New application of total phenolic acid extract of trollius chinensis bunge

Abstract

The invention relates to the technical field of biological medicines, discloses a novel application of a trollflower total phenolic acid extract, and provides an application of the trollflower total phenolic acid extract in preparation of a medicine for treating acnes, and the preparation process of the trollflower total phenolic acid extract comprises the following steps: (1) adding a 50-90% ethanol solution into trollflower for reflux extraction, filtering and collecting filtrate; (2) concentrating the filtrate to obtain a concentrated solution with the solid content of 0.1-0.4 g / mL; and (3) adjusting the pH value of the concentrated solution to 2-6, loading the concentrated solution on a macroporous resin adsorption column, eluting with water, then eluting with a 20-80% ethanol solution, and concentrating and drying the collected ethanol eluent. The invention provides a novel application of the total phenolic acid extract of trollflower in preparation of a medicine for treating acne, and the total phenolic acid extract of trollflower has a very high inhibition rate on propionibacterium acnes and staphylococcus aureus; a rat acne model is constructed to further verify that the trollius chinensis bunge total phenolic acid extract has a good curative effect on acne treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a novel application of a total phenolic acid extract from golden lotus. Background Technology

[0002] Acne, also known as acne vulgaris or simply "pimples," is a common chronic inflammatory skin disease. In traditional Chinese medicine, it's called "pimples." The incidence rate among adolescent males and females can reach as high as 54%. The main clinical manifestations of acne include comedones, papules, pustules, cysts, and nodules. It not only affects the skin health of patients but also negatively impacts the psychological and social lives of adolescents. The causes of acne are complex. Abnormal androgen levels, excessive sebum secretion, abnormal keratinization of cells around the hair follicles, bacterial infection, psychological stress, and immune abnormalities can all influence the onset or worsen the condition. Drug treatment primarily uses retinoic acid, antibiotics, or hormones to reduce sebum secretion, inhibit Propionibacterium acnes, and reduce inflammation.

[0003] Most existing acne medications cause adverse reactions such as redness, dryness, and burning, and long-term use can lead to bacterial resistance. Therefore, there is an urgent need to find safer and more effective drugs, and natural remedies, with their high efficacy and low toxicity, offer a new option for acne treatment.

[0004] Golden lotus is a plant belonging to the genus *Trollius* in the family Ranunculaceae. Trollius chinensis The dried flowers of *Trollius chinensis* (also known as golden lotus), first recorded in *Compendium of Materia Medica Supplement*, have been shown by modern pharmacology to possess anti-inflammatory, antibacterial, antioxidant, antiviral, and antitumor effects. Phenolic acids are among its main active components, primarily protopterocaryonic acid and veratronic acid. However, there are currently no reports on the effects of total phenolic acids from *Trollius chinensis* on inhibiting *Propionibacterium acnes* and treating acne. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of existing acne treatment drugs, which mostly cause adverse reactions such as skin redness, dryness, and burning, and long-term use can lead to bacterial resistance. This invention provides a new application of the total phenolic acid extract of golden lotus.

[0006] To achieve the above objectives, the present invention provides an application of total phenolic acid extract from *Trollius chinensis* in the preparation of a medicament for treating acne, wherein the total phenolic acid extract from *Trollius chinensis* is prepared according to the following preparation steps: (1) Add 50-90% ethanol solution to the golden lotus and reflux extract, filter and collect the filtrate; (2) The collected filtrate is concentrated to obtain a concentrated solution with a solid content of 0.1-0.4 g / mL; (3) Adjust the pH of the concentrate to 2-6, then load it onto a macroporous resin adsorption column. After adsorption equilibrium is reached, first wash with water, then wash with 20-80% ethanol solution, collect the ethanol eluent, and then concentrate and dry it.

[0007] Preferably, the drug is a tablet, capsule, oral liquid, injection, cream, or gel.

[0008] Preferably, in step (1), the weight ratio of the golden lotus flower to the ethanol solution is 1:10-50.

[0009] Preferably, in step (1), the reflux extraction conditions include: a temperature of 50-90℃ and a time of 30-150min.

[0010] Preferably, in step (3), the packing material in the macroporous resin adsorption column is selected from HPD100 type macroporous resin, NKA-9 type macroporous resin, HPD400 type macroporous resin, D101 type macroporous resin and AB-8 type macroporous resin, and preferably HPD100 type macroporous resin.

[0011] Preferably, the sample loading amount of the macroporous resin adsorption column is 20-50g of golden lotus for every 100g of macroporous resin.

[0012] Preferably, in step (3), the elution flow rate of the ethanol solution is 1-4 BV / h, and the elution volume is 1-8 BV.

[0013] Preferably, the main components of the total phenolic acid extract of the golden lotus are guava acid, trans-cinnamic acid, caffeic acid, veratric acid, arbutin, quercetin, protopterocaryonic acid, arbutin-2''-O-β-D-xylanopyranoside and 6'''-HMG-2''-O-β-L-galactoside arbutin.

[0014] Preferably, the total phenolic acid extract of the golden lotus further contains vitexin, hyperoside, harvestin-2''-O-β-D-xylanopyranoside, 2''-O-(2'''-methylbutyryl)vitexin, 7-2''-O-(2'''-methylbutyryl)harvestin, 2''-O-feruloylvitexin, 6'''-HMG-2''-O-β-L-galactosideharvestin, 6''-malonylastragalin, 6''-malonyl cosmosiin, isochlorogenic acid A, ferulic acid, vanillic acid, p-hydroxycinnamic acid and 2'''-methylbutyrylharvestin.

