African cardamom seed extract for initiating inflammatory resolution mechanisms, its preparation method and application

Abstract

This invention provides an African cardamom seed extract for initiating an inflammation-reducing mechanism, its preparation method, and its application, belonging to the field of cosmetic technology. The preparation method involves washing, drying, and pulverizing African cardamom seeds into granules, then mixing them with water and a complex enzyme to extract active substances. After enzyme inactivation, the extract is filtered to obtain a purified extract. Adding glycerin yields a stable African cardamom seed extract. The African cardamom seed extract prepared using this method has no organic reagent residues and possesses the effects of initiating an inflammation-reducing mechanism and shortening the duration of skin inflammation. It can be applied in cosmetics to achieve anti-inflammatory and soothing purposes.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to an African cardamom seed extract for initiating an inflammation resolution mechanism, its preparation method, and its application. Background Technology

[0002] Skin inflammation is an uncomfortable skin condition characterized by redness, swelling, ulceration, itching, burning, and general discomfort. It doesn't appear and disappear suddenly, but rather develops over time, primarily consisting of three phases: the initial inflammation phase, the spreading inflammation phase, and the resolution inflammation phase. In the initial inflammation phase, initiating factors, including but not limited to pathogens, endogenous damage, and environmental exposure factors (UV rays, pollutants, allergens), trigger the inflammatory response. This leads to the production of various primary inflammatory cytokines in response to stimuli. During the spreading inflammation phase, pro-inflammatory factors allow the inflammatory response to spread, producing secondary inflammatory cytokines and increasing vascular permeability. Immune cells migrate and infiltrate inflamed tissue, neutralizing pathogens or stimuli. In the resolution inflammation phase, de-inflammatory factors increase, inhibiting the production of pro-inflammatory factors. Macrophages are induced to phagocytose apoptotic neutrophils, clearing pathogens and stimuli and restoring tissue homeostasis.

[0003] Once inflammation begins, the process is not reversed or suddenly disappears; it requires a continuous process. Skin inflammation can cause many unpleasant sensations, such as itching, redness, stinging, and even weaken the skin barrier. Therefore, many cosmetic ingredients and products are dedicated to improving skin inflammation. These improvements can be summarized in two dimensions: the intensity of inflammation and its duration. Currently, many cosmetics on the market can reduce the intensity of skin inflammation by inhibiting some common cellular inflammatory signaling pathways and suppressing the production of pro-inflammatory factors. For example, Chinese patent CN118286138A discloses an anti-allergic and soothing composition that uses active ingredients from saffron to inhibit the production of pro-inflammatory factors; Chinese patent CN118557587A discloses a method for alleviating skin inflammation by using gentianin to reduce the content of pro-inflammatory factors in tissues.

[0004] However, shortening the duration of skin inflammation is a challenge. This is because once inflammation occurs, pro-inflammatory factors or irritants remain in the inflamed tissue, exerting their pro-inflammatory effects. Even if the production of new pro-inflammatory factors is inhibited, existing factors will not be cleared until the inflammation subsides. Therefore, developing cosmetic ingredients that can shorten the duration of skin inflammation is a new research direction for those skilled in the art.

[0005] Historically, inflammation was thought to resolve as a natural, passive process: over time, pro-inflammatory factors gradually break down or are engulfed by macrophages, decreasing their concentration and causing inflammation to subside naturally. However, extensive research over the past few decades has shown that inflammation resolution is a programmed, active process, and a deficiency in any component can lead to overactive, uncontrolled chronic inflammation. Once the inflammation resolution mechanism is activated, inflammation rapidly enters a resolution phase: pro-inflammatory factors decrease, anti-inflammatory factors increase, macrophages engulf existing pro-inflammatory factors and irritants, and ultimately, the inflammation ends and symptoms disappear. If the inflammation resolution mechanism is not activated in time, the inflammation remains in the spreading phase, effectively prolonging its duration. Furthermore, prolonged inflammation can transform into chronic inflammation, leading to persistent autoimmune responses, tissue fibrosis, inflammatory aging, and other problems. This also signifies a further prolongation of the inflammatory duration. Therefore, the ability to activate the inflammation resolution mechanism is crucial for shortening the duration of skin inflammation.

