Application of sparganolide B in preparation of medicine for preventing or treating acne

Abstract

The invention discloses an application of sparganolide B in preparation of a medicine for preventing or treating acne. According to the present invention, the sparganolide B can significantly inhibit the protein expression levels of TLR2 and caspase1, inhibit the protein expression levels of inflammatory factor media TNF alpha and IL-1beta, and alleviate the inflammatory reaction induced by propionibacterium acnes, can be used as the selective drug for preventing or treating acne, and fills the blank of the sparganolide B in the treatment of acne or other skin inflammations.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of trifoliolide B in preparing drugs for preventing or treating acne. Background Art

[0002] Black Trigonum, also known as Trigonum, Light Trigonum, and Red Pugen, belongs to the family Sparganium. Black Trigonum is the tuber of Sparganium stoloniferum Buch-Ham, a plant of the family Sparganium. Its peeled and dried tuber is the traditional Chinese medicine Trigonum, which tastes pungent, bitter, and neutral in nature. It has the effects of removing blood stasis, promoting menstruation, breaking blood and eliminating symptoms. Trigonolide B, also known as Black Trigonolide B, is an extract from the tuber of Black Trigonum. Its structure was identified by nuclear magnetic resonance spectroscopy and X-ray crystallography as 8,5-dihydroxy-4-phenyl-5,2′-oxidized isocoumarin, with a molecular formula of C 15 H8O 5。

[0003] Trigonella lactone B is a polyphenolic compound with xanthone and isocoumarin, among which xanthone has antioxidant, immunomodulatory, cholesterol-lowering and other functions, while isocoumarin has anticoagulant, antitumor and anti-inflammatory effects. In recent years, it has been found that it can be used to treat a variety of diseases. The application number is 201110110987.9, and the invention name is "Application of Trigonella lactone B as TLR2 and TLR4 antagonist in pharmaceutical manufacturing". It explains from the perspective of mechanism of action that Trigonella lactone B is a selective TLR2 and TLR4 antagonist for the treatment of atherosclerosis, ischemia-reperfusion, tumors, chronic inflammatory bowel disease, diabetes, Alzheimer's disease, chronic obstructive pulmonary disease, sepsis, systemic lupus erythematosus, rheumatoid arthritis and other inflammatory diseases related to TLR signaling or autoimmune diseases.

[0004] Acne is the most common chronic inflammatory disease of the hair follicles and sebaceous glands in dermatology. It can cause post-inflammatory pigmentation and even leave permanent scars, which has a serious adverse impact on the patient's psychology and social life. The occurrence of acne is closely related to factors such as androgen levels and excessive sebum secretion, blockage of the hair follicle sebaceous gland ducts, bacterial infection and inflammatory response.

[0005] Existing literature has disclosed that the pathogenesis of acne is closely related to TLR2. For example, "Traditional Chinese and Western Medicine Treatment of Acne" reported that "TLR2 is a subtype of the TLRs family and shares common structural features with other TLR family members. It is closely related to the pathogenesis of acne. TLR2 can recognize peptidoglycan, the main component of the cell wall of Propionibacterium acnes, and can form a heterodimer with TLR6 with the participation of CD14, transmit signals through downstream inflammatory factors, stimulate cascade effects, and initiate inflammatory immune responses. Studies have also found that TLR2 levels are positively correlated with the severity of acne. Inhibiting the expression of TLR2 may prevent the further development of acne and avoid the formation of severe acne and scars" (see "Traditional Chinese and Western Medicine Treatment of Acne", Chen Zhiwei et al., page 11, Hubei Science and Technology Press, 20180131). Although the TLR2 pathway is closely related to acne, there is no literature report on whether TLR2 inhibitors can be used to treat acne.