[0015] The second aspect of this invention provides the application of total phenolic acid extract from golden lotus in the preparation of skin care products that improve acne.

[0016] The third aspect of this invention provides the application of total phenolic acid extract from golden lotus in the preparation of in vitro inhibitors of Propionibacterium acnes.

[0017] Preferably, the total phenolic acid extract of the golden lotus is prepared according to the following preparation steps: (1) Add 50-90% ethanol solution to the golden lotus and reflux extract, filter and collect the filtrate; (2) The collected filtrate is concentrated to obtain a concentrated solution with a solid content of 0.1-0.4 g / mL; (3) Adjust the pH of the concentrate to 2-6, then load it onto a macroporous resin adsorption column. After adsorption equilibrium is reached, first wash with water, then wash with 20-80% ethanol solution, collect the ethanol eluent, and then concentrate and dry it.

[0018] This invention provides novel applications for the total phenolic acid extract of *Trollius chinensis*, particularly in the preparation of drugs for treating acne, skincare products for improving acne, and in the preparation of in vitro inhibitors of *Propionibacterium acnes*. Experiments show that the total phenolic acid extract of *Trollius chinensis* described in this invention is effective against *Propionibacterium acnes* (…). C.acnes ) and Staphylococcus aureus ( S.aureus It exhibits a high inhibition rate, with inhibition zone diameters of 17.9 mm and 21.2 mm against Propionibacterium acnes and Staphylococcus aureus, respectively, both exceeding the inhibition zone diameter of veratril at the same concentration. However, its inhibitory effect on Propionibacterium acnes has not yet been reported.

[0019] Given that *Propionibacterium acnes* and *Staphylococcus aureus* are the main pathogens causing acne, the total phenolic acid extract of *Trollius chinensis* prepared in this invention can be used not only for in vitro antibacterial purposes for non-disease treatment but also for clinical treatment of acne. Furthermore, this invention also established a rat acne model to further verify the good efficacy of the total phenolic acid extract of *Trollius chinensis* in treating acne. Attached Figure Description

[0020] Figure 1 The total phenolic acid extract of golden lotus and veratric acid prepared in Example 1 of this invention are... C. acnes, S. aureus The inhibition zone; where A is the solvent control (DMSO); B is veratric acid; and C is the total phenolic acid extract of golden lotus prepared in Example 1. **** P <0.0001; Figure 2 The total phenolic acid extracts of *Trollius chinensis* prepared in Example 1 and Comparative Example 1 of this invention are effective against... C. acnes, S. aureus The inhibition zone; where A is the solvent control (DMSO); B is the total phenolic acid extract of *Trollius chinensis* prepared in Comparative Example 1; and C is the total phenolic acid extract of *Trollius chinensis* prepared in Example 1. **** P<0.0001; Figure 3 These are the ear thickness measurement results of rats in each group in Test Example 3 of this invention; among them, compared with the blank group, #### P <0.0001; compared with the model group, **** P <0.0001; Figure 4 These are morphological and H&E staining images of the surface of the rat ears in each group of test examples 3 of this invention; where the magnification is 100x. Figure 5 These are the results of the detection of inflammatory factors (IL-6, TNF-α, IL-17A, IL-10) in the serum of rats in each group of test examples 3 of this invention; among them, compared with the blank group, # P <0.05, ## P <0.01, ### P <0.001, #### P <0.0001; compared with the model group, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Detailed Implementation

[0021] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] In this invention, all ethanol concentrations are volume concentrations, such as 40% ethanol, which means that 100 ml of ethanol aqueous solution contains 40 ml of ethanol; BV in this invention refers to column volume.

[0024] This invention provides the application of total phenolic acid extract from *Trollius chinensis* in the preparation of a medicament for treating acne, wherein the total phenolic acid extract from *Trollius chinensis* is prepared according to the following preparation steps: (1) Add 50-90% ethanol solution to the golden lotus and reflux extract, filter and collect the filtrate; (2) The collected filtrate is concentrated to obtain a concentrated solution with a solid content of 0.1-0.4 g / mL; (3) Adjust the pH of the concentrate to 2-6, then load it onto a macroporous resin adsorption column. After adsorption equilibrium is reached, first wash with water, then wash with 20-80% ethanol solution, collect the ethanol eluent, and then concentrate and dry it.

[0025] The purification principle of the macroporous adsorption resin used in this invention is as follows: Macroporous resin adsorption is a common purification technique for traditional Chinese medicine, widely used in the extraction and separation of various components including total phenolic acids, total flavonoids, total alkaloids, terpenes, and polysaccharides. The principle is to first selectively adsorb chemical molecules of suitable molecular weight using van der Waals forces and hydrogen bonds between macroporous adsorption resin molecules. Then, based on the solubility and polarity of the chemical molecules, appropriate solvents are used for elution, thereby selectively separating the target analyte from the macroporous resin. Since different traditional Chinese medicinal materials contain different types of phenolic acid compounds, the molecular weight, intermolecular van der Waals forces, polarity, and solubility in organic solvents also vary. Therefore, different purification conditions are required for different traditional Chinese medicinal materials. Among these, the resin type and elution solvent are the most critical. Different types of resins have different van der Waals forces, resulting in different adsorption and desorption capacities for the target analyte. Furthermore, elution solvents of different polarities directly determine whether the target analyte can be eluted from the resin. This invention, through screening purification conditions, not only effectively separates the total phenolic acids from golden lotus, but also achieves high product purity and yield.

