Application of alkaloid compounds in the preparation of ultraviolet protection agents

Abstract

The present invention discloses the application of alkaloid compounds in the preparation of ultraviolet protection agents, and relates to the fields of cosmetics and pharmaceutical technology. The alkaloid compounds are compounds described in any one of the following (1)-(4): (1) Epi-aszonalenin A; (2) a salt or ester of the Epi-aszonalenin A; (3) Asterrelenin; (4) a salt or ester of the Asterrelenin. The present invention has found that the alkaloid compounds Epi-aszonalenin A and Asterrelenin have no cytotoxicity to human skin cells, and can remove ultraviolet-induced intracellular reactive oxygen species, protect collagen and eliminate inflammatory factors, so they have broad application prospects in the preparation of ultraviolet protection agent products.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics and medicine, and in particular to the application of alkaloid compounds in the preparation of ultraviolet protection agents. Background Art

[0002] Skin photoaging is a chronic damage process caused primarily by ultraviolet radiation (UV) from the sun. As the body's primary defense barrier, the skin plays a vital role in protecting it from external radiation. The UV rays in sunlight consist of three different wavelengths: UVA, UVB, and UVC. Of the three wavelengths, UVC is almost entirely absorbed by the ozone layer in the atmosphere. Compared to UVA, UVB has a higher energy level and can directly penetrate the epidermis and shallow dermis, thereby promoting skin damage and premature aging.

[0003] The absorption of light by the skin, especially UVB radiation, leads to the activation of a range of reactive oxygen species (ROS). These include superoxide anions (·O 2 - ), hydroxyl radical (·OH), singlet oxygen ( 1 O 2 ) and hydrogen peroxide (H 2 O 2 ). ROS are a class of highly reactive oxidants produced by cells under normal conditions and are essential for regulating intracellular signaling cascades and physiological processes. However, excessive ROS generation can exceed the body's antioxidant defense capacity, leading to many harmful effects, including inflammatory responses, DNA damage, and impaired integrity of extracellular matrix (ECM) proteins.

[0004] ROS generated by UVB irradiation can activate the MAPK signaling pathway by promoting the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinase. This, in turn, upregulates the activator of transcription factor activator protein 1 (AP-1), stimulates the development of matrix metalloproteinases (MMPs), and leads to the degradation of the extracellular matrix and collagen in skin cells. MMP-1 is the key enzyme for collagen breakdown. The MAPK signaling cascade further stimulates nuclear factor κB (NF-κB), activating the gene transcription and expression of inflammation-related enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby promoting the release of inflammatory cytokines. Therefore, active molecules that can scavenge intracellular reactive oxygen species induced by ultraviolet rays, protect collagen, and eliminate inflammation are of great value for the development of ultraviolet protectants and have unique advantages over various ultraviolet absorbers, the main functional components in current sunscreen products. Therefore, the present invention aims to explore alkaloid compounds that can scavenge intracellular reactive oxygen species induced by ultraviolet rays, protect collagen, and eliminate inflammation, so as to provide technical support for the development of ultraviolet protectants and related cosmetics and drugs. Summary of the Invention

[0005] The object of the present invention is to provide the application of alkaloid compounds in the preparation of ultraviolet protectants to solve the problems existing in the above-mentioned prior art. The present invention's research found that the alkaloid compounds Epi-aszonalenin A and Asterrelenin have no cytotoxicity to human skin cells and can scavenge intracellular reactive oxygen species induced by ultraviolet rays, protect collagen, and eliminate inflammatory factors. Therefore, they have broad application prospects in the preparation of ultraviolet protectant products.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the application of alkaloid compounds in the preparation of ultraviolet protectants, and the alkaloid compounds are compounds described in any one of the following (1)-(4):

[0008] (1) Epi-aszonalenin A;

[0009] (2) The salt or ester of Epi-aszonalenin A;

[0010] (3) Asterrelenin;

[0011] (4) The salt or ester of Asterrelenin;

[0012] The structural formula of Epi-aszonalenin A is:

[0013]

[0014] The structural formula of Asterrelenin is:

[0015]

[0016] The present invention also provides an ultraviolet protection agent, the active ingredients of which include alkaloid compounds;

[0017] The alkaloid compound is a compound described in any one of the following (1)-(4):

[0018] (1) Epi-aszonalenin A;

[0019] (2) a salt or ester of Epi-aszonalenin A;

[0020] (3) Asterrelenin;

[0021] (4) A salt or ester of Asterrelenin;

[0022] The structural formula of the Epi-aszonalenin A is:

[0023]

[0024] The structural formula of Asterrelenin is:

[0025]

[0026] The present invention also provides the use of the above-mentioned ultraviolet protection agent in the preparation of a product for inhibiting ultraviolet-induced skin damage;

[0027] The product is a cosmetic or a medicine.

