A coffee fruit peel extract, a preparation method thereof based on micro-molecular cluster water, and application thereof in cosmetics or food

Abstract

The present application belongs to the technical field of cosmetic and food raw materials, and particularly relates to a coffee peel extract, a preparation method of micro-molecular cluster water and application of the coffee peel extract in cosmetics or food. 17 The micro-molecular cluster water with O-NMR half-peak width less than 100 Hz is subjected to negative pressure cavitation extraction to obtain an extract material; the extract material is subjected to solid-liquid separation and then subjected to adsorption through a macroporous adsorption resin column to obtain an elution liquid; and the elution liquid is concentrated to obtain the coffee peel extract. The coffee peel extract provided by the present application is not only rich in active ingredients, but also has excellent effects of promoting the skin penetration of active ingredients, and has the characteristics of high safety, no irritation and promotion of digestion and absorption. The coffee peel extract can be applied in cosmetics or food without relying on or reducing the use of traditional chemical penetration enhancers, and can be widely applied in cosmetics or food, thereby having significant economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics and food ingredients technology, specifically relating to a coffee fruit peel extract, a method for preparing micro-molecular cluster water, and their application in cosmetics or food. Background Technology

[0002] Coffee cherry peel is a major byproduct of coffee bean processing and is usually discarded as waste. Studies have shown that coffee cherry peel contains abundant natural active ingredients such as polysaccharides, chlorogenic acid, and caffeine, possessing potential skincare benefits including antioxidant and anti-inflammatory properties. However, traditional extraction processes (such as hot water extraction and organic solvent extraction) suffer from low efficiency, high energy consumption, potential damage to heat-sensitive components, and solvent residue risks, limiting the high-value development and utilization of coffee cherry peel and its application in the safety-critical cosmetics field.

[0003] On the other hand, in cosmetic formulations, the transdermal absorption efficiency of active ingredients is a key factor determining their final efficacy. Many active ingredients (such as whitening and anti-aging ingredients) have difficulty effectively penetrating the skin's stratum corneum barrier due to their molecular weight or polarity. Currently, chemical penetration enhancers (such as azone and Tween) are often added to improve permeability, but some penetration enhancers may pose a risk of skin irritation, and long-term use may damage the skin barrier, which contradicts the trend of cosmetics pursuing safety and gentleness.

[0004] In summary, existing technologies lack methods for efficiently and gently enriching active ingredients from coffee fruit peels; they also lack plant extracts that do not rely on traditional chemical penetration enhancers and possess excellent biocompatibility and the ability to promote transdermal penetration of active ingredients. Summary of the Invention

[0005] The purpose of this invention is to provide a coffee fruit peel extract, a method for preparing micro-molecular cluster water, and its application in cosmetics or food. The coffee fruit peel extract (also known as micro-molecular coffee fruit peel cell fluid) provided by this invention is not only rich in active ingredients, but also has excellent effects in promoting the transdermal penetration of active ingredients. At the same time, it also has the characteristics of promoting digestion and absorption, high safety, and non-irritation. When applied to cosmetics or food, it can reduce or eliminate the need for traditional chemical penetration enhancers, and can be widely used in cosmetics or food, with significant economic benefits.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing coffee pericarp extract based on micro-molecular cluster water, comprising the following steps: Coffee fruit peel raw material and first micro-molecular cluster water were extracted using negative pressure cavitation to obtain the extract material. The first micro-molecular cluster water... 17The O-NMR full width at half maximum (FWHM) is less than 100 Hz, and the conditions for negative pressure cavitation extraction include: negative pressure of -0.075 to -0.08 MPa and extraction temperature of 2 to 25 °C. The extract material is subjected to solid-liquid separation to obtain an extract solution; The extract was adsorbed onto a macroporous adsorption resin column, and then eluted with a sodium chloride solution comprising NaCl and second micro-molecular cluster water. 17 The O-NMR full width at half maximum (FWHM) was less than 100 Hz, and the eluent was obtained. The solution was concentrated to obtain coffee fruit peel extract.

[0007] Preferably, the first micro-molecule cluster water and the second micro-molecule cluster water 17 The half-peak width of the O-NMR is less than 70 Hz; the preparation methods of the first and second micro-molecule cluster water include: activating and filtering water through physical packing materials; the physical packing materials include one or more of the following: sand ceramic, activated carbon, ion exchange resin, calcium sulfate, negative potential spheres, tourmaline ceramic spheres, maifanite spheres and KDF-55C.

[0008] Preferably, the conditions for negative pressure cavitation extraction also include: the particle size of the coffee fruit peel raw material is 1~20mm, the negative pressure cavitation time is 40~65min, the liquid-to-material ratio is 4:1~8:1, and the extraction is performed once.

[0009] Preferably, the solid-liquid separation is performed by filtration using a sieve, wherein the mesh size of the sieve used for filtration is 60 mesh and 80 mesh respectively; the macroporous adsorption resin filling the macroporous adsorption resin column includes one or more of X-5, AB-8, NK-2, NKA-2, NK-9, D312, D001, D101 and WLD.

[0010] Preferably, the macroporous adsorption resin column is made of D312 acrylic macroporous resin and D001 styrene macroporous resin used in series. The elution includes a first-stage elution and a second-stage elution; the molar concentration of NaCl in the sodium chloride solution used in the first-stage elution is 0.25~0.35 mol / L, and the molar concentration of NaCl in the sodium chloride solution used in the second-stage elution is 0.05~0.15 mol / L.

[0011] Preferably, the concentration is a membrane concentration; the solid content of the coffee fruit peel extract is 6~7 mg / mL.

[0012] This invention provides a coffee pericarp extract prepared by the preparation method described in the above technical solution.

[0013] This invention provides the application of the coffee fruit peel extract described above in cosmetics or food.

[0014] Preferably, the cosmetic is a formulation that promotes the transdermal penetration of active ingredients; the food is a formulation used to promote digestion and absorption.

[0015] This invention provides a cosmetic product comprising an active ingredient and the coffee fruit peel extract described in the above technical solution; the active ingredient comprises one or more of ferulic acid, dipotassium glycyrrhizate, niacinamide, and oligopeptides.

