A preparation method for extracting a whitening and anti-aging active ingredient verbascoside from a lemon verbena fermentation substrate

Abstract

The present application relates to the technical field of fermentation, and especially relates to a preparation method for extracting whitening and anti-aging active ingredient verbascoside from lemon verbena fermentation substrate, the separation method of the present application is three times of separation and purification, first, macroporous resin D101 is used, then, silica gel column chromatography resin is used, then, dextran gel resin is used, and finally, monomeric compound is obtained through recrystallization; the content of verbascoside in the initial raw material is increased from 1.57-4.11% in the initial lemon verbena stem and leaf to 2.83-7.75%, so that the content of lemon verbena in the starting raw material is increased by 2-5 times; the present application uses three-step separation and purification, so that monomeric compound verbascoside with a monomer purity of 90.42-99.17% can be obtained; the present application increases the final yield by 2-3 times, from the existing 3-5% to 7.28-11.33%, so that the technical problems of the preparation method in the prior art, such as high price, low content, complex separation and purification process, low recovery rate and low monomer purity, are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation, and particularly relates to a preparation method for extracting a whitening and anti-aging active ingredient, verbascoside, from a fermentation substrate of lemon verbena. BACKGROUND

[0002] In the cosmetic and health product industries, finding natural ingredients with whitening and anti-aging activity has always been a research hotspot. Verbascoside, as a natural active ingredient with significant whitening and anti-aging effects, has received extensive attention due to its unique pharmacological activity and safety. Verbascoside is usually extracted by a preparation method.

[0003] However, the existing preparation method has problems such as high price, low content, complex separation and purification process, low recovery rate, and low monomer purity. SUMMARY

[0004] The present application relates to the technical field of fermentation, and particularly relates to a preparation method for extracting a whitening and anti-aging active ingredient, verbascoside, from a fermentation substrate of lemon verbena.

[0005] To achieve the above-mentioned purpose, a preparation method for extracting a whitening and anti-aging active ingredient, verbascoside, from a fermentation substrate of lemon verbena is adopted, which comprises the following steps:

[0006] Step one, first, break and cut the lemon verbena branches and leaves according to the proportion to prepare lemon verbena short branches for standby;

[0007] Step two, then immerse the lemon verbena short branches in citric acid water and ferment for 36-72 hours, centrifuge at high speed and filter, discard the centrifugal liquid, and obtain fermentation dregs;

[0008] Step three, then extract twice with ethanol with a concentration of 85%, collect the two extraction liquids, recover ethanol under reduced pressure and vacuum, dilute with water to a concentration of ethanol below 10%, and obtain a concentrated liquid for standby;

[0009] Step four, load the concentrated liquid onto D101 macroporous resin by wet column loading, after loading, saturate adsorption for 45 minutes, elute impurities with pure water, elute to colorless and odorless to 10 column volumes, and the flow rate is 1 VB / hours;

[0010] Step five, then elute 4-8 column volumes with ethanol aqueous solution with a concentration of 60%, the flow rate is V=0.5 VB / H, and collect the eluate, concentrate the eluate under reduced pressure and vacuum, and recover ethanol to obtain a concentrated extract;

[0011] Step six, the concentrated extract 200 mesh silica gel is mixed evenly, dried at room temperature, and then subjected to dry pressure silica gel column chromatography. Vacuum is used to dry the silica gel. Acetone and ethyl acetate are used as eluents. The elution is performed for 6-10 column volumes. The flow rate is V=1.0 VB / H, and the pressure is p=0.008 kpa. The eluate is collected, concentrated under reduced pressure and vacuum, and dried to obtain a white powder.

[0012] Step seven, the white powder is dissolved in ethanol with a concentration of 10%. After filtration, the solution is subjected to Sephadex LH-20 gel resin. The saturation adsorption is performed for 30 minutes. Then, ethanol with a concentration of 75% is used as an eluent for 4-6 column volumes. The flow rate is V=0.5 VB / H. The eluate is collected. Half of the ethanol is recovered under reduced pressure and vacuum. After cooling and standing for 24 hours, the crystals are obtained by filtration. The crystals are verbascoside.

