African amomum kravanh seed extract for starting inflammation fading mechanism as well as preparation method and application of African amomum kravanh seed extract

Abstract

The invention provides an African amomum kravanh seed extract for starting an inflammation fading mechanism as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. The preparation method comprises the following steps: cleaning, drying and crushing African amomum kravanh seeds into particles, mixing the particles with water and compound enzyme to extract active substances, inactivating the enzyme, filtering to obtain a refined extracting solution, and adding glycerol to obtain the stable African amomum kravanh seed extract. The African amomum kravanh seed extract obtained by the preparation method has no organic reagent residue, has the effects of starting an inflammation fading mechanism and shortening the duration time of skin inflammation, and can be applied to cosmetics to achieve the anti-inflammatory and soothing purposes.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and particularly relates to an African cardamom seed extract for initiating an inflammation-relief mechanism, and a preparation method and application thereof. Background Art

[0002] Skin inflammation is an uncomfortable skin condition with symptoms including redness, swelling, ulcers, itching, burning and discomfort. Skin inflammation is not a phenomenon that appears and disappears suddenly, but a process that develops over time, mainly including the initial stage of inflammation, the spread stage and the resolution stage. In the initial stage of inflammation, the initiating factors of inflammation, including but not limited to pathogens, endogenous damage, and exposure factors in the environment (UV, pollutants, allergens) trigger the initiation of inflammatory responses. And produce a variety of primary inflammatory cytokines to respond to stimuli. During the spread of inflammation, proinflammatory factors will cause the inflammatory response to spread and produce secondary inflammatory cytokines, leading to increased vascular permeability. Immune cells migrate and infiltrate inflamed tissues, which can neutralize pathogens or stimuli. In the resolution stage of inflammation, inflammatory resolution factors increase and inhibit the production of proinflammatory factors. Induce macrophages to phagocytose apoptotic neutrophils, remove pathogens and stimuli, and restore tissue homeostasis.

[0003] Once inflammation begins, the process will not be reversed or disappear suddenly, but needs to go through the whole process. Skin inflammation can bring many unpleasant feelings, such as itching, redness, tingling, and even weakening of the skin barrier. Therefore, many cosmetic raw materials or cosmetic products are committed to improving skin inflammation. These improvements can be summarized into two dimensions, one is the intensity of inflammation, and the other is the duration of inflammation. At present, many cosmetics on the market can reduce the intensity of skin inflammation by inhibiting the production of proinflammatory factors by inhibiting some common cell inflammation signaling pathways. For example, Chinese patent CN118286138A discloses an anti-allergic and soothing composition, which uses the active ingredients in saffron to inhibit the production of proinflammatory factors; Chinese patent CN118557587A discloses a method of using gentiopicroside to reduce the content of proinflammatory factors in tissues to relieve skin inflammation.

[0004] However, shortening the duration of skin inflammation is a difficult problem. After inflammation occurs, the pro-inflammatory factors or stimuli that have been produced will always exist in the inflamed tissue and play a pro-inflammatory role. Even if the production of new pro-inflammatory factors is suppressed, the existing pro-inflammatory factors will not be cleared until the inflammation subsides. Therefore, how to develop cosmetic raw materials that can shorten the duration of skin inflammation is a new research and development direction for those skilled in the art.

[0005] Historically, people believed that the resolution of inflammation was a natural passive process. As time passed, pro-inflammatory factors slowly decomposed or were slowly engulfed by macrophages, the concentration of pro-inflammatory factors decreased, and the inflammation naturally subsided. However, a large number of studies in the past few decades have shown that the resolution of inflammation is a programmed active process, and the lack of any of its components may lead to overactive, uncontrolled chronic inflammation. After the inflammation resolution mechanism is activated, the inflammation quickly enters the resolution period, the pro-inflammatory factors decrease, the anti-inflammatory factors increase, and the macrophages engulf the existing pro-inflammatory factors and irritants, and finally the inflammation ends and the symptoms of inflammation disappear. If the inflammation resolution mechanism cannot be activated in time, the inflammation will remain in the spreading period, which is equivalent to the extension of the inflammation time. In addition, a long-term inflammatory state will turn into chronic inflammation, leading to persistent autoimmune reactions, tissue fibrosis, inflammatory aging and other problems. At the same time, it also means a further extension of the inflammation time. Therefore, whether the inflammation resolution mechanism can be activated becomes the key to shortening the duration of skin inflammation.