[0016] This invention provides a method for preparing coffee pericarp extract (also known as "micro-molecular coffee pericarp cell sap") based on micro-molecular cluster water, comprising the following steps: extracting coffee pericarp raw material and a first micro-molecular cluster water using negative pressure cavitation to obtain the extract material, wherein the first micro-molecular cluster water... 17 The O-NMR full width at half maximum (WHM) is less than 100 Hz. The negative pressure cavitation extraction conditions include: negative pressure of -0.075 to -0.08 MPa and extraction temperature of 2 to 25°C. The extract material is subjected to solid-liquid separation to obtain an extract. The extract is then adsorbed onto a macroporous adsorption resin column and eluted with a sodium chloride solution comprising NaCl and second-micro-cluster water. 17 An O-NMR half-peak width of less than 100 Hz was obtained, yielding an eluent; the eluent was then concentrated to obtain a coffee pericarp extract. Firstly, this invention employs... 17Using micro-molecular cluster water with a half-peak width of less than 100 Hz in O-NMR as the extraction solvent, this invention differs from the commonly used ordinary macromolecular cluster water in existing technologies. It has high reactivity, low surface tension, improved fluidity and wettability, and significantly enhanced permeation and diffusion capabilities. This invention combines negative pressure cavitation extraction. The high permeability and low surface tension of the micro-molecular cluster water allow it to quickly penetrate into the cell wall micro-cracks and cell interior generated by negative pressure cavitation, significantly shortening the path and time for active ingredients to diffuse from the cell to the solvent, enhancing the mass transfer efficiency of cavitation extraction, and solving the technical problems of ordinary macromolecular cluster water's inability to enter micropores and slow mass transfer. At the same time, this invention optimizes the pressure conditions of negative pressure cavitation. The local extreme environment generated by negative pressure cavitation can further activate the reactivity of micro-molecular cluster water, increasing its solubility capacity for active ingredients in coffee pericarps, and avoiding the problems of incomplete dissolution and high residue of active ingredients in traditional ordinary water extraction. Secondly, this invention uses micro-molecular cluster water as an extraction solvent to achieve solid-liquid separation of the extract. Due to the high wettability and high dispersibility of the micro-molecular cluster water, the active ingredients in the extract can be uniformly dispersed, preventing them from agglomerating and clogging the resin pores. At the same time, its low viscosity and high mass transfer rate characteristics can promote the rapid diffusion of active ingredients into the resin channels, improving the resin's adsorption kinetics and saturated adsorption capacity. In addition, the micro-molecular cluster water has weak selectivity for dissolving water-soluble impurities such as polysaccharides and proteins, which can reduce the competition between impurities and target active ingredients for resin adsorption sites, significantly improving adsorption purity. Third, the eluent used in the elution stage of this invention is a sodium chloride solution prepared with micro-molecular cluster water as the solvent. This small-molecule cluster water can quickly penetrate deep into the resin pores, efficiently replacing the adsorbed active ingredients, thus solving the problem of ordinary water's inability to elute deep-layer components and incomplete elution. Simultaneously, the micro-molecular cluster water reduces the surface tension of the eluent, synergistically disrupting the hydrophobic and hydrogen bonding interactions between the active ingredients and the resin with sodium chloride, further improving elution efficiency and resolution. Furthermore, the elution process is gentle and does not damage the structure of the active ingredients, ensuring the high purity and high bioactivity of the final extract. Therefore, this invention, through the combination of micro-molecular cluster water, negative pressure cavitation extraction, and macroporous adsorption resin, achieves a synergistic effect of enhanced extraction medium characteristics, improved physical field cell wall disruption, and precise adsorption separation. The resulting micro-molecular coffee pericarp cell sap is not only rich in active ingredients but also possesses excellent efficacy in promoting transdermal penetration of active ingredients, while also promoting digestion and absorption, high safety, and non-irritating properties.

[0017] In summary, compared with the prior art, the micro-molecular coffee pericarp cell sap prepared by this invention has the following beneficial effects: (1) Excellent transdermal penetration enhancement effect Permeability experiments using the Franz diffusion cell verified that, with ferulic acid as a model drug, the coffee pericarp extract (also known as micro-molecular coffee pericarp cell sap) prepared in this invention exhibits a superior transdermal permeation-enhancing effect on ferulic acid compared to commonly used permeation enhancers such as 3% water-soluble azone solution and 0.5% Tween solution. Furthermore, its permeation-enhancing effect is significantly better than that of coffee pericarp extract prepared using traditional extraction processes (the heating extraction process used in Comparative Example 1). This indicates that the coffee pericarp extract prepared in this invention can effectively help active ingredients penetrate the skin barrier and enhance the efficacy of cosmetic formulations.

[0018] (2) Promotes digestion and absorption Efficacy evaluation using zebrafish as a model organism showed that after using the coffee pericarp extract prepared according to this invention, the lipid staining intensity of blood vessels from the intestine to the tail of zebrafish was significantly higher than that of the normal control group, and the difference was statistically significant (P<0.05). This demonstrates that the coffee pericarp extract prepared according to this invention has the effect of promoting digestion and absorption under the experimental conditions.

[0019] (3) High safety and non-irritating Cytotoxicity tests using the CCK-8 assay showed that, within a concentration range of 0–100 mg / mL, the coffee pericarp extract prepared in this invention exhibited no cytotoxicity against either HaCaT (hydroepiandrotic epidermal cells) or HSF (hymenal fibroblasts). Furthermore, according to the IS-CAM criteria for the chicken embryo chorioallantoic membrane assay, the coffee pericarp extract prepared in this invention had an irritation score of 0.07, classifying it as “non-irritating,” thus demonstrating its safety for use in cosmetics.

[0020] (4) Stable physicochemical properties and clearly defined active ingredients Characterization revealed that the coffee pericarp extract prepared by this invention is a brownish-red transparent liquid with a characteristic odor. 17 The half-peak width of the O-NMR spectrum was 64.353 Hz, confirming the structural characteristics of the micro-molecular coffee pericarp cell sap of this invention as micro-molecular cluster water. Simultaneously, the coffee pericarp extract prepared by this invention is rich in polysaccharides (2.42 mg / mL) and contains active ingredients such as caffeine and chlorogenic acid. The coffee pericarp extract product prepared by this invention exhibits excellent microbiological indicators (total colony count, total mold and yeast counts are all <10 CFU / mL) and stable physicochemical properties.

[0021] (5) The process is mild, green and efficient. The preparation method provided by this invention employs a low-temperature physical extraction and purification process throughout, avoiding the damage to heat-sensitive components caused by high temperatures. Utilizing the high permeability of micro-molecular cluster water and negative pressure cavitation technology, the extraction efficiency and purity of the target active ingredient are significantly improved. Attached Figure Description

[0022] Figure 1 A flowchart illustrating the preparation process of coffee pericarp extract based on micro-molecular cluster water provided by this invention; Figure 2 NMR in Example 1 17 O half-width peak profile; Figure 3 For comparative example 1 NMR- 17 O half-width peak profile; Figure 4 This is the HPLC chromatogram for caffeine content detection in Example 1; Figure 5 The HPLC chromatogram for caffeine content detection in Comparative Example 1 is shown. Figure 6 This is the HPLC chromatogram of caffeine reference standard; Figure 7 This is the HPLC chromatogram for the detection of chlorogenic acid content in Example 1; Figure 8 The HPLC chromatogram for the determination of chlorogenic acid content in Comparative Example 1; Figure 9 This is the HPLC chromatogram of chlorogenic acid reference standard; Figure 10 The HPLC chromatogram of the ferulic acid standard sample in Test Example 3; Figure 11 The HPLC chromatogram of ferulic acid from Example 1 in Test Example 3; Figure 12 The HPLC chromatogram of ferulic acid from Comparative Example 1 in Test Example 3; Figure 13 Typical images showing the intensity of fat staining in the blood vessels from the intestine to the tail of zebrafish after each treatment group. Detailed Implementation

[0023] This invention provides a method for preparing coffee pericarp extract based on micro-molecular cluster water, comprising the following steps: Coffee fruit peel raw material and first micro-molecular cluster water were extracted using negative pressure cavitation to obtain the extract material. The first micro-molecular cluster water... 17 The O-NMR full width at half maximum (FWHM) is less than 100 Hz, and the conditions for negative pressure cavitation extraction include: negative pressure of -0.075 to -0.08 MPa and extraction temperature of 2 to 25 °C. The extract material is subjected to solid-liquid separation to obtain an extract solution; The extract was adsorbed onto a macroporous adsorption resin column, and then eluted with a sodium chloride solution comprising NaCl and second micro-molecular cluster water. 17 The O-NMR full width at half maximum (FWHM) was less than 100 Hz, and the eluent was obtained. The solution was concentrated to obtain coffee fruit peel extract.