[0026] When preparing acne treatment drugs using the total phenolic acid extract of *Trollius chinensis*, the extract can be formulated into various preparations suitable for clinical use according to pharmaceutical methods well known in the art. In some embodiments, the drug may be tablets, capsules, oral liquids, injections, creams, gels, liquid preparations, etc.

[0027] In a preferred embodiment, the drug is a topical skin preparation, such as a cream, gel, or liquid preparation.

[0028] In this invention, the golden lotus used in step (1) is a medicinal material of golden lotus.

[0029] In some embodiments, the drug may also contain other commonly used acne treatment drugs, and the efficacy may be further improved by combining the total phenolic acid extract of golden lotus with other acne treatment drugs. These pharmaceutical uses of adding the total phenolic acid extract of golden lotus and using it for acne are all within the scope of protection of this invention.

[0030] In this invention, in step (1), the concentration of the ethanol solution used for reflux extraction is 50-90%, specifically, for example, the concentration of the ethanol solution can be 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, or 90%. To further improve the yield of the total phenolic acid extract of *Trollius chinensis*, in a preferred embodiment, the concentration of the ethanol solution used for reflux extraction in step (1) is 58-62%.

[0031] In a preferred embodiment, in step (1), the weight ratio of the golden lotus flower to the ethanol solution is 1:10-50, specifically, for example, 1:10, 1:15, 1:18, 1:20, 1:22, 1:25, 1:30, 1:40 or 1:50.

[0032] In a more preferred embodiment, the weight ratio of the golden lotus flower to the ethanol solution is 1:15-25.

[0033] In a preferred embodiment, in step (1), the reflux extraction temperature is 50-90℃, specifically, for example, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃ or 90℃; the reflux extraction time is 30-150min, specifically, for example, 30min, 40min, 50min, 60min, 70min, 80min, 850min, 90min, 95min, 100min, 120min or 150min.

[0034] In a more preferred embodiment, in step (1), the reflux extraction conditions include a temperature of 55-65°C and a time of 85-95 min. Under the above reflux extraction conditions, the yield of total phenolic acid extract from golden lotus is higher.

[0035] In the most preferred embodiment, in step (1), the reflux extraction conditions include a temperature of 58-62°C and a time of 88-92 min. Under the above reflux extraction conditions, the yield of total phenolic acid extract from golden lotus is the highest.

[0036] In some embodiments, in step (3), the packing material in the macroporous resin adsorption column is selected from HPD100 type macroporous resin, NKA-9 type macroporous resin, HPD400 type macroporous resin, D101 type macroporous resin and AB-8 type macroporous resin.

[0037] In a preferred embodiment, in step (3), the packing material in the macroporous resin adsorption column is HPD100 macroporous resin. By selecting HPD100 macroporous resin as the packing material in the adsorption column, the yield and purity of the total phenolic acid extract of golden lotus are increased.

[0038] In this invention, by selecting HPD100 macroporous resin as the packing material in the adsorption column and using 20-80% ethanol solution as the elution solvent, polysaccharides, pigments and other substances in the concentrate can be removed, resulting in a higher purity of the total phenolic acid extract of nasturtium.

[0039] In a preferred embodiment, in step (3), after adsorption equilibrium is reached, the adsorption is first eluted with water, and then eluted with a 30-60% ethanol solution. Specifically, the concentration of the ethanol solution used for elution can be 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0040] In some embodiments, the sample loading amount of the macroporous resin adsorption column is 20-50g of *Trollius chinensis* per 100g of macroporous resin. To further improve the yield and purity of the total phenolic acid extract of *Trollius chinensis*, in a preferred embodiment, the sample loading amount of the macroporous resin adsorption column is 30-40g of *Trollius chinensis* per 100g of macroporous resin.

[0041] In a preferred embodiment, in step (3), the elution flow rate of the ethanol solution is 1-4 BV / h, and the elution volume is 1-8 BV. More preferably, in step (3), the elution flow rate of the ethanol solution is 1-3 BV / h, and the elution volume is 3-5 BV.

[0042] In this invention, the main components of the total phenolic acid extract of the golden lotus are guava acid, trans-cinnamic acid, caffeic acid, veratric acid, arbutin, quercetin, protopanaxanoic acid, arbutin-2''-O-β-D-xylanopyranoside and 6'''-HMG-2''-O-β-L-galactoside arbutin; Furthermore, the total phenolic acid extract of the golden lotus also contains vitexin, hyperoside, harvestinoin-2''-O-β-D-xylanopyranoside, 2''-O-(2'''-methylbutyryl)vitexin, 7-2''-O-(2'''-methylbutyryl)harvestinoin, 2''-O-feruloylvitexin, 6'''-HMG-2''-O-β-L-galactosideharvestinoin, 6''-malonylastragalin, 6''-malonyl cosmosiin, isochlorogenic acid A, ferulic acid, vanillic acid, p-hydroxycinnamic acid and 2'''-methylbutyrylharvestinoin.