[0006] In the process of initiating the inflammation resolution mechanism, a core class of initiating factors is called SPM (specialized pro-resolving mediators). SPM includes lipoxins, resolvins, protectins, maresins, etc. Among them, LXA4 (lipoxin A4) is a very important SPM.

[0007] On the other hand, in the process of initiating the inflammation resolution mechanism, SPM needs to bind to a series of specific receptors to play an inflammation resolution role. Among these receptors, FPR2 (formyl peptide receptor 2) is a very important receptor. FPR2 can recognize the invasion of microorganisms or the initiation factors of inflammation, and further initiate the immune and inflammatory processes. In this link, formyl peptides from mitochondria and bacteria, peptides of surface proteins of bacteria or viruses, or endogenous proteins in the human body, such as SAA (serum amyloid A) and Aβ42 (β-amyloid protein, amyloid β-protein), can activate the pro-inflammatory effect of FPR2. However, FPR2 has different binding sites. In addition to binding to the above-mentioned 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 different ligands it binds to.

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

[0009] At present, many cosmetic raw materials on the market are derived from ginger plants. However, different specific varieties and different extraction parts have different effects. African cardamom (AFRAMOMUM MELEGUETA), as a kind of ginger plant, has relatively few related studies. At present, no research has found that African cardamom has the effect of activating the mechanism of skin inflammation resolution, or increasing the expression of SPM and LXA4, or regulating FPR2.

[0010] At present, the common plant extract processes on the market involve high-temperature boiling, high-temperature crushing, organic solvent extraction and other processes. High temperature environment often destroys the effective ingredients in plants. Organic solvent extraction can extract water-insoluble components in plants, and these components often have higher biological activity. However, organic solvents are prone to environmental pollution, and the residues of organic solvents in cosmetic raw materials will cause damage to the skin. If organic solvents are not used and only water is used as the extraction medium, the active ingredients in the dissolved plants cannot be well extracted. Summary of the invention

[0011] In view of the problems existing in the prior art, the present invention provides an African cardamom seed extract for initiating an inflammation resolution mechanism, and a preparation method and application thereof. The African cardamom seed extract obtained by the preparation method of the present invention has the effects of increasing the expression of LXA4 and regulating the sensitivity of FPR2, thereby initiating an inflammation resolution mechanism, shortening the duration of skin inflammation, and achieving the purpose of anti-inflammatory and soothing.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing an African cardamom seed extract for initiating an inflammation-resolving mechanism comprises the following steps: S1. Take African cardamom seeds, wash with water and dry until there is no moisture on the surface, and store in a refrigerator at 4°C. When using, crush them at low temperature into African cardamom seed particles; S2, mixing the African cardamom seed particles in step S1 with the compound enzyme and water in a formula amount into a reaction kettle, and stirring to obtain a crude extract; S3, deactivating the composite enzyme in the crude extract of S2, and filtering to obtain a refined extract; S4. Add glycerol to the refined extract in S3, and seal the extract in subpackages to obtain African cardamom seed extract.

[0013] Preferably, the particle size of the particles in step S1 is 8-14 mesh, and the temperature during pulverization is 0-60°C.

[0014] Preferably, the formula amount described in step S2 is, by mass: 10-20 parts of African cardamom seed particles, 1-5 parts of complex enzyme and 80-90 parts of water.