[0024] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0025] This invention uses negative pressure cavitation extraction to extract coffee fruit peel raw material and first micro-molecular cluster water, obtaining an extract material. The first micro-molecular cluster water... 17 The O-NMR half-peak width is less than 100 Hz, and the conditions for negative pressure cavitation extraction include: negative pressure of -0.075 to -0.08 MPa and extraction temperature of 2 to 25℃.

[0026] In this invention, the coffee pericarp raw material is preferably dried coffee pericarp raw material. The moisture content of the coffee pericarp raw material before drying is 70-80%. The moisture content of the dried coffee pericarp raw material is ≤10%. The particle size of the coffee pericarp raw material is preferably 1-20 mm, more preferably 5-15 mm.

[0027] In this invention, the first micro-molecule cluster water... 17 The half-peak width of the O-NMR is preferably less than 70 Hz, more preferably 60-68 Hz. The preparation method of the first micro-molecular cluster water preferably includes: activating and filtering water through a physical packing material to obtain the first micro-molecular cluster water. The water can be one or more of tap water and purified water. The physical packing material preferably includes one or more of sand ceramic, activated carbon, ion exchange resin, calcium sulfate, negative potential spheres, tourmaline ceramic spheres, maifanite spheres, and KDF-55C, more preferably one or more of tourmaline ceramic spheres, maifanite spheres, and KDF-55C. The activation filtration is preferably carried out in a packed column, the packed column being filled with the above-mentioned physical packing material. In a specific embodiment of the present invention, the preparation method of the first micro-molecular cluster water may include: filtering water sequentially through a first packed column, a second packed column, and a third packed column to obtain the first micro-molecular cluster water. The packing material in the first packed column can be tourmaline ceramic spheres. The packing material in the second packed column can be maifanite spheres. The packing material in the third packed column can be KDF-55C. The volume ratio of the filler in the first filled column, the filler in the second filled column, and the filler in the third filled column is preferably 1:1:1.

[0028] In this invention, the negative pressure cavitation extraction can be performed in a CNC ultrasonic cleaner. Preferably, the coffee fruit peel raw material and the first micro-molecular cluster water are mixed in the extraction tank of the CNC ultrasonic cleaner, and then the negative pressure cavitation extraction is performed.

[0029] In this invention, the preferred conditions for negative pressure cavitation extraction include: a negative pressure of -0.075 to -0.08 MPa; an extraction temperature of 2 to 25°C, preferably 2 to 10°C, more preferably 3 to 8°C, and in this embodiment, 5°C; a negative pressure cavitation time of 40 to 65 min, more preferably 55 to 60 min; and a liquid-to-solid ratio of 4:1 to 8:1, more preferably 5:1 to 7:1. The liquid-to-solid ratio is the ratio of the volume (mL) of the first micro-molecular cluster water to the mass (g) of the coffee fruit peel raw material. The extraction is preferably performed once.

[0030] In this invention, the negative pressure cavitation extraction utilizes the strong cavitation effect and mechanical vibration caused by the bubbles generated by negative pressure cavitation to accelerate the rapid rupture of the cell walls of coffee fruit peel raw materials, and accelerate the release, diffusion and dissolution of intracellular components of coffee fruit peel raw materials into the solvent medium, thereby improving extraction efficiency.

[0031] After obtaining the extract, the present invention performs solid-liquid separation to obtain an extract. In this invention, the solid-liquid separation is preferably performed using a sieve. The sieve can be a stainless steel sieve. The mesh size of the sieve used for filtration is preferably 60 mesh and then 80 mesh, meaning the solid-liquid separation preferably includes: filtering the extract through a 60-mesh sieve and then an 80-mesh sieve sequentially to obtain the extract.

[0032] After obtaining the extract, the present invention adsorbs the extract onto a macroporous adsorption resin column, and then elutes it with a sodium chloride solution, wherein the sodium chloride solution comprises NaCl and second micro-molecular cluster water, and the second micro-molecular cluster water comprises NaCl and second micro-molecular cluster water. 17 The half-maximum width of the O-NMR was less than 100 Hz, and the eluent was obtained.

[0033] In this invention, the second micro-molecule cluster water... 17The half-peak width of the O-NMR is preferably less than 70 Hz, more preferably 60-68 Hz. The preparation method of the second micro-molecular cluster water preferably includes: activating and filtering water through a physical packing material, wherein the water can be one or more of tap water, plant water, and purified water, to obtain the second micro-molecular cluster water. The physical packing material preferably includes one or more of sand ceramic, activated carbon, ion exchange resin, calcium sulfate, negative potential spheres, tourmaline ceramic spheres, maifanite spheres, and KDF-55C, more preferably one or more of tourmaline ceramic spheres, maifanite spheres, and KDF-55C. The activation filtration is preferably carried out in a packed column, wherein the packed column is filled with the above-mentioned physical packing material. In a specific embodiment of the present invention, the preparation method of the second micro-molecular cluster water may include: filtering water sequentially through a first packed column, a second packed column, and a third packed column to obtain the second micro-molecular cluster water. The first packed column is filled with tourmaline ceramic spheres. The second packed column is filled with maifanite spheres. The third packed column is filled with KDF-55C. The volume ratio of the filler in the first filled column, the filler in the second filled column, and the filler in the third filled column is preferably 1:1:1.

[0034] In this invention, the macroporous adsorption resin filled in the macroporous adsorption resin column preferably includes one or more of X-5, AB-8, NK-2, NKA-2, NK-9, D312, D001, D101, and WLD, and in the embodiments, it can be D312 and D001. In this invention, the macroporous adsorption resin column preferably consists of a first macroporous adsorption resin column and a second macroporous adsorption resin column connected in series. The first macroporous adsorption resin column is preferably filled with D312 acrylic macroporous resin. The second macroporous adsorption resin column is preferably filled with D001 styrene-based macroporous resin. The volume ratio (mL) of D312 acrylic macroporous resin filled in the first macroporous adsorption resin column to the mass (g) of the coffee fruit peel raw material is preferably 3-4:1. The volume ratio (mL) of D001 styrene-based macroporous resin filled in the second macroporous adsorption resin column to the mass (g) of the coffee fruit peel raw material is preferably 3-4:1.

