7-acetyl-lushanrubescensin A for use in the preparation of an anti-inflammatory cosmetic or pharmaceutical product, and compositions containing the same and methods of preparation

Abstract

The application discloses application of 7-acetyl-lushanrubescensin A in preparation of anti-inflammatory cosmetics or medicines, and a composition containing the same and a preparation method, and belongs to the field of natural medicine research and development. The compound 7-acetyl-lushanrubescensin A is separated from isodon lichiangensis, can significantly inhibit release of NO and IL-6 inflammatory factors of RAW264.7 cells induced by LPS, and has better effect than a positive control dexamethasone in a concentration range of 1-3 mu g / mL, and has obvious anti-inflammatory activity. The 7-acetyl-lushanrubescensin A compound can be used as an anti-inflammatory active agent in skin inflammation care and / or inflammation repair cosmetics or medicines, and provides a new natural active ingredient source for care of inflammatory skin.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine development technology, and in particular relates to the application of 7-acetyl-lushanrubescensin A in the preparation of anti-inflammatory cosmetics or pharmaceuticals, as well as compositions containing it and preparation methods. Background Technology

[0002] *Isodon calcicolus* (Handel-Mazzetti) H. Hara is a perennial herb belonging to the genus *Isodon* in the family Lamiaceae. It is mainly distributed in northwestern Yunnan Province. While there are folk records of its medicinal use, research on its chemical composition and biological activity is still insufficient. Currently, there are no reports on the compound 7-acetyl-lushanrubescensin A from *Isodon calcicolus*, nor are there any reports on its extraction, purification, pharmacological activity, or application in cosmetics or pharmaceuticals.

[0003] Based on this, this application innovatively develops a compound 7-acetyl-lushanrubescensin A extracted from *Gnaphalium affine*, which has a well-defined structure and significant anti-inflammatory effects, and studies its anti-inflammatory activity, providing a reliable basis for the practical significance and application value of this compound. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new use for 7-acetyl-lushanrubescensin A compound in cosmetics or pharmaceuticals, so that it can exert anti-inflammatory activity and has practical application value.

[0005] To address the aforementioned technical problems, this invention provides the application of 7-acetyl-lushanrubescensin A in the preparation of anti-inflammatory cosmetics or pharmaceuticals.

[0006] Further improvements showed that 7-acetyl-lushanrubescensin A could inhibit LPS-induced release of NO and / or IL-6 inflammatory factors from macrophages.

[0007] In specific embodiments, cytotoxicity tests showed that 7-acetyl-lushanrubescensin A had a CV90 (concentration at which cell viability decreased to 90%) of 14.188 μg / mL in RAW264.7 macrophages. Anti-inflammatory activity tests showed that 7-acetyl-lushanrubescensin A, within a concentration range of 1-3 μg / mL, significantly inhibited LPS-induced release of NO and IL-6 in RAW264.7 cells, with better effects than the positive control dexamethasone (10 μM). In another embodiment, an anti-inflammatory essence containing 0.001% 7-acetyl-lushanrubescensin A was prepared, which significantly inhibited LPS-induced release of NO and IL-6 in an in vitro cell model, with an inhibition rate of 28.3% ± 3.4% for NO and 34.1% ± 4.1% for IL-6 (p < 0.01), significantly better than the blank matrix group. The blank matrix group showed no significant inhibitory effect, further confirming that the anti-inflammatory activity originates from compound 7-acetyl-lushanrubescensin A. Therefore, 7-acetyl-lushanrubescensin A possesses anti-inflammatory efficacy.

[0008] Further improvements include making the anti-inflammatory cosmetics into skin inflammation care cosmetics and / or inflammation repair cosmetics.

[0009] Further improvements include making the anti-inflammatory cosmetics into aqueous solutions, serums, lotions, creams, masks, freeze-dried powders, or sprays; and making the anti-inflammatory drugs into topical solutions, ointments, plasters, pastes, or patches.

[0010] Further improvements were made, and 7-acetyl-lushanrubescensin A was extracted and purified from *Gnaphalium affine*, with the molecular formula C. 30 H 40 O 11 CAS number 110325-80-9.