[0006] At present, many researchers are considering using Chinese medicine components to inhibit or treat acne. The application number is 201910676883.0, and the invention name is "A Chinese medicine preparation for treating acne caused by phlegm and blood stasis and its preparation method", which discloses that a Chinese medicine preparation composed of Curcuma, Bupleurum, Trillium, etc. is used to treat acne caused by phlegm and blood stasis; the application number is 201510736208.4, and the invention name is "A medicine for treating cystic acne", which specifically discloses that a medicine composed of Radix Rehmanniae, Taraxaci, Herba Prunellae, and Herba Trillium, etc. is used to treat cystic acne; the application number is 201811607494.4, and the invention name is "A Chinese medicine composition for treating acne caused by spleen deficiency and dampness accumulation and its preparation method", which specifically discloses that a Chinese medicine composition composed of cinnamon twig, cortex moutan, Poria, Trillium, Herba Prunellae, etc. is used to treat acne caused by spleen deficiency and dampness accumulation. The application number is 202310624807.1 and the invention name is "A Trillium extract complex and its preparation method and application", which specifically discloses the application of a Trillium extract complex composed of Trillium extract, Coptis chinensis extract, Glycyrrhiza glabra extract and a base material in inhibiting skin acne.

[0007] In summary, as a selective TLR2 and TLR4 antagonist, trilobite B can be used to treat atherosclerosis, ischemia-reperfusion, tumors, chronic inflammatory bowel disease, diabetes, Alzheimer's disease, chronic obstructive pulmonary disease, sepsis, systemic lupus erythematosus, and rheumatoid arthritis, but it has not been found to be used to treat acne, nor has any other TLR2 inhibitor been reported to be used to treat acne. Although trilobite or trilobite extracts combined with other traditional Chinese medicine components can be used to treat phlegm and blood stasis type, cystic type, and spleen deficiency and dampness type acne, because the traditional Chinese medicine components are a mixture of multiple drugs, the specific mechanism of action is unclear, and the specific role of trilobite or trilobite extracts in the treatment of acne is unknown. It is even more unknown whether trilobite B, as a purified product, has a therapeutic effect on acne. Therefore, there is currently no literature reporting that trilobite B can be used for the prevention and treatment of acne. Summary of the invention

[0008] In view of the above-mentioned defects of the prior art, the present invention provides the use of trifoliolide B in the preparation of drugs for preventing or treating acne, filling the gap of trifoliolide B in the treatment of acne; in addition, the present invention also provides the use of trifoliolide B or a composition containing trifoliolide B in the preparation of cosmetics for preventing or improving acne, and trifoliolide B is used in cosmetics to prevent or improve acne.

[0009] The first aspect of the present invention provides the use of trifolinate B or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing trifolinate B in the preparation of a drug for preventing or treating acne, wherein the drug inhibits the TLR2-mediated signaling pathway, reduces the protein expression levels of the inflammatory mediators IL-1β, TLR2 and caspase1, and thereby inhibits the inflammatory response of acne.

[0010] Trigonella lactone B, its structural formula is as follows:

[0011]

[0012] Trigonella lactone B can simultaneously inhibit the three signaling pathways of IL-1β, TLR2 and caspase1, reduce the protein expression levels of inflammatory mediators IL-1β, TLR2 and caspase1, thereby inhibiting the inflammatory response of acne and achieving a therapeutic effect on acne.

[0013] The following components can be compounded with trifoliolide B. The compounding method is to mix the following components with trifoliolide B in a certain way and apply them to the skin surface or in the body; the following components and trifoliolide B can also be applied to the human body successively to improve the effect of preventing or treating acne.

[0014] 1) Rhein, chemical formula is C 15 H 10O5 can significantly inhibit the growth of sebaceous gland spots and the proliferation of sebaceous gland cells. It can also significantly inhibit the lipid synthesis and secretion of sebaceous gland cells, thereby inhibiting the vigorous secretion of sebaceous glands, thereby treating diseases with vigorous sebaceous gland secretion such as acne and seborrheic alopecia.

[0015] 2) Chlamydomonas oligosaccharides have the function of regulating the flora, specifically inhibiting the growth and proliferation of harmful bacteria and promoting the growth and proliferation of probiotics. In addition to Staphylococcus aureus, harmful bacteria include but are not limited to Propionibacterium acnes and Malassezia, and in addition to Staphylococcus aureus, probiotics include but are not limited to Bifidobacterium, etc. Chlamydomonas oligosaccharides can also promote the proliferation and migration of skin epidermal-related cells, relieve skin inflammation caused by chemical irritants such as surfactants, inhibit the expression of inflammation-promoting factors TNF-α, IL-1β, IL-8mRNA, and promote the expression of inflammation-suppressing factor IL-13; relieve skin inflammation by promoting cell autophagy.