[0035] In this invention, the preferred method for loading the extract includes: mixing the extract with the macroporous adsorption resin packed in the first macroporous adsorption resin column, filtering to obtain the mixed macroporous adsorption resin, and packing the mixed macroporous adsorption resin into the first macroporous adsorption resin column; further mixing the filtered extract with the macroporous adsorption resin packed in the second macroporous adsorption resin column, and packing the mixed macroporous adsorption resin into the second macroporous adsorption resin column.

[0036] In this invention, the elution preferably includes sequentially performing a first-stage elution and a second-stage elution. Before the elution, a pre-wash may be performed to remove impurities. The molar concentration of NaCl in the sodium chloride solution used in the first-stage elution is preferably 0.25~0.35 mol / L, and in the examples, it can be 0.3 mol / L. The molar concentration of NaCl in the sodium chloride solution used in the second-stage elution is preferably 0.05~0.15 mol / L, and in the examples, it can be 0.1 mol / L. The ratio of the total volume of the sodium chloride solution used in the first and second-stage elutions to the column volume of the first macroporous adsorption resin column is preferably 3:1.

[0037] In this invention, the flow rate of the sodium chloride solution during the elution process is preferably 1~3 BV / h, and in the examples it can be 2 BV / h.

[0038] After obtaining the eluent, the present invention concentrates the eluent to obtain coffee pericarp extract. In this invention, the concentration is preferably membrane concentration. The membrane concentration can be reverse osmosis membrane concentration. The pore size of the reverse osmosis membrane is preferably ≤1 nm.

[0039] In this invention, the solid content of the coffee pericarp extract is preferably 6-7 mg / mL. 17 The preferred half-peak width of O-NMR is less than 65 Hz.

[0040] This invention provides a coffee pericarp extract prepared by the method described in the above technical solution. In this invention, the coffee pericarp extract, i.e., micromolecular coffee pericarp cell sap, contains polysaccharides, caffeine, and chlorogenic acid; wherein the polysaccharide content is preferably ≥2 mg / mL, and in the example, it is 2.42 mg / mL; the caffeine content is preferably ≥600 mg / mL, and in the example, it is 639 mg / mL; the chlorogenic acid content is preferably 200 mg / mL, and in the example, it is 212 mg / mL.

[0041] This invention provides the application of the coffee fruit peel extract described above in cosmetics or food.

[0042] In this invention, the cosmetic is a preparation that promotes the transdermal penetration of active ingredients, and the food is a preparation for promoting digestion and absorption.

[0043] This invention provides a cosmetic product comprising an active ingredient and the coffee fruit peel extract described in the above technical solution; the active ingredient comprises one or more of ferulic acid, dipotassium glycyrrhizate, niacinamide, and oligopeptides.

[0044] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1: Preparation of coffee pericarp extract (hereinafter referred to as "micro-molecular coffee pericarp cell sap") This embodiment illustrates the small-scale preparation method of micro-molecular coffee pericarp cell sap provided by the present invention: (1) Preparation of micro-molecular cluster water: Tap water was taken and passed sequentially through three packed columns containing 10L tourmaline ceramic balls, 10L maifanite balls, and 10L KDF-55C packing material for activation filtration to prepare micro-molecular cluster water. 17 5L of micro-molecular cluster water with a half-peak width of O-NMR <100Hz, for later use.

[0046] (2) Negative pressure cavitation extraction: Weigh 500g of dried coffee fruit peel raw material, add 2.5L of micro-molecular cluster water obtained in step (1) (liquid-to-material ratio 5:1), and place it in the extraction tank of a CNC ultrasonic cleaner. Set the negative pressure to -0.075 MPa and the temperature to 5℃, and perform negative pressure cavitation extraction for 60 min.

[0047] (3) Filtration: After extraction, the extract is released and filtered through 60-mesh and 80-mesh stainless steel screens in sequence to obtain about 2.3L of filtrate.

[0048] (4) Macroporous resin adsorption purification: The filtrate obtained in step (3) is mixed with D312 acrylic macroporous resin, filtered, and the mixed D312 acrylic macroporous resin is loaded into the first chromatography column. The filtered filtrate is then mixed with D001 styrene macroporous resin and loaded into the second chromatography column. The volume ratio of D312 acrylic macroporous resin used in the first chromatography column to the mass ratio of coffee fruit peel raw material used in step (2) is 3:1, and the volume ratio of D001 styrene macroporous resin used in the second chromatography column to the mass ratio of coffee fruit peel raw material used in step (2) is 3:1. After adsorption, 0.3 mol / L and 0.1 mol / L NaCl solutions are prepared with micro-molecular cluster water. The total volume of the NaCl solution is in the ratio of the column volume of the first chromatography column to the column volume of the first chromatography column. After pre-washing with water, elution was carried out sequentially with 0.3 mol / L NaCl solution and 0.1 mol / L NaCl solution at the same flow rate (2 BV / h), and the eluent was collected to obtain an eluent rich in chlorogenic acid, caffeine and polysaccharides.

[0049] (5) Concentration: The eluent is concentrated using a reverse osmosis membrane with a pore size of 1 nm. The solid content is concentrated to 6~7 mg / mL to obtain 1.48 kg of micro-molecular coffee pericarp cell slurry.

[0050] Comparative Example 1: Preparation of Traditional Coffee Pericarp Extract This comparative example illustrates the traditional method for preparing coffee fruit peel extract.

[0051] (1) Weigh 500g of dried coffee fruit peel raw material and add it to the mixture prepared in Example 1. 17 2.5 L of water (liquid-to-solid ratio 5:1) with a half-peak width of O-NMR <100 Hz was added to the extraction vessel. Extraction was carried out at 85-100℃ for 60 min.

[0052] (2) Filtration: After extraction, the extract is released and filtered through 60-mesh and 80-mesh stainless steel screens to obtain filtrate.

[0053] (3) Macroporous resin adsorption purification: The filtrate obtained in step (2) was mixed with D312 acrylic macroporous resin, filtered, and the mixed D312 acrylic macroporous resin was loaded into the first chromatography column. The filtered filtrate was then mixed with D001 styrene macroporous resin and loaded into the second chromatography column. The volume ratio of D312 acrylic macroporous resin used in the first chromatography column to the mass ratio of coffee fruit peel raw material used in step (2) was 3:1, and the volume ratio of D001 styrene macroporous resin used in the second chromatography column to the mass ratio of coffee fruit peel raw material used in step (2) was 3:1. After adsorption, 0.3 mol / L and 0.1 mol / L NaCl solutions were prepared with micro-molecular cluster water. The total volume of the NaCl solution was in the ratio of the column volume of the first chromatography column to the column volume of the first chromatography column. After pre-washing with water, elution was carried out sequentially with 0.3 mol / L NaCl solution and 0.1 mol / L NaCl solution at the same flow rate (2 BV / h), and the eluent was collected to obtain an eluent rich in chlorogenic acid, caffeine and polysaccharides.