[0011] As a further improvement of the present invention, the present invention also includes a method for preparing the compound 7-acetyl-lushanrubescensin A, wherein the compound is extracted and purified from *Hemiberlesia lataniae*, and the extraction and purification method is as follows:

[0012] (1) Take the dried aerial parts of the limestone tea plant, crush them, and extract them twice with 75% ethanol at room temperature using ultrasonic extraction for 3 hours each time. The solvent used is 8 times and 6 times the weight of the medicinal material, respectively. Combine the extracts and concentrate them under reduced pressure to obtain the total extract.

[0013] (2) After suspending the total extract in an appropriate amount of water, it was loaded onto a D101 macroporous adsorption resin column and eluted sequentially with water, 40% ethanol and 80% ethanol gradients, with each gradient eluting for 3 column volumes. The 80% ethanol eluent was collected and concentrated under reduced pressure to obtain the 80% ethanol eluent fraction.

[0014] (3) Take the 80% ethanol eluent, dissolve it in a chloroform-methanol mixture with a volume ratio of 1:1, mix it into 100-200 mesh silica gel, evaporate the solvent, grind it into a uniform powder, pack it into the top of a pre-equilibrated silica gel column, and elute with a gradient of n-hexane / ethyl acetate system, wherein the gradient of the n-hexane / ethyl acetate system is 100:0, 50:50, 0:100 in sequence, collect one fraction every 50 mL, monitor it by TLC chromatography, and combine the target fractions containing 7-acetyl-lushanrubescensin A to obtain target fraction I; wherein the developing solvent of the TLC chromatography is: dichloromethane-methanol 20:1, and the colorimetric reagent is: iodine vapor;

[0015] (4) The target fraction I was subjected to medium-pressure reversed-phase C18 column chromatography with gradient elution of water / methanol system, wherein the methanol concentration was 30%→100%, the flow rate was 5mL / min, and one fraction was collected every 10mL. The target fraction containing 7-acetyl-lushanrubescensin A was collected to obtain target fraction II.

[0016] (5) Purify the target fraction II by HPLC. HPLC chromatographic conditions: Agilent ZORBOX EclipsePlus C 18 The chromatographic column (4.6×250mm, 5μm) was used with a mobile phase of 0.05% formic acid-water (A) and 0.05% formic acid-acetonitrile (B), gradient elution (0-20min: 10%-95%B, 20-25min: 95%B), flow rate of 1.0mL / min, detection wavelength of 254nm, column temperature of 25℃, and injection volume of 20μL. The target peak fraction of 7-acetyl-lushanrubescensin A was collected, concentrated under reduced pressure, and lyophilized to obtain compound 7-acetyl-lushanrubescensin A with a purity greater than 98%.

[0017] As a further improvement of the present invention, the present invention also provides an anti-inflammatory composition comprising 7-acetyl-lushanrubescensin A, and a cosmetic or pharmaceutically acceptable matrix, carrier and / or excipient.

[0018] Further improvements were made, with the concentration of 7-acetyl-lushanrubescensin A in the composition being 0.01-3 μg / mL.

[0019] Further improvements were made, and 7-acetyl-lushanrubescensin A was extracted and purified from *Hemiberlesia lataniae*.

[0020] With this design, the present invention has at least the following advantages:

[0021] This invention isolates compound 7-acetyl-lushanrubescensin A from *Gnaphalium affine* and discovers for the first time that it has anti-inflammatory activity. It can be used as an anti-inflammatory active agent in anti-inflammatory cosmetics or pharmaceuticals at extremely low concentrations, providing a new source of natural active ingredients for the care of inflammatory skin. Attached Figure Description

[0022] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Figure 1 This is the HPLC purity chromatogram of the 7-acetyl-lushanrubescensin A compound extracted in this invention.

[0024] Figure 2 This is the 1H NMR spectrum of the 7-acetyl-lushanrubescensin A compound extracted in this invention.

[0025] Figure 3 This is the carbon NMR spectrum of the 7-acetyl-lushanrubescensin A compound extracted in this invention.

[0026] Figure 4 This is a schematic diagram illustrating the inhibitory effect of the 7-acetyl-lushanrubescensin A compound of this invention on LPS-induced NO release in RAW264.7 cells.

[0027] Figure 5 This is a schematic diagram illustrating the inhibitory effect of the 7-acetyl-lushanrubescensin A compound of this invention on LPS-induced IL-6 release in RAW264.7 cells.

