WO2021208947A1 - Coffee silverskin extract, preparation method therefor and use thereof - Google Patents

Abstract

Disclosed is a method for preparing a coffee silverskin extract, comprising the following steps: (1) taking coffee silverskin, adding an extraction solvent, carrying out ultrasonic extraction, then carrying out heating for extraction, and filtering same to obtain a crude extraction liquid; (2) concentrating the crude extraction liquid to obtain an extract; (3) loading the extract on macroporous adsorption resin, firstly eluting same with water, then eluting same with ethanol, and collecting an ethanol eluate; (4) concentrating the ethanol eluate and drying same to obtain the desired product. In the provided method for preparing a coffee silverskin extract provided, caffeine and chlorogenic acid can be effectively extracted. The obtained coffee silverskin extract has sun protection and after-sun repair effects in cosmetic use.

Description

Coffee silver bark extract and preparation method and application thereof Technical field

This application belongs to the technical field of plant extraction, and specifically relates to a coffee silver bark extract and a preparation method and application thereof.

Background technique

Coffee silver skin refers to the outer skin of coffee beans, which is one of the by-products produced during coffee roasting. The main components of coffee silver bark are polysaccharides, cellulose, protein, caffeine, chlorogenic acid, protocatechuic acid and so on. Among them, protocatechuic acid, caffeine and chlorogenic acid have clear biological activities, caffeic acid and chlorogenic acid have antibacterial and antioxidant activities, and caffeine has anti-aging, improving microcirculation and hair growth activities.

At present, coffee silver skins are mainly used in feed, biomass fuels, etc., which belong to the primary utilization of raw materials and the utilization rate is not high. There are still a large amount of coffee silver skins that are treated as waste without further processing.

Summary of the invention

This application provides a coffee silver bark extract and a preparation method and application thereof. The preparation method of the coffee silver bark extract can effectively extract caffeine and chlorogenic acid. The coffee silver bark extract is used in cosmetics applications. It has sun protection and after-sun repairing effects.

In the first aspect, this application provides a method for preparing coffee silver bark extract, which includes the following steps:

(1) Take coffee silver bark, add extraction solvent, ultrasonically extract, then heat and reflux under normal pressure, and filter to obtain crude extract; (Because the coffee silver bark has a layer of smooth wax on the surface, if a single extraction method is used, the solvent It is difficult to fully immerse the substance, resulting in insufficient extraction of the active ingredients. Therefore, two extraction methods are used in combination in this application.)

(2) Concentrating the crude extract to obtain an extract;

(3) Load the extract on the macroporous adsorption resin, eluted with water first, and then eluted with ethanol, and collect the ethanol eluate; and

(4) Concentrate and dry the ethanol eluate to obtain it.

Preferably, the extraction solvent in the step (1) is ethanol. The effective active substances in coffee silver bark, caffeine, chlorogenic acid, protocatechuic acid, and caffeic acid are all easily soluble in ethanol. In this application, the above-mentioned components are used as the screening indicators for the extraction solvent, which can extract effective active substances to a greater extent, and ethanol is used as the extraction solvent, which is safe, environmentally friendly, and easy to recycle. Preferably, the volume percentage concentration of the ethanol is 50% to 90%. Particularly preferably, the volume percentage concentration of ethanol is controlled at 80%, the caffeine and chlorogenic acid extracted under higher concentration of ethanol have a higher extraction rate, and the higher concentration of ethanol reduces the dissolution of macromolecular substances, making it easier to follow up Of filtering.

Preferably, the temperature of ultrasonic extraction in the step (1) is 25°C-50°C, and the time of ultrasonic extraction is 30-90 minutes. The surface of the coffee silver bark is waxy, and ultrasonic-assisted extraction facilitates the solvent to penetrate the material to dissolve the target component.

Preferably, the temperature of heating extraction in the step (1) is 60°C to 90°C, particularly preferably 75°C to 80°C for heating extraction; the number of heating extraction is 1 to 5 times, each heating extraction The time is 1 to 5 hours; the heating extraction method adopts atmospheric reflux extraction. Two reflux extractions can fully extract the effective active substances.

Preferably, the filtering in the step (1) is performed with a 200-300 mesh filter. Particularly preferably, the filtration adopts hot suction filtration to remove the large particulate matter aggregated by impurities such as polysaccharides, cellulose, and protein in the coffee silver bark. Because the impurity particles are larger, a 300-mesh filter with larger pores is used.

Preferably, the concentration in the step (2) is carried out by means of concentration under reduced pressure, and the temperature of concentration is 60°C to 70°C.

Preferably, in the step (3), the macroporous adsorption resin adopts two types of D-101 and LS-46D for mixed use, and the ethanol eluted with ethanol is carried out with an ethanol solution with a volume percentage concentration of 70% to 90%. The mass ratio of the two types of macroporous adsorption resins, LS-46D and D-101, is 1:0.5-2, which are used to separate and purify the active components in the coffee extract. Because caffeine belongs to alkaloids, and chlorogenic acid belongs to polyphenols. According to the different properties of the two active substances, mixed use of non-polar resin D101 and mid-polar resin LS-46D can fully enrich caffeine and chlorogenic acid at the same time .