[0028] The skin damage includes increased active oxygen content in skin tissue cells, skin collagen degradation or skin inflammation.

[0029] The cosmetics include, but are not limited to, sunscreen, face cream, lotion or lipstick.

[0030] The drugs include but are not limited to sunscreen drugs for external use on the skin.

[0031] The present invention also provides the use of an alkaloid compound in the preparation of a product for inhibiting ultraviolet-induced skin damage, wherein the alkaloid compound is any one of the following (1) to (4):

[0032] (1) Epi-aszonalenin A;

[0033] (2) a salt or ester of Epi-aszonalenin A;

[0034] (3) Asterrelenin;

[0035] (4) A salt or ester of Asterrelenin;

[0036] The structural formula of the Epi-aszonalenin A is:

[0037]

[0038] The structural formula of Asterrelenin is:

[0039]

[0040] Furthermore, the product is a cosmetic or a medicine.

[0041] The present invention also provides a cosmetic for inhibiting ultraviolet-induced skin damage, wherein the active ingredient includes an alkaloid compound;

[0042] The alkaloid compound is a compound described in any one of the following (1)-(4):

[0043] (1) Epi-aszonalenin A;

[0044] (2) a salt or ester of Epi-aszonalenin A;

[0045] (3) Asterrelenin;

[0046] (4) A salt or ester of Asterrelenin;

[0047] The structural formula of the Epi-aszonalenin A is:

[0048]

[0049] The structural formula of Asterrelenin is:

[0050]

[0051] Furthermore, the cosmetic also includes cosmetically acceptable auxiliary materials.

[0052] The cosmetically acceptable auxiliary materials include common flavors and fragrances, pigments and pigments, preservatives and antioxidants, surfactants, water-soluble polymers, whitening agents, moisturizers or oils in the cosmetics field.

[0053] The present invention also provides a drug for inhibiting ultraviolet-induced skin damage, wherein the active ingredient includes an alkaloid compound;

[0054] The alkaloid compound is a compound described in any one of the following (1)-(4):

[0055] (1) Epi-aszonalenin A;

[0056] (2) a salt or ester of Epi-aszonalenin A;

[0057] (3) Asterrelenin;

[0058] (4) A salt or ester of Asterrelenin;

[0059] The structural formula of the Epi-aszonalenin A is:

[0060]

[0061] The structural formula of Asterrelenin is:

[0062]

[0063] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0064] Pharmaceutically acceptable excipients include, but are not limited to, binders, lubricants, disintegrants, diluents, solubilizers, stabilizers, or suspending agents.

[0065] The present invention discloses the following technical effects:

[0066] The present invention has found that the alkaloid compounds Epi-aszonalenin A and Asterrelenin have no cytotoxicity to human skin cells, and can remove UV-induced intracellular reactive oxygen species, protect collagen and eliminate inflammatory factors, so they have broad application prospects in the preparation of UV protective agent products. The present invention provides strong technical support for the development of UV protective agents that can remove UV-induced intracellular reactive oxygen species, protect collagen and eliminate inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0068] Figure 1 is the H NMR spectrum of Epi-aszonalenin A;

[0069] Figure 2 is the carbon NMR spectrum of Epi-aszonalenin A;

[0070] Figure 3 is the H NMR spectrum of Asterrelenin;

[0071] Figure 4 is the carbon NMR spectrum of Asterrelenin;

[0072] Figure 5 The results of the cytotoxicity experiments of Epi-aszonalenin A and Asterrelenin are shown in Figure 1. (a) is the result of the HaCaT cell experiment; (b) is the result of the BJ cell experiment; in (a) and (b), A-1 is Epi-aszonalenin A, and A-2 is Asterrelenin;