[0028] Figure 6 This invention presents a schematic diagram showing the inhibition rate of LPS-induced NO and IL-6 release from RAW264.7 cells by a cosmetic containing 7-acetyl-lushanrubescensin A. Detailed Implementation

[0029] Several typical embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be particularly noted that the embodiments shown in the drawings are merely illustrative representations of the present invention and are not intended to limit the scope of protection of the present invention. The present invention can be implemented through various methods, and the embodiments described herein are intended to fully illustrate the technical principles of the present invention and ensure that those skilled in the art can fully understand the technical boundaries of the present invention. Specific embodiments are described below:

[0030] Example 1: Extraction, separation and structural identification of 7-acetyl-lushanrubescensin A

[0031] 1.1 Instruments and Materials

[0032] Instruments: Rotary evaporator (BUCHI R-300), freeze dryer (Labconco), medium-pressure preparative chromatograph (Büchi Sepacore), high performance liquid chromatograph (Agilent 1260 Infinity II), mass spectrometer (Agilent 6530LC / Q-TOF), nuclear magnetic resonance spectrometer (Bruker AVANCE III 600MHz).

[0033] Reagents: Ethanol (analytical grade), n-hexane (analytical grade), ethyl acetate (analytical grade), dichloromethane (analytical grade), methanol (analytical grade), acetonitrile (chromatographic grade), formic acid (chromatographic grade).

[0034] Chromatographic materials: D101 macroporous adsorption resin (Tianjin Haoju), column chromatography silica gel (100-200 mesh, 200-300 mesh, Qingdao Ocean), medium-pressure reversed-phase C18 packing material (YMC*GEL ODS-A-HG), HPLC column (Agilent ZORBOX EclipsePlus C18, 4.6×250mm, 5μm).

[0035] Medicinal material: The above-ground parts of Isodon calcicolus (Handel-Mazzetti) H. Hara were collected in October 2022 from Jianchuan County, Dali Prefecture, Yunnan Province. They were identified by Zhang Jun of Kunming Zhifen Biotechnology Co., Ltd. as plants of the genus Isodon in the Lamiaceae family.

[0036] 1.2 Extraction and coarse separation

[0037] Take 0.38 kg of dried aerial parts of *Hedysarum sylvestris*, pulverize them, and extract them twice with 75% ethanol at room temperature using ultrasonic extraction for 3 hours. The solvent volume used is 8 times and 6 times the weight of the medicinal material, respectively. Combine the extracts and concentrate them to dryness under reduced pressure at 55℃ to obtain the total extract.

[0038] The total extract was suspended in an appropriate amount of water and loaded onto a D101 macroporous adsorption resin column (column diameter × column height: 2.1 cm × 14 cm). Elution was performed sequentially with water, 40% ethanol, 80% ethanol, and 95% ethanol, with three column volumes eluted for each gradient. The 80% ethanol eluent was collected and concentrated to dryness under reduced pressure to obtain extract P0473-M003.

[0039] 1.3 Silica gel column chromatography separation

[0040] The above-mentioned P0473-M003 extract was separated by silica gel column chromatography. The specific procedure was as follows: 15.79 g of the active fraction was dissolved in an appropriate amount of chloroform-methanol mixed solution (1:1), then mixed into 100-200 mesh silica gel. The solvent was evaporated, and the mixture was ground into a uniform powder. The mixed silica gel was packed into the top of a pre-equilibrated silica gel column (200-300 mesh silica gel, column inner diameter 2.5 cm, silica gel amount 50 g). A gradient elution system of n-hexane / ethyl acetate (100:0→50:50→0:100) was used, collecting one fraction every 50 mL, and monitoring the elution by TLC.

[0041] The developing solvent in TLC chromatography is dichloromethane-methanol 20:1, and the colorimetric reagent is iodine vapor.

[0042] The target fraction A4 was obtained by combining fractions containing the target component with a spot Rf value of approximately 0.6 using TLC chromatography.

[0043] 1.4 Medium-pressure reversed-phase C18 purification

[0044] The target fraction A4 was subjected to medium-pressure reversed-phase C18 column chromatography (column diameter 2.0 cm, column height 25 cm) with a water / methanol gradient elution system (methanol from 30% to 100% linear gradient, flow rate 5 mL / min), and one fraction was collected every 10 mL. The target component was also collected by TLC chromatography to obtain the target fraction A4B1.

