obtain phase A;

[0032] (2) While stirring continuously, the verbena extract is slowly added to phase A to ensure complete and uniform dispersion; finally, phenoxyethanol preservative is added and stirred until completely dissolved and uniform to obtain an anti-inflammatory and soothing cosmetic.

[0033] Furthermore, the mass ratio of deionized water, glycerol, panthenol, and 1% sodium hyaluronate solution in phase A is 88:5:2.0:5.

[0034] Furthermore, in step (2), the mass ratio of phase A, verbena extract, and phenoxyethanol preservative is 95:3:2. Attached Figure Description

[0035] Figure 1 This is a diagram of the stem and leaves of the blocky verbena in Embodiment 1 of the present invention;

[0036] Figure 2The extract of verbena in Example 1 of the present invention; wherein (A) is an ethanol extract; and (B) is a crude extract of verbena.

[0037] Figure 3 This is the LC-MS / MS TIC detection chromatogram of the ethyl acetate fraction of Verbena officinalis alcohol extract in this invention (POS: positive ion mode, NEG: negative ion mode);

[0038] Figure 4 This is a schematic diagram of the GNPS molecular network analysis of the ethyl acetate fraction of the verbena alcohol extract of this invention;

[0039] Figure 5 This is a structural diagram of the GNPS molecular network of the ethyl acetate fraction of the verbena alcohol extract of this invention.

[0040] Figure 6 Compound I of the present invention 1 H NMR spectrum;

[0041] Figure 7 Compound I of the present invention 13 C NMR spectrum;

[0042] Figure 8 Compound III of the present invention 1 H NMR spectrum;

[0043] Figure 9 Compound II of the present invention 1 H NMR spectrum;

[0044] Figure 10 Compound III of the present invention 13 C NMR spectrum;

[0045] Figure 11 Compound 1V of the present invention 1 H NMR spectrum;

[0046] Figure 12 The results of cytotoxicity tests on RAW 264.7 macrophages are as follows: the crude extract and fractions of verbena from Example 1 of the present invention, and the verbena extract from Comparative Example 1.

[0047] Figure 13 The results of the test on NO production in LPS-induced RAW264.7 cells are as follows: the crude extract and fractions of Verbena officinalis from Example 1 of this invention and the extract of Verbena officinalis from Comparative Example 1.

[0048] Figure 14The results of cytotoxicity tests of compounds I-IV, Vo1-6, Vo1-14, Vo1-29, Vo1-32, Vo1-33, Vo1-38, Vo1-39, Vo1-41, and L-NMMA of the present invention on RAW 264.7 macrophages are as follows;

[0049] Figure 15 The results of testing the NO production in LPS-induced RAW264.7 cells by compounds I-IV, Vo1-6, Vo1-14, Vo1-29, Vo1-32, Vo1-33, Vo1-38, Vo1-39, Vo1-41, and L-NMMA of this invention are shown. Detailed Implementation

[0050] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0051] Example 1: Preparation of Verbena officinalis extract

[0052] (1) 50 kg of verbena aboveground parts were crushed to obtain verbena stems and leaves in chunks, wherein the length of the crushed verbena stems and leaves was 0.5 cm to 2 cm. Figure 1 As shown, after soaking in 300 kg of 95% ethanol solution for 1 hour, the mixture was refluxed three times. The reflux extraction temperatures were 80℃, 85℃, and 85℃ respectively, and the reflux extraction times were 3 hours, 2 hours, and 2 hours respectively. The filtrates from the three reflux extractions were combined and concentrated under reduced pressure at a temperature of 45℃~50℃ and a vacuum degree of 30mbar~60mbar to obtain an ethanol extract, as shown. Figure 2 As shown in (A);

[0053] (2) 2.5 kg of ethanol extract was dispersed in 20.0 L of deionized water to obtain a mixed solution. This solution was then extracted four times with 20 L of petroleum ether each time, yielding a petroleum ether phase and a remaining mixture. The petroleum ether phase was concentrated under reduced pressure to recover the solvent, yielding 197.4 g of petroleum ether extract, designated as MP. The remaining mixture was extracted four times with 20 L of ethyl acetate each time. The solvent was then concentrated under reduced pressure to recover the ethyl acetate phase, yielding 345.0 g of ethyl acetate extract, designated as M-E'. The petroleum ether extract MP and the ethyl acetate extract M-E' were combined to obtain verbena crude extract ME, as shown below. Figure 2 As shown in (B);

[0054] (3) Take 280.0g of verbena crude extract ME and pass it through normal phase silica gel column chromatography (200-300 mesh; Qingdao Ocean Chemical Co., Ltd.). Then, use petroleum ether-acetone elution buffer for gradient elution (V / V = 50:1; 40:1; 30:1; 20:1; 15:1; 10:1; 8:1; 4:1; 2:1; 1:1). By TLC analysis, eight fractions M1 to M8 were separated. Fraction M2, fraction M5 and fraction M6 are verbena extract.