[0073] Figure 6 Epi-aszonaleninA and Asterrelenin have an important role in the regulation of UVB-induced HaCaT cells and H 2 O 2 Figure 2 shows the protective effect of stimulated BJ cell viability; (a) is the result of HaCaT cell experiment; (b) is the result of BJ cell experiment; in (a) and (b), A-1 is Epi-aszonalenin A, and A-2 is Asterrelenin;

[0074] Figure 7 The cytofluorescence images show the inhibitory effects of Epi-aszonaleninA and Asterrelenin on ROS in HaCaT cells induced by UVB; A-1 is Epi-aszonaleninA, and A-2 is Asterrelenin;

[0075] Figure 8 The fluorescence intensity statistical diagram of the inhibitory effect of Epi-aszonaleninA and Asterrelenin on ROS in HaCaT cells induced by UVB; A-1 is Epi-aszonaleninA, and A-2 is Asterrelenin;

[0076] Fig. 9The figure shows the effect of Epi-aszonaleninA and Asterrelenin in inhibiting the nuclear translocation of p65 in HaCaT cells induced by UVB; (a) is the experimental result of Epi-aszonaleninA; (b) is the experimental result of Asterrelenin; A-1 is Epi-aszonaleninA, and A-2 is Asterrelenin;

[0077] Fig.10 The immunoblot images of Epi-aszonaleninA (A) and Asterrelenin (B) on the expression of iNOS protein in HaCaT cells induced by UVB; A-1 is Epi-aszonaleninA, and A-2 is Asterrelenin;

[0078] Fig.11 The relative concentration diagram of Epi-aszonalenin A and Asterrelenin on the expression of iNOS protein in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonalenin A, and Alkaloid 2 is Asterrelenin;

[0079] Fig.12 The immunoblot images of Epi-aszonalenin A (A) and Asterrelenin (B) on the expression of COX-2 protein in HaCaT cells induced by UVB; A-1 is Epi-aszonalenin A, and A-2 is Asterrelenin;

[0080] Fig.13 The relative concentration diagram of Epi-aszonaleninA and Asterrelenin on COX-2 protein expression in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonaleninA, and Alkaloid 2 is Asterrelenin;

[0081] Fig.14 The immunoblot images of Epi-aszonaleninA (A) and Asterrelenin (B) on the expression of IL-6 protein in HaCaT cells induced by UVB; A-1 is Epi-aszonaleninA, and A-2 is Asterrelenin;

[0082] Fig.15The relative concentration diagram of Epi-aszonaleninA and Asterrelenin on the expression of IL-6 protein in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonaleninA, and Alkaloid 2 is Asterrelenin;

[0083] Fig.16 The immunoblot images of Epi-aszonaleninA (A) and Asterrelenin (B) on the expression of IL-1β protein in HaCaT cells induced by UVB; A-1 is Epi-aszonaleninA, and A-2 is Asterrelenin;

[0084] Fig.17 The relative concentration diagram of Epi-aszonaleninA and Asterrelenin on the expression of IL-1β protein in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonaleninA, and Alkaloid 2 is Asterrelenin;

[0085] Fig.18 The immunoblot images of Epi-aszonalenin A (A) and Asterrelenin (B) on the phosphorylation level of p65 in HaCaT cells induced by UVB; A-1 is Epi-aszonalenin A, A-2 is Asterrelenin, and p-p65 is phosphorylated p65;

[0086] Fig.19 The relative concentration diagram of Epi-aszonalenin A and Asterrelenin on the expression of p65 protein in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonalenin A, and Alkaloid 2 is Asterrelenin;

[0087] Fig. 20 The immunoblot images of Epi-aszonaleninA (A) and Asterrelenin (B) on the phosphorylation level of ERK in HaCaT cells induced by UVB; A-1 is Epi-aszonalenin A, A-2 is Asterrelenin, and p-ERK is phosphorylated ERK;

[0088] Fig.21The relative concentration diagram of Epi-aszonaleninA and Asterrelenin on the phosphorylation level of ERK in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonaleninA, and Alkaloid 2 is Asterrelenin;