Preferably, the drying in the step (4) is carried out by freeze-drying, and the freezing temperature of the freeze-drying is -70°C to -50°C, and the vacuum degree is less than 20Pa.

In a second aspect, the present application also provides a coffee silver bark extract prepared by the preparation method. The coffee bark extract contains ≥10% caffeine and ≥12% chlorogenic acid by weight.

In the third aspect, the application also provides an application of coffee silver bark extract in the preparation of cosmetics, and the amount of coffee silver bark extract in the cosmetic is 0.1wt% to 100wt%.

The beneficial effects of this application are:

(1) This application adopts the preparation method of ultrasonic extraction (secondary heating and refluxing) coffee silver bark, macroporous resin separation and purification, and vacuum freeze-drying machine freeze-drying preparation method to prepare coffee silver bark extract. The obtained coffee silver bark extract has high-efficiency biological activity. The extraction rates of caffeine and chlorogenic acid in the coffee silver bark extract are both greater than 80%, and the proportions are 11.5% and 14.3%, respectively.

(2) In cosmetic applications, coffee silver bark extract, as a natural sunscreen ingredient, has a strong absorption of ultraviolet rays and can reduce the damage of ultraviolet rays to the skin. It has strong sun protection and post-sun repair effects. The verification results of sun protection and post-sun repair of coffee silver skin are as follows:

Sun protection test results:

(a) Ultraviolet absorption spectrum scanning test results: The coffee silver bark extract with a concentration of 0.084mg/mL exhibits strong light absorption in the ultraviolet range, with the maximum absorption peak of UVA at 325nm and absorbance of 1.088; for UVB The maximum absorption peak for UVC is at 278nm, and the absorbance is 1.465; the maximum absorption peak for UVC is at 233nm, and the absorbance is 1.831. In addition, the light absorption capacity of coffee silver bark extract has good light stability in the ultraviolet range. Under the same concentration, the absorption peak of UVA is at 325nm and the absorbance is 1.022; the maximum absorption peak for UVB is at 280nm and the absorbance is 1.234; The maximum absorption peak for UVC is at 233nm, and the absorbance is 1.505. The light absorption capacity of coffee silver bark extract has good thermal stability in the ultraviolet range. Under the same concentration, the maximum absorption peak of UVA is at 325nm and the absorbance is 1.029; the maximum absorption peak for UVB is at 280nm and the absorbance is 1.222; The maximum absorption peak of UVC is at 233nm, and the absorbance is 1.431. See Figure 2, Figure 3, Figure 4.

(b) UV protection test results: the test model using UV color test paper at 0.05%, 0.1%, 0.15%, 0.2% concentration, the coffee silver bark extract solution has UV protection effect at different concentrations, 0.15% concentration Coffee silver bark extract solution has a strong UV protection effect. See attached figure 5.

Test results of repairing effect after sun exposure:

(a) The detection result of the removal of active oxygen ROS: the MFI (mean fluorescence intensity) value of coffee silver bark extract is 2052116.167, and the MFI (mean fluorescence intensity) value of the positive control is 708897.767. The results show that the ability of 31.25μg/mL coffee silver bark extract to remove ROS can be higher than that of the 0.05% positive control. It can effectively inhibit the synergistic effect of UVB and ROS, and can reduce UV-induced ROS damage to the skin. See attached figure 6.

(b) DPPH clearance test results: the highest DPPH clearance rate of the coffee silver bark extract at a concentration of 0.15 mg/mL is as high as 90%, and the highest DPPH clearance rate of the positive control at a concentration of 0.1 mg/mL is 97%. The results show that the DPPH scavenging rate of coffee silver bark extract is slightly weaker than that of the positive control, but it still has a strong antioxidant capacity at low concentrations, effectively alleviating the damage of UV-induced free radicals to the skin. See attached figure 7.

(c) Test results of reducing power: the highest reducing power of coffee silver bark extract at a concentration of 1 mg/mL is as high as 97%, and the highest reducing power of a positive control at a concentration of 0.1 mg/mL is 100%. The results show that the reducing power of coffee silver bark extract is slightly weaker than that of the positive control, but it still has a strong antioxidant capacity at low concentrations, effectively alleviating UV damage to the skin. See attached figure 8.

Description of the drawings

Figure 1 is a process flow diagram of the coffee silver bark extract of the application;

Figure 2 is a graph of the UV absorption spectrum scanning test result of the coffee silver bark extract of the application at the wavelength of 200-800nm;

Fig. 3 is a graph showing the scanning test results of ultraviolet absorption spectrum of the coffee silver bark extract of the present application after light treatment at the detection wavelength of 200-800nm;

Figure 4 is a graph of the UV absorption spectrum scanning test result of the coffee silver bark extract of the application after heat treatment at the detection wavelength of 200-800nm;

Figure 5 is a diagram of the detection results of the UV protection test of the coffee silver bark extract of the application;

Figure 6 is a diagram of the detection results of the application of coffee silver bark extract to remove ROS;

Figure 7 is a diagram showing the detection results of the application of coffee silver bark extract to remove DPPH;

Fig. 8 is a graph of the detection result of the reducing power test of the coffee silver bark extract of the application.

Detailed ways

In order to enable those skilled in the art to understand the production process and technical effects of this application in detail, specific production examples are used below to further introduce the application and technical effects of this application.