[0054] (4) Concentration: Vacuum decompression concentration or open-air concentration is used to concentrate the eluent to a solid content of 6~7 mg / mL, thus obtaining coffee fruit peel extract, 1.42 kg.

[0055] Test Example 1: Characterization of the properties of coffee pericarp cell sap Objective: To characterize the physicochemical properties and content of characteristic substances of coffee pericarp extract prepared in Example 1 and Comparative Example 1 as test samples.

[0056] method: (1) Physicochemical properties Soluble solids content: Tested according to the method specified in GB / T 14415; NMR- 17 O Half-width peak: According to T / BJWA 005—2022 "Water Quality Standard" 17"Determination of Half-Peak Width of O-NMR by Nuclear Magnetic Resonance Method"; (2) Content of characteristic substances Polysaccharide content (sulfuric acid phenol method); Preparation of the standard curve: Accurately transfer 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, and 0.9 mL of 0.1 mg / mL D-anhydrous glucose standard solution into colorimetric tubes, respectively. Add water to a final volume of 1.0 mL, then add 1 mL of 5% phenol sequentially. After shaking well, add 5.0 mL of concentrated sulfuric acid at a uniform rate. Incubate at room temperature for 20 min, and measure the absorbance at 486 nm. Plot a standard curve for glucose with polysaccharide concentration on the x-axis and absorbance on the y-axis. Use 1 mL of purified water as a blank control instead of glucose solution.

[0057] Determination of polysaccharide content in samples: Accurately transfer 1 mL of sample and dilute with water 100 times to obtain the test solution. Take 1 mL of the test solution, add 1 mL of 5% (v / v) phenol aqueous solution, shake well, and add 5.0 mL of concentrated sulfuric acid (98%) at a uniform rate. React at room temperature for 20 min, and measure the absorbance at a wavelength of 486 nm. Calculate the mass concentration of polysaccharides in the test sample according to the standard curve, and calculate the polysaccharide concentration in the sample according to the following formula based on the mass concentration of polysaccharides in the test sample.

[0058] Chlorogenic acid and caffeine content 1. Principle: Chlorogenic acid and caffeine are detected at a wavelength of 254nm using high performance liquid chromatography with a UV detector. Quantification is performed based on the peak area using the external standard method.

[0059] 2. Instruments and equipment: High performance liquid chromatograph: Shimadzu LC-2030 PLUS.

[0060] 3. Reagents and solutions: 3.1 Reagents: Methanol (chromatographic grade), purified water, methanol (analytical grade), phosphoric acid (analytical grade); 3.2 Standard substances: caffeine reference standard, chlorogenic acid reference standard.

[0061] 4. Chromatographic conditions: Instrument: Shimadzu LC2030 Plus, with octadecylsilane-bonded silica gel as the packing material; Mobile phase: Aqueous solution of phosphoric acid with H3PO4 content of 0.1 wt% (76% by volume): methanol (24% by volume) eluted isocratically; Flow rate: 1.0 mL / min; Column temperature: 25℃; Detection wavelength: 254nm; Analysis time: 20 min; Injection volume: 10 μL.

[0062] 5. Preparation of standard products: Accurately weigh 2 mg each of chlorogenic acid and caffeine reference standards (accurate to 0.01 mg), dissolve them in 0.1% phosphoric acid water-methanol (volume ratio of 80:20) solution, and then dilute to 10 mL to obtain a 200 μg / mL chlorogenic acid and caffeine reference standard solution.

[0063] 6. Sample processing: Accurately weigh an appropriate amount of sample, dilute to 10 mL with 0.1% phosphoric acid solution, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0064] 7. Determination Method: Accurately pipette 10 μL of the test solution and inject it into the liquid chromatograph. Analyze under chromatographic conditions. Qualitatively determine the content of chlorogenic acid and caffeine in the sample based on retention time, and quantitatively calculate the content based on peak area using the external standard method. Calculate the content of chlorogenic acid and caffeine in the sample using the following formula: ; in: w represents the content (%). S 样 The peak area of ​​the sample; C 对 Reference standard concentration; V dilution factor; S 对 The peak area of ​​the reference standard; M represents the sample size.

[0065] result: (1) Physicochemical properties: The solid content of the micro-molecular coffee pericarp cell sap prepared in Example 1 was 4.12 mg / mL; the solid content of the traditional coffee pericarp extract prepared in Comparative Example 1 was 4.39 mg / mL.

[0066] Figure 2 NMR analysis of micro-molecular coffee pericarp cell sap prepared in Example 1 17 O half-width peak profile Figure 3 NMR analysis of traditional coffee pericarp extract prepared for Comparative Example 1 17 O half-width peak spectrum. NMR- of micro-molecular coffee pericarp cell sap prepared in Example 1. 17 The half-width of the O peak was 64.353 Hz; the NMR of the traditional coffee pericarp extract prepared in Comparative Example 1 was... 17 The half-amplitude peak width is 91.88 Hz.

[0067] (2) Content of characteristic substances: Figure 4The HPLC chromatogram for caffeine content detection in the micro-molecular coffee pericarp cell sap prepared in Example 1 is shown below. Figure 5 HPLC chromatogram for caffeine content detection in traditional coffee pericarp extract prepared in Comparative Example 1. Figure 6 This is the HPLC chromatogram of caffeine reference standard. Figure 7 The image shows the HPLC chromatogram for detecting chlorogenic acid content in the micro-molecular coffee pericarp cell sap prepared in Example 1. Figure 8 HPLC chromatogram for the determination of chlorogenic acid content in the traditional coffee fruit peel extract prepared in Comparative Example 1. Figure 9 This is the HPLC chromatogram of chlorogenic acid reference standard.

[0068] Depend on Figures 4-9 It can be seen that: the polysaccharide content in the micro-molecular coffee pericarp cell sap prepared in Example 1 was 2.42 mg / mL; the caffeine content was 639 mg / L; and the chlorogenic acid content was 212 mg / L. The polysaccharide content in the traditional coffee pericarp extract prepared in Comparative Example 1 was 1.83 mg / mL; the caffeine content was 247 mg / L; and the chlorogenic acid content was 77 mg / L.

[0069] Test Example 2 and the effect of commonly used penetration enhancers on percutaneous penetration Objective: To conduct a Franz diffusion cell permeability experiment using ferulic acid as a model drug, and to compare and evaluate the transdermal permeation-enhancing effects of the micro-molecular coffee pericarp cell sap prepared in Example 1 and two commonly used permeation enhancers, water-soluble azone and Tween, on ferulic acid.

[0070] Principle: This test case is based on the transdermal absorption mechanism, using ferulic acid as a model drug and a Franz dispersion pool to simulate the skin drug delivery environment. Ferulic acid formulations containing different penetration enhancers were placed in the supply chamber and brought into contact with simulated skin (Bama miniature pig skin), while the receiving chamber maintained physiological conditions through constant temperature circulation. The penetration enhancers altered the structure of the stratum corneum, promoting the permeation of ferulic acid into the receiving chamber. By measuring the cumulative permeation of ferulic acid in the receiving chamber at different time points, the in vitro transdermal permeation enhancement behavior of each sample was analyzed.