[0089] Fig. 22 The immunoblot images of Epi-aszonalenin A (A) and Asterrelenin (B) on the phosphorylation level of JNK in HaCaT cells induced by UVB; A-1 is Epi-aszonalenin A, A-2 is Asterrelenin, and p-JNK is phosphorylated JNK;

[0090] Fig.23 The relative concentration diagram of Epi-aszonaleninA and Asterrelenin on the phosphorylation level of JNK in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonaleninA, and Alkaloid 2 is Asterrelenin;

[0091] Fig.24 The immunoblot images of Epi-aszonalenin A (A) and Asterrelenin (B) on the phosphorylation level of p38 in HaCaT cells induced by UVB; A-1 is Epi-aszonalenin A, A-2 is Asterrelenin, and p-p38 is phosphorylated p38;

[0092] Fig.25 The relative concentration diagram of Epi-aszonalenin A and Asterrelenin on the phosphorylation level of p38 in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonalenin A, and Alkaloid 2 is Asterrelenin;

[0093] Fig.26 The immunoblot images of Epi-aszonaleninA (A) and Asterrelenin (B) on the phosphorylation level of c-Fos in HaCaT cells induced by UVB; A-1 is Epi-aszonaleninA, A-2 is Asterrelenin, and pc-Fos is phosphorylated c-Fos;

[0094] Fig. 27The relative concentration diagram of Epi-aszonalenin A and Asterrelenin on the phosphorylation level of c-Fos in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonaleninA, and Alkaloid 2 is Asterrelenin;

[0095] Fig.28 The immunoblot images of Epi-aszonalenin A (A) and Asterrelenin (B) on the phosphorylation level of c-Jun in HaCaT cells induced by UVB; A-1 is Epi-aszonalenin A, A-2 is Asterrelenin, and pc-Jun is phosphorylated c-Jun;

[0096] Fig.29 The relative concentration diagram of Epi-aszonalenin A and Asterrelenin on the phosphorylation level of c-Jun in HaCaT cells induced by UVB; wherein, Alkaloid 1 is Epi-aszonaleninA, and Alkaloid 2 is Asterrelenin;

[0097] Fig.30 Epi-aszonalenin A (A) and Asterrelenin (B) for H 2 O 2 Effect diagram of MMP-1 protein expression in stimulated BJ cells (immunoblotting); A-1 is Epi-aszonalenin A, A-2 is Asterrelenin;

[0098] Fig.31 Epi-aszonalenin A and Asterrelenin for H 2 O 2 Relative concentration of MMP-1 protein expression in stimulated BJ cells; Alkaloid 1 is Epi-aszonalenin A and Alkaloid 2 is Asterrelenin. DETAILED DESCRIPTION

[0099] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0100] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0101] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0102] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0103] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0104] Aspergillus terreus C23-3 used in the following examples has been deposited in Guangdong Microbiological Culture Collection Center on January 22, 2018, with the deposit number GDMCC No. 60316, and is disclosed in patent publication CN108165500A.

[0105] Example 1 Preparation of Epi-aszonalenin A and Asterrelenin

[0106] Ⅰ. Fermentation

[0107] Aspergillus terreus C23-3 was activated in a potato sucrose peptone agar medium and cultured at 28°C for 7 days. Spores were collected to prepare a suspension, which was inoculated into 3L triangular flasks, each containing 1L of potato sucrose peptone medium, and collected after 28 days of static fermentation, and mycelium and fermentation liquid were separated by filtration.

[0108] The composition of the potato sucrose peptone agar medium is: 200 mL / L potato juice, 20 g / L sea salt, 20 g / L sucrose, 5 g / L peptone, 20 g / L agar, pH 7.2 ± 0.2. The inoculation amount of the potato sucrose peptone agar medium during activation is 2 inoculation loops per 100 mL of medium. The composition of the potato sucrose peptone medium is: 200 mL / L potato juice, 20 g / L sea salt, 20 g / L sucrose, 5 g / L peptone, ZnCl 2 10 mM, pH 7.2 ± 0.2. The inoculation volume during fermentation was 5 vol%.

[0109] The preparation method of potato juice is as follows: peel the potatoes, wash and chop them, add deionized water, heat to boil for 30 minutes, filter, boil 200 mL of potato juice for every 200 g of potatoes, and freeze for storage.