[0015] Preferably, the composite enzyme in step S2 is composed of cellulase, acid xylanase, pectinase, galactosidase, protease, glycosyltransferase and hydroxyl oxidase in a mass ratio of 1-2:1-2:1-2:1-2:1-1.5:1-1.5:1-1.5.

[0016] Preferably, the enzyme activity of the complex enzyme in step S2 is: cellulase 1000-1500U / mL, acid xylanase 1000-2000U / mL, pectinase 1500-2000U / mL, galactosidase 1000-1500U / mL, protease 800-1200U / mL, glycosyltransferase 500-800U / mL and hydroxyl oxidase 500-800 U / mL.

[0017] Preferably, the glycosyltransferase is SQ-GR6 extractase.

[0018] The substrate of the glycosyltransferase includes common monosaccharides such as glucose. The monosaccharide can be transferred to the polyphenol to form a glycosidic bond, thereby increasing the solubility of the polyphenol and gingerol in water. The use of other brands and types of glycosyltransferases in the extraction process is also regarded as the protection content of the present invention.

[0019] Preferably, the hydroxyl oxidase is SQ-P200 extractase.

[0020] Hydroxyl oxidase has a wide range of substrate adaptability and can identify a variety of different polyphenol molecules, converting the hydroxyl groups on the polyphenol molecules into carboxyl groups, thereby increasing the solubility of polyphenols and gingerol in water. If other brands and models of hydroxyl oxidase are used in the extraction process, they are also regarded as the protection content of the present invention.

[0021] Preferably, the stirring condition in step S2 is stirring at 25-30° C. for 3-6 hours.

[0022] Preferably, the conditions for inactivating the complex enzyme in step S3 are inactivation at 50-60° C. for 30-60 min.

[0023] Preferably, the filtering condition in step S3 is to first filter through 0.5 μm and then filter through 0.22 μm.

[0024] Preferably, the mass ratio of the refined extract to glycerol in step S4 is 2-4:6-8; more preferably 3:7.

[0025] The African cardamom seed extract obtained by the above method contains a variety of polyphenols and polyphenol derivatives, as well as gingerol as active ingredients. 6-gingerol is used as a calibration substance to control the quality of the final extract, and the set quality standard is: 6-gingerol in the African cardamom seed extract solution is greater than 50 micrograms / gram (μg / g).

[0026] The present invention also provides the African cardamom seed extract prepared by the above preparation method.

[0027] The present invention also provides a composition of the African cardamom seed extract.

[0028] The present invention also provides the use of the African cardamom seed extract in preparing products with anti-inflammatory and soothing effects.

[0029] Specifically, the anti-inflammatory and soothing product is a cosmetic.

[0030] The technical effects achieved by the present invention are: (1) In the process of preparing the African cardamom seed extract, the present invention adopts a low-temperature composite enzyme method for aqueous phase extraction, thereby avoiding the destruction of the active ingredients by high temperature.

[0031] (2) The African cardamom seed extract obtained by the preparation method of the present invention has the ability to activate the inflammation-resolving mechanism and can be safely used in anti-inflammatory and soothing cosmetics.

[0032] (3) The composite enzyme of the present invention ionizes and glycosides components that are poorly soluble in water, making them soluble in water. Therefore, they can be extracted with pure water without the need for organic solvents. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are the fluorescence staining images of the positive control group 2, the LXA4 control group, the test group 1 and the test group 2, wherein the green fluorescence represents the IL-8 protein, the red fluorescence represents the LXA4 protein, and the blue fluorescence represents the cell nucleus. DETAILED DESCRIPTION

[0034] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0035] The following sources of raw materials are provided for illustrative purposes: African cardamom: originated from the Republic of Ivory Coast in western Africa; Cellulase, acid xylanase, pectinase, galactosidase, protease: Novozyme Corp.; SQ-GR6 extraction enzyme, SQ-P200 extraction enzyme: Proteus SA; Human skin epidermal cells HaCaT, human fibroblasts HSF: CLS Cell Lines Service GmbH, Germany; Keratinocyte growth medium (KGM), fetal bovine serum BSA, DMEM high glucose medium, cell culture related reagents: Gibco; ELISA kits, immunofluorescence staining-related reagents, basic chemical reagents, lysis buffer, related proteases, and cell wall fragments of Pseudomonas aeruginosa and Staphylococcus aureus were from ThermoFisher Scientific, Hyclone, and SIGMA Aldrich.

