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

Abstract

The invention relates to the technical field of fermentation, in particular to a preparation method for extracting a whitening and anti-aging active ingredient verbascoside from a lemon verbena fermentation substrate, the separation method comprises three times of separation and purification, macroporous resin D101 is added firstly, silica gel column chromatography resin is added secondly, dextran gel resin is added secondly, and the verbascoside is obtained. And finally recrystallizing to obtain a monomeric compound. According to the invention, the content of verbascoside in the initial raw material is increased from 1.57-4.11% of stems and leaves of the initial verbena citriodora to 2.83-7.75%, so that the content of verbascoside in the initial raw material is increased by 2-5 times; according to the method disclosed by the invention, three steps of separation and purification are adopted, so that a monomeric compound verbascoside with the monomer purity of 90.42-99.17% can be obtained; according to the method, the final yield is increased by 2-3 times, and is increased to 7.28-11.33% from the existing 3-5%, so that the technical problems of high price, low content, complex separation and purification procedures, low recovery rate and low monomer purity of the preparation method in the prior art are solved.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, and in particular to a preparation method for extracting verbascoside, a whitening and anti-aging active ingredient, from a lemon verbena fermentation substrate. Background Art

[0002] In the cosmetics and health care products industry, the search for natural ingredients with whitening and anti-aging activities has always been a hot topic. 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. The preparation method of verbascoside is usually used to extract verbascoside.

[0003] However, the existing preparation methods have the problems of high price, low content, complicated separation and purification process, low recovery rate and low monomer purity. Summary of the invention

[0004] The object of the present invention is to provide a preparation method for extracting verbascoside, a whitening and anti-aging active ingredient, from a lemon verbena fermentation substrate, aiming to solve the technical problems of the preparation method in the prior art, namely, high price, low content, complex separation and purification process, low recovery rate and low monomer purity.

[0005] To achieve the above object, the present invention adopts a preparation method for extracting verbascoside, a whitening and anti-aging active ingredient, from a lemon verbena fermentation substrate, comprising the following steps: Step 1: First, take lemon verbena branches and leaves in proportion, crush and cut them into sections to prepare short branches of lemon verbena for later use; Step 2: Submerge the short branches of lemon verbena in citric acid water, ferment for 36-72 hours, centrifuge at high speed and filter, discard the centrifuge liquid, and obtain fermentation residue; Step 3: extract twice with 85% ethanol, collect the two extracts, recover the ethanol under reduced pressure, add water to dilute to an ethanol concentration of less than 10%, and obtain a concentrated solution for standby use; Step 4: Add the above concentrate to D101 macroporous resin by wet column loading. After loading, saturated adsorption for 45 minutes, and then elute impurities with pure water until the impurities are colorless and odorless for 10 column volumes at a flow rate of 1VB / hours. Step 5: Elute with 60% ethanol aqueous solution for 4-8 column volumes at a flow rate of V=0.5VB / H, collect the eluate, concentrate the eluate under reduced pressure and vacuum, and recover ethanol to obtain a concentrated extract; Step 6: Mix the concentrated extract with 200 mesh silica gel, dry it at room temperature, and perform dry pressure silica gel column chromatography. Use vacuum to remove air from the silica gel, use acetone and ethyl acetate as eluent, elute for 6-10 column volumes, flow rate V=1.0VB / H, pressure p=0.008kpa, collect the eluate, concentrate and dry it under reduced pressure to obtain an off-white powder; Step 7, dissolve the off-white powder with 10% ethanol, filter, apply it to Sephadex LH-20, saturate adsorb for 30 minutes, then elute with 75% ethanol for 4-6 column volumes at a flow rate of V=0.5VB / H, collect the eluate, recover half of the ethanol under reduced pressure, cool and stand for crystallization for 24 hours, and filter to obtain crystals, which are verbascoside.

[0006] Wherein, in step 1, the branches and leaves of lemon verbena are crushed and cut into segments to obtain short branches of lemon verbena for standby use, and the length of the crushed and cut segments is 0.3-0.5 cm.