[0110] Ⅱ. Extraction

[0111] The mycelium was soaked in methanol overnight, ultrasonically extracted for 30 min (dichloromethane: methanol = 2:1), and the liquid was collected by filtration, and this process was repeated three times; the fermentation broth was extracted three times with ethyl acetate; the extracts were combined and concentrated to obtain a crude extract.

[0112] III. Product separation and purification

[0113] The crude extract was gradient eluted with a normal pressure silica gel column (the eluent was n-hexane phase, n-hexane: ethyl acetate = 4:1, n-hexane: ethyl acetate = 2:1, n-hexane: ethyl acetate = 1:1, n-hexane: ethyl acetate = 1:2, ethyl acetate, ethyl acetate: methanol = 20:1, ethyl acetate: methanol = 10:1, ethyl acetate: methanol = 5:1, ethyl acetate: methanol = 3:1, ethyl acetate: methanol = 1:1 and methanol), and the obtained eluent was eluted with a reduced pressure silica gel column (the eluent was dichloromethane: methanol = 1:1); the obtained eluent was separated by liquid chromatography (Lisure HP Plus 50D, the chromatography column was SinoChrom ODS-AP produced by Dalian Yilite Company, 15μm, 20.0mm×250mm, the mobile phase was 60% methanol-40% water, and the flow rate of the mobile phase was 5mL / min), and a compound meeting the following characteristics was prepared:

[0114] Analytical liquid chromatography analysis, t R =5.74min

Epi-aszonaleninA

Asterrelenin

[0115] The proton spectrum of Epi-aszonalenin A: 1 H NMR (700 MHz, CDCl 3 )δ6.04(1H,s,H-2),7.32(1H,dd,J=7.4,1.8Hz,H-5),7.10(1H,m,H-6),7.20(1H,m,H-7),7.84(1H,m,H-8),3.05(1H, d,J=13.1Hz,H-10a),2.49(1H,dd,J=13.1,9.7Hz,H-10b),4.02(1H,d,J=9.7Hz,H-11),6.75(1H,d,J=8.0Hz,H-15),7. 39(1H,td,J=7.2,1.4Hz,H-16),7.20(1H,m,H-17),7.98(1H,d,J=7.0Hz,H-18),5.81(1H,dd,J=17.4,10.9Hz,H-23), 5.10(1H,d,J=17.4Hz,H-24),5.07(1H,d,J=10.9Hz,H-24),1.11(3H,s,H-25),0.88(3H,s,H-26),2.66(3H,s,H-28); such as Figure 1 shown.

[0116] The carbon spectrum of Epi-aszonalenin A: 13 C NMR (176 MHz, CDCl 3 )δ81.48(C-2),61.25(C-3),133.39(C-4),126.79(C-5),123.79(C-6),128.69(C-7),119.4 9(C-8),141.95(C-9),29.84(C-10),57.13(C-11),171.34(C-12),136.18(C-14),121.06(C- 15),131.87(C-16),124.98(C-17),131.59(C-18),125.3(C-19),166.75(C-20),40.94(C-22),143.56(C-23),114.73(C-24),22.42(C-25),23.26(C-26),170.43(C-27),23.46(C-28); Figure 2 shown.

[0117] The Hydrogen Spectrum of Asterrelenin: 1 H NMR (700MHz, CD 3 OD)δ6.20(1H,s,H-2),7.40(1H,dd,J=7.7,1.3Hz,H-5),7.07(1H,td,J=7.6,0.8Hz,H-6),7.17(1H,td,J=7.7,1 .3Hz,H-7),7.64(1H,d,J=5.8Hz,H-8),3.59(1H,d,J=13.6Hz,H-10),2.48(1H,d,J=13.6Hz,H-10),4.14(1H,br s,11-OH),10.11(1H,br s,13-NH),6.94(1H,d,J=8.1Hz,H-15),7.42(1H,td,J=7.6,1.7Hz,H-16), 7.12(1H,td,J=7.8,1.1Hz,H-17),7.85(1H,dd,J=7.9,1.1Hz,H-18),5.90( 1H,dd,J=17.3,10.8Hz,H-23),5.11(1H,d,J=17.3Hz,H-24),5.09(1H,d,J =10.8Hz,H-24),1.10(3H,s,H-25),0.88(3H,s,H-26),2.61(3H,s,H-28); such as Figure 3 shown.