[0036] LXA4, IL-8 and PGF2 related antibodies were from Abcam, USA.

[0037] 1. Preparation of African cardamom seed extract Example 1 Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 10g of complex enzyme. The composition of the complex enzyme is cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme, and the mass ratio is 1:1:1:1:1:1:1. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. 2000 g of glycerol was added to the refined extract, and the extract was packaged and sealed in a sterile room to obtain the African cardamom seed extract.

[0038] Example 2 The mixture was prepared according to the method described in Example 1, wherein the amount of the complex enzyme used was 30 g.

[0039] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 30g of complex enzyme. The composition of the complex enzyme is cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme, and the mass ratio is 1:1:1:1:1:1:1. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. 2000 g of glycerol was added to the refined extract, and the extract was packaged and sealed in a sterile room to obtain the African cardamom seed extract.

[0040] Example 3 The composite enzyme was prepared according to the method described in Example 2, wherein the mass ratio of cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme was 1.5:1.5:1.5:1:1:1:1.

[0041] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 30g of complex enzyme. The composition of the complex enzyme is cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme, and the mass ratio is 1.5:1.5:1.5:1:1:1:1. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. 2000 g of glycerol was added to the refined extract, and the extract was packaged and sealed in a sterile room to obtain the African cardamom seed extract.

[0042] Example 4 The composite enzyme was prepared according to the method described in Example 2, wherein the mass ratio of cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme was 1:1:1:1.5:1.5:1:1.

[0043] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 30g of complex enzyme. The composition of the complex enzyme is cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme, and the mass ratio is 1:1:1:1.5:1.5:1:1. Continue stirring at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. 2000 g of glycerol was added to the refined extract, and the extract was packaged and sealed in a sterile room to obtain the African cardamom seed extract.

[0044] Example 5 The composite enzyme was prepared according to the method described in Example 2, wherein the mass ratio of cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme was 1:1:1:1:1:1.5:1.5.

[0045] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 30g of complex enzyme. The composition of the complex enzyme is cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme, and the mass ratio is 1:1:1:1:1:1.5:1.5. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. 2000 g of glycerol was added to the refined extract, and the extract was packaged and sealed in a sterile room to obtain the African cardamom seed extract.

[0046] Comparative Example 1 The composite enzyme was prepared according to the method described in Example 1, wherein the composite enzyme consisted of α-amylase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme in a mass ratio of 1:1:1:1:1:1:1:1.

[0047] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 10g of complex enzyme. The composition of the complex enzyme is α-amylase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme, and the mass ratio is 1:1:1:1:1:1:1. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. 2000 g of glycerol was added to the refined extract, and the extract was packaged and sealed in a sterile room to obtain the African cardamom seed extract.

[0048] Comparative Example 2 The composite enzyme was prepared according to the method described in Example 1, wherein the mass ratio of the composite enzyme consisting of galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme was 1:1:1:1.

[0049] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 10g of complex enzyme. The composition of the complex enzyme is galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme in a mass ratio of 1:1:1:1. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. Add 2000g of glycerol to the refined extract, and seal it in a sterile room to obtain African cardamom seed extract.

[0050] Comparative Example 3 The composite enzyme was prepared according to the method described in Example 1, wherein the composite enzyme consisted of cellulase, acid xylanase and pectinase in a mass ratio of 1:1:1.