[0016] 3) Total alkaloids of Coptis chinensis and / or volatile oil of Costusroot. Berberine, the main active ingredient in total alkaloids of Coptis chinensis, has the function of regulating TLRs expression and inhibiting the release of inflammatory factors. Volatile oil of Costusroot is an important biologically active ingredient of Costusroot, and also has anti-inflammatory and antibacterial effects. The combination of total alkaloids of Coptis chinensis and volatile oil of Costusroot can significantly improve swelling, reduce the degree of keratinization of the epidermis, reduce inflammatory response, and has preventive and therapeutic effects on acne.

[0017] 4) Homophytosine, whose chemical name is 4',5,7-trihydroxy-6-methoxyflavone, is a flavonoid compound that is widely found in plants such as Xinjiang Saussurea involucrata, Artemisia annua and Plantago asiatica. It has multiple biological activities and pharmacological effects such as anti-inflammatory, antioxidant and anti-mutagenic, and has an excellent inhibitory effect on rosacea.

[0018] 5) Retinoyl phytosphingosine is a product obtained by the condensation of retinoic acid and phytosphingosine. It has excellent effects in repairing the skin barrier, promoting skin tissue healing, anti-aging, anti-inflammatory, and anti-acne.

[0019] In some embodiments, the dose of trifoliolide B is positively correlated with the effect of inhibiting acne inflammatory response.

[0020] In some embodiments, the drug is a drug that reduces the expression level of TNFα.

[0021] In some embodiments, the drug for preventing or treating acne contains trifoliolide B as a main ingredient.

[0022] The second aspect of the present invention provides a drug for preventing or treating acne, wherein the drug is a preparation made from trifolinate B or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing trifolinate B and a pharmaceutically acceptable excipient.

[0023] In some embodiments, the drug is administered parenterally.

[0024] In some embodiments, the drug is administered via silica sponge spicules.

[0025] The siliceous sponge spicule in application number 202310560808.4 is used to wrap triangular lactone B on the surface of the siliceous sponge spicule. Without excessively increasing the diameter of the siliceous sponge spicule, the microenvironment of the relevant position can also be improved on the basis of forming a microchannel.

[0026] In some embodiments, the drug is in the form of an injection, a liniment, a nanopowder, an ointment, or a gel.

[0027] The injections here are injected with water-light needles. As an injectable skin care therapy, water-light needles use a special instrument called "water-light gun" to inject the required beauty injections into the dermis just below the epidermis. Water-light needles are mainly injected with hyaluronic acid, which can make the skin hydrated and bright after injection. Water-light needles can also inject other nutrients, such as type I collagen, type II collagen, type VII collagen, biopolysaccharide gum, sheep placenta extract, L-C stock solution, grape seed extract, etc.; amino acid substances such as glycine, alanine, proline, hydroxylysine, hydroxyproline, etc. can be injected; coenzyme substances such as Q10, niacinamide and vitamin B2, etc. can be injected; anti-allergic substances such as otamine, dipotassium glycyrrhizinate, Stephania tetrandra extract, rhamnose, biopolysaccharide Glycoside-2, Centella asiatica extract, Glycyrrhiza glabra root extract, Chamomile flower extract, Rosemary extract, ceramide, etc.; antioxidant substances can be injected, such as anthocyanins, superoxide dismutase, water-soluble vitamin E, tea polyphenols, retinol, glutathione reductase, glutathione peroxidase, glutathione S-transferase, tea polyphenols, etc.; sunscreen substances can be injected, such as glabridin, rutin, and polydatin; moisturizing substances can be injected, such as glycerol, trehalose, panthenol, 1,3-butylene glycol, 1,2-propylene glycol, 1,2-hexanediol, betaine, etc.

[0028] Liniment is a liquid preparation for external use that is rubbed on the skin surface or applied with a soft brush. Liniment can also be applied on a flannel cloth to cover the skin surface. Due to rubbing, some drug components in the liniment can be absorbed through the skin. Liniments with different purposes have different dispersion media. Liniments that protect and moisturize the skin often use oil as a dispersion medium, such as bone-clearing detergent; analgesic and anti-inflammatory liniments often use dimethyl sulfoxide diluent as a dispersion medium to increase penetration, such as compound dexamethasone liniment. There are also liniments that use glacial acetic acid as a dispersion medium, such as minoxidil liniment. Cosolvents such as glycerin, vegetable oil, methanol, propylene glycol, ethanol, etc. may also be added to the liniment; binders such as corn starch, povidone, polyethylene glycol, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, gelatin, gum arabic, sodium alginate, etc. may also be added; stabilizers such as sodium alginate, citric acid, pregelatinized starch, povidone, sodium carboxymethyl cellulose, hydroxypropyl cellulose, glycerin, hydroxypropyl methylcellulose, etc. may also be added. The trilobalide B in the present application may be dispersed in a dimethyl sulfoxide diluent, in which a cosolvent and / or a stabilizer is mixed.