[0118] The Carbon Spectrum of Asterrelenin: 13 C NMR (176MHz, CD 3 OD)δ81.19(C-2),57.13(C-3),132.60(C-4),127.09(C-5),123.29(C-6),127.87(C-7),118. 09(C-8),141.51(C-9),40.28(C-10),87.88(C-11),168.31(C-12),136.91(C-14),119.26(C- 15),132.59(C-16),122.87(C-17),130.21(C-18),124.43(C-19),165.93(C-20),48.62(C-22),143.71(C-23),113.85(C-24),22.30(C-25),22.75(C-26),169.61(C-27),23.41(C-28); Figure 4 shown.

[0119] The structural formula of Epi-aszonalenin A is as follows:

[0120]

[0121] The structural formula of Asterrelenin is as follows:

[0122]

[0123] Example 2 Cytotoxicity Experiment of Epi-aszonalenin A and Asterrelenin

[0124] 1. Experimental methods:

[0125] Human keratinocytes (HaCaT cells) and human skin fibroblasts (BJ cells) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The culture medium was high-glucose Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were incubated at 37°C with an incubator of 100 mL / min. 2 The cells were cultured in an incubator with a concentration of 5%. The potential toxicity of Epi-aszonalenin A and Asterrelenin to cells was determined by the cell counting kit-8 method.

[0126] Blank group: HaCaT and BJ cells were only fed with serum-free DMEM medium without alkaloid treatment, and were not exposed to UVB or H 2 O 2 Stimulate.

[0127] Sample group: 10mM stock solutions of two alkaloid compounds Epi-aszonalenin A and Asterrelenin were diluted with serum-free DMEM medium to a final concentration of 1μM and 10μM, and equal volumes were added to each well to treat cells for 2 hours. Afterwards, HaCaT cells were irradiated with UVB, while BJ cells were irradiated with H 2 O 2 Stimulate.

[0128] 2. Experimental results:

[0129] Epi-aszonalenin A and Asterrelenin acted on HaCaT cells and BJ cells at doses of 1 and 10 μM, respectively. The cell viability of each dose group was similar to that of the blank group, with no significant difference ( Figure 5 ).

[0130] Example 3 Effects of Epi-aszonalenin A and Asterrelenin on UVB-induced HaCaT cells and H2 O 2 Protection assay of stimulated BJ cell viability

[0131] 1. Experimental methods:

[0132] When HaCaT cells reached the logarithmic growth phase, they were plated in a 96-well plate (8 × 10 4 The cells were treated with different doses of two alkaloid compounds Epi-aszonalenin A and Asterrelenin for 2 hours and then irradiated with UVB. BJ cells were also treated with the same samples as HaCaT cells, except that the UVB irradiation was replaced by exposure to H 2 O 2 After 2 hours, the supernatant was discarded. After 24 hours of incubation, the absorbance was measured at a wavelength of 450 nm using a microplate reader.

[0133] Blank group: HaCaT and BJ cells were only fed with serum-free DMEM medium without alkaloid treatment, and were not exposed to UVB or H 2 O 2 Stimulate.

[0134] Control group: An equal volume of serum-free DMEM medium was added to each well, and no alkaloids were treated. HaCaT cells were irradiated with UVB to evaluate the degree of damage to cells caused by UVB. BJ cells were irradiated with H 2 O 2 Stimulation, evaluation H 2 O 2 Oxidative stress damage to BJ cells.

[0135] Sample group: 10mM stock solutions of two alkaloid compounds Epi-aszonalenin A and Asterrelenin were diluted with serum-free DMEM medium to a final concentration of 1μM and 10μM, and equal volumes were added to each well to treat cells for 2 hours. Afterwards, HaCaT cells were irradiated with UVB, while BJ cells were irradiated with H 2 O 2 Stimulate.

[0136] 2. Experimental results:

[0137] from Figure 6It can be clearly seen in (a) that UVB irradiation reduced cell survival compared to unirradiated cells. In contrast, pretreatment with 1 and 10 μM Epi-aszonalenin A and Asterrelenin significantly increased cell viability. This suggests that 1 and 10 μM Epi-aszonalenin A and Asterrelenin can effectively alleviate UVB-induced HaCaT cell damage and death and stimulate epidermal cell proliferation. Figure 6 As shown in (b), when BJ cells were exposed to 600 μM H 2 O 2 In contrast, 1 and 10 μM Epi-aszonalenin A and Asterrelenin had no significant effect on H 2 O 2 Reduced cell viability has a restorative effect.