[0055] Example 2: Preparation of cosmetics

[0056] (1) Preparation of phase A: Deionized water, glycerol, panthenol, and 1% sodium hyaluronate solution were added to a beaker in the following proportions of mass: 88%, 5%, 2.0%, and 5%, respectively. The mixture was stirred at room temperature until it was completely mixed to obtain phase A.

[0057] (2) Under continuous stirring, the fractions M2, M5 and M6 extracted in Example 1 were slowly added to phase A to ensure complete and uniform dispersion; finally, phenoxyethanol preservative (2%) was added, wherein the mass fractions of fractions M2, M5 and M6 were all 1.0%, the mass fraction of phase A was 95%, and the mass fraction of phenoxyethanol preservative was 2%. The mixture was stirred until completely dissolved and uniform to obtain an anti-inflammatory and soothing repair cosmetic.

[0058] Comparative Example 1:

[0059] (1) 50 kg of verbena aboveground parts were crushed to obtain verbena stems and leaves in chunks, with the length of the crushed verbena stems and leaves being 0.5 cm to 2 cm. After soaking in 300 kg of 95% ethanol solution for 1 h, the mixture was refluxed three times, with the reflux extraction temperatures being 80℃, 85℃, and 85℃ respectively, and the reflux extraction times being 3 h, 2 h, and 2 h respectively. The filtrates obtained from the three reflux extractions were combined and concentrated under reduced pressure at a temperature of 45℃ to 50℃ and a vacuum degree of 30 mbar to 60 mbar to obtain an ethanol extract.

[0060] (2) 2.5 kg of ethanol extract was dispersed in 20.0 L of deionized water to obtain a mixed solution. The solution was then extracted four times with petroleum ether, with 20 L of petroleum ether used each time. The remaining mixture was then extracted four times with ethyl acetate, with 20 L of ethyl acetate used each time. After removing the ethyl acetate phase extract, the solution was extracted four times with n-butanol, with 20 L of n-butanol used each time. After the solvent was recovered under reduced pressure, 791.7 g of n-butanol phase extract was obtained, which is the verbena extract, denoted as MB.

[0061] The identification process in this embodiment includes the following steps:

[0062] 1. LC-MS / MS and GNPS molecular network analysis

[0063] Weigh the dried ME components, fractions M2, M5, and M6 and prepare solutions of 2 mg / mL each using chromatographically pure methanol. Centrifuge at 12,000 rpm for 15 min and filter the supernatant through a 0.22 μm nylon filter membrane into a liquid chromatography sample vial for later use. Prepare a blank sample of methanol solution using the same steps.

[0064] 1.1. LC-MS / MS (Liquid Chromatography-Tandem Mass Spectrometry):

[0065] The chromatographic conditions are as follows:

[0066] Liquid chromatography conditions: Ultra-high performance liquid chromatography system: Agilent 1290 Infinity II UPLC; Column: Acquity ZORBAX Eclipse Plus C18 column (1.8μm, 3.0×100mm); Sample injection volume: 2μL; Mobile phase: A was water containing 0.1% formic acid (v / v), and B was methanol.

[0067] Gradient elution program: 0–15 min (15% B–60% B), 15–20 min (60% B–80% B), 20–23 min (80% B–100% B), 23–28 min B (100% B–100%), 28–29 min (100% B–15% B), 29–33 min (15% B–15% B); flow rate: 0.3 mL / min.

[0068] Mass spectrometry conditions:

[0069] Mass spectrometry system: Agilent 6560Q-TOF, ion source parameters are as follows: Gas Temp: 225℃; Gas Flow: 7L / min; Nebulizer (25psig), Sheath Gas Temp: 350℃; Sheath Gas Flow: 12L / min; Capillary Voltage (VCap): 3500V, Nozzle Voltage: 500V; Fragmentor: 400V; Skimmer (30), Octopolr RF Peak (750).