[0045] 1.5 HPLC purification and purity determination

[0046] The target fraction A4B1 was further purified by HPLC under the following chromatographic conditions: Agilent ZORBOX Eclipse Plus C. 18 The chromatographic column (4.6 × 250 mm, 5 μm) was used with a mobile phase of 0.05% formic acid-water (A) and 0.05% formic acid-acetonitrile (B), using gradient elution (0–20 min: 10%–95% B, 20–25 min: 95% B), a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, a column temperature of 25 °C, and an injection volume of 20 μL. The target peak fraction was collected, concentrated under reduced pressure, and lyophilized to obtain compound Y 13.0 mg.

[0047] A suitable amount of compound Y was dissolved in methanol and analyzed by HPLC. The purity was calculated to be 98.5% using the area normalization method. Figure 1 As shown.

[0048] 1.6 Structural Identification

[0049] Compound Y, white powder, ESI-MS m / z: 577.2645 [M+H]⁺(C 30 H 40 O 11 The calculated value is 576.2645.

[0050] ¹H-NMR (600MHz, C5D5N) and ¹³C-NMR (150MHz, C5D5N) data are shown in Table 1, and the figures are shown in the figures below. Figure 2 and Figure 3 .

[0051] Table 1. ¹H-NMR and ¹³C-NMR data for 7-acetyl-lushanrubescensin A

[0052]

[0053] As shown in the figure, compound Y is 7-acetyl-lushanrubescensin A, CAS number 110325-80-9. Its structural formula is as follows:

[0054]

[0055] Example 2: Cytotoxicity Test

[0056] 2.1 Instruments and Reagents

[0057] Instruments: CO2 cell incubator (Thermo 3111), biosafety cabinet (ESCO), inverted microscope (Olympus CKX53), microplate reader (BioTek Synergy H1).

[0058] Reagents: DMEM high glucose medium (HyClone), fetal bovine serum (Gibco), penicillin-streptomycin antibiotic (HyClone), CCK-8 kit (Dojindo), DMSO (Sigma).

[0059] Cell line: Mouse mononuclear macrophage leukemia cells (RAW264.7), purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0060] Test sample: 7-acetyl-lushanrubescensin A, prepared by separation in Example 1, with an HPLC purity of 98.5%.

[0061] 2.2 Experimental Methods

[0062] RAW264.7 cells were cultured at a rate of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, the supernatant was discarded.

[0063] Different concentrations of 7-acetyl-lushanrubescensin A solution (1, 5, 10, 50, 100, 200 μg / mL) were added and prepared with DMSO, ensuring that the final DMSO concentration in each well was ≤0.1%. A blank control group (containing cells, no drug, and an equal volume of culture medium) and a solvent control group (containing cells and 0.1% DMSO) were also included. Each concentration was tested in triplicate, and the experiment was independently repeated three times.

[0064] After culturing for another 24 hours, add 10 μL of CCK-8 reagent to each well and incubate at 37°C for 2 hours. Measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate cell viability using the following formula:

[0065] Cell viability (%) = (OD value of drug-treated group - OD value of blank group) / (OD value of solvent control group - OD value of blank group) × 100%

[0066] Plot a dose-response curve with compound concentration on the x-axis and cell viability on the y-axis, and calculate the compound concentration (CV90) corresponding to a cell viability of 90%.

[0067] 2.3 Experimental Results

[0068] The CV90 of 7-acetyl-lushanrubescensin A against RAW264.7 cells was 14.188 μg / mL. At a concentration of 10 μg / mL, the cell viability dropped to 91.2%, indicating that the compound begins to exhibit cytotoxicity at concentrations close to 10 μg / mL. The cytotoxicity of this compound needs to be monitored in subsequent experiments.

[0069] Example 3: Anti-inflammatory activity test

[0070] 3.1 Instruments and Reagents

[0071] Instruments: CO2 cell incubator (Thermo 3111), biosafety cabinet (ESCO), microplate reader (BioTekSynergy H1).

[0072] Reagents: DMEM high glucose medium (HyClone), fetal bovine serum (Gibco), penicillin-streptomycin antibiotic (HyClone), lipopolysaccharide (LPS, Sigma), mouse IL-6 ELISA kit (R&D Systems), DMSO (Sigma).

[0073] Positive control: Dexamethasone (DEX, Sigma).