[0051] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 10g of complex enzyme. The composition of the complex enzyme is cellulase, acid xylanase and pectinase, and the mass ratio is 1:1:1. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. Add 2000g of glycerol to the refined extract, and seal it in a sterile room to obtain African cardamom seed extract.

[0052] Comparative Example 4 The composite enzymes were prepared according to the method described in Example 1, wherein the composition ratio of the composite enzymes was different, specifically: cellulase, acid polysaccharidase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme, and the mass ratio thereof was 1:2.5:2.5:2.5:2:2:2.

[0053] Take African cardamom seeds, wash them with clean water, and place them in an oven at 40°C to dry until there is no moisture on the surface. Use a low-temperature grinder to crush them, and the crushed African cardamom seed particles are sieved through a 10-mesh sieve. Take 100g of the sieved African cardamom seed particles, add 800g of water, and 10g of complex enzyme. The proportions of the complex enzymes are different, and the mass ratios of cellulase, acid xylanase, pectinase, galactosidase, protease, SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme are 1:2.5:2.5:2.5:2:2:2. Stir continuously at 25°C for 5h to obtain a crude extract. The crude extract is heated to 60°C and maintained for 60min to inactivate the complex enzyme. The inactivated crude extract is transferred to a filtration device, first filtered once using a 0.5μm filter membrane, and then filtered twice using a 0.22μm filter membrane, and the filtrate obtained is a refined extract. 2000 g of glycerol was added to the refined extract, and the extract was packaged and sealed in a sterile room to obtain the African cardamom seed extract.

[0054] 2. Effect Test The content of active ingredients in African cardamom seed extract was tested by conventional HPLC method with 6-gingerol as the calibration substance and 6-gingerol standard as the external standard. The results are shown in Table 1.

[0055] Table 1

[0056] When using low-temperature composite enzyme method to extract active substances from African cardamom seeds, the choice of composite enzyme type is very important.

[0057] Both cellulase and acid xylanase can be used to decompose the cell walls of plant cells and release the active ingredients in the cells. Pectinase can decompose the galacturonic acid rich in African cardamom seeds. Galactase and protease can separate the active ingredients from the protein and polysaccharide complex, promote the release and dissolution of the active ingredients, and also have the effect of stabilizing the extract. Most of the active ingredients in African cardamom seed cells are difficult to dissolve in water, have low bioavailability and are easy to precipitate. SQ-GR6 extraction enzyme and SQ-P200 extraction enzyme can use the acid radical ions, glucose and mannose in the plant cells themselves to ionize and glycosylate the insoluble polyphenols and gingerol components to form a state that can be soluble in water. Therefore, an extract with stable properties can be obtained without the need for organic solvents.

[0058] Comparative Example 1 used α-amylase to replace cellulase, and Comparative Example 2 only used galactosidase, protease, SQ-GR6 extraction enzyme, and SQ-P200 extraction enzyme. Finally, the content of 6-gingerol in the extracts of Comparative Examples 1-2 was <1 μg / g. In view of the characteristics of the cells of African cardamom seeds, whether other enzymes are used to replace or only a few of them are used, the cell wall of African cardamom seeds cannot be destroyed, and the active substances therein cannot be extracted.

[0059] In Comparative Example 3, only cellulase, acid xylanase and pectinase were used, and the content of 6-gingerol in the final extract was only 9 μg / g, and flocculent precipitation began to appear after the extract was left to stand for 2 hours.

[0060] That is, the cellulase, acid xylanase, pectinase, galactosidase, protease, glycosyltransferase and hydroxyl oxidase of the present invention have a coordinated and increasing effect, and none of them can be missing.

[0061] Although the seven enzymes of the present invention were used in Comparative Example 4, their proportions were not within the protection scope of the present invention, and the content of 6-gingerol in the final extract was only 13 μg / g.

[0062] In Examples 1-5 within the scope of protection claimed by the present invention, the content of 6-gingerol in the final extract is 54-68 μg / g, and there is no precipitation after standing.