Example 1

A preparation method of coffee silver bark extract includes the following steps:

(1) Crushing: the coffee silver skin is crushed to obtain powdered coffee silver skin.

(2) Extraction: Mix coffee silver bark powder with 60% ethanol solution at a material-to-liquid ratio of 1:20, and heat and reflux for one extraction at atmospheric pressure. The extraction temperature is 78°C, and the heating time is 2h. The net is filtered to obtain a crude extract.

(3) Concentration: The crude extract is first concentrated under reduced pressure using a decompression concentrator, the pressure is 0.65Pa, the temperature is 50-70°C, and the concentration until the alcohol concentration is significantly slowed down. The open is concentrated to the extract at a temperature of Boil and concentrate to 2 times the feeding amount.

(6) Separation and purification: add the extract to a macroporous resin column filled with wet resin, the resin model is AB-8, the amount of resin is 2 times the amount of feed; wash with water, the amount of washing is 4 times the column volume; 60 % Ethanol elution, the volume of the alcohol wash is 4 times the column volume; after the alcohol wash, the alcohol wash is collected.

(7) Concentration: First use a vacuum concentrator to concentrate the crude extract under reduced pressure, the pressure is 0.65Pa, the temperature is 50-70℃, and the concentration is less than 10%, then continue to open and concentrate until the extract, and the temperature is boiling. , Concentrated to 2 times the feeding amount.

(8) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours, the freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 2

A preparation method of coffee silver bark extract includes the following steps:

(1) Crushing: the coffee silver skin is crushed to obtain powdered coffee silver skin.

(2) Extraction: Mix coffee silver bark powder with 75% ethanol solution according to the material-to-liquid ratio of 1:12, heat and ultrasonically extract for 30 minutes, the ultrasonic frequency is 20kHz, and the material liquid temperature is 25℃～50℃ (ultrasonic The material liquid in the process will generate heat); then heat and reflux under normal pressure to extract once, the reflux temperature is 78°C, and the reflux time is 2h; filter paper is used to obtain the crude extract.

(3) Concentration: The crude extract is first concentrated under reduced pressure using a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 50-70°C, and the concentration is concentrated to an alcohol concentration of <10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 2 times the feeding amount.

(6) Separation and purification: add the extract to a macroporous resin column filled with wet resin, the resin model is AB-8, the amount of resin is 2 times the amount of feed; wash with water, the amount of washing is 4 times the column volume; 60 Elute with% ethanol, the volume of the alcohol wash is 4 times the column volume, and the alcohol wash is collected.

(7) Concentration: The crude extract is first concentrated under reduced pressure using a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 50-70°C, and the alcohol concentration is less than 10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 2 times the feeding amount.

(8) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours, the freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 3

A preparation method of coffee silver bark extract includes the following steps:

(1) Crushing: the coffee silver skin is crushed to obtain powdered coffee silver skin.

(2) Extraction: Mix coffee silver bark powder with 75% ethanol solution, and heat and reflux for 2 times at normal pressure: the ratio of material to liquid for the first reflux is 1:12, heating temperature 78℃, heating time It is 2h; the material-liquid ratio of the second reflux is 1:8, the heating temperature is 78°C, and the heating time is 1h; filter paper is used to filter and combine to obtain the crude extract.

(3) Concentration: First use a vacuum concentrator to concentrate the crude extract under reduced pressure, the pressure is 0.65Pa, the temperature is 50-70℃, and the concentration is less than 10%, and the concentration is continued until the extract is open and the temperature is boiling. , Concentrated to 2 times the feeding amount.

(6) Separation and purification: add the extract to a macroporous resin column filled with wet resin, the resin model is AB-8, the amount of resin is 2 times the amount of feed; wash with water, the amount of washing is 4 times the column volume; 60 Elute with% ethanol, and the volume of the alcohol wash is 4 times the column volume to collect the alcohol wash.

(7) Concentration: the alcohol lotion is first concentrated under reduced pressure with a decompression concentrator, the pressure is 0.65Pa, the temperature is 50-70℃, and the concentration until the alcohol concentration is significantly slowed down, the open is concentrated to the extract, and the temperature is boiling , Concentrated to 2 times the feeding amount.

(8) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours, the freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder

Example 4

A preparation method of coffee silver bark extract includes the following steps:

(1) Crushing: the coffee silver skin is crushed to obtain powdered coffee silver skin.

(2) Extraction: Mix coffee silver bark powder with 70% ethanol solution, heat and reflux for 2 times at normal pressure, the first reflux ratio is 1:12, heating temperature 78℃, heating time It is 2h, the material-to-liquid ratio of the second reflux is 1:10, the heating temperature is 78°C, and the heating time is 1h; filter cloth is used to filter to obtain the crude extract.

(3) Concentration: The crude extract is first concentrated under reduced pressure using a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 50-70°C, and the concentration is concentrated to an alcohol concentration of less than 10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 0.5 times the feeding amount.

(4) Alcohol precipitation: mix the extract and 80% ethanol for more than 6 hours, the ethanol concentration of alcohol precipitation is 80%, the amount of ethanol is 5 times the amount of extract, filter, collect the supernatant, the filter material is filter paper.