[0007] Among them, in step 2, the lemon verbena short branches are immersed in citric acid water, fermented for 36-72 hours, centrifuged at high speed and filtered, and the centrifuge liquid is discarded to obtain the fermented medicinal residue: The pH value of the citric acid used is 3-4, the speed of the high-speed centrifugation is 3500 r / min, the time is 15 min, and the filtration mesh size is 800-1000 mesh.

[0008] Among them, in step three, during the two extractions, the first extraction material-liquid ratio is 1:8, the extraction temperature is 90°C, and the Soxhlet reflux extraction is 2 hours; the second extraction material-liquid ratio is 1:6, the extraction temperature is 90°C, and the extraction time is 1 hour.

[0009] Wherein, in step three, water is added to dilute the mixture to an ethanol concentration of less than 10%, and when the concentrated solution is obtained for standby use, the temperature T≤70°C and the pressure≥0.1kpa.

[0010] Wherein, in step five, when recovering ethanol, the temperature T≤70°C and the pressure≥0.1kpa.

[0011] Wherein, in step six, when dry method is applied to pressurized silica gel column chromatography, the mesh size is 100 mesh silica gel, the diameter-to-height ratio is ≥10:1, and the acetone:ethyl acetate ratio used as the acetone-ethyl acetate eluent is 3:1.

[0012] The invention discloses a preparation method for extracting verbascoside, a whitening and anti-aging active ingredient, from a lemon verbena fermentation substrate. The raw material of the invention is the fermented lemon verbena fermentation substrate drug residue, which is different from other raw materials. The extraction solvent used in the invention is weak acid alcohol extraction (pH=4-5), which is different from other water and ethanol. The separation method of the present invention is a three-step separation and purification method, firstly applying macroporous resin D101, then applying silica gel column chromatography resin, then applying dextran gel resin, and finally recrystallizing to obtain a monomer compound; The present invention increases the content of verbascoside in the initial raw material from 1.57-4.11% of the initial lemon verbena stems and leaves to 2.83-7.75%, thereby increasing the content of the lemon verbena in the starting raw material by 2-5 times; The present invention uses three steps of separation and purification to obtain a monomer compound verbascoside with a monomer purity of 90.42-99.17%; The present invention increases the final yield by 2-3 times, from the existing 3-5% to 7.28-11.33%.

[0013] In this way, the technical problems of the prior art preparation method, such as high price, low content, complicated separation and purification process, low recovery rate and low monomer purity, are solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 It is a step flow chart of Example 1 of the present invention.

[0016] Figure 2 It is a step flow chart of Example 2 of the present invention. DETAILED DESCRIPTION

[0017] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0018] The present invention provides a preparation method for extracting verbascoside, a whitening and anti-aging active ingredient, from a lemon verbena fermentation substrate, comprising the following steps: Step 1: First, take lemon verbena branches and leaves in proportion, crush and cut them into sections to prepare short branches of lemon verbena for later use; Step 2: Submerge the short branches of lemon verbena in citric acid water, ferment for 36-72 hours, centrifuge at high speed and filter, discard the centrifuge liquid, and obtain fermentation residue; Step 3: extract twice with 85% ethanol, collect the two extracts, recover the ethanol under reduced pressure, add water to dilute to an ethanol concentration of less than 10%, and obtain a concentrated solution for standby use; Step 4: Add the above concentrate to D101 macroporous resin by wet column loading. After loading, saturated adsorption for 45 minutes, and then elute impurities with pure water until the impurities are colorless and odorless for 10 column volumes at a flow rate of 1VB / hours. Step 5: Elute with 60% ethanol aqueous solution for 4-8 column volumes at a flow rate of V=0.5VB / H, collect the eluate, concentrate the eluate under reduced pressure and vacuum, and recover ethanol to obtain a concentrated extract; Step 6: Mix the concentrated extract with 200 mesh silica gel, dry it at room temperature, and perform dry pressure silica gel column chromatography. Use vacuum to remove air from the silica gel, use acetone and ethyl acetate as eluent, elute for 6-10 column volumes, flow rate V=1.0VB / H, pressure p=0.008kpa, collect the eluate, concentrate and dry it under reduced pressure to obtain an off-white powder; Step 7, dissolve the off-white powder with 10% ethanol, filter, apply it to Sephadex LH-20, saturate adsorb for 30 minutes, then elute with 75% ethanol for 4-6 column volumes at a flow rate of V=0.5VB / H, collect the eluate, recover half of the ethanol under reduced pressure, cool and stand for crystallization for 24 hours, and filter to obtain crystals, which are verbascoside.