[0029] Nano powder, with the same dosage, has more active ingredients and a wider contact area with the skin, and the treatment effect is better. Nano powder can be applied to the skin surface by spraying, or it can be dispersed by adding solvent and applied to the skin surface as a liniment.

[0030] Ointment is a semi-solid or nearly solid dosage form for external use made of matrix and drug. It protects the wound surface, lubricates the skin and has local treatment effects. It can also penetrate the skin or mucous membrane to have a systemic treatment effect. Ointments are divided into ointments and plasters. Ointments are semi-solid dosage forms for external use made by mixing drugs, fine powder of traditional Chinese medicine, extracts of traditional Chinese medicine and suitable matrix; plasters are formed by applying drugs and viscous matrix to the backing material. The matrix in the ointment includes: O / W emulsion matrix, W / O emulsion matrix, water-absorbent ointment matrix, animal fat, vegetable oil, and hydrocarbon matrix. Some additives are also added to the ointment, such as surfactants, which increase the solubility of the drug and the wettability of the skin, help the drug to disperse, and promote drug penetration; such as penetration enhancers, such as dimethyl sulfoxide, azone, etc., which accelerate the penetration of drugs through the skin, increase the permeability of local medications, and increase the percutaneous absorption of drugs; other additives include preservatives, cosolvents, emulsifiers, antioxidants, thickeners, skin penetration enhancers, etc. The trifoliolide B in the present application is mixed with a base for ointment to prepare an ointment, and dimethyl sulfoxide is added at the same time, which can not only increase the dispersion effect of trifoliolide B, but also accelerate the penetration effect of the drug and enhance the percutaneous absorption of the drug.

[0031] Gel, or gel preparation, requires the drug component and carrier to be assembled into a drug-carrier complex, and then the excipient ingredients are added. Commonly used drug carriers include silk fibroin, fibrin, globulin, etc. Commonly used excipient ingredients include chitosan and its derivatives, cellulose and its derivatives, gelatin, sodium hyaluronate, polyvinyl pyrrolidone, cyclodextrin, sodium alginate, etc.

[0032] The third aspect of the present invention provides the use of trifoliolide B or a composition containing trifoliolide B in the preparation of cosmetics for preventing or improving acne.

[0033] In some embodiments, the cosmetic is selected as a skin care product.

[0034] As a preferred embodiment, the cosmetics include cleansing milk, toner, lotion, emulsion, facial mask, facial cream, essence, and essential oil.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The trifoliolide B in the present invention can significantly inhibit the expression levels of inflammatory mediators IL-1β, TLR2 and caspase1 proteins, reduce the inflammatory response induced by Propionibacterium acnes, and can be used as an optional drug for preventing or treating acne, filling the gap of trifoliolide B in the treatment of acne or other skin inflammations.

[0037] (2) The trifoliate lactone B in the present invention is derived from the traditional Chinese medicine Trifoliate lactone and has high safety. In addition, since trifoliate lactone B can also be used for chemical synthesis, the problem of a small amount of trifoliate lactone B extracted from traditional Chinese medicine is avoided, which facilitates the realization of industrial production and the widespread promotion of trifoliate lactone B.

[0038] (3) The trifoliolide B in the present invention can be used in a variety of preparation forms, such as microneedle injection, nanopowder spraying, etc. Trifoliolide B enters cells and inhibits the inflammatory response of Propionibacterium acnes to alleviate acne symptoms. A variety of preparation forms allow patients to flexibly choose the dosage form according to their own conditions, avoiding the conventional single application method of skin patients.