(5) Concentration: The crude extract is first concentrated under reduced pressure using a decompression concentrator at a pressure of 0.65 Pa and a temperature of 50-70°C. Concentration until the alcohol concentration is significantly slowed down. The open is concentrated to the extract at a temperature of Boil and concentrate to 0.5 times the feeding amount.

(6) Separation and purification: add the extract into a macroporous resin column filled with wet resin, the resin model is SP825L, the amount of resin is 2 times the amount of material; wash with water, the amount of washing is 5 times the column volume; 80% ethanol Alcohol washing, the amount of alcohol washing is 5 times the column volume, and the alcohol washing liquid is collected.

(7) Concentration: The alcohol lotion is first concentrated under reduced pressure with a decompression concentrator, the pressure is 0.65Pa, the temperature is 50-70°C, and the alcohol concentration is less than 10%. The open is concentrated to the extract, and the temperature is boiling. , Concentrated to 0.5 times the feeding amount.

(8) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours, the freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 5

A preparation method of coffee silver bark extract includes the following steps:

(1) Crushing: the coffee silver skin is crushed to obtain powdered coffee silver skin.

(2) Extraction: Mix coffee silver bark powder with 70% ethanol solution, heat and reflux for 2 times at normal pressure, the first reflux ratio is 1:12, heating temperature 78℃, heating time It is 2h, the material-to-liquid ratio of the second reflux is 1:10, the heating temperature is 78°C, and the heating time is 1h; filter cloth is used to filter to obtain the crude extract.

(3) Concentration: The crude extract is first concentrated under reduced pressure using a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 50-70°C, and the concentration is concentrated to an alcohol concentration of <10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 0.5 times the feeding amount.

(4) Alcohol precipitation: mix the extract and 80% ethanol for more than 6 hours, the ethanol concentration of the alcohol precipitation is 80%, the amount of ethanol is 5 times the amount of extract, filter, collect the supernatant, the filter material is filter paper.

(5) Concentration: The filtrate is first concentrated under reduced pressure with a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 50-70°C, and the concentration is concentrated to an alcohol concentration of <10%. The open mouth is concentrated to the extract, the temperature is boiling, and the concentration is 1 times the feeding amount.

(6) Separation and purification: add the extract to a macroporous resin column filled with wet resin, the resin model is D101, and the amount of resin is 1.5 times the amount of feed; wash with water, the amount of washing is 4 times the column volume; 80% ethanol For elution, the alcohol wash volume is 5 times the column volume to collect the alcohol wash.

(7) Concentration: The alcohol lotion is first concentrated under reduced pressure with a decompression concentrator, the pressure is 0.65Pa, the temperature is 50-70℃, and the alcohol concentration is less than 10%. Continue to open and concentrate to the extract, and the temperature is normal pressure Boil and concentrate to 1 times the feeding amount.

(8) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours, the freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 6

(1) Ultrasonic extraction: mix coffee silver bark with 80% ethanol solution, sonicate for 60min, heat reflux for extraction 2 times, the first reflux ratio is 1:12, reflux time is 2h, The material-to-liquid ratio of the secondary reflux is 1:15, and the reflux time is 2h; a 300-mesh filter is used for filtration to obtain a crude extract.

(2) Concentration: the crude extract is first concentrated under reduced pressure with a decompression concentrator, the pressure is 0.65Pa, the temperature is 50-70℃, and the concentration is less than 10%, and the concentration is continued until the extract is open and the temperature is boiling. Concentrate to 1 times the feeding amount.

(3) Separation and purification: add the extract to a resin column filled with wet resin, the resin model is LS-46D, and the amount of resin is 1.5 times the feed amount; wash with water and wash with 4 times the column volume; wash with 80% ethanol The amount of eluent is 5 times the column volume, and the alcohol washing liquid is collected.

(4) Concentration: The alcohol washing liquid is first concentrated under reduced pressure with a vacuum concentrator, the pressure is 0.65Pa, the temperature is 60-90°C, and the alcohol concentration is less than 10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 1 times the feeding amount.

(5) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours. The freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 7

(1) Extraction: Mix coffee silver bark with 80% ethanol solution, heat and reflux under normal pressure for 2 times, the first reflux ratio is 1:12, reflux time is 2h, and the second time The material-to-liquid ratio of the stream is 1:15, and the reflux time is 2h; a 300-mesh filter is used for filtration to obtain a crude extract.

(3) Concentration: The crude extract is first concentrated under reduced pressure with a decompression concentrator, the pressure is 0.65Pa, the temperature is 60℃, and the concentration is less than 10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrated to 1 times the amount of material to get the extract.

(6) Separation and purification: add the extract to a macroporous resin column filled with wet resin, the resin model is LS-46D, the amount of resin is 1.5 times the amount of feed; wash with water, the amount of washing is 3 times the column volume; 80 % Ethanol elution, the volume of the alcohol wash is 5 times the column volume; after the alcohol wash, the alcohol wash is collected.

(7) Concentration: the alcohol lotion is first concentrated under reduced pressure with a decompression concentrator, the pressure is 0.65Pa, the temperature is 50-70℃, and the concentration until the alcohol concentration is significantly slowed down, the open is concentrated to the extract, and the temperature is boiling , Concentrated to 1 times the feeding amount.