[0006] In the process of initiating inflammatory resolution mechanisms, a core class of initiating factors is called SPM (specialized pro-resolving mediators). SPMs include lipoxins, resolvins, protectins, and maresins. Among them, LXA4 (lipoxin A4) is a very important type of SPM.

[0007] On the other hand, in initiating the inflammation resolution mechanism, SPM needs to bind to a series of specific receptors to exert its anti-inflammatory effect. Among these receptors, FPR2 (formylpeptide receptor 2) is a very important one. FPR2 can recognize microbial invasion or initiating factors of inflammation, further initiating immune responses and inflammatory processes. In this process, mitochondrial and bacterial formylpeptides, peptides of bacterial or viral surface proteins, or endogenous human proteins, such as SAA (serum amyloid A) and Aβ42 (β-amyloid β-protein), can all activate the pro-inflammatory effect of FPR2. However, FPR2 has different binding sites. In addition to binding to the aforementioned pro-inflammatory factors, FPR2 can also bind to SPM, thereby exerting an anti-inflammatory effect and initiating the inflammation resolution mechanism. In other words, FPR2 will trigger different downstream events depending on the ligand it binds to.

[0008] However, in the initial and spreading phases of inflammation, the amount of pro-inflammatory factors (such as SAA1) is often 2-3 orders of magnitude higher than that of anti-inflammatory factors (such as LXA4). Therefore, SAA1 suppresses the binding of LXA4 to FPR2 and the transduction of downstream signals.

[0009] Currently, many cosmetic ingredients on the market are derived from ginger family plants. However, different specific varieties and different extracted parts have different effects. African cardamom (AFRAMOMUM MELEGUETA), as a member of the ginger family, has relatively little research. Currently, no studies have found that African cardamom has the effect of initiating skin inflammation resolution mechanisms, increasing SPM and LXA4 expression levels, or regulating FPR2.

[0010] Currently, common plant extract processing methods involve high-temperature boiling, high-temperature pulverization, and organic solvent extraction. High-temperature environments often destroy the active ingredients in plants. Organic solvent extraction can extract water-insoluble components from plants, and these components often have higher bioactivity. However, organic solvents easily cause environmental pollution, and their residues in cosmetic raw materials can harm the skin. If only water is used as the extraction medium without organic solvents, it is difficult to effectively extract and dissolve the active ingredients in the plants. Summary of the Invention

[0011] This invention addresses the problems existing in the prior art by providing an African cardamom seed extract for initiating an inflammatory resolution mechanism, its preparation method, and its application. The African cardamom seed extract obtained by the preparation method of this invention has the effects of increasing LXA4 expression and regulating FPR2 sensitivity, thereby initiating an inflammatory resolution mechanism, shortening the duration of skin inflammation, and achieving anti-inflammatory and soothing effects.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] A method for preparing African cardamom seed extract for initiating an inflammatory resolution mechanism includes the following steps:

[0014] S1. Take African cardamom seeds, wash them with clean water, and dry them until there is no moisture on the surface. They can be stored in the refrigerator at 4℃. When using, grind them into African cardamom seed granules at a low temperature.

[0015] S2. Mix the African cardamom seed granules described in step S1 with the compound enzyme and water, and put them into the reaction vessel. Stir to obtain a crude extract.

[0016] S3. The crude extract described in S2 is inactivated by a compound enzyme and filtered to obtain a purified extract.

[0017] S4. Add glycerin to the refined extract described in S3, dispense and seal to obtain African cardamom seed extract.

[0018] Preferably, the particle size in step S1 is 8-14 mesh, and the crushing temperature is 0-60℃.

[0019] Preferably, the formula amount in step S2 is as follows, by weight: 10-20 parts African cardamom seed granules, 1-5 parts compound enzyme and 80-90 parts water.

[0020] Preferably, the complex enzyme in step S2 consists of cellulase, acidic xylanase, pectinase, galactosidase, protease, glycosyltransferase, and hydroxy oxidase in a mass ratio of 1-2:1-2:1-2:1-2:1-1.5:1-1.5:1-1.5.