[0043] In one specific embodiment, the total phenolic acid extract of *Trollius chinensis* contains purslane, vitexin, quercetin, hyperoside, purslane-2''-O-β-L-galactoside, purslane-2''-O-β-D-xylanopyranoside, 2''-O-(2'''-methylbutyryl)vitexin, 7-2''-O-(2'''-methylbutyryl)purslane, 6'''-HMG-2''-O-β-L-galactoside vitexin, 2''-O-feruloyl vitexin, 6'''-HMG-2''-O-β-L-galactoside purslane, 6''-malonyl astragalin, and 6''-malonyl Cosmosiin, isochlorogenic acid A, veratric acid, caffeic acid, ferulic acid, trans-cinnamic acid, guava acid, protoporphyric acid, vanillic acid, p-hydroxycinnamic acid, and 2'''-methylbutyryl cinnamic acid.

[0044] This invention also proposes the application of total phenolic acid extract from *Trollius chinensis* in the preparation of skincare products for improving acne. As mentioned above, the total phenolic acid extract from *Trollius chinensis* exhibits a high inhibition rate against *Propionibacterium acnes* and *Staphylococcus aureus*, which cause acne; therefore, this extract can be used to improve acne.

[0045] This invention also proposes the application of total phenolic acid extract from *Trollius chinensis* in the preparation of in vitro inhibitors of *Propionibacterium acnes*. Experiments show that the total phenolic acid extract from *Trollius chinensis* exhibits a high inhibition rate against *Propionibacterium acnes*; therefore, it can be used to prepare in vitro inhibitors of *Propionibacterium acnes*.

[0046] In the above applications, the total phenolic acid extract of the golden lotus is prepared according to the following preparation steps: (1) Add 50-90% ethanol solution to the golden lotus and reflux extract, filter and collect the filtrate; (2) The collected filtrate is concentrated to obtain a concentrated solution with a solid content of 0.1-0.4 g / mL; (3) Adjust the pH of the concentrate to 2-6, then load it onto a macroporous resin adsorption column. After adsorption equilibrium is reached, first wash with water, then wash with 20-80% ethanol solution, collect the ethanol eluent, and then concentrate and dry it.

[0047] It is understood that the specific preparation process of the total phenolic acid extract of the golden lotus is the same as that described in the previous embodiment (e.g., the packing material in the macroporous resin adsorption column, reflux extraction conditions, etc.), and will not be repeated here.

[0048] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0049] The raw materials involved in the following embodiments and test examples include: Golden lotus medicinal material: Golden lotus was purchased from Anguo Traditional Chinese Medicine Market in Hebei Province, batch number: 20231014; NKA-9 type macroporous resin, HPD400 type macroporous resin, D101 type macroporous resin, and HPD100 type macroporous resin were all purchased from Cangzhou Maoquan New Material Technology Co., Ltd., batch number: 231120. Propionibacterium acnes: China Center for Type Culture Collection, accession number CCTCAB201; Staphylococcus aureus was purchased from Shanghai Beikangnuote Biotechnology Co., Ltd., with accession number ATCC6538; Veratronic acid: purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number: DN1127BA13; BHI culture medium: purchased from Qingdao Haibo Biotechnology Co., Ltd., batch number: 20241111; LB medium: purchased from Qingdao Haibo Biotechnology Co., Ltd., batch number: 20250103; Retinoic acid cream: purchased from Chongqing Huabang Pharmaceutical Co., Ltd., batch number: 240060002.

[0050] Example 1 This embodiment illustrates the total phenolic acid extract of *Trollius chinensis* and its preparation method according to the present invention, which includes the following steps: (1) Using dried golden lotus as the extraction raw material, ethanol was added at 20 times the weight of the raw material for reflux extraction. The ethanol concentration was 60%, the reflux temperature was 60℃, and the reflux time was 90min. After reflux, the mixture was filtered and the filtrate was collected. (2) The collected filtrate was concentrated under reduced pressure to obtain a concentrated solution with a solid content of 0.3 g / mL; (3) Adjust the pH of the concentrate to 3 with 1 mol / L hydrochloric acid, and then load it onto an HPD100 macroporous resin adsorption column with a column diameter-to-height ratio of 1:6. The sample loading amount is 36 g of golden lotus medicinal material for every 100 g of resin. Load the column at a flow rate of 2 BV / h. After the sample loading is completed and the dynamic adsorption equilibrium is reached, first elute with water at a flow rate of 3 BV / h and an elution volume of 3 BV. Then elute with 40% ethanol solution at a flow rate of 2 BV / h and an elution volume of 4 BV. Collect the ethanol eluent. (4) After concentrating the ethanol eluent, freeze-dry it to obtain the total phenolic acid extract of golden lotus.

[0051] Example 2 This embodiment illustrates the total phenolic acid extract of *Trollius chinensis* and its preparation method according to the present invention, which includes the following steps: (1) Using dried golden lotus as the extraction raw material, ethanol was added at 20 times the weight of the raw material for reflux extraction. The ethanol concentration was 60%, the reflux temperature was 60℃, and the reflux time was 90min. After reflux, the mixture was filtered and the filtrate was collected. (2) The collected filtrate was concentrated under reduced pressure to obtain a concentrated solution with a solid content of 0.1 g / mL; (3) Adjust the pH of the concentrate to 4 with 1 mol / L hydrochloric acid, and then load it onto an NKA-9 macroporous resin adsorption column with a column diameter-to-height ratio of 1:6. The sample loading amount is 24 g of golden lotus medicinal material for every 100 g of resin. Load the column at a flow rate of 1 BV / h. After loading the sample, first elute with water at a flow rate of 4 BV / h and an elution volume of 2 BV. Then elute with 60% ethanol solution at a flow rate of 2 BV / h and an elution volume of 4 BV. Collect the ethanol eluent. (4) After concentrating the ethanol eluent, freeze-dry it to obtain the total phenolic acid extract of golden lotus.