(8) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours, the freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 8

(1) Ultrasonic extraction: mix the coffee silver bark with 80% ethanol solution, sonicate at room temperature for 60 minutes at a frequency of 20kHz; then heat and reflux for extraction 2 times, the first reflux ratio of material to liquid is 1:12, The reflux time is 2h; the material-to-liquid ratio of the second reflux is 1:15, and the reflux time is 2h; a 300-mesh filter is used for filtration to obtain a crude extract.

(2) Concentration: The crude extract is first concentrated under reduced pressure using a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 50-70°C, and the concentration is concentrated to an alcohol concentration of <10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 1 times the feeding amount.

(3) Separation and purification: add the extract into the macroporous resin column filled with wet resin, the resin models are LS-46D and D101 mixed at a ratio of 1:1 (mass ratio), and the amount of resin is 2 times the amount of feed; Wash with water, the amount of water washing is 3 times the column volume; 80% ethanol elution, the amount of alcohol washing is 5 times the column volume, and the alcohol washing liquid is collected.

(4) Concentration: First use a vacuum concentrator to concentrate the crude extract under reduced pressure at a pressure of 0.65Pa, a temperature of 50-70°C, and concentrate until the alcohol concentration is less than 10%, and continue to open and concentrate until the extract, and the temperature is boiling. , Concentrated to 1 times the feeding amount.

(5) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours. The freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 9

(1) Ultrasonic extraction: mix the coffee silver bark with an ethanol solution with a concentration of 80%, ultrasound at room temperature for 30 minutes at a frequency of 20kHz; then heat and reflux for 5 times, the first reflux ratio of material to liquid is 1:12, The reflux time is 2h; the material-to-liquid ratio of the second to fifth reflux is 1:15, and the reflux time is 2h; a 300-mesh filter is used for filtration to obtain a crude extract.

(2) Concentration: The crude extract is first concentrated under reduced pressure using a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 70°C, and the concentration is concentrated to an alcohol concentration of <10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 1 times the feeding amount.

(3) Separation and purification: add the extract into the macroporous resin column filled with wet resin, the resin model is LS-46D and D101 mixed at 1:0.5 (mass ratio), and the amount of resin is 2 times the feeding amount; proceed Wash with water, the amount of water washing is 3 times the column volume; elution with 50% ethanol, the amount of alcohol washing is 5 times the column volume, and the alcohol washing liquid is collected.

(4) Concentration: First use a vacuum concentrator to concentrate the crude extract under reduced pressure at a pressure of 0.65Pa, a temperature of 50°C, and concentrate until the alcohol concentration is <10%, and continue to open and concentrate until the extract is boiled and concentrated. To 1 times the feeding amount.

(5) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours. The freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

Example 10

(1) Ultrasonic extraction: mix the coffee silver bark with an ethanol solution with a concentration of 80%, ultrasound at room temperature for 90 minutes at a frequency of 20kHz; then heat and reflux for two extractions, the first reflux ratio of material to liquid is 1:12, The reflux time is 2h; the material-to-liquid ratio of the second reflux is 1:15, and the reflux time is 2h; a 300-mesh filter is used for filtration to obtain a crude extract.

(2) Concentration: The crude extract is first concentrated under reduced pressure using a vacuum concentrator, the pressure is 0.65 Pa, the temperature is 70°C, and the concentration is concentrated to an alcohol concentration of <10%. Continue to open and concentrate until the extract, the temperature is boiling, and concentrate to 1 times the feeding amount.

(3) Separation and purification: add the extract to the macroporous resin column filled with wet resin, the resin model is LS-46D and D101 mixed at 1:2 (mass ratio), and the amount of resin is twice the amount of feed; Wash with water, the amount of water washing is 3 times the column volume; 90% ethanol elution, the amount of alcohol washing is 5 times the column volume, and the alcohol washing liquid is collected.

(4) Concentration: First use a vacuum concentrator to concentrate the crude extract under reduced pressure at a pressure of 0.65Pa, a temperature of 50-70°C, and concentrate until the alcohol concentration is less than 10%, and continue to open and concentrate until the extract, and the temperature is boiling. , Concentrated to 1 times the feeding amount.

(5) Freeze-drying: freeze-dry the extract in a vacuum freeze-drying machine for more than 24 hours. The freezing temperature of freeze-drying is -70℃～-50℃, and the vacuum degree is less than 20Pa to prepare coffee silver bark extract freeze-dried powder .

The extraction rates of caffeine and chlorogenic acid in the coffee silver bark extract prepared in the examples and their content in the coffee silver bark extract freeze-dried powder were tested respectively, and the test results are included in Table 1 below.

The extraction ratio is the ratio between the content of caffeine and chlorogenic acid in the coffee silver bark extract and the content of caffeine and chlorogenic acid in the raw material of coffee silver bark. The content of caffeine and chlorogenic acid was determined by high performance liquid chromatography. The conditions of caffeine analysis chromatography are: mobile phase: water (A) (≥99.9%: methanol (B) (≥99.9%)=76:24; chromatographic column: 4.6mm×150mm, 5μm C18; flow rate: 1mL/min Column temperature: 25℃; Detection wavelength: 272nm. The conditions of chlorogenic acid analysis chromatography are: Mobile phase: 0.085% phosphoric acid solution (A): Acetonitrile (B) (≥99.9%) = 13:87; Column: 4.6 mm×250mm, 5μm C18; Flow rate: 1mL/min; Column temperature: 30℃; Injection volume: 10μL; Detection wavelength: 327nm.