[0019] Wherein, in step 1, the branches and leaves of lemon verbena are crushed and cut into segments to obtain short branches of lemon verbena for standby use, and the length of the crushed and cut segments is 0.3-0.5 cm.

[0020] Among them, in step 2, the lemon verbena short branches are immersed in citric acid water, fermented for 36-72 hours, centrifuged at high speed and filtered, and the centrifuge liquid is discarded to obtain the fermented medicinal residue: The pH value of the citric acid used is 3-4, the speed of the high-speed centrifugation is 3500 r / min, the time is 15 min, and the filtration mesh size is 800-1000 mesh.

[0021] Among them, in step three, during the two extractions, the first extraction material-liquid ratio is 1:8, the extraction temperature is 90°C, and the Soxhlet reflux extraction is 2 hours; the second extraction material-liquid ratio is 1:6, the extraction temperature is 90°C, and the extraction time is 1 hour.

[0022] Wherein, in step three, water is added to dilute the mixture to an ethanol concentration of less than 10%, and when the concentrated solution is obtained for standby use, the temperature T≤70°C and the pressure≥0.1kpa.

[0023] Wherein, in step five, when recovering ethanol, the temperature T≤70°C and the pressure≥0.1kpa.

[0024] Wherein, in step six, when dry method is applied to pressurized silica gel column chromatography, the mesh size is 100 mesh silica gel, the diameter-to-height ratio is ≥10:1, and the acetone:ethyl acetate ratio used as the acetone-ethyl acetate eluent is 3:1. Example

[0025] See also Figure 1 , Figure 1 It is a step flow chart of Example 1 of the present invention.

[0026] In this embodiment, the preparation method of extracting the whitening and anti-aging active ingredient verbascoside from the lemon verbena fermentation substrate is as follows: S1, take lemon verbena branches and leaves 2.50kg, adopt physical crushing and cut length of 0.3-0.5cm, make lemon verbena short branches, obtain 2.37kg, loss 5.2%, submerge lemon verbena short branches in pH=3-4 citric acid water, ferment for 72 hours, high-speed centrifugation (speed 3500r / min, 15min) and filter (800-1000 mesh), discard the centrifuge, and obtain fermentation residues; S2, the above-mentioned fermented medicinal residues were extracted twice with 85% ethanol, the first extraction material-liquid ratio was 1:8, the extraction temperature was 90 ° C, and the Soxhlet reflux extraction was 2 hours; the second extraction material-liquid ratio was 1:6, the extraction temperature was 90 ° C, and the extraction time was 1 hour; the two extracts were collected, and the ethanol was recovered under reduced pressure (temperature T≤70 ° C, pressure ≥0.1kpa), and water was added to dilute to an ethanol concentration of less than 10%, and a concentrated solution was obtained for standby use; S3, the above concentrate is loaded onto D101 macroporous resin, and the column is loaded by wet method. After loading, saturated adsorption is performed for 45 minutes, and impurities are eluted with pure water until colorless and odorless for 10 column volumes, and the flow rate is 1VB / hours; then eluted with 60% ethanol aqueous solution for 4-8 column volumes, V=0.5VB / H, and the eluate is collected. The eluate is concentrated under reduced pressure and vacuum, and ethanol is recovered (temperature T≤70℃, pressure≥0.1kpa), and 1.896kg of concentrated extract is obtained, with a water content of 33%, and the yield after macroporous resin is 1.27kg; S4, the extract was mixed with 200 mesh silica gel, dried at room temperature (25 ° C), and then chromatographed on a pressurized silica gel column (100 mesh silica gel, diameter-to-height ratio ≥ 10:1) by dry method, and the air in the silica gel was evacuated by vacuum, and acetone ethyl acetate eluent (acetone: ethyl acetate = 3:1) was applied for elution for 6-10 column volumes, flow rate V = 1.0VB / H, pressure p = 0.008kpa, the eluate was collected, and the solvent was recovered under reduced pressure to obtain an off-white powder, 0.54kg of mixed crystals; S5. Dissolve the mixed crystalline sample in a low-alcohol solvent (10% CH3CH2OH) by heating, filter (800-1000 mesh), apply it to dextran gel resin (model: Sephadex LH-20, diameter-height ratio ≥10:1), saturate adsorb for 30 min, elute with 75% ethanol for 4-6 column volumes, flow rate V=0.5VB / H, collect the eluate, recover half of the ethanol by vacuum pressure, cool and let stand for crystallization for 24 h, and filter to obtain 0.227 kg of crystals. Example