[0074] Cell line: RAW264.7 cells (mouse mononuclear macrophage leukemia cells), purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0075] Test sample: 7-acetyl-lushanrubescensin A, prepared by separation in Example 1, with an HPLC purity of 98.5%.

[0076] 3.2 Experimental Methods

[0077] RAW264.7 cells were cultured at a rate of 2.5 × 10⁻⁶. 5 100 μL of the cells were seeded at a density of 100 cells / mL in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0078] The experiment was divided into the following groups:

[0079] Blank control group: Contains no LPS or drugs, and contains an equal volume of culture medium;

[0080] Model control group: Contains LPS (1 μg / mL), does not contain drugs;

[0081] Positive control group: containing LPS (1 μg / mL) + dexamethasone (10 μM);

[0082] 7-acetyl-lushanrubescensin Group A: containing LPS (1 μg / mL) + 7-acetyl-lushanrubescensin A (1, 3, 10 μg / mL);

[0083] Each compound group was first pre-incubated with different concentrations of the test compound for 1 hour, followed by stimulation with LPS (1 μg / mL) for 24 hours. Each group had 3 replicates, and the experiment was independently repeated 3 times.

[0084] After the culture is completed, collect the cell culture supernatant.

[0085] NO detection: Take 50 μL of supernatant into a new 96-well plate, add an equal volume of Griess reagent, incubate at room temperature in the dark for 10 minutes, and measure the absorbance (OD value) at 540 nm wavelength using an ELISA reader. Calculate the NO concentration based on the sodium nitrite standard curve.

[0086] IL-6 detection: The IL-6 level in the supernatant was detected by ELISA following the instructions of the kit.

[0087] The inhibition rates of NO and IL-6 are calculated using the following formula:

[0088] Inhibition rate (%) = (Inflammatory mediator level in the model control group - Inflammatory mediator level in the drug treatment group) / (Inflammatory mediator level in the model control group - Inflammatory mediator level in the blank control group) × 100%

[0089] 3.3 Experimental Results

[0090] 3.3.1 Inhibitory effect on NO release

[0091] The inhibitory effect of 7-acetyl-lushanrubescensin A on LPS-induced NO release in RAW264.7 cells is as follows: Figure 4 As shown.

[0092] from Figure 4 The NO concentration in the blank control group was 7.2 ± 1.2 μM, and the NO concentration in the model control group (LPS 1 μg / mL) was 20.5 ± 2.1 μM. The NO concentration in the positive control group treated with dexamethasone (10 μM) was 15.8 ± 1.8 μM, with an inhibition rate of 35.3%.

[0093] At concentrations of 1, 3, and 10 μg / mL, 7-acetyl-lushanrubescensin A inhibited NO release at concentrations of 12.8 ± 1.5 μM, 4.5 ± 1.0 μM, and 0.8 ± 0.5 μM, respectively, with inhibition rates of 57.9%, 120.3% (66.2% based on 100% saturation inhibition), and 148.1% (94.0% based on 100% saturation inhibition). Notably, at the 10 μg / mL concentration, cell state visibly deteriorated significantly (this concentration is close to the CV90 value of 14.188 μg / mL), and NO concentration approached zero. This indicates that the significant decrease in NO release at this concentration is likely primarily due to cell death rather than the compound's inherent anti-inflammatory activity. Therefore, the inhibition rate data at this concentration is outlier and cannot be considered valid evidence of the compound's anti-inflammatory activity. However, 7-acetyl-lushanrubescensin A is within the safe range of use at a concentration of 1 μg / mL, and its inhibition rate of NO (57.9%) is significantly better than that of the positive control dexamethasone (35.3%).

[0094] 3.3.2 Inhibitory effect on IL-6 release

[0095] The inhibitory effect of 7-acetyl-lushanrubescensin A on LPS-induced IL-6 release in RAW264.7 cells is as follows: Figure 5 As shown.

[0096] The IL-6 concentration in the blank control group was 4.5 ± 1.2 pg / mL, and the IL-6 concentration in the model control group (LPS 1 μg / mL) was 390.5 ± 25.3 pg / mL. The IL-6 concentration in the positive control group treated with dexamethasone (10 μM) was 190.8 ± 18.5 pg / mL, with an inhibition rate of 51.7%.