[0039] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The present invention adopts the western blot method to measure the TLR2, IL-1β, and Caspase-1 proteins under different experimental conditions;

[0041] Figure 2This is a bar chart of statistical analysis of TLR2 protein expression levels under different experimental conditions measured by western blot method in the present invention, where * indicates significant difference (P < 0.05), and ** indicates extremely significant difference (P < 0.01);

[0042] Figure 3 It is a bar chart of the statistical analysis of the IL-1β protein expression level under different experimental conditions determined by the western blot method in the present invention, wherein ns means no significant difference, * means significant difference (P < 0.05), and ** means extremely significant difference (P < 0.01);

[0043] Figure 4 The present invention adopts the western blot method to determine the expression level of Caspase-1 protein under different experimental conditions. The ns indicates no significant difference, the * indicates a significant difference (P < 0.05), and the ** indicates a very significant difference (P < 0.01).

[0044] Figure 5 In the mouse experiment of the present invention, Propionibacterium acnes was used to induce ear inflammation in mice, and then trilobacide B was applied. After 48 hours of administration, the degree of redness and swelling caused by ear inflammation in mice was measured;

[0045] Figure 6 In the mouse experiment of the present invention, Propionibacterium acnes was used to induce ear inflammation in mice, and then trifoliolide B was applied. After 48 hours of administration, the expression of inflammatory factors in the mouse ears was measured. DETAILED DESCRIPTION

[0046] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0047] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0048] "Traditional Chinese and Western Medicine Treatment of Acne" discloses that TLR2 is closely related to the pathogenesis of acne, and inhibiting the expression of TLR2 may prevent the further development of acne and avoid the formation of severe acne and scars. According to the records of "Traditional Chinese and Western Medicine Treatment of Acne", inhibiting the TLR2 pathway may prevent the development of acne and avoid the aggravation of acne, and can be used for the prevention and treatment of acne.

[0049] However, the records in the existing literature are contrary to this. In the literature records in the past 10 years, there has been no report on the use of TLR2 inhibitors to treat acne. For example, Yumiko Imai disclosed an oxidized phospholipid OxPAPC in "Identification of Oxidative Stress and Toll-like Receptor 4 Signaling as a Key Pathway of Acute Lung Injury", which can inhibit TLR2 and TLR4-mediated inflammatory responses for acute lung injury, but did not disclose that it can be used to treat acne. For another example, Pragnesh Mistry disclosed a TLR2 inhibitor C29 in "Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain", application number 202310852839.7, for the treatment of chronic migraine. For example, application number 202310682470.X discloses a TLR2 inhibitor, taspine-derived compound, which is used to regulate abnormally activated immune responses in the intestine and alleviate the symptoms of inflammatory bowel disease.

[0050] Therefore, although the TLR2 pathway is closely related to the pathogenesis of acne, inhibiting the TLR2 signaling pathway alone may not achieve the therapeutic effect on acne.

[0051] In addition, B. Dreno further disclosed in "Understanding innate immunity and inflammation inacne: implications for management" that "Qin et al. provided evidence that P. acnes contributes to the inflammatory response seen in acne by triggering the activation of the NLRP3-inflammasome in antigen-presenting cells, subsequently leading to enhanced IL-1b secretion…Further, bacterial phagocytosis has anessential role in NF-kb dependent That is, the document records that the inflammatory response of acne is not limited to the TLR2-mediated pathway. IL-1β, NLRP3 inflammasome, cytokines, chemokines, immune cells and oxidative stress can all induce the inflammatory response of acne. Inhibiting any one of these signaling pathways alone will have little effect on the treatment of acne, which also proves that there is no document recording that TLR2 inhibitors can be used to treat acne.

[0052] As a selective TLR2 and TLR4 antagonist, triangularis lactone B can be used to treat atherosclerosis, ischemia-reperfusion, tumors, chronic inflammatory bowel disease, diabetes, Alzheimer's disease and other diseases, but has not yet been found to be useful for treating acne. There are literature reports that triangularis lactone or triangularis lactone extracts combined with other traditional Chinese medicine components can be used to treat acne, but because there are many components involved and the mechanism of action is unclear, the role of triangularis lactone B is unknown, and whether triangularis lactone B has a therapeutic effect on acne is unknown. Currently, there is no research on the use of triangularis lactone B as an anti-acne drug.

[0053] During the process of developing the range of diseases treated by trifolinate B, the inventors of the present application accidentally discovered that trifolinate B has a therapeutic effect on acne by inhibiting three signaling pathways: IL-1β, TLR2 and caspase1.