[0021] Preferably, the enzyme activities of the complex enzymes in step S2 are: cellulase 1000-1500 U / mL, acidic xylanase 1000-2000 U / mL, pectinase 1500-2000 U / mL, galactosidase 1000-1500 U / mL, protease 800-1200 U / mL, glycosyltransferase 500-800 U / mL, and hydroxy oxidase 500-800 U / mL.

[0022] Preferably, the glycosyltransferase is an SQ-GR6 extractant.

[0023] Glycosyltransferases, whose substrates include common monosaccharides such as glucose, can transfer monosaccharides to polyphenols to form glycosidic bonds, thereby increasing the solubility of polyphenols and gingerol in water. The use of other brands and types of glycosyltransferases in the extraction process is also considered part of the scope of this invention.

[0024] Preferably, the hydroxy oxidase is an SQ-P200 extractant.

[0025] Hydroxy oxidases have a wide substrate adaptability, capable of recognizing various polyphenol molecules and converting hydroxyl groups on polyphenol molecules into carboxyl groups, thereby increasing the solubility of polyphenols and gingerol in water. The use of other brands and models of hydroxy oxidases during the extraction process is also considered within the scope of protection of this invention.

[0026] Preferably, the stirring conditions in step S2 are stirring at 25-30°C for 3-6 hours.

[0027] Preferably, the inactivation conditions for the complex enzyme in step S3 are 50-60℃ for 30-60 min.

[0028] Preferably, the filtration conditions in step S3 are: first pass through a 0.5μm filter, then pass through a 0.22μm filter.

[0029] Preferably, the mass ratio of the purified extract to glycerol in step S4 is 2-4:6-8; more preferably, it is 3:7.

[0030] The African cardamom seed extract obtained by the above method contains various polyphenols and their derivatives, as well as gingerol. Using 6-gingerol as a standard substance, the quality control of the final extract was performed, with the set quality standard being: 6-gingerol greater than 50 micrograms / gram (μg / g) in the African cardamom seed extract solution.

[0031] The present invention also provides an African cardamom seed extract prepared by the above preparation method.

[0032] The present invention also provides a composition of the above-mentioned African cardamom seed extract.

[0033] The present invention also provides the application of the above-mentioned African cardamom seed extract in the preparation of products with anti-inflammatory and soothing effects.

[0034] Specifically, the anti-inflammatory and soothing products are cosmetics.

[0035] The technical effects achieved by this invention are:

[0036] (1) In the process of preparing African cardamom seed extract, the present invention uses low temperature complex enzyme method for aqueous phase extraction, which avoids the destruction of active ingredients by high temperature.

[0037] (2) The African cardamom seed extract obtained by the preparation method of the present invention has the ability to initiate the inflammation reduction mechanism and can be safely applied to anti-inflammatory and soothing cosmetics.

[0038] (3) The complex enzyme of the present invention ionizes and glycosylates components that are difficult to dissolve in water, so that they can be dissolved in water and can be extracted with pure water without the need for organic solvents. Attached Figure Description

[0039] Figure 1 The images show the fluorescence staining of positive control group 2, LXA4 control group, test group 1, and test group 2. Green fluorescence represents IL-8 protein, red fluorescence represents LXA4 protein, and blue fluorescence represents cell nuclei. Detailed Implementation

[0040] It is worth noting that the raw materials used in this invention are all commercially available products, and their sources are not specifically limited.

[0041] The following sources of raw materials are provided as examples:

[0042] African cardamom: Originates from the Republic of Catalonia in West Africa;

[0043] Cellulase, acidic xylanase, pectinase, galactosidase, protease: Novozyme Corp.;

[0044] SQ-GR6 extractant and SQ-P200 extractant: Proteus SA;

[0045] Human skin epidermal cells (HaCaT), human fibroblasts (HSF): CLS Cell Lines Service GmbH, Germany;

[0046] Keratinocyte growth medium (KGM), fetal bovine serum BSA, DMEM high glucose medium, cell culture-related reagents: Gibco;

[0047] ELISA kits, immunofluorescence staining reagents, basic chemical reagents, lysis buffers, related proteases, and cell wall fragments of Pseudomonas aeruginosa and Staphylococcus aureus were obtained from ThermoFisher Scientific, Hyclone, and SIGMA Aldrich.

[0048] The LXA4, IL-8, and PGF2-related antibodies are from Abcam, Inc., USA.