[0097] At concentrations of 1, 3, and 10 μg / mL, 7-acetyl-lushanrubescensin A resulted in IL-6 concentrations of 135.2 ± 15.6 pg / mL, 10.8 ± 3.2 pg / mL, and 2.5 ± 0.8 pg / mL, with inhibition rates of 66.1%, 98.4%, and 100.5% (99.5% based on saturation inhibition rate of 100%), respectively. The 10 μg / mL concentration showed a significant deterioration in cell state, and the substantial decrease in IL-6 concentration is likely primarily attributed to cell death; therefore, the inhibition rate at this concentration is outlier and cannot be considered a reliable indicator of the compound's anti-inflammatory activity. In contrast, 7-acetyl-lushanrubescensin A at concentrations of 1 μg / mL and 3 μg / mL (66.1% and 98.4%, respectively) showed significantly higher inhibition rates against IL-6 than the positive control dexamethasone (51.7%).

[0098] 3.4 Discussion of Results

[0099] The above results indicate that 7-acetyl-lushanrubescensin A significantly inhibited the release of LPS-induced inflammatory mediators NO and IL-6 from RAW264.7 cells within a safe concentration range of 1-3 μg / mL, and its effect was superior to that of the positive control dexamethasone (10 μM). At a concentration of 1 μg / mL, the inhibition rates against NO and IL-6 were 57.9% and 66.1%, respectively; at a concentration of 3 μg / mL, the inhibition rates against both NO and IL-6 were close to 100%. At a concentration of 10 μg / mL, due to its proximity to its CV90 value (14.188 μg / mL), the cell state deteriorated significantly. The inhibitory activity at this concentration is mainly attributed to cell death caused by cytotoxicity, rather than the compound's own anti-inflammatory effect.

[0100] Since NO is a pro-inflammatory mediator produced by iNOS catalysis, and IL-6 is a key pro-inflammatory cytokine in the inflammatory response, both play important roles in the inflammatory cascade. The effective inhibition of NO and IL-6 release by compound 7-acetyl-lushanrubescensin A suggests that it may exert its anti-inflammatory effects by regulating the iNOS pathway and inhibiting the transcription of inflammatory factors. Due to its low CV90 of only 14.188 μg / mL, the dosage must be strictly controlled in practical applications; a concentration not exceeding 3 μg / mL is recommended.

[0101] Example 4: Evaluation of the in vitro anti-inflammatory activity of a cosmetic composition containing 7-acetyl-lushanrubescensin A.

[0102] 4.1 Preparation of an anti-inflammatory serum containing 7-acetyl-lushanrubescensin A

[0103] 4.1.1 Formulation Composition

[0104] Based on a total mass of 100g, the anti-inflammatory serum containing 7-acetyl-lushanrubescensin A consists of the following components: 0.001g of 7-acetyl-lushanrubescensin A (equivalent to 10μg / mL in 100g of serum), 5.0g of glycerin, 3.0g of 1,3-butanediol, 0.1g of sodium hyaluronate, 1.0g of trehalose, 0.5g of 1,2-hexanediol, 0.5g of p-hydroxyacetophenone, with the remainder being deionized water to bring the total to 100g.

[0105] 4.1.2 Preparation method

[0106] Mix deionized water, glycerin, 1,3-butanediol, and sodium hyaluronate, heat to 80-85℃ and stir until dissolved. Cool to 45℃ and add 7-acetyl-lushanrubescensin A and trehalose, stirring until completely dissolved. Finally, cool to 40℃ and add 1,2-hexanediol and p-hydroxyacetophenone, stirring until dissolved to obtain an anti-inflammatory serum containing 7-acetyl-lushanrubescensin A.

[0107] Meanwhile, a blank matrix without 7-acetyl-lushanrubescensin A was prepared as a control. Except for the absence of 7-acetyl-lushanrubescensin A, the other components and preparation methods were exactly the same.