Table 1 Test results of the extraction rate of caffeine and chlorogenic acid of coffee silver bark extract and the content of coffee bark extract freeze-dried powder

Test items	Caffeine extraction rate (%)	Chlorogenic acid extraction rate (%)	Caffeine content (%)	Chlorogenic acid content (%)
Example 1	33.9	22.9	2.20	1.83
Example 2	63.09	24.41	4.65	1.90
Example 3	56.94	32.88	4.25	2.06

Example 4	90	91.58	8.80	6.50
Example 5	90	91.58	10.06	9.57
Example 6	94.28	90.3	11.48	11.55
Example 7	40.74	52.17	5.30	4.85
Example 8	94.28	90.3	11.51	14.3

To verify the sun protection and post-sun repair effects of the coffee silver bark extract of this application, the specific steps are as follows:

(1) Carry out UV absorption spectrum scanning test and UV protection test of coffee silver bark extract;

(2) Carry out the ROS scavenging test, DPPH free radical scavenging test, and free radical reduction ability test of coffee silver bark extract.

The above detection methods have been explored and tested, and the experimental results are relatively accurate.

(1) Ultraviolet absorption spectrum scanning test: use 50% methanol as a blank control and 50% methanol as a solvent to prepare a sample into a solution, use an ultraviolet detector to measure the untreated sample at the same concentration, and undergo 4 hours of light treatment And the sample after 4 hours of heat treatment has the absorption peak of the ultraviolet absorption spectrum in the detection wavelength range of 200nm-800nm, and the absorbance value is used for comparison.

Ultraviolet protection test: (a) The detection model is ultraviolet light discoloration test paper, the stimulating condition is UVB (254-365nm), and the detection index is the degree of discoloration of the test paper; (b) Set experimental group 1 as blank test paper, and experimental group 2 as classics 80 % Ethanol smeared (solvent blank group) test paper, experimental group 3 is a test paper smeared with sample solutions of different concentrations prepared with 80% ethanol.

(2) ROS removal test: blank control is BC, negative control is NC, positive control is PC (VE), stimulation condition is UVB (300mJ/cm ² ), detection index is ROS, detection model is keratinocytes, detection method For flow cytometry.

DPPH free radical removal test:

(a) Experimental reagents and preparation methods

DPPH methanol solution: Take 7.88mg of DPPH and dilute to 100mL with methanol, and store in a dark place;

VC solution (0.1mg/mL): accurately weigh out 10mg of ascorbic acid and dilute to 100mL;

Sample solution: Take 15mg of sample and dilute to 10.00mL.

(b) Testing process

Experimental group: Take 0.050mL, 0.100mL, 0.250mL, 0.500ml samples in a deep well plate, make up to 0.500mL with distilled water, add 2.0mL DPPH methanol solution, mix well, place in the dark for 30 minutes, shake continuously, measure 520nm The absorbance value at the place.

Blank group: Take the same volume of sample solution as the experimental group from the deep well plate, make up to 0.500 mL with distilled water, and add 2.0 mL of methanol solution. After mixing, place in a dark place for 30 minutes, shake continuously, and measure the absorbance at 520nm.

Control group: 0.500 mL of distilled water was added to 2.0 mL of DPPH methanol solution. After mixing, place in a dark place for 30 minutes, shake continuously, and measure the absorbance at 520nm.

Positive control group: Take 0.050mL, 0.100mL, 0.250mL, 0.500mL VC solution in a test tube, and make up to 0.500mL with distilled water.

Two 48-well deep-well plates are used as a group, and each group is set with a control group: after the gradient sample is added, the experimental group is added with DPPH methanol solution, and the control group uses anhydrous methanol instead of DPPH methanol solution. After the reaction in the deep-well plate is completed, take 200 μL from each well to the microtiter plate for detection.

DPPH clearance rate (%)=(A0-A1+A2)/A0*% (where A0 is the absorbance value of the control group, A1 is the absorbance value of the experimental group, and A2 is the absorbance value of the blank group)

Free radical reduction ability test:

(a) Experimental reagents and configuration methods

Phosphate buffer (pH=6.6):

Reagent A: Weigh 7.16g Na ₂ HPO ₄ ·12H ₂ O and dilute the volume to 100 mL with distilled water; Reagent B: Weigh 3.12 g NaH ₂ PO ₄ ·2H ₂ O and dilute the volume to 100 mL with distilled water; Or take KH ₂ PO ₄ , Weigh 2.72g, and dilute the volume to 100mL; take 37.5mL of Reagent A and 62.5mL of Reagent B, mix well, measure the pH value with a pH meter, and adjust the pH to 6.6 with Reagent A or B.

Potassium ferricyanide solution (1%): Weigh 0.5 g of potassium ferricyanide and dilute to 50 mL with water.

Trichloroacetic acid solution (10%): Weigh 2.5 g of trichloroacetic acid and dilute to 25 mL with water.

Ferric chloride (0.1%): Weigh 0.166g FeCl ₃ and dilute to 100 mL with water.