[0013] In step one, the lemon verbena branches and leaves are crushed and cut into segments to prepare lemon verbena short branches for use. The crushing and cutting length is 0.3-0.5 cm.

[0014] In step two, the lemon verbena short branches are submerged in citric acid water for fermentation for 36-72 hours. High-speed centrifugation and filtration are performed. The centrifugate is discarded to obtain fermented dregs.

[0015] The pH value of citric acid used is 3-4. The high-speed centrifugation speed is 3500 r / min, and the time is 15 min. The filtration mesh size is 800-1000 mesh.

[0016] In step three, in the two extractions, the first extraction is performed at a solid-liquid ratio of 1:8, an extraction temperature of 90°C, and a Soxhlet reflux extraction time of 2 hours. The second extraction is performed at a solid-liquid ratio of 1:6, an extraction temperature of 90°C, and an extraction time of 1 hour.

[0017] In step three, when the water is added for dilution to obtain a concentrated solution with an ethanol concentration of less than 10% for standby use, the temperature T is ≤70°C, and the pressure ≥0.1 kpa.

[0018] In step five, when the ethanol is recovered, the temperature T is ≤70°C, and the pressure ≥0.1 kpa.

[0019] In step six, when the dry pressure silica gel column chromatography is performed, the mesh size is 100 mesh silica gel, and the diameter-height ratio is ≥10:1. Acetone and ethyl acetate are used as eluents. The acetone: ethyl acetate ratio is 3:1.

[0020] The present application discloses a preparation method for extracting the whitening and anti-aging active ingredient verbascoside from the fermentation substrate of lemon verbena. The raw material used in the present application is the fermented lemon verbena fermentation substrate dregs, which is different from other raw materials. The extraction solvent used in the present application is weak acid alcohol extraction (pH=4-5), which is different from water and ethanol.

[0021] The separation method described in the application is three times of separation and purification, first on macroporous resin D101, second on silica gel column chromatography resin, on Sephadex gel resin, and finally recrystallization to obtain a monomer compound;

[0022] The application increases the content of verbascoside in the initial raw material from 1.57-4.11% of the initial lemon verbena stem and leaf to 2.83-7.75%, which increases the content of lemon verbena in the starting raw material by 2-5 times;

[0023] The application uses three-step separation and purification to obtain a monomer compound verbascoside with a monomer purity of 90.42-99.17%;

[0024] The application increases the final yield by 2-3 times, from the existing 3-5% to 7.28-11.33%.

[0025] In this way, the technical problems of the prior art preparation method, such as high price, low content, complex separation and purification process, low recovery rate, and low monomer purity, are solved. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0027] Figure 1 is a step flow chart of embodiment 1 of the application.

[0028] Figure 2 is a step flow chart of embodiment 2 of the application. DETAILED DESCRIPTION

[0029] The embodiments of the application will be described in detail below, and the examples of the embodiments are shown in the drawings. The embodiments described below by referring to the drawings are exemplary and are intended to explain the application, and cannot be understood as a limitation of the application.

[0030] The application provides a preparation method for extracting the whitening and anti-aging active ingredient verbascoside from lemon verbena fermentation substrate, comprising the following steps:

[0031] Step one, first, take lemon verbena branches and leaves in proportion, crush and cut them, and prepare lemon verbena short branches for use;

[0032] Step two, then the lemon verbena short branches with citric acid water immersion, and fermentation 36-72 hours, high speed centrifugation and filtration, discard the centrifugal liquid, get fermented dregs;

[0033] Step three, then extracted twice with 85% ethanol, collect the two times of extract, vacuum recovery of ethanol, dilution with water to the ethanol concentration is less than 10%, get concentrated liquid for use;

[0034] Step four, the above concentrated liquid on D101 macroporous resin, wet column, after the column, saturated adsorption 45 minutes, with pure water elution impurities, elution to no color and no smell to 10 column volumes, flow rate 1VB / hours;

[0035] Step five, again with 60% ethanol elution 4-8 column volumes, flow rate V=0.5VB / H, and collect the eluate, vacuum concentration and recovery of ethanol, get concentrated extract;

[0036] Step six, the concentrated extract 200 mesh silica gel, drying after drying, dry method on pressure silica gel column chromatography, vacuum dry silica gel hollow air, eluent with acetone ethyl acetate, elution 6-10 column volumes, flow rate V=1.0VB / H, pressure p=0.008kpa, collect the eluate, vacuum concentration and drying, get white powder;

[0037] Step seven, the white powder with concentration of 10% ethanol solution, after filtration, on sephadex gel resin Sephadex LH-20, saturated adsorption 30 minutes, then eluted with 75% ethanol 4-6 column volumes, flow rate V=0.5VB / H, collect the eluate, vacuum recovery of half of the ethanol, cooling and crystallization 24h, filtration to get crystal is verbascoside.