[0049] I. Preparation of African Cardamom Seed Extract

[0050] Example 1

[0051] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder, and the pulverized seeds were passed through a 10-mesh sieve. 100g of the sieved African cardamom seeds were taken and mixed with 800g of water and 10g of a compound enzyme. The compound enzyme consisted of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1:1:1:1:1:1:1. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device. It was first filtered through a 0.5μm filter membrane, and then filtered again through a 0.22μm filter membrane. The resulting filtrate was the purified extract. Add 2000g of glycerol to the purified extract, and dispense and seal in a sterile room to obtain African cardamom seed extract.

[0052] Example 2

[0053] Prepared according to the method described in Example 1, wherein the amount of the complex enzyme used is 30g.

[0054] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder, and the pulverized seeds were passed through a 10-mesh sieve. 100g of the sieved African cardamom seeds were taken and mixed with 800g of water and 30g of a compound enzyme. The compound enzyme consisted of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1:1:1:1:1:1:1. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device. It was first filtered through a 0.5μm filter membrane, and then filtered again through a 0.22μm filter membrane. The resulting filtrate was the purified extract. Add 2000g of glycerol to the purified extract, and dispense and seal in a sterile room to obtain African cardamom seed extract.

[0055] Example 3

[0056] Prepared according to the method described in Example 2, wherein the complex enzyme is composed of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant and SQ-P200 extractant in a mass ratio of 1.5:1.5:1.5:1:1:1:1.

[0057] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder, and the pulverized seeds were passed through a 10-mesh sieve. 100g of the sieved African cardamom seeds were added to 800g of water and 30g of a compound enzyme. The compound enzyme consisted of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1.5:1.5:1.5:1:1:1:1. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device. It was first filtered through a 0.5μm filter membrane, and then filtered again through a 0.22μm filter membrane. The resulting filtrate was the purified extract. Add 2000g of glycerol to the purified extract, and dispense and seal in a sterile room to obtain African cardamom seed extract.

[0058] Example 4

[0059] Prepared according to the method described in Example 2, wherein the complex enzyme consists of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant and SQ-P200 extractant in a mass ratio of 1:1:1:1.5:1.5:1:1.

[0060] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100g of the sieved seeds were added to 800g of water and 30g of a compound enzyme. The compound enzyme consisted of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1:1:1:1.5:1.5:1:1. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device. It was first filtered through a 0.5μm membrane and then through a 0.22μm membrane to obtain the purified extract. Add 2000g of glycerol to the purified extract, and dispense and seal in a sterile room to obtain African cardamom seed extract.

[0061] Example 5

[0062] Prepared according to the method described in Example 2, wherein the complex enzyme consists of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant and SQ-P200 extractant in a mass ratio of 1:1:1:1:1:1.5:1.5.

[0063] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder, and the pulverized seeds were passed through a 10-mesh sieve. 100g of the sieved African cardamom seeds were added to 800g of water and 30g of a compound enzyme. The compound enzyme consisted of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1:1:1:1:1:1.5:1.5. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device. It was first filtered through a 0.5μm filter membrane, and then filtered again through a 0.22μm filter membrane. The resulting filtrate was the purified extract. Add 2000g of glycerol to the purified extract, and dispense and seal in a sterile room to obtain African cardamom seed extract.

[0064] Comparative Example 1

[0065] Prepared according to the method described in Example 1, wherein the complex enzyme consists of α-amylase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant and SQ-P200 extractant in a mass ratio of 1:1:1:1:1:1:1:1.

[0066] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100g of the sieved seeds were added to 800g of water and 10g of a compound enzyme. The compound enzyme consisted of α-amylase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1:1:1:1:1:1:1. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device. It was first filtered through a 0.5μm membrane and then through a 0.22μm membrane to obtain the purified extract. Add 2000g of glycerol to the purified extract, and dispense and seal in a sterile room to obtain African cardamom seed extract.

[0067] Comparative Example 2

[0068] Prepared according to the method described in Example 1, wherein the complex enzyme is composed of galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1:1:1:1.