VC solution (560μg/mL): Weigh 28mg of VC solid and dilute to 50mL with phosphate buffer.

Sample processing: Take 15 mg of the sample and dilute it to 10 mL with phosphate buffer for testing.

(b) Testing process

Experimental group: Take 25μL, 50μL, 125μL, and 250μL samples in the deep well plate, and make up the volume to 250μL with phosphate buffer; add 250μL phosphate buffer and 250μL potassium ferricyanide solution to each group of samples, mix Homogenize, water bath at 50℃ for 20min, take it out, add 250μL of trichloroacetic acid solution, and mix well. Add 1000μL of distilled water and 200μL of ferric chloride solution and mix quickly. Measure the absorbance of the solution at 700nm.

Blank group: Take 25μL, 50μL, 125μL, and 250μL samples in the deep well plate, and make up the volume to 250μL with phosphate buffer; add 250μL phosphate buffer and 250μL potassium ferricyanide solution to each group of samples, mix Homogenize, water bath at 50℃ for 20min, take it out, add 250μL of trichloroacetic acid solution, and mix well. Add 1200μL of distilled water and mix quickly. Measure the absorbance of the solution at 700nm.

Positive control group: Take the positive control sample-560g/mL VC solution 25μL, 50μL, 125μL, 250μL, and make up to 250μL with phosphate buffer. The rest of the operation is the same as the experimental group.

Positive control blank group: Take the positive control samples-560g/mL VC solution 25μL, 50μL, 125μL, 250μL, and make up to 250μL with phosphate buffer. The rest of the operation is the same as the blank group.

Generally, two 48-well deep-well plates are used as a group, and a control group and a control blank group are set for each group. After the gradient samples are added and completed, the reagents are added in the detailed order and kept warm. Take 200μL from each well for detection in the microtiter plate.

Reducing power (%)=(A1-A1 ₀ )/(A2-A2 ₀ )*% (where: A1 is the absorbance measured by the sample group or positive control group, A1 ₀ is the absorbance measured by the sample blank group; A2 is The absorbance value of the sample measured in the fourth group _{of VC; A2 0} is the absorbance value of the sample measured in the blank group of the fourth group of VC.)

When performing the ultraviolet absorption spectrum scanning test and ultraviolet protection test of coffee silver bark extract, the concentration of the coffee extract in the ultraviolet absorption spectrum scanning test is 0.084 mg/mL. Spectral scanning is performed in the wavelength range of 200nm-800nm. By adopting the above technical scheme, the ultraviolet absorption capacity, the light stability of the ultraviolet absorption capacity and the thermal stability of the ultraviolet absorption capacity of the sample are tested.

When the coffee silver bark extract ultraviolet absorption spectrum scanning test and the ultraviolet protection test are performed, the concentration of the coffee bark extract solution in the ultraviolet protection test is 0.05%, 0.1%, 0.15%, 0.2%, respectively. The detection model is ultraviolet Discoloration test paper, the stimulation condition is UVB (254-365nm), the detection index is the degree of discoloration of the test paper, the detection method is to smear the sample solution on the surface of the test paper, and observe and compare the influence of different concentrations of samples on the discoloration effect of the test paper. By adopting the above technical scheme, testing the UV protection ability of the coffee silver bark extract under different concentrations can verify the sunscreen effect of the coffee bark extract.

When performing the ROS scavenging test, DPPH free radical scavenging test and free radical reduction ability test of coffee silver bark extract, the concentration of coffee silver bark extract in the ROS scavenging test is 0.31%, the blank control is BC; the negative control is NC; positive The control is PC (VE) with a concentration of 0.05%. The stimulation condition is UVB (300mJ/cm ² ), the detection index is ROS, the detection model is keratinocytes, and the detection method is flow cytometry. By adopting the above technical scheme, the ability of coffee silver bark extract to remove ROS compared with the positive control is tested. The combined action of UVB and ROS causes DNA damage. Testing the coffee bark extract's ability to remove ROS can verify that the coffee silver bark extract has an effect on the sun. Repair effect after damaged skin.

When performing the ROS scavenging test, DPPH free radical scavenging test, and free radical reduction ability test of coffee silver bark extract, the concentration of coffee silver bark extract in the DPPH free radical scavenging test is 0.15mg/mL, and the positive control is 0.1mg/ mL VC solution, the color reagent is 0.0788mg/mL DPPH solution, the detection instrument is a microplate reader, and the detection wavelength is 520nm. By adopting the above technical scheme, the ability of coffee silver bark extract to scavenge DPPH free radicals was tested and compared with a positive control to verify the antioxidant capacity of coffee bark extract.

During the ROS removal test, DPPH free radical removal test, and free radical reduction ability test of coffee silver bark extract, the concentration of coffee silver bark in the free radical reduction ability test is 1 mg/mL, and the positive control is 0.56 mg/mL VC solution , The color reagent is 1.66mg/mL ferric chloride solution, the detection instrument is a microplate reader, and the detection wavelength is 700nm. By adopting the above technical scheme, the ability of coffee silver bark extract to reduce free radicals is tested, and compared with the positive control, the degree of reducing red blood salt to yellow blood salt is tested to reflect the strength of its reducing power, that is, the negative side verifies coffee Antioxidant ability of silver bark extract.