[0070] Primary mass spectrometry (MS) data were acquired in both positive and negative ion modes, ranging from 100 m / z to 1250 m / z, with a collision energy of 40 eV. Secondary mass spectrometry (MS / MS) data were acquired in data-dependent mode (DDA), ranging from 50 m / z to 1250 m / z. The test results are as follows: Figure 3 As shown, the ethyl acetate fraction of the verbena ethanol extract exhibited different compound response characteristics in positive and negative ion modes. 78 compounds were identified in the positive ion mode, and 38 compounds were identified in the negative ion mode.

[0071] 1.2. GNPS Molecular Network Analysis

[0072] The acquired LC-MS / MS data were converted to mzML format using MSConvert software, and then uploaded to the GNPS platform via WinSCP for molecular network analysis (https: / / gnps.ucsd.edu). The parameters were set as follows: Precursor ion mass tolerance (0.02), MS / MS fragment ion tolerance (0.02), cosinescore ≥ 0.6, and matched peaks ≥ 5. Data matching was performed in the GNPS spectral database with matching parameters of cosinescore ≥ 0.5 and matched peaks ≥ 4. Figure 4 As shown. Using Qualitative Navigator B.08.00 software, total ion chromatograms and UV absorption spectra of each component were extracted. Compounds were identified by combining molecular network analysis with comparisons with literature reports. The structures of some compounds are shown below. Figure 5 As shown in the figure, the analysis results indicate that 78 compounds were identified in the positive ion mode, including 21 flavonoids (3 isoflavones and 4 flavonoid glycosides), 12 alkaloids, 12 terpenoids (6 diterpenoids, 1 triterpenoid, 2 iridoid glycosides, 3 sesquiterpenoids and monoterpenoids), 5 phenylethanoid glycosides, 6 coumarins, 1 naphthoquinone, 3 stigmonesans, 5 phenolic acids, and 13 other compounds. In the negative ion mode, 38 compounds were identified, including 21 flavonoids (2 isoflavones and 6 flavonoid glycosides), 5 phenylethanoid glycosides, 2 alkaloids, 2 quinones, and 8 other compounds. A total of 109 compounds were identified in both positive and negative ion modes, including 36 flavonoids, 14 alkaloids, 10 phenylethanoid glycosides, 3 anthraquinones or naphthoquinones, and 46 other types of compounds.

[0073] 2. Extraction, structural identification, and application of compounds

[0074] 2.1 The extraction steps for compounds I to III are as follows:

[0075] (1) The ethyl acetate fraction M5 was subjected to chromatography on a dextran gel LH-20 (Sephadex LH-20, GE-Healthcare) column A, eluted with a 1:1 volume ratio of dichloromethane-methanol mixture. The eluted components were analyzed by TLC thin-layer chromatography to separate them into 8 fractions M51-M58. Fraction M58 was subjected to chromatography on a normal phase silica gel column B (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.), eluted with a 10000:200:1 volume ratio of dichloromethane-acetone-acetic acid mixture. Compound I (0.001%-0.003% of the crude verbena extract ME) and compound III (0.001%-0.003% of the crude verbena extract ME) were collected after purification by the normal phase silica gel column.

[0076] (2) Fraction M6 was subjected to reversed-phase silica gel ODS column C chromatography with gradient elution using a methanol-water mixture with a volume ratio of 3:2-1:1. The eluent collected from the methanol-water mixture with a volume ratio of 3:2 was then subjected to normal-phase silica gel column D chromatography (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.) with isocratic elution using a dichloromethane-acetone mixture with a volume ratio of 40:1. The collected eluent was analyzed by TLC thin-layer chromatography to separate it into 7 fraction pairs (M621-M627). Fraction M625 was subjected to normal-phase silica gel column E chromatography (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.) with a volume ratio of 3:2-1:1. The M627 fraction was purified by chromatography on a normal-phase silica gel column (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.), eluted with a dichloromethane-ethyl acetate mixture at a volume ratio of 15:1, and the purified compound IV (0.010%-0.015% of the crude verbena extract ME) was collected.