[0052] Example 3 This embodiment illustrates the total phenolic acid extract of *Trollius chinensis* and its preparation method according to the present invention, which includes the following steps: (1) Using dried golden lotus as the extraction raw material, ethanol was added at 20 times the weight of the raw material for reflux extraction. The ethanol concentration was 60%, the reflux temperature was 60℃, and the reflux time was 90min. After reflux, the mixture was filtered and the filtrate was collected. (2) The collected filtrate was concentrated under reduced pressure to obtain a concentrated solution with a solid content of 0.2 g / mL; (3) Adjust the pH of the concentrate to 5 with 1 mol / L hydrochloric acid, and then load it onto an HPD400 macroporous resin adsorption column with a column diameter-to-height ratio of 1:6. The sample loading amount is 48 g of golden lotus medicinal material for every 100 g of resin. Load the column at a flow rate of 3 BV / h. After loading the sample, first elute with water at a flow rate of 4 BV / h and an elution volume of 4 BV. Then elute with 50% ethanol solution at a flow rate of 2 BV / h and an elution volume of 4 BV. Collect the ethanol eluent. (4) After concentrating the ethanol eluent, freeze-dry it to obtain the total phenolic acid extract of golden lotus.

[0053] Example 4 This embodiment illustrates the total phenolic acid extract of *Trollius chinensis* and its preparation method according to the present invention, which includes the following steps: (1) Using dried golden lotus as the extraction raw material, ethanol was added at 20 times the weight of the raw material for reflux extraction. The ethanol concentration was 60%, the reflux temperature was 60℃, and the reflux time was 90min. After reflux, the mixture was filtered and the filtrate was collected. (2) The collected filtrate was concentrated under reduced pressure to obtain a concentrated solution with a solid content of 0.4 g / mL; (3) Adjust the pH of the concentrate to 2 with 1 mol / L hydrochloric acid, and then load it onto a D101 macroporous resin adsorption column with a column diameter-to-height ratio of 1:6. The sample loading amount is 30 g of golden lotus medicinal material for every 100 g of resin. Load the column at a flow rate of 1 BV / h. After loading the sample, first elute with water at a flow rate of 4 BV / h and an elution volume of 2 BV. Then elute with 30% ethanol solution at a flow rate of 2 BV / h and an elution volume of 5 BV. Collect the ethanol eluent. (4) After concentrating the ethanol eluent, freeze-dry it to obtain the total phenolic acid extract of golden lotus.

[0054] Example 5 This embodiment illustrates the total phenolic acid extract of *Trollius chinensis* and its preparation method according to the present invention, which includes the following steps: (1) Using dried golden lotus as the extraction raw material, ethanol was added at 20 times the weight of the raw material for reflux extraction. The ethanol concentration was 60%, the reflux temperature was 60℃, and the reflux time was 90min. After reflux, the mixture was filtered and the filtrate was collected. (2) The collected filtrate was concentrated under reduced pressure to obtain a concentrated solution with a solid content of 0.3 g / mL; (3) Adjust the concentrated solution to pH 3, and then load it onto an HPD100 macroporous resin adsorption column with a column diameter-to-height ratio of 1:6. The sample loading amount is 36g of golden lotus medicinal material for every 100g of resin. Load the column at a flow rate of 3BV / h. After loading the sample, first elute with water at a flow rate of 4BV / h and an elution volume of 4BV. Then elute with 50% ethanol solution at a flow rate of 2BV / h and an elution volume of 3BV. Collect the ethanol eluent. (4) After concentrating the ethanol eluent, freeze-dry it to obtain the total phenolic acid extract of golden lotus.

[0055] Comparative Example 1 The method described in Example 1 is implemented, except that purification is not performed, i.e., steps (2) and (3) are not performed. Specifically, the method includes the following steps: (1) Using dried golden lotus as the extraction raw material, ethanol was added at 20 times the weight of the raw material for reflux extraction. The ethanol concentration was 60%, the reflux temperature was 60℃, and the reflux time was 90min. After reflux, the mixture was filtered and the filtrate was collected. (2) After concentrating the filtrate, freeze-dry it to obtain the total phenolic acid extract of golden lotus.

[0056] Test Example 1 This test case was used to detect the total phenolic acid content in the total phenolic acid extracts of *Trollius chinensis* obtained from the examples and comparative examples, and to calculate the yield. The results are shown in Table 1 below.

[0057] (1) The purity test method for total phenolic acid extract of golden lotus is as follows: The crucibles used were dried at 105°C to constant weight, and the weight of the crucibles was recorded as m0. A suitable amount of liquid was taken from the ethanol eluent collected in step (3) of Examples 1-5 and placed in the crucibles. The liquid was dried at 105°C to constant weight, and the total weight was measured as m1. The total phenolic acid content in the ethanol eluent was determined by the Prussian blue method. The purity of the total phenolic acid extract of the golden lotus was m / (m1-m0)×100%. When the total phenolic acid extract of the golden lotus of Comparative Example 1 was tested, the filtrate in step (1) was taken.