[0038] In step one, the lemon verbena branches and segments are broken and cut, and the lemon verbena short branches are prepared for use, the length of the broken and cut is 0.3-0.5cm.

[0039] In step two, the lemon verbena short branches are immersed in citric acid water and fermented for 36-72 hours, high speed centrifugation and filtration, and the centrifugal liquid is discarded to obtain fermented dregs.

[0040] The pH value of citric acid used is 3-4, the speed of high speed centrifugation is 3500r / min, the time is 15min, and the filter mesh is 800-1000 mesh.

[0041] In step three, the ratio of the first extraction is 1:8, the extraction temperature is 90℃, and the Soxhlet reflux extraction time is 2 hours; the ratio of the second extraction is 1:6, the extraction temperature is 90℃, and the extraction time is 1 hour.

[0042] In step three, the temperature T≤70℃, and the pressure ≥0.1kpa when the concentrated solution is obtained by diluting with water to an ethanol concentration of less than 10%.

[0043] In step five, the temperature T≤70℃, and the pressure ≥0.1kpa when the ethanol is recovered.

[0044] In step six, the diameter-height ratio is ≥10:1 when the dry method is used to perform the pressure silica gel column chromatography, and the mesh size is 100 mesh silica gel. Example

[0045] Please refer to Figure 1 , Figure 1 is the step flow chart of Example 1 of the present application.

[0046] In this example, the preparation method of the whitening and anti-aging active ingredient verbascoside from lemon verbena fermentation substrate is as follows:

[0047] S1, take lemon verbena branches and leaves 2.50kg, adopt physical crushing and cutting length 0.3-0.5cm, prepare lemon verbena short branches, get 2.37kg, loss 5.2%, use pH=3~4 citric acid water to immerse the lemon verbena short branches, and ferment for 72 hours, high-speed centrifugation (speed 3500r / min, 15min) and filtration (800-1000 mesh), discard the centrifugal liquid, and get the fermentation dregs;

[0048] S2, extract the above fermented dregs with 85% ethanol twice, the ratio of the first extraction is 1:8, the extraction temperature is 90℃, and the Soxhlet reflux extraction time is 2 hours; the ratio of the second extraction is 1:6, the extraction temperature is 90℃, and the extraction time is 1 hour; collect the two extraction liquids, recover the ethanol under reduced pressure and vacuum (temperature T≤70℃, pressure ≥0.1kpa), dilute with water to an ethanol concentration of less than 10%, and get the concentrated solution for standby;

[0049] S3, the concentrated liquid is loaded onto D101 macroporous resin by wet loading method. After loading, the saturated adsorption is performed for 45 minutes. Impurities are eluted with purified water until the eluent is colorless and odorless. The elution is performed for 10 column volumes at a flow rate of 1 VB / hours. Then, 60% ethanol aqueous solution is used to elute for 4-8 column volumes at a flow rate of 0.5 VB / H, and the eluate is collected. The eluate is concentrated under reduced pressure and vacuum, and ethanol is recovered (temperature T≤70℃, pressure≥0.1 kpa). The concentrated extract 1.896 kg is obtained, which contains 33% of water, and the yield after macroporous resin is 1.27 kg;

[0050] S4, the extract is mixed with 200 mesh silica gel at room temperature (25℃), and then dried. The dried silica gel is loaded onto a pressure silica gel column (100 mesh silica gel, diameter-height ratio≥10:1) by dry method. The silica gel is vacuum dried to remove air. Acetone and ethyl acetate (acetone: ethyl acetate=3:1) are used as eluent to elute for 6-10 column volumes at a flow rate of 1.0 VB / H and a pressure of 0.008 kpa. The eluate is collected, and the solvent is recovered under reduced pressure to obtain a white powder-like mixture of 0.54 kg.