[0054] Test Example 1 Determination process of the effect of trilobacinolone B on the inflammatory response caused by Propionibacterium acnes:

[0055] 1. Cultivate THP-1 monocytes. THP-1 monocytes were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. The culture medium used was 1640 culture medium containing 10% FBS (fetal bovine serum). The brands of 1640 culture medium (Cat. No. C11875500BT) and FBS (Cat. No. 10091148) were both Thermo;

[0056] 2. 2*10 6 THP-1 monocytes were evenly divided into six-well cell culture plates, and 100 ng / ml phorbol myristate ester (PMA) was added to induce differentiation for 24 h, the supernatant was discarded, and fresh culture medium was replaced;

[0057] 3. The subjects were randomly divided into 6 groups: (1) normal group, (2) model group, (3) low-dose trilobite B group, (4) medium-dose trilobite B group, (5) high-dose trilobite B group, and (6) DMSO group;

[0058] 4. Add DMSO solution containing trifolinate B (purchased from Sigma, catalog number SML-1767) to (3) trifolinate B low-dose group, (4) trifolinate B medium-dose group, and (5) trifolinate B high-dose group, respectively, wherein the final concentrations of groups (3), (4), and (5) are 5 μM, 50 μM, and 100 μM, respectively. Add the same amount of DMSO to (6) DMSO group and incubate for 4 h;

[0059] 5. After 4 hours, Propionibacterium acnes inactivated by high temperature and high pressure steam was added to groups (2), (3), (4), and (5) at a ratio of 1:100 for THP-1 monocytes and Propionibacterium acnes to induce inflammatory response. After 48 hours, total cell protein was extracted and the protein expression levels of TLR2, IL-1β, and Caspase-1 were determined by western blot, and the significant differences were calculated. The results are shown in Table 1. Figure 1 , 2 , 3, and 4.

[0060] like Figure 1 The first row indicates the volume of DMSO added to different groups; the second row indicates the concentration of trilobacin B added to different groups; the third row indicates whether Propionibacterium acnes is added, - indicates that Propionibacterium acnes is not added, + indicates that Propionibacterium acnes is added; the fourth row indicates the blot of TLR2 protein; the fifth row indicates the blot of IL-1β protein; the sixth row indicates the blot of Caspase-1 protein, and the seventh row (Tublin) indicates the blot of the internal control. Figure 1 The corresponding blots of TLR2 protein, IL-1β protein, and Caspase-1 protein in the six groups, respectively. Among them, Propionibacterium acnes was added to the model group (corresponding to the second column), and the grayscale of the blots of TLR2 protein, IL-1β protein, and Caspase-1 protein were all darker, indicating that the content of TLR2 protein, IL-1β protein, and Caspase-1 protein was high; the low-dose group of trilobite B (corresponding to the third column, 5μM), the medium-dose group of trilobite B (corresponding to the fourth column) In the high-dose group (corresponding to the fifth column, 50 μM) and the high-dose group (corresponding to the fifth column, 100 μM), the grayscale corresponding to the TLR2 protein, IL-1β protein, and Caspase-1 protein blots gradually became lighter due to the increase in the dosage of trifoliolide B, indicating that the presence of trifoliolide B has an inhibitory effect on Propionibacterium acnes, and with the increase in the dosage of trifoliolide B, the inhibitory effect on Propionibacterium acnes gradually increases, and the contents of TLR2 protein, IL-1β protein, and Caspase-1 protein decrease successively.

[0061] like Figure 2 , 3, 4. The model group (P.acnes) had the highest protein contents of TLR2, IL-1β, and Caspase-1, indicating that after the addition of Propionibacterium acnes, Propionibacterium acnes induced an inflammatory response, which significantly increased the protein contents of TLR2, IL-1β, and Caspase-1; while the corresponding protein contents of TLR2, IL-1β, and Caspase-1 in the low-dose group, medium-dose group, and high-dose group of trifoliolide B were significantly lower than those in the model group, indicating that trifoliolide B has an inhibitory effect on the inflammatory response induced by Propionibacterium acnes, and with the increase of the concentration of trifoliolide B, the protein contents of TLR2, IL-1β, and Caspase-1 decreased significantly. The dosage of trifoliolide B is positively correlated with the effect of inhibiting the inflammatory response of acne within a certain range.