[0058] (2) The yield test method is as follows: When testing the total phenolic extract of the golden lotus in Examples 1-5, the test method is as follows: the total phenolic acid content of the ethanol eluent obtained in step (3) is determined by the Prussian blue method to be a mg, the loading volume of the macroporous resin adsorption column in step (3) is b mL, the mass concentration of the concentrated solution loaded in step (3) is d g / mL, and the yield is: a / (b×d). When testing the total phenolic acid extract of *Trollius chinensis* from Comparative Example 1, the test method was as follows: the total phenolic acid content of the filtrate from step (1) was determined by the Prussian blue method to be a1 mg, the mass of *Trollius chinensis* used during extraction was a2 g, and the yield was a 1 / a2.

[0059] Table 1: Purity and Yield of Total Phenolic Acid Extract from Golden Lotus

[0060] As shown in Table 1, the purity of the total phenolic acid extracts from *Trollius chinensis* in Examples 1-5 was approximately 41-61%, with a yield of approximately 28-45%. In contrast, the purity of the crude extract of total phenolic acids from *Trollius chinensis* obtained in Comparative Example 1 was only 20.41%. This indicates that purifying the filtrate obtained from the extraction significantly improved the purity of the total phenolic acid extract from *Trollius chinensis*. Furthermore, Example 1 demonstrated the best overall effect, with the highest purity and a relatively high yield.

[0061] Test Example 2 Test Example 2 is used to illustrate the composition of the total phenolic acid extract of the golden lotus prepared in Example 1.

[0062] The total phenolic acid extract of the golden lotus obtained in Example 1 was analyzed by UPLC-Q-TOF-MS / MS. Chromatographic conditions: An Agilent Zorbax Extend C18 column (2.1 mm × 100 mm, 1.8 μm) was used with a gradient elution of acetonitrile (phase A) and 0.1% formic acid aqueous solution (phase B) as the mobile phase (0–1.0 min, 5% A; 1.0–3 min, 5%–10% A; 3–4 min, 10%–15% A; 4–15 min, 15%–20% A; 15–25 min, 20%–25% A; 25–35 min, 25%–30% A; 35–45 min, 30%–35% A; 45–50 min, 35%–40% A; 50–52 min, 40%–5% A; 52–57 min, 5%–5% A), column temperature 35 °C, flow rate 0.3 mL / min. -1 The injection volume was 5.0 µL.

[0063] Mass spectrometry conditions: Electrospray ionization (ESI) was used to acquire data from the test solution in both positive and negative ion modes. The relevant mass spectrometry parameters were as follows: nozzle voltage 1000V in positive mode and 1500V in negative mode; capillary voltage 4000V in positive mode and 3500V in negative mode; dry gas temperature 320℃; segmented voltage 150V; cone voltage 65V; sheath gas temperature 280℃; MS data were acquired using full scan mode, with a mass scan range of m / z 100~1500Da. The test results are shown in Table 2 below.

[0064] Table 2. Analytical results of total phenolic acid extract from *Trollius chinensis* by UPLC-Q-TOF-MS / MS.

[0065] As shown in Table 2, 23 chemical components were identified in the total phenolic acid extract of the golden lotus, including: ferulic acid, piscidic acid, trans-cinnamic acid, caffeic acid, veratric acid, p-coumaric acid, and vanillic acid. acid), orientin-2''-O-β-L-galactopyranosylorientin, orientin-2''-O-β-D-xylopyranoside, orientin, hyperoside, 6'''-HMG-2''-O-β-L-galactopyranoside orientin, vitexin, 6''-malonyl astragalin, quercetin, 6'''-HMG-2''-O-β-L-galactopyranoside vitexin vitexin, 6''-malonyl cosmosiin, proglobefloweryacid, sochlorogenic acid A, 2'''-Methylbutyryl orientin, 2''-O-Feruloylvitexin, 2''-O-(2'''-methylbutyryl)vitexi, 7-2''-O-(2'''-methylbutyryl)orientin; Meanwhile, the main components of the total phenolic acid extract of golden lotus are guava acid, trans-cinnamic acid, caffeic acid, veratric acid, arbutin, quercetin, protopanaxanoic acid, arbutin-2''-O-β-D-xylanopyranoside and 6'''-HMG-2''-O-β-L-galactoside arbutin.

[0066] Test Example 3 This test example 3 is used to illustrate the efficacy evaluation of the total phenolic acid extract of golden lotus obtained in Example 1 of the present invention.

[0067] 1. Study on the in vitro antibacterial activity of total phenolic acid extract from golden lotus. Microbial colonization on the skin surface is one of the main factors in the development of acne. Propionibacterium acnes (… C.acnes Staphylococcus aureus ( S.aureus ) is the main pathogen of acne and an important target for topical drug treatment. Therefore, by measuring the total phenolic acids of nasturtium, the effects on acne can be investigated. C.acnes , S.aureus The size of the inhibition zone is used to reflect the antibacterial activity of total phenolic acids in golden lotus.

[0068] 1.1 Preparation of bacterial suspension Will C.acnes Inoculate into BHI medium and culture anaerobically at 37°C until the logarithmic growth phase. S.aureus Inoculate into LB medium and incubate at 37°C until the logarithmic growth phase.