[0108] 4.2 Evaluation of in vitro anti-inflammatory activity

[0109] 4.2.1 Experimental Methods

[0110] RAW264.7 cells were cultured at a rate of 2.5 × 10⁻⁶. 5 100 μL of the cells were seeded at a density of 100 cells / mL in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0111] The experiment was divided into the following groups:

[0112] Blank control group: Contains no LPS or drugs, and contains an equal volume of culture medium;

[0113] Model control group: Contains LPS (1 μg / mL), does not contain drugs;

[0114] Positive control group: containing LPS (1 μg / mL) + dexamethasone (10 μM);

[0115] Blank matrix group: containing LPS (1μg / mL) + blank matrix (diluted 1000 times with the same basic formula as the serum);

[0116] Serum group: containing LPS (1 μg / mL) + serum containing 0.001% 7-acetyl-lushanrubescensin A (diluted 1000 times in the same manner as the serum to make the final concentration of 7-acetyl-lushanrubescensin A equivalent to 0.01 μg / mL).

[0117] Each group was first incubated with the sample to be tested for 1 hour, and then stimulated with LPS (1 μg / mL) for 24 hours. Each group had 3 replicates, and the experiment was independently repeated 3 times.

[0118] After culture, the cell culture supernatant was collected, and the NO content was detected by the Griess method, and the IL-6 level was detected by the ELISA method.

[0119] 4.2.2 Experimental Results

[0120] The results are as follows Figure 6 As shown, the blank matrix group showed an inhibition rate of 3.2%±2.2% on LPS-induced NO release and an inhibition rate of 4.1%±3.0% on IL-6 release, which were not significantly different from the model control group (p>0.05). The serum containing 7-acetyl-lushanrubescensin A showed an inhibition rate of 28.3%±3.4% on NO and an inhibition rate of 34.1%±4.1% on IL-6 (p<0.01), which were close to the positive control group. This indicates that the active ingredient in the serum is 7-acetyl-lushanrubescensin A, and that this formula can effectively exert an anti-inflammatory effect.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

Application of 1,7-acetyl-lushanrubescensin A in the preparation of anti-inflammatory cosmetics or pharmaceuticals.

2. The application of 7-acetyl-lushanrubescensin A according to claim 1 in the preparation of anti-inflammatory cosmetics or pharmaceuticals, characterized in that, 7-acetyl-lushanrubescensin A can inhibit LPS-induced release of NO and / or IL-6 inflammatory factors from macrophages.

3. The application of 7-acetyl-lushanrubescensin A according to claim 1 in the preparation of anti-inflammatory cosmetics or pharmaceuticals, characterized in that, The anti-inflammatory cosmetics are skin inflammation care cosmetics and / or inflammation repair cosmetics.

4. The application of 7-acetyl-lushanrubescensin A according to claim 3 in the preparation of anti-inflammatory cosmetics or pharmaceuticals, characterized in that, The anti-inflammatory cosmetic is an aqueous solution, serum, lotion, cream, mask, freeze-dried powder, or spray; or the anti-inflammatory drug is a topical solution, ointment, plaster, paste, or patch.

5. The application of 7-acetyl-lushanrubescensin A according to claim 1 in the preparation of anti-inflammatory cosmetics or pharmaceuticals, characterized in that, 7-acetyl-lushanrubescensin A was extracted and purified from *Hemiberlesia lataniae*.