[0077] Compounds I-IV appear black under a 254 nm UV lamp, compound II appears yellow-green under a 254 nm UV lamp, and dark purple under a 365 nm UV lamp. A 10% sulfuric acid-ethanol solution was used as a colorimetric reagent and showed yellow spots after heating on a TLC plate.

[0078] 2.2 Extraction of other compounds

[0079] The specific steps are as follows:

[0080] (1) The ethyl acetate fraction M1 was subjected to chromatography on a normal phase silica gel column G (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.) with gradient elution using a petroleum ether:ethyl acetate mixture with a volume ratio of 50:1–10:1. The eluted components were analyzed by TLC thin-layer chromatography to separate them into 9 fractions M11-M19. Fraction M14 was subjected to chromatography on a normal phase silica gel column H (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.) with a petroleum ether:ethyl acetate mixture of 20:1. The eluted components were analyzed by TLC thin-layer chromatography to separate them into 8 fractions M141-M148. Fraction M14 was subjected to chromatography on a normal phase silica gel column I (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.) with a dichloromethane:ethyl acetate mixture of 20:1. The purified compound Vo1-41 (accounting for 0.004%–0.006% of the crude verbena extract ME) was collected.

[0081] (2) The ethyl acetate fraction M4 was subjected to dextran gel LH-20 (Sephadex LH-20, GE-Healthcare) column chromatography, eluted with a 1:1 volume ratio of dichloromethane:methanol mixture. The eluted components were analyzed by TLC to separate them into 7 fractions M41-M47. Fraction M45 was further analyzed by dextran gel LH-20 (Sephadex LH-20, GE-Healthcare) column chromatography. LH-20 (GE-Healthcare) column K chromatography, eluted with a 2:1:1 petroleum ether:dichloromethane:methanol mixture. The eluted components were analyzed by TLC thin-layer chromatography to separate into 7 fractions M451-M457. Fraction M456 was purified by normal-phase silica gel column L (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.), eluted with a 60:1 dichloromethane:methanol mixture. The purified compound Vo1-39 (accounting for 0.004%-0.006% of the crude verbena extract ME) was collected.

[0082] (3) The M6 ​​fraction was subjected to reversed-phase silica gel ODS column chromatography with gradient elution using a methanol-water mixture with a volume ratio of 3:2-1:1. The eluted components were analyzed by TLC thin-layer chromatography and separated into 6 fractions M61-M66. The M61 fraction was subjected to normal-phase silica gel column chromatography (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.) with isocratic elution using a dichloromethane-acetone mixture with a volume ratio of 40:1. The collected eluent fraction was subjected to normal-phase silica gel column chromatography (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.) with elution using a dichloromethane-ethyl acetate mixture with a volume ratio of 15:1. The compounds Vo1-32 (0.020%-0.025% of the crude verbena extract ME) and Vo1-38 (0.005%-0.007% of the crude verbena extract ME) were collected after purification by normal-phase silica gel column chromatography. Fraction M62 was purified by normal-phase silica gel column chromatography (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.), eluted with a 40:1 volume ratio of dichloromethane-acetone mixture, and the purified compound Vo1-14 (0.02%-0.023% of the crude verbena extract ME) was collected. Fraction M64 was purified by dextran gel chromatography on a Sephadex LH-20 (GE-Healthcare) column, eluted with a 1:1 volume ratio of dichloromethane-methanol mixture, and the purified compound Vo1-6 (0.050%-0.052% of the crude verbena extract ME) was collected.

[0083] (3) The M7 fraction was subjected to chromatography on a dextran gel LH-20 (Sephadex LH-20, GE-Healthcare) column Q, eluted with methanol solution. The eluted components were analyzed by TLC thin-layer chromatography and separated into 7 fractions M71-M77. The M74 fraction was subjected to chromatography on a normal phase silica gel column R (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.), eluted isocratically with a 10:1 volume ratio of dichloromethane-methanol mixture. The compounds Vo1-33 (0.008%-0.010% of the crude verbena extract ME) and Vo1-29 (0.002%-0.003% of the crude verbena extract ME) were collected after purification by the normal phase silica gel column.