[0051] S5, the mixture of the crystal-like sample is dissolved by heating with a low alcohol solvent (10% CH3CH2OH). After filtration (800-1000 mesh), the sample is loaded onto a Sephadex LH-20 gel column (diameter-height ratio≥10:1). The saturated adsorption is performed for 30 min. 75% ethanol is used to elute for 4-6 column volumes at a flow rate of 0.5 VB / H. The eluate is collected, and half of the ethanol is recovered under reduced pressure and vacuum. After cooling and standing for 24 h, the crystal 0.227 kg is obtained by filtration. Example

[0052] Please refer to Figure 2 , Figure 2 is the step flow chart of Example 2 of the present application.

[0053] In this example, the preparation method of the whitening and anti-aging active ingredient verbascoside extracted from the fermentation substrate of lemon verbena is as follows:

[0054] S1, 3.17 kg of lemon verbena branches and leaves are physically broken and cut into 0.3-0.5 cm long to obtain 3.01 kg of lemon verbena short branches. The lemon verbena short branches are immersed in pH=3-4 citric acid water, and fermented for 72 hours. High-speed centrifugation (4000 r / min, 15 min) and filtration (800-1000 mesh) are performed, and the centrifugate is discarded to obtain fermentation residues;

[0055] S2. Extract the fermented residue twice with 85% ethanol. The first extraction ratio is 1:8, the extraction temperature is 90℃, and the extraction is carried out by Soxhlet reflux for 2 hours. The second extraction ratio is 1:6, the extraction temperature is 90℃, and the extraction time is 1 hour. Collect the two extracts, and recover the ethanol under reduced pressure (temperature T≤70℃, pressure≥0.1kpa). Dilute with water to an ethanol concentration of less than 10% to obtain a concentrated solution for later use.

[0056] S3. Load the above concentrated solution onto D101 macroporous resin using a wet column loading method. After loading, allow it to saturate for 45 minutes. Elute impurities with purified water until the solution is colorless and odorless, up to 10 column volumes, at a flow rate of 1VB / hour. Then elute with 60% ethanol aqueous solution for 4-8 column volumes, V=0.5VB / H, and collect the eluent. Concentrate the eluent under reduced pressure and vacuum, and recover the ethanol (temperature T≤70℃, pressure≥0.1kPa), yielding 2.40kg of concentrated extract with a water content of 27%, and a macroporous resin yield of 1.75kg.

[0057] S4. Mix the above extract with 200-mesh silica gel, dry it at room temperature (25℃), and then perform dry chromatography on a pressurized silica gel column (100-mesh silica gel, aspect ratio ≥10:1). Vacuum the silica gel to remove air, and elute with acetone and ethyl acetate (acetone:ethyl acetate = 3:1). Elute for 6-10 column volumes at a flow rate V = 1.0VB / H and a pressure p = 0.008 kPa. Collect the eluent, recover the solvent under reduced pressure, and obtain an off-white powder and 0.691 kg of mixed crystals.

[0058] S5. The above mixed crystalline sample was dissolved by heating in a low-alcohol solvent (10% CH3CH2OH), filtered, and then saturated with dextran gel resin (model: Sephadex LH-20, aspect ratio ≥10:1) for 30 min. It was then eluted with 75% ethanol for 4-6 column volumes at a flow rate V=0.5VB / H. The eluent was collected, and half of the ethanol was recovered under reduced pressure. After cooling and standing for crystallization for 24 h, 0.301 kg of crystals were obtained by filtration.

[0059] Comparative example:

[0060] The preparation steps for the comparative example are as follows:

[0061] S1. Take 2.50 kg of lemon verbena branches and leaves, and use physical crushing and cutting into sections of 0.3-0.5 cm in length to obtain 2.37 kg of lemon verbena short branches, with a loss of 5.2%. Soak the lemon verbena short branch pieces in citric acid water with pH=3~4 and ferment for 72 hours. Centrifuge at high speed (4000 r / min, 15 min) and filter (800-1000 mesh). Discard the centrifuged liquid to obtain fermented residue.