[0062] In addition, by calculating Figure 2 The significant difference levels of the corresponding TLR2 protein contents in the low-dose group, medium-dose group, and high-dose group of triangular lactone B relative to the model group, and the significant difference levels of the model group relative to the control group showed that the P value of the model group relative to the control group was 0.0062, the P value of the low-dose group of triangular lactone B relative to the model group was 0.031, the P value of the medium-dose group of triangular lactone B relative to the model group was 0.0118, and the P value of the high-dose group of triangular lactone B relative to the model group was 0.0085. It can be seen that the corresponding TLR2 protein contents in the low-dose group, medium-dose group, and high-dose group of triangular lactone B were significantly different from those in the model group.

[0063] By calculation Figure 3 The significant difference levels of IL-1β protein content in the low-dose group, medium-dose group and high-dose group of triangular lactone B relative to the model group, and the significant difference levels of the model group relative to the control group showed that the P value of the model group relative to the control group was 0.0038, the P value of the low-dose group of triangular lactone B relative to the model group was 0.8478, the P value of the medium-dose group of triangular lactone B relative to the model group was 0.0236, and the P value of the high-dose group of triangular lactone B relative to the model group was 0.0171. It can be seen that the corresponding IL-1β protein content in the medium-dose group and high-dose group of triangular lactone B was significantly different from that in the model group.

[0064] By calculation Figure 4The significant difference levels of the corresponding Caspase-1 protein contents in the low-dose group, medium-dose group and high-dose group of triangular lactone B relative to the model group, and the significant difference levels of the model group relative to the control group showed that the P value of the model group relative to the control group was 0.0241, the P value of the low-dose group of triangular lactone B relative to the model group was 0.1225, the P value of the medium-dose group of triangular lactone B relative to the model group was 0.0381, and the P value of the high-dose group of triangular lactone B relative to the model group was 0.0496. It can be seen that the corresponding Caspase-1 protein contents in the medium-dose group and high-dose group of triangular lactone B were significantly different from those in the model group.

[0065] In summary, trilobacinolone B can simultaneously inhibit the three signaling pathways of IL-1β, TLR2 and caspase1, thereby inhibiting the inflammatory response caused by Propionibacterium acnes and can be used in anti-acne drugs.

[0066] Experimental Example 2: Experimental process of mice using trilobacinol B to inhibit inflammatory response caused by Propionibacterium acnes:

[0067] 1. Propionibacterium acnes (P. acnes) was inoculated into the reinforced clostridial medium (Oxoid, Basingstoke, UK) at -80°C and cultured anaerobically at 37°C until OD600 reached the middle of the logarithmic growth phase.

[0068] 2. Collect the bacteria, centrifuge, wash three times with PBS (pH 7.2), and resuspend in PBS.

[0069] 3. 2.5X10 7 CFU P.acnes (20 μL) was intradermally injected into the left and right ears of the experimental group mice. After the injection of P.acnes, the right ear was smeared with trifolinate B (DMSO solution containing 50 μM trifolinate B, the smear volume was 100 μL), and the left ear was smeared with an equal amount of DMSO. The group injected with PBS was used as the blank group, the left ear as the control group, and the right ear as the experimental group.

[0070] 4. Measure the thickness of mouse ears at 48 hours. After 48 hours, kill the mice and take the homogenate of the mice ears to detect the changes of cytokines IL-1β and TNFα using ELISA Kit.

[0071] The results are as follows Figure 5 , 6 shown.

[0072] like Figure 5A is a picture of the left and right ears of some mice. Propionibacterium acnes (P.acnes) was injected into the left and right ears of the mice to induce an inflammatory and swollen phenotype. After the injection, trilobacide B was applied to the right ear, and an equal volume of DMSO was applied to the left ear. After 48 hours, it can be seen that the degree of redness and swelling in the right ear is lower than that in the left ear.

[0073] like Figure 5 B. The ear thickness of all mice in the control group and the experimental group was counted and their average values ​​were taken. It can be seen that the ear thickness of mice in the experimental group was significantly lower than that in the control group, with a significant difference. It can be seen that for acne caused by Propionibacterium acnes, applying trilobite B can reduce the degree of redness and swelling and has a certain therapeutic effect.