[0084] Compound Vo1-6 showed no fluorescence under UV light at 254 and 365 nm, but developed purplish-red spots upon heating with a 10% sulfuric acid-ethanol solution on a TLC plate. Vo1-14 exhibited black fluorescence at 254 nm UV light and developed black spots upon heating with 10% sulfuric acid-ethanol. Vo1-14 also showed black fluorescence under UV light at both 254 and 365 nm, and developed yellow spots upon iodine fuming. Vo1-32 and Vo1-33 showed purple fluorescence under UV light at 254 nm and bright blue fluorescence at 365 nm, and developed orange spots upon heating with 10% sulfuric acid-ethanol. Vo1-38, Vo1-39, and Vo1-41 showed black fluorescence under UV light at 254 nm and developed light brown spots upon heating with 10% sulfuric acid-ethanol.

[0085] 2.3 Structural identification of compounds I-IV

[0086] Testing steps:

[0087] The testing instrument was a Bruker AVANCE III-400 NMR spectroscopy unit at 400 MHz, and the deuteration reagent was provided by Ningbo Cuiying Chemical Technology Co., Ltd. 2 mg of sample was dried to remove water, dissolved in 0.5 mL of the deuteration reagent, and then loaded into the NMR tube for detection. The results are as follows: Figures 6-11 As shown.

[0088] (1) Structural identification of compound I

[0089] Figure 6 of 1 The 1H NMR spectrum showed characteristic signals of 5,7-dihydroxy substitution on ring A of flavonoids in compound I [δ]. H 6.18(s,H-6) and 6.43(s,H-8)] and characteristic proton signals of ring C-3 in ring B [δ H 6.65(s,H-3)]; a group of aromatic proton signals [δ H 7.40(m,H-2',H-6') and 6.89(d,J=8.4Hz,H-5')], where [δ H The coupling constant of 6.89 is 8.4 Hz, indicating the presence of ortho-coupled aromatic ring protons. Based on the integral area, it is inferred that the compound has a trisubstituted benzene ring. Figure 7 of 13 The 12C NMR spectrum showed that compound I had fifteen carbon signals, including one carbonyl carbon signal [δ]. C 181.7(C-4)], a characteristic signal of a flavonoid C-2 hydroxyl carbon [δ C [164.2(C-2)] Based on literature data, compound I was identified as luteolin.

[0090] (2) Structural identification of compound II

[0091] Figure 8 As shown, 1 The 1H NMR spectrum showed that compound II had a set of proton signals from a para-substituted aromatic ring [δ]. H 7.92 (d, J = 8.4 Hz, H-2', H-6') and 6.92 (d, J = 8.4 Hz, H-3', H-5')]. Similar to compound I. 1 H NMR data comparison showed that ring II lacked the C-6 proton signal of ring A [δ]. H 6.20(1H,s)], while the C-8 proton signal [δ H [6.58(s,H-8)] is basically consistent with I, suggesting that this compound has a 5,6,7-trihydroxy-substituted ring A. [δ] H [6.78(s,C-3)] is the characteristic signal of the C-3 hydrogen proton in flavonoids. Based on literature data, compound II was identified as scutellarein.

[0092] (3) Structural identification of compound II

[0093] Figure 9 of 1 The 1H NMR spectrum showed that compound II had a set of ABX system aromatic ring proton signals [δ]. H 7.47 (dd, J = 8.4, 2.4 Hz, H-6'), 7.43 (d, J = 1.8 Hz, H-2'), and 6.91 (d, J = 8.4 Hz, H-5')], two hydroxymethyl proton signals [δ H 3.95(s,3'-OCH3) and 3.88(s,6-OCH3)]. Figure 10 In 13 The 12C NMR spectrum showed that compound II had seventeen carbon signals, including one carbonyl carbon signal [δ]. C 184.2(C-4)], a characteristic signal of a flavonoid C-2 hydroxyl carbon [δ C Based on the data, compound III was identified as jaceosidin.

[0094] (4) Structural identification of compound IV

[0095] Figure 11 of 1 Comparison of H NMR data with those of compound IV revealed that the two data were essentially identical. The data comparison showed that compound IV was missing only one methoxy group [δ]. H[3.95 (s, 3'-OCH3)]. Based on the data, compound IV was identified as Nepetin.

[0096] 3. Anti-inflammatory activity study

[0097] 3.1 Study on the anti-inflammatory activity of verbena extract

[0098] a. After thawing frozen mouse RAW 264.7 macrophages (Wuhan Pronosei Life Science Technology Co., Ltd.), they were inoculated into DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) solution and cultured in a constant temperature incubator (DHP-9162; Shanghai Yiheng Scientific Instruments Co., Ltd.) at 37℃ and 5% CO2.