[0027] See also Figure 2 , Figure 2 It is a step flow chart of Example 2 of the present invention.

[0028] In this embodiment, the preparation method of extracting the whitening and anti-aging active ingredient verbascoside from the lemon verbena fermentation substrate is as follows: S1, taking 3.17kg of lemon verbena branches and leaves, using physical crushing and cutting into segments with a length of 0.3-0.5cm, preparing lemon verbena short branches, obtaining 3.01kg, immersing the lemon verbena short branches in pH=3-4 citric acid water, and fermenting for 72 hours, high-speed centrifugation (speed 4000r / min, 15min) and filtering (800-1000 mesh), discarding the centrifuge, and obtaining fermentation residues; S2, the above-mentioned fermented medicinal residues were extracted twice with 85% ethanol, the first extraction material-liquid ratio was 1:8, the extraction temperature was 90 ° C, and the Soxhlet reflux extraction was 2 hours; the second extraction material-liquid ratio was 1:6, the extraction temperature was 90 ° C, and the extraction time was 1 hour; the two extracts were collected, the reduced pressure vacuum was recovered (temperature T≤70 ° C, pressure ≥0.1kpa) to collect ethanol, and water was added to dilute to an ethanol concentration of less than 10% to obtain a concentrated solution for standby use; S3, the above concentrate is loaded onto D101 macroporous resin, and the column is loaded by wet method. After loading, saturated adsorption is performed for 45 minutes, and impurities are eluted with pure water until colorless and odorless for 10 column volumes, and the flow rate is 1VB / hours; then eluted with 60% ethanol aqueous solution for 4-8 column volumes, V=0.5VB / H, and the eluate is collected. The eluate is concentrated under reduced pressure and vacuum, and ethanol is recovered (temperature T≤70℃, pressure≥0.1kpa), and 2.40kg of concentrated extract is obtained, with a water content of 27%, and the yield after macroporous resin is 1.75kg; S4. Mix the above extract with 200 mesh silica gel, dry it at room temperature (25°C), and then dry it on a pressurized silica gel column chromatography (100 mesh silica gel, diameter-to-height ratio ≥10:1). Use vacuum to evacuate the air in the silica gel, and add acetone ethyl acetate eluent (acetone: ethyl acetate = 3:1) for 6-10 column volumes at a flow rate of V = 1.0VB / H and a pressure of p = 0.008 kPa. Collect the eluate and recover the solvent under reduced pressure to obtain an off-white powder and 0.691 kg of mixed crystals.