[0138] Example 4 Experiment on the inhibition of ROS generation by Epi-aszonalenin A and Asterrelenin

[0139] 1. Experimental methods:

[0140] DCFH-DA was used to detect the accumulation of reactive oxygen species. HaCaT cells were pretreated with 1 and 10 μM doses of Epi-aszonalenin A and Asterrelenin for 2 hours, respectively, and then UVB irradiated to induce ROS generation. After 24 hours, the cells were rinsed three times with phosphate buffered saline (PBS), incubated with DCFH-DA (10 μM) for 30 minutes, and the fluorescence intensity was observed under an inverted fluorescence microscope.

[0141] Blank group: HaCaT was only added with serum-free DMEM medium, and was not treated with alkaloid compounds and was not irradiated with UVB.

[0142] Control group: An equal volume of serum-free DMEM medium was added to each well, and no alkaloid compound treatment was performed. HaCaT cells were irradiated with UVB to evaluate the degree of cell damage caused by UVB irradiation.

[0143] Sample group: 10mM stock solutions of two alkaloid compounds Epi-aszonalenin A and Asterrelenin were diluted with serum-free DMEM medium to a final concentration of 1μM and 10μM, and equal volumes were added to each well to treat cells for 2 hours. Afterwards, HaCaT cells were irradiated with UVB.

[0144] 2. Experimental results:

[0145] After UVB irradiation, the intracellular ROS content in the model group increased significantly, while Epi-aszonalenin A and Asterrelenin significantly inhibited the generation of ROS. In addition, it was found that the ROS generation in the high-dose group (10 μM) of Epi-aszonalenin A and Asterrelenin was comparable to that in the blank group. This indicates that both Epi-aszonalenin A and Asterrelenin can effectively remove excess ROS in damaged HaCaT cells ( Figure 7 and Figure 8 ).

[0146] Example 5 Experiment on the inhibition of p65 protein nuclear translocation by Epi-aszonalenin A and Asterrelenin

[0147] 1. Experimental methods:

[0148] HaCaT cells (1×10 6 Cells were seeded in glass-bottomed culture dishes (Φ20 mm) with 100 μl of 4% paraformaldehyde (4% paraformaldehyde) at 4°C for 30 min after treatment with Epi-aszonalenin A and Asterrelenin, respectively, followed by UVB irradiation. The cells were then fixed with 4% paraformaldehyde at 4°C for 30 min, permeabilized with 0.2% triton X-100, and treated with 5% bovine serum albumin (BSA) for 1 h. The next day, the cells were stained with fluorophore-conjugated antibodies for two hours, labeled with 4′,6-diamidino-2-phenylindole (DAPI), and imaged with a confocal microscope.

[0149] Blank group: HaCaT was only added with serum-free DMEM medium, and was not treated with alkaloid compounds and was not irradiated with UVB.

[0150] Control group: An equal volume of serum-free DMEM medium was added to each well, and no alkaloid compound treatment was performed. HaCaT cells were irradiated with UVB to evaluate the degree of cell damage caused by UVB irradiation.

[0151] Sample group: 10mM stock solutions of two alkaloid compounds Epi-aszonalenin A and Asterrelenin were diluted with serum-free DMEM medium to a final concentration of 1μM and 10μM, and equal volumes were added to each well to treat cells for 2 hours. Afterwards, HaCaT cells were irradiated with UVB.

[0152] 2. Experimental results:

[0153] Immunofluorescence staining results showed that Epi-aszonalenin A and Asterrelenin could significantly inhibit the nuclear translocation of p65 under UVB stimulation ( Fig. 9 ).