[0062] S2, the above fermented dregs are extracted twice with 85% ethanol, the first extraction is carried out at a material-liquid ratio of 1:8, an extraction temperature of 90 DEG C, and a Soxhlet reflux extraction time of 2 hours; the second extraction is carried out at a material-liquid ratio of 1:6, an extraction temperature of 90 DEG C, and an extraction time of 1 hour; the two extraction solutions are collected, ethanol is recovered under reduced pressure and vacuum (temperature T≤70 DEG C, pressure≥0.1 kpa), and water is added to dilute the ethanol to a concentration of less than 10%;

[0063] S3, the above concentrated solution is subjected to wet column chromatography on D101 macroporous resin (Xi'an Lanxiao Science and Technology), after completion of the column chromatography, saturated adsorption is carried out for 45 minutes, impurities are eluted with pure water until the eluent is colorless and odorless, the elution is carried out for 10 column volumes at a flow rate of 1 VB / hours; then, 25% ethanol aqueous solution is used to elute for 4-8 column volumes at a flow rate of 0.5 VB / H, and 60% ethanol aqueous solution is used to elute for 4-6 column volumes; finally, all the eluent is collected; the eluent is concentrated under reduced pressure and vacuum, and ethanol is recovered (temperature T≤70 DEG C, pressure≥0.1 kpa), to obtain 1.9 kg of concentrated extract, with a water content of 33%, and a macroporous resin post-yield of 1.4 kg;

[0064] S4, the above extract is mixed with 200-mesh silica gel, dried at room temperature (25 DEG C), and then subjected to dry column chromatography on a silica gel column (100-mesh silica gel, diameter-height ratio≥10:1); air in the silica gel is vacuum-dried, and the silica gel is eluted with acetone-ethyl acetate (acetone: ethyl acetate=3:1) as an eluent; the elution is carried out for 6-10 column volumes at a flow rate of 1.0 VB / H and a pressure of 0.008 kpa; the eluent is collected, and the solvent is recovered under reduced pressure, to obtain 0.62 kg of white powder-like mixed crystals;

[0065] S5, the above mixed crystal sample is dissolved in a low-alcohol solvent (10% CH3CH2OH) and heated, filtered (800-1000 mesh), and then subjected to column chromatography on Sephadex gel resin (type: Sephadex LH-20, diameter-height ratio≥10:1); saturated adsorption is carried out for 30 min, 75% ethanol is used to elute for 4-6 column volumes at a flow rate of 0.5 VB / H, the eluent is collected, and half of the ethanol is recovered under reduced pressure and vacuum; after cooling and standing for crystallization for 24 h, the crystals are filtered, to obtain 0.342 kg of crystals.

[0066] The preparation method for extracting the whitening and anti-aging active ingredient verbascoside from fermented lemon verbena substrate according to the application uses fermented lemon verbena substrate dregs as raw materials, which are different from other raw materials; and a weak acid alcohol extraction (pH=4-5) is used as an extraction solvent, which is different from water and ethanol;

[0067] The separation method according to the application is a three-time separation and purification method, which comprises the following steps: first, column chromatography on macroporous resin D101; second, column chromatography on silica gel column chromatography resin; third, column chromatography on Sephadex gel resin; and finally, recrystallization to obtain a monomeric compound;

[0068] The application increases the content of verbascoside in the initial raw material, from 1.57-4.11% of the initial lemon verbena stem and leaf, to 2.83-7.75%, which increases the content of lemon verbena in the starting raw material by 2-5 times;

[0069] The application uses three-step separation and purification to obtain monomeric compound verbascoside with a monomer purity of 90.42-99.17%;

[0070] The application increases the final yield by 2-3 times, from the existing 3-5% to 7.28-11.33%.

[0071] In this way, the technical problems of the prior art preparation method, such as high price, low content, complex separation and purification process, low recovery rate, and low monomer purity, are solved.

[0072] The above only discloses a preferred embodiment of the application, and of course cannot limit the scope of the right of the application, and those skilled in the art can understand that all or part of the above-mentioned embodiments are implemented, and equivalent changes made according to the claims of the application still belong to the scope covered by the application.