[0099] b. The toxicity of Verbena officinalis extract to mouse RAW 264.7 macrophages was detected using the CCK-8 assay. Mouse RAW 264.7 macrophages were seeded at a density of 5000 cells / well in 96-well plates (11510; LABSELECT). After cell adhesion, 100 μL of the ME component, fragments M1-M8, and MB component prepared in Example 1 were added to each well to replace the medium (DMSO as solvent, final concentration <0.1%, initial screening concentration 50 μg / mL), and the cells were treated at 37°C for 24 hours. Subsequently, 10 μL of CCK-8 solution was added to each well, centrifuged, and cultured at 37°C for 1 hour. The OD value of each well was measured at 450 nm to assess cell viability. The control group was replaced with fresh medium (10% fetal bovine serum and 1% penicillin-streptomycin DMEM medium) without any added drugs; the blank group consisted of cell-free wells with the same volume of medium added. The cell viability of each group was calculated using the formula shown in Equation ①. The results are as follows: Figure 12 As shown:

[0100]

[0101] c. The concentration of nitrite in the culture supernatant was detected using the Griess reagent method to determine the NO production level. Mouse RAW 264.7 macrophages were cultured at 8 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of cells / well in 96-well plates and pre-cultured for 12 hours to ensure full adhesion. 90 μL of the test sample (DMSO as solvent, final concentration <0.1%, initial screening concentration 50 μg / mL) was added for pretreatment for 2 hours, followed by stimulation with 10 μL of lipopolysaccharide (LPS, 10 μg / mL) (Beyotime Biotechnology Co., Ltd.). After 22 hours of culture, 80 μL of supernatant was collected, and 100 μL of an equal volume of a mixture of Griess A and Griess B reagents (Beyotime Biotechnology Co., Ltd.) was added. The cells were incubated at 37°C for 10 min, and the OD value of each well was measured at 540 nm. The control group consisted of cell-free wells containing an equal volume of culture medium (10% fetal bovine serum and 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) DMEM medium). The calculation formula is shown in Equation ②, and the results are as follows. Figure 13 As shown:

[0102]

[0103] Depend on Figure 12 , 13 The results indicate that the crude extract of Verbena officinalis ME and its sub-fractions M2, M5, and M6 possess certain anti-inflammatory activities.

[0104] 3.2 Determination of the anti-inflammatory activity of compounds I–IV

[0105] The toxicity of verbena crude extract compounds, including compounds I-IV, to mouse RAW264.7 macrophages was detected using the CCK8 assay. The specific steps were as follows:

[0106] (1) RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin solution in a humidified incubator at 37°C and 5% CO2. Compounds I-IV, as well as compounds isolated from verbena extract (Vo1-6, Vo1-14, Vo1-29, Vo1-32, Vo1-33, Vo1-38, Vo1-39, Vo1-41), and the positive control L-NMMA (Beijing Solarbio) were dissolved in DMSO and then diluted with complete medium to the required final concentration (40 μg / mL). A control group was set up as a blank control without the compounds.

[0107] (2) RAW264.7 cells (1.0×10⁻⁶) were used to prepare the cells. 4Cells were seeded per well in 96-well plates and pre-cultured for 12 h. The medium was then replaced with fresh medium (100 μL per well) containing compounds I–IV, Vo1-6, Vo1-14, Vo1-29, Vo1-32, Vo1-33, Vo1-38, Vo1-39, Vo1-41, and L-NMMA, respectively, and cultured for another 24 h. Cell viability was then assessed using CCK-8 reagent, and absorbance was measured at 450 nm.

[0108] The concentration of nitrite in the culture supernatant was detected using the Griess reagent method to determine the NO production level, thereby detecting the anti-inflammatory activity of compounds in the crude verbena extract, including compounds I-IV. The specific steps were as follows: RAW264.7 cells (5.0 × 10⁻⁶) were... 4 Each well was incubated with compounds I-IV, Vo1-6, Vo1-14, Vo1-29, Vo1-32, Vo1-33, Vo1-38, Vo1-39, Vo1-41, and the positive control L-NMMA at a concentration of 40 μg / mL for 1 h, followed by stimulation with LPS (10 μg / mL). After 23 h, 50 μL of the supernatant was collected and mixed with 100 μL of Griess reagent. After incubation at 37 °C for 10 min, the absorbance was measured at 540 nm using a microplate reader. The results are shown below. Figure 14 , Figure 15 As shown.