[0069] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder, and the pulverized seeds were passed through a 10-mesh sieve. 100g of the sieved African cardamom seeds were taken and mixed with 800g of water and 10g of a compound enzyme. The compound enzyme consisted of galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant in a mass ratio of 1:1:1:1. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device and filtered once using a 0.5μm filter membrane, followed by a second filtration using a 0.22μm filter membrane. The resulting filtrate was the purified extract. 2000g of glycerol was added to the purified extract, and the mixture was aliquoted and sealed in a sterile environment to obtain the African cardamom seed extract.

[0070] Comparative Example 3

[0071] Prepared according to the method described in Example 1, wherein the complex enzyme consists of cellulase, acidic xylanase, and pectinase in a mass ratio of 1:1:1.

[0072] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder, and the pulverized seeds were passed through a 10-mesh sieve. 100g of the sieved African cardamom seeds were taken and mixed with 800g of water and 10g of a compound enzyme. The compound enzyme consisted of cellulase, acidic xylanase, and pectinase in a mass ratio of 1:1:1. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the compound enzyme. The inactivated crude extract was transferred to a filtration device and filtered once using a 0.5μm filter membrane, followed by a second filtration using a 0.22μm filter membrane. The resulting filtrate was the purified extract. 2000g of glycerol was added to the purified extract, and the mixture was dispensed and sealed in a sterile environment to obtain the African cardamom seed extract.

[0073] Comparative Example 4

[0074] Prepared according to the method described in Example 1, wherein the composition ratio of the complex enzyme is different, specifically: cellulase, acidic polysaccharide enzyme, pectinase, galactosidase, protease, SQ-GR6 extractant and SQ-P200 extractant, with a mass ratio of 1:2.5:2.5:2.5:2:2:2.

[0075] African cardamom seeds were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The seeds were then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100g of the sieved seeds were added to 800g of water and 10g of a complex enzyme. The proportions of the complex enzymes varied, with the mass ratio of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant being 1:2.5:2.5:2.5:2:2:2. The mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C and held for 60 minutes to inactivate the complex enzymes. The inactivated crude extract was transferred to a filtration device. It was first filtered through a 0.5μm membrane and then through a 0.22μm membrane to obtain the purified extract. Add 2000g of glycerol to the purified extract, and dispense and seal in a sterile room to obtain African cardamom seed extract.

[0076] II. Effect Test

[0077] The content of active ingredients in African cardamom seed extract was determined using 6-gingerol as the standard substance. Conventional HPLC method was used with 6-gingerol standard as external standard to test the content of active ingredients in African cardamom seed extract. The results are shown in Table 1.

[0078] Table 1

[0079]

[0080] The selection of complex enzymes is crucial for extracting active substances from African cardamom seeds using a low-temperature complex enzyme method.

[0081] Cellulase and acidic xylanase can both break down plant cell walls, releasing intracellular active ingredients. Pectinase can break down galacturonic acid, which is abundant in African cardamom seeds. Galactase and protease can detach active ingredients from protein and polysaccharide complexes, promoting their release and dissolution, while also stabilizing the extract. Most of the active ingredients within African cardamom seed cells are poorly soluble in water, have low bioavailability, and are prone to precipitation. SQ-GR6 and SQ-P200 extractants can utilize the acid radicals, glucose, and mannose within plant cells to ionize and glycosylate insoluble polyphenols and gingerol, forming water-soluble components. Therefore, a stable extract can be obtained without the need for organic solvents.

[0082] Comparative Example 1 used α-amylase instead of cellulase, while Comparative Example 2 used only galactosidase, protease, SQ-GR6 extractant, and SQ-P200 extractant. The content of 6-gingerol in the extracts of both Comparative Examples 1 and 2 was <1 μg / g. Given the characteristics of African cardamom seed cells, neither replacing them with other enzymes nor using only a few enzymes can break down the cell walls, thus preventing the extraction of active substances.

[0083] Comparative Example 3 used only cellulase, acidic xylanase and pectinase, and the content of 6-gingerol in the final extract was only 9 μg / g, and flocculent precipitate began to appear after the extract was left to stand for 2 hours.

[0084] That is, the cellulase, acid xylanase, pectinase, galactosidase, protease, glycosyltransferase and hydroxyoxidase of the present invention have a synergistic effect and none of them can be omitted.

[0085] Although Comparative Example 4 used the seven enzymes of the present invention, their proportions were not within the scope of protection of the present invention, and the content of 6-gingerol in the final extract was only 13 μg / g.