[0074] like Figure 6 This is a statistical chart of the experimental results of ELISA Kit detecting changes in cytokines IL-1β and TNFα, that is, mice were killed 48 hours later, and the mouse ears were homogenized and ELISA Kit was used to detect changes in cytokines IL-1β and TNFα. Among them, Figure 6 A is the change result of TNFα, Figure 6 B is the change result of IL-1β. It can be seen that compared with the control group applied with DMSO, the IL-1β and TNFα of the experimental group applied with trilobacide B were significantly reduced, and the IL-1β and TNFα levels of the experimental group were only slightly higher than those of the blank group without Propionibacterium acnes injection, which had a significant therapeutic effect.

[0075] The present invention provides the use of trifoliolide B or its pharmaceutically acceptable salt or a pharmaceutical composition containing trifoliolide B in preparing a drug for preventing or treating acne. The drug can significantly reduce the protein expression levels of TLR2, caspase1 and IL-1β; and can also significantly reduce the level of TNFα, thereby inhibiting the inflammatory response of acne.

[0076] The present invention also provides a medicine for preventing or treating acne, which is a preparation made from trifoliolide B or its pharmaceutically acceptable salt or a pharmaceutical composition containing trifoliolide B and pharmaceutically acceptable excipients.

[0077] Trigonella lactone B can be used as an active ingredient alone and added with excipients to prepare anti-acne drugs. The drug can be administered parenterally, such as injecting Trigonella lactone B into the dermis under the epidermis with a water light needle, or dissolving Trigonella lactone B in DMSO to prepare a liniment and applying it on the skin surface, or preparing Trigonella lactone B into a nano powder and spraying it on the skin surface. Under this preparation method, the same amount of Trigonella lactone B has a wider contact area with the skin and a better therapeutic effect.

[0078] Trigonella lactone B can also be combined with other components to treat acne. The components that can be combined include emodin, which can inhibit the growth of sebaceous gland spots, the proliferation of sebaceous gland cells, the synthesis and secretion of sebaceous gland cells, and the vigorous secretion of sebaceous glands; or total alkaloids of Coptis chinensis, which can regulate the expression of TLRs and inhibit the release of inflammatory factors; or volatile oil of Aucklandia lappa, which has anti-inflammatory and antibacterial effects; or psyllium, which has multiple biological activities and pharmacological effects such as anti-inflammatory, antioxidant, and anti-mutation; or retinoyl phytosphingosine, which has excellent effects in repairing the skin barrier, promoting skin tissue healing, anti-aging, anti-inflammatory, and anti-acne.

[0079] Also provided is the use of trifoliolide B or a composition containing trifoliolide B in cosmetics for preventing or improving acne. Trifoliolide B or a composition containing trifoliolide B can be used in cosmetics to prevent or improve acne. The cosmetics herein can be skin care products, including cleansing milk, toner, lotion, emulsion, facial mask, facial cream, essence, essential oil.

[0080] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Use of trifolinate B or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing trifolinate B in the preparation of a drug for preventing or treating acne, wherein the drug inhibits the TLR2-mediated signaling pathway, reduces the protein expression levels of the inflammatory mediators IL-1β, TLR2 and caspase1, and thereby inhibits the inflammatory response of acne.

2. The use according to claim 1, characterized in that The dose of trifolinate B was positively correlated with its effect in inhibiting acne inflammatory response.

3. The use according to claim 1, characterized in that The drug is a drug for reducing the expression level of TNFα.

4. The use according to claim 1, characterized in that The acne prevention or treatment drug contains trifoliolide B as a main component.

5. A drug for preventing or treating acne, characterized in that: The medicine is a preparation made from trifoliolide B or its pharmaceutically acceptable salt or a pharmaceutical composition containing trifoliolide B and pharmaceutically acceptable excipients.

6. The drug for preventing or treating acne according to claim 5, characterized in that: The medicine is in the form of an injection, an ointment, a nano powder, an ointment or a gel.

7. Use of trifoliolide B or a composition containing trifoliolide B in the preparation of cosmetics for preventing or improving acne.

8. The use according to claim 7, characterized in that The cosmetics are selected as skin care products.

9. The use according to claim 7, characterized in that The cosmetics include cleansing milk, toner, lotion, emulsion, facial mask, facial cream, essence and essential oil.

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