[0109] Depend on Figure 14 , 15 The results indicate that compounds I to IV possess certain anti-inflammatory activity and are key components for quality control of the anti-inflammatory function of verbena extract.

[0110] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for extracting verbena extract, characterized in that, Includes the following steps: (1) Obtain the tuberous verbena stems and leaves, add ethanol solution for reflux extraction to obtain ethanol extract, and then concentrate the ethanol extract under reduced pressure to obtain ethanol extract. (2) The ethanol extract is dissolved in water to obtain a mixed solution, and extracted with petroleum ether to obtain a petroleum ether phase and a remaining mixture. The petroleum ether phase is concentrated under reduced pressure to obtain a petroleum ether extract. The remaining mixture is extracted with ethyl acetate and concentrated under reduced pressure to obtain an ethyl acetate extract. The petroleum ether extract and the ethyl acetate extract are mixed to obtain a crude extract of verbena. (3) The crude extract of verbena was subjected to normal phase silica gel column chromatography and then eluted to obtain verbena extract.

2. The method for extracting verbena extract according to claim 1, characterized in that, Step (1) Before performing the reflux extraction, the tuberous verbena stems and leaves need to be soaked in the ethanol solution for 1h to 2h; the reflux extraction is performed 3 times, with temperatures of 78℃ to 82℃, 83℃ to 87℃, and 83℃ to 87℃ respectively; and the time is 2h to 3h, 1h to 2h, and 1h to 2h respectively. The volume concentration of the ethanol solution is 85% to 95%; the mass ratio of the ethanol solution to the stems and leaves of the verbena is 1:5 to 7.

3. The method for extracting verbena extract according to claim 1, characterized in that, The vacuum concentration in step (1) is carried out at a temperature of 45℃~50℃ and a vacuum degree of 30mbar~60mbar.

4. The method for extracting verbena extract according to claim 1, characterized in that, In step (2), the volume concentration of ethanol extract in the mixed solution is 10% to 20%; the mass ratio of petroleum ether to the mixed solution is 1:50 to 100; and the mass ratio of ethyl acetate to the mixed solution is 1:40 to 50.

5. The method for extracting verbena extract according to claim 1, characterized in that, The normal-phase silica gel column mentioned in step (3) is 200-300 mesh; the elution solution is petroleum ether-acetone elution solution.

6. A verbena extract, characterized in that, It is extracted by the extraction method described in any one of claims 1 to 5.

7. The verbena extract according to claim 6, characterized in that, The verbena extract includes the following compounds: luteolin, baicalin, brown cyanidin, and eupatorium flavonoids.

8. The verbena extract according to claim 7, characterized in that, The verbena extract comprises the following compounds by mass fraction: 0.1%–0.3% luteolin, 0.2%–0.4% baicalin, 0.1%–0.3% cyanidin, and 0.3%–0.5% eupatorium flavonoids.

9. An application of verbena extract, characterized in that, The verbena extract according to any one of claims 1 to 8 may be used to prepare medicaments for treating inflammatory diseases and / or anti-inflammatory and soothing cosmetics.

10. The application of a verbena extract according to claim 9, characterized in that, The inflammatory diseases include at least one of the following: acute and chronic respiratory infections, rhinitis, pharyngitis, prostatitis, atopic dermatitis, inflammatory bowel disease, arthritis, and autoimmune diseases.

11. A verbena medicine for treating inflammatory diseases, characterized in that, The active ingredient of the drug is the verbena extract as described in any one of claims 1 to 8, and the dosage form of the drug includes at least one of tablets, capsules, oral liquids, lozenges, granules, suspensions, suppositories, injections, powder injections, pellets, sustained-release agents, and controlled-release agents.

12. An anti-inflammatory and soothing cosmetic product, characterized in that, The effective ingredient of the anti-inflammatory and soothing cosmetic is the verbena extract as described in any one of claims 1 to 8, and the anti-inflammatory and soothing cosmetic includes at least one of the following: serum, face cream, face mask, lotion, cleansing water, facial cleanser, and shampoo and conditioner.

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