[0071] Reagents: Micro-molecular coffee pericarp cell sap (prepared from Example 1), medical water-soluble azone (Zhengzhou Jinbei Chemical Co., Ltd., 20200306), Tween (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.), pH 7.4 PBS solution (Shandong Puhuifen Chemical Technology Co., Ltd., batch number 20250527), ferulic acid (Shanghai Titan Technology Co., Ltd., P3348261), Wahaha (Dali Wahaha Food Co., Ltd., batch number 20250423).

[0072] Instrument: YB-P6 intelligent transdermal assay apparatus (Tianjin Pharmacopoeia Standard Instrument Factory). The diffusion cells exhibit no mutual interference and no bubble formation. The receiving chamber has a volume of 7 mL, and the diffusion area is 2.33 cm². 2 The Franz cell diffusion cell consists of two cylindrical glass tubes joined together, with a patch sandwiched between them dividing the space into two chambers. The upper chamber is the diffusion chamber, and the lower chamber is the receiving chamber. A sampling tube is connected to the right side of the receiving chamber for sample injection, sampling, and air bubble removal.

[0073] The content of ferulic acid was determined by high performance liquid chromatography (Shimadzu, LC-2050). The chromatographic conditions were as follows: column: C18, 4.6×250 mm; flow rate: 1 mL / min; column temperature: 35℃; detection wavelength: 322 nm; injection volume: 10 μL; mobile phase: 16% acetonitrile-84% phosphate buffer (pH=2.8).

[0074] Test samples and grouping: Table 1. Study Test Grouping

[0075] Method and steps: (1) Fixation: Fix the pigskin between the diffusion chamber and the receiving chamber of the Franz cell diffusion cell, with the keratin layer of the pigskin facing the diffusion chamber and the dermis layer facing the receiving chamber. After fixing the pigskin, place the matching magnetic stir bar in the receiving chamber. Use a syringe (with a sampling needle attached to the syringe) to draw 7.0 mL of receiving solution (PBS solution, pH=7.4) and tilt it to inject it into the receiving chamber, ensuring that the dermis layer of the pigskin is in close contact with the receiving solution and that there are no air bubbles between them. Fix the Franz cell diffusion cell in the transdermal experimental apparatus, turn on the electromagnetic stirrer at a speed of 600 rpm, maintain a constant temperature water bath at (32±0.5 ℃), and ensure that there are no air bubbles in the water bath jacket.

[0076] (2) Sample loading: After the water bath temperature of the diffuser is constant, sample loading is performed. According to the grouping in Table 1, use a pipette to add 1 mL of sample to the diffusion chamber.

[0077] (3) Sampling: ① At 2h, 6h, 12h, and 24h, the receiving solution in the corresponding receiving cell was completely removed for testing, and 7.0mL of PBS receiving solution was added. ② After completing the 24h sampling, the pigskin was removed from the diffusion cell, the outer ring was cut off, and the effective contact area of ​​transdermal absorption (2.33 cm²) was retained. 2 Rinse the surface with purified water 2-3 times, dry, cut into small pieces, add 5 mL of purified water, homogenize with a high-speed disperser, centrifuge at 6000 rpm for 15 min, and collect the supernatant for testing.

[0078] (4) Determination: Take 1 mL of the sample to be tested into a disposable syringe, filter it with a 0.22 μm filter head, and take the filtrate for content determination in high performance liquid chromatography.

[0079] (5) Cumulative infiltration (i=1···n-1); Where: Q: cumulative permeation volume; V: volume of receiving liquid in the receiving chamber, 7 mL; V0: volume of each sample taken, 7 mL; Ci: drug concentration in the receiving liquid from the first to the (n-1)th sampling point; Cn: sample concentration measured at the nth sampling point.

[0080] (6) Percentage of diffusion ; Where: P: diffusion percentage; Q: cumulative permeation of the sample in the receiving chamber; Po: initial sample loading amount in the diffusion chamber.

[0081] Results: The effects of each test sample on promoting transdermal penetration of the model drug ferulic acid are shown in Table 2.

[0082] Table 2. Cumulative permeation of ferulic acid in test samples over different time periods

[0083] 2. Ferulic acid permeability of each test sample is shown in Table 3. Since the saturation loading concentration of ferulic acid is 2 mg / mL, the loading amount of ferulic acid in the cumulative permeability of the test samples is calculated using 2 mg / mL.

[0084] Table 3. Cumulative permeability of ferulic acid in the test samples

[0085] Conclusion: Under the conditions of this experiment, the micro-molecular coffee pericarp cell sap showed the best effect in promoting the transdermal permeation of ferulic acid. The transdermal permeation rates of different samples were ranked as follows: micro-molecular coffee pericarp cell sap > 3% water-soluble azone solution > 0.5% Tween solution.

[0086] Test Example 3 compared with Comparative Example 1: Transdermal penetration enhancement effect Objective: To conduct a Franz diffusion cell permeability experiment using ferulic acid as a model drug, and to compare the percutaneous permeation-promoting effects of Example 1 and Comparative Example 1 on ferulic acid.

[0087] Principle: Same as test case 2.

[0088] Reagents: Micro-molecular coffee pericarp cell sap prepared in Example 1, coffee pericarp extract prepared in Comparative Example 1, PBS solution (pH 7.4, Shandong Puhuifen Chemical Technology Co., Ltd., batch number 20250527), ferulic acid (Shanghai Titan Technology Co., Ltd., P3348261), medical water, Wahaha (Dali Wahaha Food Co., Ltd., batch number 20250423). Instruments: Same as in Experiment 2.

[0089] Table 4. Test Samples and Grouping:

[0090] Method and steps: Same as in Experiment 2.

[0091] Results: The effects of each test sample on promoting transdermal penetration of the model drug ferulic acid are shown in Table 5.

[0092] Table 5. Cumulative permeation of ferulic acid in the test samples over different time periods.

[0093] 2. Ferulic acid permeability of each test sample is shown in Table 6. Since the saturation loading concentration of ferulic acid is 2 mg / mL, the loading amount of ferulic acid in the cumulative permeability of the test samples is calculated using 2 mg / mL.

[0094] Table 6. Cumulative permeability of ferulic acid in the test samples

[0095] Figure 10 The HPLC chromatogram of the ferulic acid standard sample in Test Example 3; Figure 11 The HPLC chromatogram of ferulic acid from Example 1 in Test Example 3; Figure 12 The image shows the HPLC chromatogram of ferulic acid from Comparative Example 1 in Test Example 3. Conclusion: Under the conditions of this experiment, the micro-molecular coffee pericarp cell slurry prepared in Example 1 was more effective than the coffee pericarp extract prepared in Comparative Example 1 in promoting the transdermal penetration of ferulic acid.