6. The application of 7-acetyl-lushanrubescensin A according to claim 5 in the preparation of anti-inflammatory cosmetics or pharmaceuticals, characterized in that, The preparation method of 7-acetyl-lushanrubescensin A is as follows: (1) Take the dried aerial parts of the limestone tea plant, crush them, and extract them twice with 75% ethanol at room temperature using ultrasonic extraction for 3 hours each time. The solvent used is 8 times and 6 times the weight of the medicinal material, respectively. Combine the extracts and concentrate them under reduced pressure to obtain the total extract. (2) After suspending the total extract in an appropriate amount of water, it was loaded onto a D101 macroporous adsorption resin column and eluted sequentially with water, 40% ethanol and 80% ethanol gradients, with each gradient eluting for 3 column volumes. The 80% ethanol eluent was collected and concentrated under reduced pressure to obtain the 80% ethanol eluent fraction. (3) Take the 80% ethanol eluent, dissolve it in a chloroform-methanol mixture with a volume ratio of 1:1, mix it into 100-200 mesh silica gel, evaporate the solvent, grind it into a uniform powder, pack it into the top of a pre-equilibrated silica gel column, and elute with a gradient of n-hexane / ethyl acetate system, wherein the gradient of the n-hexane / ethyl acetate system is 100:0, 50:50, 0:100 in sequence, collect one fraction every 50 mL, monitor it by TLC chromatography, and combine the target fractions containing 7-acetyl-lushanrubescensin A to obtain target fraction I; wherein the developing solvent of the TLC chromatography is: dichloromethane-methanol 20:1, and the colorimetric reagent is: iodine vapor; (4) The target fraction I was subjected to medium-pressure reversed-phase C18 column chromatography with gradient elution of water / methanol system, wherein the methanol concentration was 30%→100%, the flow rate was 5mL / min, and one fraction was collected every 10mL. The target fraction containing 7-acetyl-lushanrubescensin A was collected to obtain target fraction II. (5) Purify the target fraction II by HPLC. HPLC chromatographic conditions: Agilent ZORBOX Eclipse Plus C 18 The chromatographic column (4.6×250mm, 5μm) was used with a mobile phase of 0.05% formic acid-water (A) and 0.05% formic acid-acetonitrile (B), gradient elution (0-20min: 10%-95%B, 20-25min: 95%B), flow rate of 1.0mL / min, detection wavelength of 254nm, column temperature of 25℃, and injection volume of 20μL. The target peak fraction of 7-acetyl-lushanrubescensin A was collected, concentrated under reduced pressure, and lyophilized to obtain compound 7-acetyl-lushanrubescensin A with a purity greater than 98%. A method for preparing compound 7,7-acetyl-lushanrubescensin A, characterized in that, The compound was extracted and purified from *Gnaphalium affine*, and the extraction and purification method was as follows: (1) Take the dried aerial parts of the limestone tea plant, crush them, and extract them twice with 75% ethanol at room temperature using ultrasonic extraction for 3 hours each time. The solvent used is 8 times and 6 times the weight of the medicinal material, respectively. Combine the extracts and concentrate them under reduced pressure to obtain the total extract. (2) After suspending the total extract in an appropriate amount of water, it was loaded onto a D101 macroporous adsorption resin column and eluted sequentially with water, 40% ethanol and 80% ethanol gradients, with each gradient eluting for 3 column volumes. The 80% ethanol eluent was collected and concentrated under reduced pressure to obtain the 80% ethanol eluent fraction. (3) Take the 80% ethanol eluent, dissolve it in a chloroform-methanol mixture with a volume ratio of 1:1, mix it into 100-200 mesh silica gel, evaporate the solvent, grind it into a uniform powder, pack it into the top of a pre-equilibrated silica gel column, and elute with a gradient of n-hexane / ethyl acetate system, wherein the gradient of the n-hexane / ethyl acetate system is 100:0, 50:50, 0:100 in sequence, collect one fraction every 50 mL, monitor it by TLC chromatography, and combine the target fractions containing 7-acetyl-lushanrubescensin A to obtain target fraction I; wherein the developing solvent of the TLC chromatography is: dichloromethane-methanol 20:1, and the colorimetric reagent is: iodine vapor; (4) The target fraction I was subjected to medium-pressure reversed-phase C18 column chromatography with gradient elution of water / methanol system, wherein the methanol concentration was 30%→100%, the flow rate was 5mL / min, and one fraction was collected every 10mL. The target fraction containing 7-acetyl-lushanrubescensin A was collected to obtain target fraction II. (5) The target fraction II was purified by HPLC. The HPLC chromatographic conditions were: Agilent ZORBOX Eclipse Plus C18 column (4.6×250mm, 5μm), mobile phase was 0.05% formic acid-water (A) and 0.05% formic acid-acetonitrile (B), gradient elution (0-20min: 10%-95%B, 20-25min: 95%B), flow rate was 1.0mL / min, detection wavelength was 254nm, column temperature was 25℃, injection volume was 20μL, the target peak fraction of 7-acetyl-lushanrubescensin A was collected, concentrated under reduced pressure and lyophilized to obtain compound 7-acetyl-lushanrubescensin A with a purity greater than 98%.

8. An anti-inflammatory composition, characterized in that, This includes 7-acetyl-lushanrubescensin A, as well as cosmetic or pharmaceutically acceptable bases, carriers, and / or excipients.

9. The anti-inflammatory composition according to claim 8, characterized in that, The concentration of 7-acetyl-lushanrubescensin A used in the composition is 0.01-3 μg / mL.

10. The anti-inflammatory composition according to claim 8, characterized in that, 7-acetyl-lushanrubescensin A was extracted and purified from *Hemiberlesia lataniae*.

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