[0069] 1.2 Antibacterial zone The total phenolic acids in *Trollius chinensis* were determined using the plate drilling method (8mm aperture). C.acnes , S.aureus The diameter of the inhibition zone was determined by a concentration of 1×10⁻⁶. 8 CFU, 100 μl of bacterial suspension was evenly spread in BHI and LB medium, and wells were punched. 100 μl of DMSO (solvent control), veratril acid solution (50 mg / ml), total phenolic acid extract solution of *Trollius chinensis* prepared in Example 1 (50 mg / ml), and total phenolic acid extract solution of *Trollius chinensis* prepared in Comparative Example 1 (50 mg / ml) were added to the wells respectively. In both Example 1 and Comparative Example 1, the total phenolic acid extract of *Trollius chinensis* and veratril acid were prepared using DMSO to form the corresponding solutions. C.acnes Cultured in an anaerobic environment at 37℃ for 48 h. S.aureus The culture was carried out in a 37 ℃ constant temperature incubator for 24 h, and the diameter d (mm) of the inhibition zone was measured using vernier calipers. The diameters of the inhibition zones of the test drugs were then compared to determine the results. An inhibition zone diameter <8 mm indicated no antibacterial effect, while >8 mm indicated antibacterial effect. All experiments were performed in triplicate. The test results are as follows: Figure 1-2 As shown.

[0070] The test results show that ( Figure 1-2 Veratril acid and the total phenolic acid extract of nasturtium prepared in Example 1, C.acnes The diameters of the inhibition zones were 11.6 mm and 17.9 mm, respectively. S.aureus The diameters of the inhibition zones were 16.8 mm and 21.2 mm, respectively; while the total phenolic acid extract of *Trollius chinensis* prepared in Comparative Example 1 showed... C.acnes and S.aureusThe diameters of the inhibition zones were 8 mm and 9.8 mm, respectively, indicating that the purified total phenolic acid extract of *Trollius chinensis* described in this invention has an inhibitory effect on the body's antibacterial activity. C.acnes , S.aureus It has a good antibacterial effect.

[0071] 2. Methods for testing the efficacy of acne treatment in rats. Acne is an inflammatory skin disease affecting the pilosebaceous unit. Its occurrence is the result of multiple factors, currently widely believed to be related to excessive sebum secretion, the proliferation of Propionibacterium acnes, and hyperkeratosis of the follicular orifice epithelium. The rat acne model is a commonly used animal model for studying the anti-inflammatory and antibacterial mechanisms of acne treatment drugs. Propionibacterium acnes is injected into the rat's ear to simulate the pathogenesis of acne inflammation, and then drugs are applied topically for treatment, allowing for the evaluation of the efficacy of drug therapy for acne.

[0072] 2.1 System and Sample Size (1) Mouse strain used: male SD rats; (2) Age of SD rats: 6 weeks old; (3) Sample size per group: 8 animals; 2.2 Experimental Procedure: (1) Grouping: 40 SD rats were randomly divided into 5 groups of 8 rats each. One group was selected as the blank group, and the remaining 4 groups were used to construct a rat auricular acne model.

[0073] (2) Modeling: Four groups of rats were subjected to modeling by subcutaneous injection of Propionibacterium acnes bacterial solution (5×10⁻⁶) into the inner surface of both ears. 7 50 μl of (cfu / ml) tretinoin cream was administered once daily for 7 consecutive days. The control group received no treatment. Successful acne model establishment was determined by the presence or absence of blackheads, pustules, redness, and discoloration. Successfully modeled SD rats were randomly divided into four groups: model group, positive control group (0.025% retinoic acid cream), low-dose total phenolic acid group from *Trollius chinensis*, and high-dose total phenolic acid group from *Trollius chinensis*.

[0074] (3) Drug administration: After the rat acne model was successfully established, in order to prevent the rats' autoimmunity from causing the ear acne to gradually heal, oleic acid was applied to the inner side of the rat's ear every day. Oleic acid was applied evenly to the inner side of both ears of the rats using a medical cotton swab, once a day, 0.5 ml each time. After absorption, the drug was applied to the acne lesions on the rat's ear. The positive control group was treated with 0.025% retinoic acid cream 1 g / (kg·d), and the low-dose and high-dose groups of total phenolic acid from golden lotus were treated with 6.25 mg / (kg·d) and 12.5 mg / (kg·d) of total phenolic acid extract from golden lotus prepared in Example 1, respectively, to the rat's ear. The model group was given a blank matrix (0.5% CMC-Na) for 14 consecutive days.

[0075] 2.3 Observation Indicators (1) Thickness of ear swelling After the treatment cycle, the thickness of the ear tissue in each group of rats was measured using calipers to compare the degree of ear swelling in each group. The thickness of the auricle in each group was used to reflect the effect of total phenolic acids from *Trollius chinensis* on the degree of swelling in the acne model. The experimental results are as follows: Figure 3 As shown.

[0076] Figure 3 The results showed that the ear thickness of rats in the blank group, model group, retinoic acid cream group, low-dose nasturtium group, and high-dose nasturtium total phenolic acid group were 0.76 mm, 2.89 mm, 1.23 mm, 1.39 mm, and 1.31 mm, respectively. This indicates that the nasturtium total phenolic acid extract provided in this invention can effectively alleviate ear swelling in rat acne models.

[0077] (2) Observation of rat ear appearance and pathology After the treatment cycle, the surface morphology of the ears and the pathological changes of the ear tissues of rats in each group were observed to determine whether the total phenolic acids of *Trollius chinensis* had the effect of preventing and treating acne. The experimental results are as follows: Figure 4 As shown.