[0096] Test Example 4: In vitro cytotoxicity evaluation of micro-molecular coffee pericarp cell sap Objective: To investigate the in vitro toxicity of micro-molecular coffee pericarp cell sap to HaCaT cells and HSF cells using CCK-8.

[0097] Principle: The skin irritation monolayer model is a cell model formed by culturing isolated skin cells in vitro. Keratinocytes and fibroblasts are the most studied skin cells. The stratum corneum, the outermost layer of the epidermis, is the first to come into contact with irritants and is the most important barrier against external irritants. It also produces a large amount of inflammatory mediators; therefore, keratinocytes can be used as a cell model for evaluating skin irritation in vitro. The HaCaT keratinocyte line has also been used to evaluate skin irritation. The HaCaT cell line is a spontaneously immortalized human keratinocyte that exhibits differentiation characteristics similar to normal human keratinocytes during immersion culture, and is very convenient to culture and passage. Fibroblasts are another important cell type in the skin besides keratinocytes, mainly found in the dermis. In skin irritation responses, fibroblasts can also secrete inflammatory mediators, affecting the inflammatory response. Due to morphological and physiological differences, there are relatively few studies on the use of fibroblasts alone in constructing in vitro models of skin irritation. Monolayer cultures of human skin keratinocytes and fibroblasts can be used to screen for skin irritants. Although there is limited research on monolayer cell models, they can still be used as a preliminary screening test for skin irritation.

[0098] The Cell Counting Kit-8 (CCK-8) is a rapid, highly sensitive assay kit based on WST-8 (water-soluble tetrazolium salt), widely used for detecting cell proliferation and cytotoxicity. WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt) is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan dye by the electron carrier 1-methoxy-5-methylphenazineonium sulfate (1-Methoxy PMS). The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same number of cells, the color intensity is linearly related to the cell number.

[0099] The optical density (OD value) of water-soluble formazan dye was measured at a wavelength of 450 nm, and the in vitro toxicity of the samples to HaCaT cells and HSF cells was evaluated.

[0100] Cells: HaCaT human immortalized epidermal cells, catalog number SCSP-5091, cell line sourced from Shanghai Cell Bank, Chinese Academy of Sciences; HSF human dermal fibroblasts, cell line sourced from Cybio (Shanghai) Biotechnology Co., Ltd.

[0101] Culture conditions: DMEM medium, fetal bovine serum, trypsin / EDTA solution (0.25% trypsin solution and 0.02 mol / L EDTA solution mixed 1:1), phosphate buffer (PBS), cell culture flasks.

[0102] Materials and instruments: 96-well plates; CCK-8 cytotoxicity assay kit (Beijing Polymer Biotechnology Co., Ltd. MF128-01); microplate reader; clean bench.

[0103] Test Groups: Table 7 Experimental Group Design

[0104] Method and steps: Before testing, the micro-molecule coffee pericarp cell sap was dissolved in primary water (sterile purified water) to a concentration of 10%, thoroughly mixed, filtered through a 0.22μm filter membrane for sterilization, and then diluted with culture medium to the test dose concentration.

[0105] The in vitro toxicity of the test samples to HaCaT cells and HSF cells was examined using CCK-8 assays. (2 × 10⁻⁶) 4 Cells were seeded at a density of 200 μL / mL into 96-well plates. After incubation for 24 h, the old culture medium was discarded. Different concentrations of the test substance diluted with culture medium were added to the 96-well plates, and incubated for 24 h. The supernatant was then discarded. A mixture of 100 μL culture medium and 10 μL CCK-8 solution was added to each well, and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using a negative control as a baseline.

[0106] When cell viability is ≥80%, the test sample at this concentration can be considered to have no effect on cell proliferation, i.e., a safe concentration for cells. Each sample group was repeated 4 times.

[0107] Cell viability = ×100%; Results: The cell proliferation and cytotoxicity of different doses of the test sample (10% micro-molecular coffee pericarp cell sap) against HaCaT and HSF were tested using the CCK-8 kit, as shown in Table 8. Table 8. Comparison of the effects of different concentrations of test samples on the survival rate of HaCaT and HSF cells.

[0108] This invention used the CCK-8 assay to determine the effect of 10% micro-molecular coffee pericarp cell sap at four concentrations on the cell viability of HaCaT and HSF cells after 24 hours of treatment. The results are shown in Table 8. The purpose of this experiment was to screen the effects of cosmetic raw materials at four concentrations on the proliferation and cytotoxicity of epidermal and dermal cells. The drug concentration at which the relative cell viability was ≥80% was considered to have no effect on cell proliferation or to be non-toxic.

[0109] Conclusion: At the highest concentration of 10% micro-molecular coffee pericarp cell slurry (100 mg / mL), the survival rates of human immortalized epidermal cells (HaCaT) in the epidermis and human skin fibroblasts (HSF) in the dermis were both ≥80%. Therefore, the safe concentration range of 10% micro-molecular coffee pericarp cell slurry for HaCaT cells was determined to be 0–100 mg / mL; and the safe concentration range for HSF cells was also determined to be 0–100 mg / mL.

[0110] Test Example 5: Evaluation of the digestive and absorptive effects of micro-molecular coffee pericarp cell sap prepared in Example 1. Objective: To evaluate the digestive and absorptive effects of the test samples using zebrafish as a model organism.

[0111] Principle: Zebrafish serve as a model organism for digestion and absorption studies: The yolk syncytial layer (YSL) of zebrafish expresses triglyceride transfer protein (MTP) during early development. This protein is involved in lipoprotein assembly and lipid transport, and its function is highly conserved in mammals. Zebrafish also possess key digestion and absorption-related proteins homologous to mammals, such as apolipoprotein E (ApoE), phospholipase A2 (PLA2), and lipoprotein lipase (LPL).

[0112] Establishment of an exogenous food absorption model: Egg yolk powder (rich in protein, fat, and carbohydrates) was used as the exogenous food to simulate the digestive process. Oil Red O is a dye specifically for staining neutral lipids (such as triglycerides), turning orange-red after staining, which facilitates microscopic observation and quantitative analysis of the amount of fat absorbed in blood vessels. Furthermore, juvenile zebrafish are completely transparent, allowing for direct observation of lipid absorption in the viscera and blood vessels without the need for dissection.

[0113] Experimental Methods and Evaluation Indicators: Zebrafish were first fed egg yolk powder, then given the test sample (such as coffee fruit peel concentrate) or a positive control (Xinghao Meitong Compound Digestive Enzyme Capsules, hereinafter referred to as Compound Digestive Enzyme Capsules, batch number 086230304, Guangdong Dingxin Pharmaceutical Technology Co., Ltd.). After treatment, Oil Red O staining was performed, and the intensity of fat staining from the intestine to the tail vessels was quantified using image analysis software to reflect fat absorption efficiency. If the fat staining intensity of the sample group was significantly higher than that of the normal control group (…), the positive control was considered a positive control. p If the value is less than 0.05, the sample is considered to have the effect of promoting digestion and absorption.