[0154] Example 6 Western blot experiment of the mechanism of Epi-aszonalenin A and Asterrelenin against UV damage

[0155] 1. Experimental methods:

[0156] Epi-aszonalenin A and Asterrelenin are respectively 2 O 2 HaCaT and BJ cells were tested under controlled conditions of stress. Proteins were extracted using RIPA buffer and quantified by BCA assay. Gel electrophoresis was performed using SDS-PAGE, and then the proteins were transferred to NC membranes. The membranes were blocked in 5% skim milk for 2 hours. Afterwards, proteins were incubated with primary antibodies overnight and with secondary antibodies for 2 hours. Subsequently, imaging was performed using an enhanced chemiluminescence detection system, and image analysis was performed using ImageJ software.

[0157] Blank group: HaCaT and BJ cells were only fed with serum-free DMEM medium without alkaloid treatment, and were not exposed to UVB or H 2 O 2 Stimulate.

[0158] Control group: An equal volume of serum-free DMEM medium was added to each well, and no alkaloids were treated. HaCaT cells were irradiated with UVB to evaluate the degree of damage to cells caused by UVB. BJ cells were irradiated with H 2 O 2 Stimulation, evaluation H 2 O 2 Oxidative stress damage to BJ cells.

[0159] Sample group: 10mM stock solutions of two alkaloid compounds Epi-aszonalenin A and Asterrelenin were diluted with serum-free DMEM medium to a final concentration of 1μM and 10μM, and equal volumes were added to each well to treat cells for 2 hours. Afterwards, HaCaT cells were irradiated with UVB, while BJ cells were irradiated with H 2 O 2 Stimulate.

[0160] 2. Experimental results:

[0161] The experimental results are as follows Figure 10-Figure 31As shown, Western blot analysis showed that H 2 O 2 In contrast, pretreatment with Epi-aszonalenin A and Asterrelenin significantly downregulated the expression of H 2 O 2 Stimulated MMP-1 production, especially at high concentrations (10μM), the expression of MMP-1 tended to normal levels. Asterrelenin pretreatment significantly inhibited UVB-mediated phosphorylation of p38, ERK and JNK. Epi-aszonalenin A also reduced the phosphorylation levels of ERK and JNK, but not p38. Notably, Asterrelenin pretreatment significantly reduced the phosphorylation levels of c-Fos and c-Jun at the highest concentration (10μM). Epi-aszonalenin A pretreatment did not significantly change the extent of c-Jun phosphorylation, while c-Fos phosphorylation was significantly inhibited only in the highest dose group (10μM). Epi-aszonalenin A and Asterrelenin inhibited the expression of COX-2, iNOS, IL-6, IL-1β and p65 in a dose-dependent manner.

[0162] From the above examples, it can be seen that the alkaloid compounds Epi-aszonalenin A and Asterrelenin provided by the present invention can significantly inhibit the release of ROS, and can effectively inhibit the activation of the MAPK pathway induced by UVB, reduce the phosphorylation levels of c-Fos and c-Jun, thereby reducing the transcription of MMP-1, and can reduce the ultraviolet-induced inflammatory response by inhibiting the nuclear translocation of p65, especially in reducing pro-inflammatory markers such as IL-6, IL-1β, COX-2 and iNOS. Therefore, Epi-aszonalenin A and Asterrelenin have broad application prospects in the preparation of ultraviolet protection products.

[0163] The embodiments described are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of an alkaloid compound in the preparation of a product for inhibiting ultraviolet-induced skin damage, characterized in that: The alkaloid compound is any one of the following (1) to (4): (1) Epi-aszonalenin A; (2) a salt or ester of Epi-aszonalenin A; (3) Asterrelenin; (4) A salt or ester of Asterrelenin; The structural formula of the Epi-aszonalenin A is: ； The structural formula of Asterrelenin is: ； The product is a cosmetic or a medicine.

2. The use according to claim 1, characterized in that: The skin damage includes skin collagen degradation or skin inflammation.

3. A cosmetic for inhibiting ultraviolet-induced skin damage, characterized in that: The active ingredients include alkaloid compounds; The alkaloid compound is any one of the following (1) to (4): (1) Epi-aszonalenin A; (2) a salt or ester of Epi-aszonalenin A; (3) Asterrelenin; (4) A salt or ester of Asterrelenin; The structural formula of the Epi-aszonalenin A is: ； The structural formula of Asterrelenin is: 。 4. The cosmetic according to claim 3, characterized in that: The cosmetic also includes cosmetically acceptable excipients.

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