[0086] Examples 1-5 within the scope of protection of this invention have a final extract containing 54-68 μg / g of 6-gingerol, with no precipitation after standing.

[0087] The effect of African cardamom seed extract on the expression of prostaglandin PGF2 and anti-inflammatory factor LXA4 was investigated by culturing human skin epidermal cells according to standard procedures. After cell resuscitation, the cells were cultured in vitro for 7 days, growing into a dense monolayer, which was confirmed by microscopy to possess keratinocyte morphology. The cells were then passaged and the cell number was adjusted to 1×10⁶. 5Cells were seeded at a density of 1 cell / mL in culture plates, and after the cells reached a confluence of about 80%, they were grouped for testing.

[0088] Negative control group 1 received no inducing agent. Positive control group 1 received phospholipase A2 as an inducing agent. The experimental group was divided into two dosage groups, with the same amount of phospholipase A2 as the inducing agent as positive control group 1, and the African cardamom seed extract obtained in Example 1 was added at 0.2% and 0.5% of the culture medium mass fraction, respectively. After cell incubation for 24 hours, the culture medium was washed off, and subsequent tests were performed. The protein expression levels of prostaglandin F2 (PGF2) and lipoxygenin A4 (LXA4) were measured using ELISA.

[0089] The protein expression level of the negative control group was set as 1, and the ratio of the protein expression levels of the positive control group and the experimental group to the negative control group was recorded as the relative expression level. Table 2 shows the effect of African cardamom seed extract on the expression levels of PGF2 and LXA4.

[0090] Table 2

[0091]

[0092] After the addition of the inducer phospholipase A2, cells entered an inflammatory state, and phospholipids in the cell membrane and intracellular space were broken down to generate arachidonic acid (AA), which further generates the pro-inflammatory factor PGF2. Therefore, compared to the negative control group 1, the expression of PGF2 protein in both the positive control group 1 and the experimental group increased accordingly. In the experimental group, the addition of African cardamom seed extract inhibited the relative expression of PGF2. Compared to the positive control group 1, PGF2 expression decreased by 27.7% in the experimental group 1 and by 55.8% in the experimental group 2.

[0093] The downstream pathway of amino acids (AA) to generate either PGF2 or LXA4 is a competitive equilibrium process. In inflammatory states, a large amount of AA is used to generate PGF2, while the production of LXA4 is relatively low. Compared to negative control group 1, positive control group 1 showed only a slight increase in LXA4 production. With the addition of African cardamom seed extract to the experimental groups, the relative expression level of LXA4 increased significantly. Compared to positive control group 1, LXA4 expression increased by 21.3% in experimental group 1 and by 47.8% in experimental group 2.

[0094] The test results indicate that African cardamom seed extract can inhibit the expression of pro-inflammatory factor PGF2 and promote the expression of anti-inflammatory factor LXA4 in human skin epidermal cells (HaCaT) under inflammatory conditions, thereby achieving a soothing and anti-inflammatory effect.

[0095] Effect of African cardamom seed extract on FPR2 sensitivity 3.1 Detection methods and procedures

[0096] Human fibroblasts were cultured according to standard procedures. After cell resuscitation, they were cultured in vitro for 7 days, growing into a dense monolayer, and their fibroblast morphology was confirmed under a microscope. The cells were then passaged and the cell number was adjusted to 1 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / mL into culture plates. Once the cell confluence reached approximately 80%, the cells were grouped for testing. The specific group settings are listed in Table 3.

[0097] Table 3

[0098]

[0099] Table 3 shows that the inducing substances were a mixture of 5 μg / mL serum amyloid-1 (SAA1) and 100 ng / mL cell wall fragments of Pseudomonas aeruginosa and Staphylococcus aureus. This combination of inducing substances can simulate the initial stage of skin inflammation. LXA4 was added at 100 ng / mL. After cell incubation for 4 hours, the culture medium was washed off, and subsequent tests were performed, using ELISA to determine protein expression levels.

[0100] 3.2 Results of IL-8 and LXA4 expression level detection

[0101] The protein expression level of the negative control group was set as 1, and the ratio of the protein expression levels of the positive control group and the test group to the negative control group was recorded as the relative expression level. Table 4 shows the effect of African cardamom seed extract on the expression levels of leukocyte mediators IL-8 and LXA4.