Sun protection test results:

(a) Ultraviolet absorption spectrum scanning test results: The coffee silver bark extract with a concentration of 0.084mg/mL exhibits strong light absorption in the ultraviolet range, with an absorption peak of UVA at 325nm and an absorbance of 1.088; for UVB The absorption peak is at 278nm, and the absorbance is 1.465; the absorption peak for UVC is at 233nm, and the absorbance is 1.831. In addition, the light absorption capacity of coffee silver bark extract has good light stability in the ultraviolet range. Under the same concentration, the absorption peak of UVA is at 325nm and the absorbance is 1.022; the absorption peak for UVB is at 280nm and the absorbance is 1.234; The absorption peak of UVC is at 233nm, and the absorbance is 1.505. The light absorption capacity of coffee silver bark extract in the ultraviolet range has good thermal stability. Under the same concentration, the absorption peak of UVA is at 325nm and the absorbance is 1.029; the absorption peak for UVB is at 280nm and the absorbance is 1.222; for UVC The absorption peak is at 233nm, and the absorbance is 1.431. See Figure 2, Figure 3, Figure 4.

(b) UV protection test results: The coffee silver bark extract solution at 0.05%, 0.1%, 0.15%, 0.2% concentration has sunscreen effect under the UV color test paper as a model. The coffee bark extract solution with a concentration of 0.15% It has obvious UV protection effect. See attached figure 5.

Test results of repairing effect after sun exposure:

(a) The detection result of the removal of active oxygen ROS: the MFI (mean fluorescence intensity) value of coffee silver bark extract is 2052116.167, and the MFI (mean fluorescence intensity) value of the positive control is 708897.767. The results show that the ability of 0.31% coffee silver bark extract to remove ROS can be higher than that of the 0.05% positive control, and can effectively reduce UV-induced ROS damage to the skin, see Figure 6.

(b) DPPH clearance test results: the highest DPPH clearance rate of the coffee silver bark extract at a concentration of 0.15 mg/mL is as high as 90%, and the highest DPPH clearance rate of the positive control at a concentration of 0.1 mg/mL is 97%. The results show that the DPPH clearance rate of coffee silver bark extract is slightly weaker than that of the positive control, but it still has a strong antioxidant capacity at a certain low concentration, and effectively relieves UV damage to the skin. See attached figure 7.

(c) Test results of reducing power: the highest reducing power of coffee silver bark extract at a concentration of 1 mg/mL is as high as 97%, and the highest reducing power of a positive control at a concentration of 0.1 mg/mL is 100%. The results show that the reducing power of coffee silver bark extract is slightly weaker than that of the positive control, but it still has a strong antioxidant capacity at a certain low concentration, effectively alleviating UV damage to the skin. See attached figure 8.

Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of this application. Although this application has been described in detail with reference to the above embodiments, those skilled in the art should understand that this application can still be modified or equivalently replaced. Any modification or partial replacement without departing from the spirit and scope of this application shall be covered by the scope of the claims of this application.

Claims (10)

1. A preparation method of coffee silver bark extract includes the following steps:

   (1) Take coffee silver bark, add extraction solvent, ultrasonic extraction, then heat extraction, filter to obtain crude extract;

   (2) Concentrating the crude extract to obtain an extract;

   (3) Load the extract on the macroporous adsorption resin, eluted with water first, then eluted with ethanol, and collect the ethanol eluate; and

   (4) Concentrate and dry the ethanol eluate to obtain it.
2. The method for preparing coffee silver bark extract according to claim 1, wherein the extraction solvent in the step (1) is ethanol.
3. The method for preparing coffee silver bark extract according to claim 1, wherein the temperature of ultrasonic extraction in step (1) is 25°C-50°C, and the time of ultrasonic extraction is 30-90 minutes.
4. The method for preparing coffee silver bark extract according to claim 1, wherein the temperature of heating and extraction in the step (1) is 60°C to 90°C; the number of heating and extraction times is 1 to 5 times, each time The heating extraction time is 1 to 5 hours; the heating extraction method adopts reflux extraction.
5. The method for preparing coffee silver bark extract according to claim 1, wherein the filtering in the step (1) is performed with a 200-300 mesh filter.
6. The method for preparing coffee silver bark extract according to claim 1, wherein the concentration in the step (2) is carried out by means of reduced pressure concentration, and the concentration temperature is 50°C to 70°C.
7. The method for preparing coffee silver bark extract according to claim 1, wherein in the step (3), the macroporous adsorption resin adopts two types of D-101 and LS-46D to be mixed and used, and it is eluted with ethanol. Ethanol is carried out with an ethanol solution with a volume percentage concentration of 50% to 90%.
8. The method for preparing coffee silver bark extract according to claim 1, wherein the drying in the step (4) is carried out by freeze-drying, and the freezing temperature of the freeze-drying is -70℃～-50℃, The vacuum degree is less than 20Pa.
9. A coffee silver bark extract prepared by the preparation method according to any one of claims 1-8, which contains 10% or more caffeine and 12% or more chlorogenic acid by weight.
10. An application of the coffee silver bark extract in the preparation of cosmetics according to claim 9, wherein the amount of the coffee silver bark extract in the cosmetic is 0.1 wt% to 100 wt%.

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