[0078] Figure 4 The results showed that the auricular tissue of the control group rats was soft, smooth, and fine. Under HE staining, the boundaries between the epidermis, dermis, and subcutaneous tissue were clear, with little infiltration of inflammatory factors. The model group rats had obvious redness and swelling of the ear, rough surface, and were covered with a certain amount of keratinized material. The pathological sections showed obvious destruction of the auricular tissue structure, blurred boundaries between layers, hyperkeratosis of the epidermis, and infiltration of a large number of inflammatory cells. Compared with the model group, the rats given total phenolic acids from golden lotus and 0.025% retinoic acid cream showed reduced redness and swelling on the surface of the ear, and reduced inflammatory infiltration in the pathological sections. All aspects were improved, indicating that the total phenolic acid extract from golden lotus described in this invention can alleviate the symptoms of rat auricular acne model.

[0079] (3) Effect of total phenolic acids from golden lotus on inflammatory factors in rat serum by ELISA Twenty-four hours after the last administration, blood was collected from the orbital cavity of rats, and serum was separated. The levels of IL-6, TNF-α, IL-17, and IL-10 in the rat serum were detected according to the ELISA kit instructions. The experimental results are as follows: Figure 5 As shown.

[0080] Figure 5The results showed that IL-6, TNF-α, IL-17, and IL-10 are inflammatory factors involved in the inflammatory response of acne. Compared with the control group, the serum levels of pro-inflammatory factors IL-6, TNF-α, and IL-17 were increased, while the level of anti-inflammatory factor IL-10 was decreased in the model group rats, indicating the presence of an inflammatory response. Compared with the model group, the levels of pro-inflammatory factors IL-6, TNF-α, and IL-17 were decreased in each treatment group, while the level of anti-inflammatory factor IL-10 was increased. This indicates that total phenolic acids from *Trollius chinensis* can alleviate the inflammatory response in the rat acne model, thereby achieving the goal of treating acne.

[0081] 4. Experimental Conclusions The total phenolic acid extract of golden lotus described in this invention has a good antibacterial effect against acne-associated bacteria in vitro, and can improve acne symptoms in rats in vivo by regulating the inflammatory response.

[0082] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. The application of total phenolic acid extract from *Trollius chinensis* in the preparation of drugs for treating acne, wherein, The total phenolic acid extract of the golden lotus was prepared according to the following preparation steps: (1) Add 50-90% ethanol solution to the golden lotus and reflux extract, filter and collect the filtrate; (2) The collected filtrate is concentrated to obtain a concentrated solution with a solid content of 0.1-0.4 g / mL; (3) Adjust the pH of the concentrate to 2-6, then load it onto a macroporous resin adsorption column. After adsorption equilibrium is reached, first wash with water, then wash with 20-80% ethanol solution, collect the ethanol eluent, and then concentrate and dry it.

2. The application according to claim 1, characterized in that, The drugs are tablets, capsules, oral liquids, injections, creams, gels, and liquid preparations.

3. The application according to claim 1, characterized in that, In step (1), the weight ratio of the golden lotus flower to the ethanol solution is 1:10-50; Preferably, in step (1), the reflux extraction conditions include: a temperature of 50-90℃ and a time of 30-150min.

4. The application according to any one of claims 1-3, characterized in that, In step (3), the packing material in the macroporous resin adsorption column is selected from HPD100 type macroporous resin, NKA-9 type macroporous resin, HPD400 type macroporous resin, D101 type macroporous resin and AB-8 type macroporous resin, preferably HPD100 type macroporous resin.

5. The application according to any one of claims 1-4, characterized in that, The loading amount of the macroporous resin adsorption column is 20-50g of golden lotus for every 100g of macroporous resin.

6. The application according to any one of claims 1-5, characterized in that, In step (3), the elution flow rate of the ethanol solution is 1-4 BV / h, and the elution volume is 1-8 BV.

7. The application according to any one of claims 1-6, characterized in that, The main components of the total phenolic acid extract of the golden lotus are guava acid, trans-cinnamic acid, caffeic acid, veratric acid, arbutin, quercetin, protopanaxanoic acid, arbutin-2''-O-β-D-xylanopyranoside and 6'''-HMG-2''-O-β-L-galactoside arbutin; Preferably, the total phenolic acid extract of the golden lotus further contains vitexin, hyperoside, harvestin-2''-O-β-D-xylanopyranoside, 2''-O-(2'''-methylbutyryl)vitexin, 7-2''-O-(2'''-methylbutyryl)harvestin, 2''-O-feruloylvitexin, 6'''-HMG-2''-O-β-L-galactosideharvestin, 6''-malonyl astragalin, 6''-malonyl cosmosiin, isochlorogenic acid A, ferulic acid, vanillic acid, p-hydroxycinnamic acid and 2'''-methylbutyrylharvestin.

8. Application of total phenolic acid extract from golden lotus in the preparation of skin care products to improve acne.

9. Application of total phenolic acid extract from golden lotus in the preparation of in vitro inhibitors of Propionibacterium acnes.

10. The application according to claim 8 or 9, characterized in that, The total phenolic acid extract of the golden lotus was prepared according to the following preparation steps: (1) Add 50-90% ethanol solution to the golden lotus and reflux extract, filter and collect the filtrate; (2) The collected filtrate is concentrated to obtain a concentrated solution with a solid content of 0.1-0.4 g / mL; (3) Adjust the pH of the concentrate to 2-6, then load it onto a macroporous resin adsorption column. After adsorption equilibrium is reached, first wash with water, then wash with 20-80% ethanol solution, collect the ethanol eluent, and then concentrate and dry it.

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