[0102] Table 4

[0103]

[0104] After adding the inducing substance and culturing for 4 hours, the cells entered the initial stage of inflammation, and the expression level of IL-8 increased significantly, indicating that the inflammation in the cells was aggravated. At this stage, simply increasing the content of the inflammation-reducing factor LXA4 did not significantly reduce the relative expression level of IL-8, and could not effectively reduce inflammation, because at this time, the amount of pro-inflammatory factors in the cells was greater, and the inflammation-reducing factor could not effectively bind to the FPR2 receptor.

[0105] In the test groups with African cardamom seed extract, the expression level of IL-8 decreased by 26.5% in test group 1 and by 49.5% in test group 2 compared to the positive control group 2; at the same time, the expression level of LXA4 increased by 85.6% in test group 1 and by 143.3% in test group 2 compared to the positive control group 2. This indicates that the addition of African cardamom seed extract significantly reduced the relative expression level of IL-8 and increased the expression level of LXA4.

[0106] 3.3 Methods and results of fluorescent staining of cells

[0107] Human fibroblasts were fixed, perforated, and blocked. After washing with PBS, they were incubated with primary and secondary antibodies in sequence, washed, and the cell nuclei were stained with DAPI and mounted. After washing, they were photographed using a fluorescence microscope. Figure 1 The effect of African cardamom seed extract on the expression of IL-8 and LXA4 proteins was shown. Green fluorescence represents IL-8 protein, red fluorescence represents LXA4 protein, and blue fluorescence represents cell nucleus.

[0108] Human fibroblasts enter an inflammatory state under the influence of inducing substances, with a significant increase in IL-8 expression. At this point, adding LXA4 alone cannot inhibit the progression of inflammation, but the simultaneous addition of LXA4 and African cardamom seed extract significantly reduces green fluorescence and increases red fluorescence.

[0109] 3.4 Summary of the test on the effect of African cardamom seed extract on FPR2 sensitivity

[0110] African cardamom seed extract can allosterically regulate the FPR2 receptor, inhibiting FPR2's sensitivity to pro-inflammatory factors and enhancing its sensitivity to the anti-inflammatory factor LXA4. Therefore, even at low LXA4 levels, FPR2 can bind to LXA4, thereby transforming its downstream effect into inhibiting inflammatory signaling pathways and initiating inflammation resolution mechanisms.

[0111] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing African cardamom seed extract for initiating an inflammatory resolution mechanism, characterized in that, Includes the following steps: S1. Take African cardamom seeds, wash, dry, and crush them to obtain African cardamom seed granules; S2. Mix the African cardamom seed granules described in step S1 with the compound enzyme and water, and stir to obtain a crude extract. S3. The crude extract described in S2 is subjected to complex enzyme inactivation and filtered to obtain African cardamom seed extract; The complex enzyme described in step S2 consists of cellulase, acidic xylanase, pectinase, galactosidase, protease, SQ-GR6 extractant and SQ-P200 extractant in a mass ratio of 1-2:1-2:1-2:1-2:1-1.5:1-1.5:1-1.

5. The formula amount described in step S2 is as follows, by weight: 10-20 parts African cardamom seed granules, 1-5 parts compound enzyme, and 80-90 parts water.

2. The preparation method according to claim 1, characterized in that, The African cardamom seed granules mentioned in step S1 have a particle size of 8-14 mesh; The pulverization temperature in step S1 is 0℃-60℃.

3. The preparation method according to claim 1, characterized in that, The stirring conditions described in step S2 are: stirring at 25-30℃ for 3-6 hours.

4. The preparation method according to claim 1, characterized in that, The inactivation conditions for the complex enzyme in step S3 are 50-60℃ for 30-60 minutes.

5. The African cardamom seed extract obtained by the preparation method according to any one of claims 1-4.

6. A composition containing the African cardamom seed extract of claim 5.

7. The use of the African cardamom seed extract according to claim 5 in the preparation of products with anti-inflammatory and soothing effects.

8. The application as described in claim 7, characterized in that, The anti-inflammatory and soothing products mentioned are cosmetics.

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