[0114] This evaluation method is applicable to the detection of the digestive and absorptive effects of food, requiring the sample to be soluble in water or to be prepared into a suspension that is uniformly dispersed in water.

[0115] Zebrafish: Wild AB strain zebrafish, 5 days post-fertilization (5dpf), 30 fish per group, raised and bred according to the standard breeding methods of this laboratory.

[0116] Materials and instruments: (1) Reagents: Egg yolk powder; PBS phosphate buffer (pH=7.4); methylcellulose; water, deionized water or water of equivalent purity; Oil Red O; compound digestive enzyme capsules; 6-well microplate; ice, ice box, etc.

[0117] (2) Instruments: Stereo microscope; analytical balance; fluorescence stereo microscope; micro homogenizer; biochemical incubator; and other conventional laboratory instruments and equipment.

[0118] Test Groups: Table 9 Experimental Group Design

[0119] Method and steps: Wild-type AB zebrafish (5 dpf) were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). All groups were first given egg yolk powder dissolved in water, washed off after 3 hours, and then the powder was dissolved in water again. The positive control group received compound digestive enzyme capsules at a concentration of 135 μg / mL (0.135 mg / mL). A blank control group (normally fed group, given only egg yolk powder, without any other samples) was also set up. Each beaker had a volume of 25 mL. After treatment at 28℃ for 16 hours, egg yolk powder was added again to all groups, and treatment continued at 28℃ for another 8 hours. Oil Red O was then used for whole-body fat staining. After staining, 10 zebrafish were randomly selected from each experimental group and photographed. Data was collected using image processing software, and the staining intensity of fat from the intestine to the tail vessels was analyzed. The statistical analysis results of this index were used to evaluate the digestive and absorptive effects of the samples. Statistical results are expressed as mean ± SD. Statistical analysis was performed, and P < 0.05 was considered statistically significant between groups.

[0120] Judgment criteria: The MTC of the sample compared with that of normal zebrafish was measured, and the intensity of fat staining in the blood vessels from the zebrafish intestine to the tail was used as an indicator to evaluate the sample's effect on promoting digestion and absorption.

[0121] result: 1. Maximum Tolerable Concentration (MTC) Under the conditions of this experiment, the MTC of the effect of micro-molecular coffee pericarp cell sap in promoting digestion and absorption was 1.25 mg / mL.

[0122] Table 10 Results of the experiment on the concentration of each group that promotes digestion and absorption (n=30)

[0123] 2. Evaluation of the effect of micro-molecular coffee pericarp cell fluid on promoting digestion and absorption Figure 13 Typical images showing the intensity of fat staining in the blood vessels from the intestine to the tail of zebrafish after each treatment group.

[0124] Table 11. Evaluation results of the efficacy of each group in promoting digestion and absorption (n = 10)

[0125] Note: Compared with the normal control group, p<0.05 p<0.01 p<0.001 The staining intensity of blood vessel fat in the intestines to tail of zebrafish treated with the positive control group compound digestive enzyme capsules was significantly higher than that in the normal control group (P<0.05). The staining intensity of blood vessel fat in the intestines to tail of zebrafish treated with micro-molecular coffee pericarp cell sap was also significantly higher than that in the normal control group (P<0.05), indicating that micro-molecular coffee pericarp cell sap promotes digestion and absorption in zebrafish under the experimental conditions.

[0126] Conclusion: Under the experimental conditions, the coffee pericarp concentrate sample has the effect of promoting digestion and absorption.

[0127] As can be seen from the above embodiments, the present invention provides a method for preparing coffee pericarp extract based on micro-molecular cluster water, and the application of the prepared coffee pericarp extract in the fields of cosmetics and food. The coffee pericarp extract prepared by the present invention is obtained by using micro-molecular cluster water prepared by physical activation filtration as a solvent, combined with negative pressure cavitation extraction, macroporous resin purification and other processes. It is not only rich in active ingredients, but also has excellent effects in promoting the transdermal penetration of active ingredients, while also having the characteristics of promoting digestion and absorption, high safety and non-irritation.

[0128] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing coffee pericarp extract based on micro-molecular cluster water, characterized in that, Includes the following steps: Coffee fruit peel raw material and first micro-molecular cluster water were extracted using negative pressure cavitation to obtain the extract material. The first micro-molecular cluster water... 17 The O-NMR full width at half maximum (FWHM) is less than 100 Hz, and the conditions for negative pressure cavitation extraction include: negative pressure of -0.075 to -0.08 MPa and extraction temperature of 2 to 25 °C. The extract material is subjected to solid-liquid separation to obtain an extract solution; The extract was adsorbed onto a macroporous adsorption resin column, and then eluted with a sodium chloride solution comprising NaCl and second micro-molecular cluster water. 17 The O-NMR full width at half maximum (FWHM) was less than 100 Hz, and the eluent was obtained. The solution was concentrated to obtain coffee fruit peel extract.

2. The preparation method according to claim 1, characterized in that, The first and second micro-molecule cluster water 17 The half-peak width of the O-NMR is less than 70 Hz; the preparation methods of the first and second micro-molecule cluster water include: activating and filtering water through physical packing materials; the physical packing materials include one or more of the following: sand ceramic, activated carbon, ion exchange resin, calcium sulfate, negative potential spheres, tourmaline ceramic spheres, maifanite spheres and KDF-55C.

3. The preparation method according to claim 1, characterized in that, The conditions for negative pressure cavitation extraction also include: the particle size of the coffee fruit peel raw material is 1~20mm, the negative pressure cavitation time is 40~65min, the liquid-to-material ratio is 4:1~8:1, and the extraction is performed once.

4. The preparation method according to claim 1, characterized in that, The solid-liquid separation is achieved by filtration using a sieve, with the sieve mesh size being 60 mesh and 80 mesh respectively; the macroporous adsorption resin filling the macroporous adsorption resin column includes one or more of the following types: X-5, AB-8, NK-2, NKA-2, NK-9, D312, D001, D101, and WLD.

5. The preparation method according to claim 1, characterized in that, The macroporous adsorption resin column is composed of D312 acrylic macroporous resin and D001 styrene macroporous resin used in series. The elution includes a first-stage elution and a second-stage elution; the molar concentration of NaCl in the sodium chloride solution used in the first-stage elution is 0.25~0.35 mol / L, and the molar concentration of NaCl in the sodium chloride solution used in the second-stage elution is 0.05~0.15 mol / L.

6. The preparation method according to claim 1, characterized in that, The concentration is a membrane concentration; the solids content of the coffee fruit peel extract is 6~7 mg / mL.

7. The coffee pericarp extract prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the coffee fruit peel extract according to claim 7 in cosmetics or food.

9. The application according to claim 8, characterized in that, The cosmetic is a preparation that promotes the transdermal penetration of active ingredients; the food is a preparation used to promote digestion and absorption.

10. A cosmetic product, characterized in that, It includes the active ingredients and the coffee fruit peel extract as described in claim 7; the active ingredients include one or more of ferulic acid, dipotassium glycyrrhizate, nicotinamide, and oligopeptides.

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