Claims

1. A method for preparing verbascoside, a whitening and anti-aging active ingredient extracted from lemon verbena fermentation substrate, characterized in that, Includes the following steps: Step 1: First, take lemon verbena branches and leaves according to the proportion, crush and cut them into sections to obtain lemon verbena short branches for later use. Step 2: Then, soak the short twigs of lemon verbena in citric acid water and ferment for 36-72 hours. Centrifuge at high speed and filter, discard the centrifuged liquid, and obtain the fermented residue. Step 3: Then extract twice with 85% ethanol, collect the two extracts, recover the ethanol under reduced pressure and vacuum, dilute with water to an ethanol concentration of less than 10%, and obtain a concentrated solution for later use. Step 4: Load the above concentrate onto D101 macroporous resin using a wet loading method. After loading is complete, allow it to saturate for 45 minutes. Then, wash away impurities with pure water until the solution is colorless and odorless to 10 column volumes. The flow rate is 1VB / hour. Step 5: Elute with 60% ethanol aqueous solution for 4-8 column volumes at a flow rate V = 0.5VB / H, collect the eluent, concentrate the eluent under reduced pressure and vacuum, and recover the ethanol to obtain concentrated extract. Step 6: Mix the concentrated extract with 200-mesh silica gel, dry it at room temperature, and then perform dry loading and pressurized silica gel column chromatography. Vacuum the silica gel to remove air, and elute with acetone and ethyl acetate for 6-10 column volumes at a flow rate of V = 1.0VB / H and a pressure of p = 0.008 kPa. Collect the eluent, concentrate it under reduced pressure, and dry it to obtain an off-white powder. Step 7: Dissolve the off-white powder in 10% ethanol, filter, and then adsorb onto Sephadex LH-20 dextran gel resin for 30 minutes until saturation. Then elute with 75% ethanol for 4-6 column volumes at a flow rate of V = 0.5VB / H. Collect the eluent, recover half of the ethanol under reduced pressure, cool and allow to crystallize for 24 hours, and then filter to obtain the crystals, which are verbascoside.

2. The method for preparing verbascoside, a whitening and anti-aging active ingredient extracted from lemon verbena fermentation substrate as described in claim 1, is characterized in that... In step one, the lemon verbena branches and leaves are crushed and cut into sections to obtain short lemon verbena branches for later use. The crushing and cutting length is 0.3-0.5cm.

3. The method for preparing verbascoside, a whitening and anti-aging active ingredient extracted from lemon verbena fermentation substrate as described in claim 2, is characterized in that... In step two, the short twigs of lemon verbena are submerged in citric acid water and fermented for 36-72 hours. After high-speed centrifugation and filtration, the centrifuged liquid is discarded, yielding the fermented residue. The citric acid used has a pH of 3-4, the high-speed centrifugation speed is 3500 r / min, the time is 15 min, and the filtration mesh size is 800-1000 mesh.

4. The method for preparing verbascoside, a whitening and anti-aging active ingredient extracted from lemon verbena fermentation substrate as described in claim 3, is characterized in that... In step three, during the two extractions, the first extraction had a material-to-liquid ratio of 1:8, an extraction temperature of 90℃, and was performed by Soxhlet reflux extraction for 2 hours; the second extraction had a material-to-liquid ratio of 1:6, an extraction temperature of 90℃, and an extraction time of 1 hour.

5. The method for preparing verbascoside, a whitening and anti-aging active ingredient extracted from lemon verbena fermentation substrate as described in claim 4, is characterized in that... In step three, water is added to dilute the ethanol concentration to below 10% to obtain a concentrated solution for later use. The temperature T ≤ 70℃ and the pressure ≥ 0.1 kPa are as follows.

6. The method for preparing verbascoside, a whitening and anti-aging active ingredient extracted from lemon verbena fermentation substrate as described in claim 5, is characterized in that... In step five, when recovering ethanol, the temperature T ≤ 70℃ and the pressure ≥ 0.1 kPa.

7. The method for preparing verbascoside, a whitening and anti-aging active ingredient extracted from lemon verbena fermentation substrate as described in claim 6, is characterized in that... In step six, during dry loading and pressurized silica gel column chromatography, the silica gel is 100 mesh with a diameter-to-height ratio ≥10:1, and the acetone-to-ethyl acetate eluent ratio is 3:1.

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