[0063] Effect of African cardamom seed extract on the expression of prostaglandin PGF2 and anti-inflammatory factor LXA4 Human skin epidermal cells were cultured according to the standard. After cell recovery, they were cultured in vitro for 7 days and grew into a dense monolayer. Microscopic confirmation showed that they had the morphology of keratinocytes. Subculture and adjust the cell number to 1×10 5 The cells were inoculated into the culture well plate at a density of 100 / mL, and the cells were grouped and tested when the cell confluence reached about 80%.

[0064] Negative control group 1, no inducing substance was added. Positive control group 1, phospholipase A2 was added as an inducing substance. The test group was divided into two dose groups, and the same amount of phospholipase A2 as the positive control group 1 was added as an inducing substance, and the African cardamom seed extract obtained in Example 1 was added at 0.2% and 0.5% of the mass fraction of the culture medium, respectively. The cells were incubated for 24 hours, and the culture medium was washed away for subsequent testing. The protein expression levels of prostaglandin F2 (PGF2) and lipoxin A4 (LXA4) were determined by ELISA method.

[0065] The protein expression level of the negative control group was set to 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 2 shows the effect of African cardamom seed extract on the expression levels of PGF2 and LXA4.

[0066] Table 2

[0067] After adding the inducing substance phospholipase A2, the cells entered an inflammatory state, the phospholipids in the cell membrane and inside the cell were broken down to produce arachidonic acid (AA), and further produced the pro-inflammatory factor PGF2. Therefore, compared with the negative control group 1, the PGF2 protein expression in the positive control group 1 and the experimental group increased accordingly. When African cardamom seed extract was added to the experimental group, the relative expression of PGF2 was inhibited. Compared with the positive control group 1, the expression of PGF2 decreased by 27.7% in the experimental group 1 and 55.8% in the experimental group 2.

[0068] The downstream pathway of AA to generate PGF2 or LXA4 is a competitive balance process. Under inflammatory conditions, a large amount of AA is used to generate PGF2, while the amount of LXA4 generated is small. Compared with the negative control group 1, the amount of LXA4 generated in the positive control group 1 was only slightly increased. When African cardamom seed extract was added to the experimental group, the relative expression of LXA4 increased significantly. Compared with the positive control group 1, the expression of LXA4 increased by 21.3% in the experimental group 1 and 47.8% in the experimental group 2.

[0069] The test results show that African cardamom seed extract can inhibit the expression of pro-inflammatory factor PGF2 and promote the expression of anti-inflammatory factor LXA4 when human skin epidermal cells HaCaT are in an inflammatory state, thereby achieving soothing and anti-inflammatory effects.

[0070] Effect of African cardamom seed extract on FPR2 sensitivity 3.1 Detection method and process Human fibroblasts were cultured according to the standard. After cell recovery, they were cultured in vitro for 7 days and grew into a dense monolayer. Microscopic confirmation of fibroblast morphology was performed. The cell number was adjusted to 1×10 5 The cells were inoculated into the culture well plate at a density of 100 cells / mL, and grouped for testing when the cell confluence reached about 80%. The specific group settings are listed in Table 3.

[0071] Table 3

[0072] The inducing substances in Table 3 are 5 μg / mL serum amyloid protein 1 (SAA1) and 100 ng / ml of a mixture of cell wall fragments of Pseudomonas aeruginosa and Staphylococcus aureus. The use of this inducing substance combination can simulate the initial stage of skin inflammation. The amount of LXA4 added is 100 ng / ml. The cells were incubated for 4 hours, and the culture medium was washed away for subsequent testing. The protein expression was determined using the ELISA method.

[0073] 3.2 Detection results of IL-8 and LXA4 expression The protein expression level of the negative control group was set to 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.