[0029] S5. Dissolve the mixed crystalline sample by heating it with a low-alcohol solvent (10% CH3CH2OH), filter it, apply it to dextran gel resin (model: Sephadex LH-20, diameter-height ratio ≥10:1), saturate adsorb for 30 min, elute it with 75% ethanol for 4-6 column volumes, flow rate V=0.5VB / H, collect the eluate, recover half of the ethanol under reduced pressure, cool and let it stand for crystallization for 24 h, and then filter to obtain 0.301 kg of crystals. Comparative Example: The comparative example preparation steps are as follows: S1, take lemon verbena branches and leaves 2.50kg, adopt physical crushing and cut length of 0.3-0.5cm, make lemon verbena short branches, obtain 2.37kg, loss 5.2%, submerge lemon verbena short branches in pH=3-4 citric acid water, ferment for 72 hours, high-speed centrifugation (speed 4000r / min, 15min) and filter (800-1000 mesh), discard the centrifuge, and obtain fermentation residues; S2, extract the above fermented medicinal residue twice with 85% ethanol, the first extraction material-liquid ratio is 1:8, the extraction temperature is 90 ° C, and the Soxhlet reflux extraction is 2 hours; the second extraction material-liquid ratio is 1:6, the extraction temperature is 90 ° C, and the extraction time is 1 hour; collect the two extracts, reduce the pressure and vacuum to recover the ethanol, (temperature T≤70 ° C, pressure ≥0.1kpa) dilute with water to an ethanol concentration of less than 10%; S3, the above concentrate was loaded with D101 macroporous resin (Xi'an Lanxiao Technology), and the column was loaded by wet method. After loading, saturated adsorption was performed for 45 minutes, and impurities were eluted with pure water until colorless and odorless for 10 column volumes, with a flow rate of 1VB / hours; then eluted with 25% ethanol aqueous solution for 4-8 column volumes, V=0.5VB / H, and then eluted with 60% ethanol aqueous solution for 4-6 column volumes, and finally all the eluent was collected; the eluent was concentrated under reduced pressure and vacuum, and ethanol was recovered (temperature T≤70°C, pressure≥0.1kpa), and 1.9kg of concentrated extract was obtained, with a water content of 33%, and the yield after macroporous resin was 1.4kg; S4, the extract was mixed with 200 mesh silica gel, dried at room temperature (25 ° C), and then chromatographed on a pressurized silica gel column (100 mesh silica gel, diameter-to-height ratio ≥ 10:1) by dry method, and the air in the silica gel was evacuated by vacuum, and acetone ethyl acetate eluent (acetone: ethyl acetate = 3:1) was applied for elution for 6-10 column volumes, flow rate V = 1.0VB / H, pressure p = 0.008kpa, the eluate was collected, and the solvent was recovered under reduced pressure to obtain an off-white powder, 0.62kg of mixed crystals; S5. Dissolve the mixed crystalline sample in a low-alcohol solvent (10% CH3CH2OH) by heating, filter (800-1000 mesh), apply it to dextran gel resin (model: Sephadex LH-20, diameter-height ratio ≥10:1), adsorb saturatedly for 30 min, elute with 75% ethanol for 4-6 column volumes, flow rate V=0.5VB / H, collect the eluate, recover half of the ethanol by vacuum pressure, cool and let stand for crystallization for 24 h, and filter to obtain 0.342 kg of crystals.

[0030] The preparation method of the present invention for extracting the whitening and anti-aging active ingredient verbascoside from a lemon verbena fermentation substrate is used. The raw material of the present invention is the fermented lemon verbena fermentation substrate residue, which is different from other raw materials. The extraction solvent used in the present invention is weak acid alcohol extraction (pH=4-5), which is different from other water and ethanol. The separation method of the present invention is a three-step separation and purification method, firstly applying macroporous resin D101, then applying silica gel column chromatography resin, then applying dextran gel resin, and finally recrystallizing to obtain a monomer compound; The present invention increases the content of verbascoside in the initial raw material from 1.57-4.11% of the initial lemon verbena stems and leaves to 2.83-7.75%, thereby increasing the content of the lemon verbena in the starting raw material by 2-5 times; The present invention uses three steps of separation and purification to obtain a monomer compound verbascoside with a monomer purity of 90.42-99.17%; The present invention increases the final yield by 2-3 times, from the existing 3-5% to 7.28-11.33%.

[0031] In this way, the technical problems of the prior art preparation method, such as high price, low content, complicated separation and purification process, low recovery rate and low monomer purity, are solved.