[0074] Table 4

[0075] After adding the inducing substance and culturing for 4 hours, the cells entered the initial stage of inflammation, and the expression of IL-8 increased significantly, indicating that the inflammation of the cells was aggravating. At this stage, only the content of the inflammation-reducing factor LXA4 was increased, and the relative expression of IL-8 did not decrease significantly, and the inflammation could not be effectively resolved, because at this time, the amount of pro-inflammatory factors in the cells was greater, and the inflammation-reducing factors could not effectively bind to the FPR2 receptor.

[0076] In the test group with African cardamom seed extract, the expression of IL-8 decreased by 26.5% in test group 1 and 49.5% in test group 2 relative to the positive control group 2; at the same time, the expression of LXA4 increased by 85.6% in test group 1 and 143.3% in test group 2 relative to the positive control group 2. This shows that when African cardamom seed extract is added, the relative expression of IL-8 is significantly reduced and the expression of LXA4 is increased.

[0077] 3.3 Methods and results of fluorescent staining of cells Human fibroblasts were fixed, permeabilized, 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 sealed. After washing, they were photographed using a fluorescence microscope. Figure 1 This is the effect of African cardamom seed extract on the expression of IL-8 and LXA4 proteins, where the green fluorescence represents IL-8 protein, the red fluorescence represents LXA4 protein, and the blue fluorescence represents the cell nucleus.

[0078] Human fibroblasts enter an inflammatory state under the action of inducing substances, and IL-8 expression increases significantly. At this time, adding LXA4 alone cannot inhibit the progression of inflammation, but adding LXA4 and African cardamom seed extract at the same time significantly reduces green fluorescence and increases red fluorescence.

[0079] 3.4 Summary of the test on the modulation of FPR2 sensitivity by Aframomum melegueta seed extract African cardamom seed extract can allosterically regulate the FPR2 receptor, inhibit the sensitivity of FPR2 to pro-inflammatory factors, and enhance the sensitivity of FPR2 to the anti-inflammatory factor LXA4. Therefore, even when the content of LXA4 is low, FPR2 and LXA4 can be combined, thereby transforming the downstream effect to inhibit the inflammatory signaling pathway and initiating the mechanism of inflammation resolution.

[0080] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an African cardamom seed extract for initiating an inflammation-resolving mechanism, characterized in that: The following steps are involved: S1, washing, drying, and crushing African cardamom seeds to obtain African cardamom seed particles; S2, mixing the African cardamom seed particles in step S1 with the compound enzyme and water, and stirring to obtain a crude extract; S3, deactivating the composite enzyme in the crude extract of S2, and filtering to obtain the African cardamom seed extract; Wherein, the complex enzyme in step S2 is composed of cellulase, acid xylanase, pectinase, galactosidase, protease, glycosyltransferase and hydroxyl oxidase.

2. The preparation method according to claim 1, characterized in that The particle size of the African cardamom seed particles in step S1 is 8-14 mesh; The pulverizing temperature in step S1 is 0°C-60°C.

3. The preparation method according to claim 1, characterized in that: The formula amount described in step S2 is, by mass: 10-20 parts of African cardamom seed particles, 1-5 parts of complex enzyme and 80-90 parts of water.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the cellulase, acid xylanase, pectinase, galactosidase, protease, glycosyltransferase and hydroxyl oxidase is 1-2:1-2:1-2:1-2:1-1.5:1-1.5:1-1.

5.

5. The preparation method according to claim 1, characterized in that: The stirring condition in step S2 is: stirring at 25-30° C. for 3-6 hours.

6. The preparation method according to claim 1, characterized in that: The condition for inactivating the complex enzyme in step S3 is inactivation at 50-60° C. for 30-60 min.

7. The African cardamom seed extract obtained by the preparation method according to any one of claims 1 to 6.

8. A composition comprising the African cardamom seed extract according to claim 7.

9. Use of the African cardamom seed extract according to claim 7 in the preparation of anti-inflammatory and soothing products.

10. The use according to claim 9, characterized in that: The anti-inflammatory and soothing product is a cosmetic.

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