[0032] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for extracting the whitening and anti-aging active ingredient verbascoside from a lemon verbena fermentation substrate, characterized in that: The following steps are involved: Step 1: First, take lemon verbena branches and leaves in proportion, crush and cut them into sections to prepare short branches of lemon verbena for later use; Step 2: Submerge the short branches of lemon verbena in citric acid water, ferment for 36-72 hours, centrifuge at high speed and filter, discard the centrifuge liquid, and obtain fermentation residue; Step 3: extract twice with 85% ethanol, collect the two extracts, recover the ethanol under reduced pressure, add water to dilute to an ethanol concentration of less than 10%, and obtain a concentrated solution for standby use; Step 4: Add the above concentrate to D101 macroporous resin by wet column loading. After loading, saturated adsorption for 45 minutes, and then elute impurities with pure water until the impurities are colorless and odorless for 10 column volumes at a flow rate of 1VB / hours. Step 5: Elute with 60% ethanol aqueous solution for 4-8 column volumes at a flow rate of V=0.5VB / H, collect the eluate, concentrate the eluate under reduced pressure and vacuum, and recover ethanol to obtain a concentrated extract; Step 6: Mix the concentrated extract with 200 mesh silica gel, dry it at room temperature, and perform dry pressure silica gel column chromatography. Use vacuum to remove air from the silica gel, use acetone and ethyl acetate as eluent, elute for 6-10 column volumes, flow rate V=1.0VB / H, pressure p=0.008kpa, collect the eluate, concentrate and dry it under reduced pressure to obtain an off-white powder; Step 7, dissolve the off-white powder with 10% ethanol, filter, apply it to Sephadex LH-20, saturate adsorb for 30 minutes, then elute with 75% ethanol for 4-6 column volumes at a flow rate of V=0.5VB / H, collect the eluate, recover half of the ethanol under reduced pressure, cool and stand for crystallization for 24 hours, and filter to obtain crystals, which are verbascoside.

2. The method for extracting the whitening and anti-aging active ingredient verbascoside from the fermentation substrate of lemon verbena as claimed in claim 1, characterized in that: In step 1, the branches and leaves of lemon verbena are crushed and cut into segments to obtain short branches of lemon verbena for standby use, and the length of the crushed and cut segments is 0.3-0.5 cm.

3. The method for extracting the whitening and anti-aging active ingredient verbascoside from the fermentation substrate of lemon verbena as claimed in claim 2, characterized in that: In step 2, the lemon verbena short branches are immersed in citric acid water and fermented for 36-72 hours, centrifuged at high speed and filtered, and the centrifuge liquid is discarded to obtain the fermented medicinal residue: The pH value of the citric acid used is 3-4, the speed of the high-speed centrifugation is 3500 r / min, the time is 15 min, and the filtration mesh size is 800-1000 mesh.

4. The method for extracting the whitening and anti-aging active ingredient verbascoside from the fermentation substrate of lemon verbena as claimed in claim 3, characterized in that: In step three, during the two extractions, the first extraction material-liquid ratio is 1:8, the extraction temperature is 90°C, and the Soxhlet reflux extraction is 2 hours; the second extraction material-liquid ratio is 1:6, the extraction temperature is 90°C, and the extraction time is 1 hour.

5. The method for extracting the whitening and anti-aging active ingredient verbascoside from the fermentation substrate of lemon verbena as claimed in claim 4, characterized in that: In step three, water is added to dilute the mixture to an ethanol concentration of less than 10% to obtain a concentrated solution for standby use, with a temperature of T≤70°C and a pressure of ≥0.1 kPa.

6. The method for extracting the whitening and anti-aging active ingredient verbascoside from the fermentation substrate of lemon verbena as claimed in claim 5, characterized in that: In step 5, when recovering ethanol, the temperature T≤70°C and the pressure≥0.1kpa.

7. The method for extracting the whitening and anti-aging active ingredient verbascoside from the fermentation substrate of lemon verbena as claimed in claim 6, characterized in that: In step 6, when dry method is used for pressurized silica gel column chromatography, the mesh size is 100 mesh silica gel, the diameter-to-height ratio is ≥10:1, and the acetone:ethyl acetate ratio used as the eluent is 3:1.

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