CN114031590A - Method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal - Google Patents
Method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal Download PDFInfo
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Abstract
The invention relates to a method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal, which comprises the following steps: (1) degreasing seabuckthorn seeds, drying, crushing and sieving; (2) pretreating the sea buckthorn seed meal by using ultrahigh pressure equipment, and then extracting the oligomeric proanthocyanidins in a gradient manner by adopting an ultrasonic extraction method: (3) carrying out centrifugal separation; (4) concentrating under high pressure; (5) separating the anthocyanin dimer by utilizing a molecular imprinting technology; (6) separating high-purity procyanidine oligomers with different polymerization degrees (the average polymerization degree is 3-4) by using a molecular sieve; (7) freeze drying; (8) the anthocyanin dimer, the trimer and the tetramer powder are compounded according to different proportions, a method for simply and efficiently depolymerizing, extracting and separating the oligomeric proanthocyanidins is established and optimized, and especially the seabuckthorn seed meal oligomeric proanthocyanidins with the average polymerization degree of less than 5 show good antioxidant activity, and the effects of the seabuckthorn seed extract in removing free radicals and inhibiting lipid peroxidation are enhanced.
Description
Technical Field
The invention belongs to the field of fine processing of sea buckthorn seeds, and particularly relates to a method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal.
Background
Hippophae rhamnoides (Hippophaerhamnoides L.) is perennial deciduous shrub or small tree of Hippophae of Elaeagnaceae. The main value part of the sea-buckthorn is the sea-buckthorn fruit, and the flesh, the leaves and the seeds of the sea-buckthorn contain rich bioactive substances, such as amino acid, flavone, polyphenol, procyanidine, terpenoids and the like; at present, researches show that the seabuckthorn seeds contain 4 monomers and 8 dimers of procyanidine, the monomers are catechin, epicatechin, catechin inedible and epigallocatechin respectively, the seabuckthorn seeds account for about 46.9 percent of the weight of seabuckthorn pomace and are resource byproducts with high yield in the seabuckthorn industry, the seabuckthorn seeds generated by seabuckthorn products only are tens of thousands of tons every year, and the efficient extraction of the oligomeric procyanidine from the seabuckthorn seeds not only has important theoretical value, but also has important significance for prolonging the seabuckthorn industrial chain, protecting the environment and effectively utilizing resources.
Procyanidins (PCs) are a general term for a large group of polyphenolic compounds widely present in plants, and two to four polymers are called Oligomeric Procyanidins (OPCs) and five or more are called Polymeric Procyanidins (PPCs) in terms of degree of polymerization; wherein the oxidation resistance is as follows: dimer > trimer > tetramer > multimer; OPC has been reported to have antioxidant, antibacterial, anti-inflammatory, anticancer, skin health-care, etc. effects; in vivo, the oxidation resistance of OPC is 50 times of VE and 20 times of VC, the water solubility of OPC is better, the OPC is widely applied to health-care food and cosmetics in recent years, and the annual sales amount reaches hundreds of millions of dollars; PPC has poor water solubility, the activity of phenolic hydroxyl is limited by steric hindrance, the PPC is difficult to be absorbed and utilized by human bodies, and only can be utilized as a low-added-value product, and meanwhile, because procyanidin is mainly high polymer in plant extraction, in order to improve the biological activity and water solubility of the seabuckthorn seed extract, the degradation of high polymer procyanidin is imperative.
The prior documents report various methods for extracting the seabuckthorn seed procyanidin, the extracted substances are only crude extracts of the seabuckthorn seed procyanidin through ultrasonic extraction and vacuum microwave drying technology in patent CN102432578B, and the procyanidin in the seabuckthorn seeds is extracted through room-temperature flash extraction, a low-temperature negative-pressure heat pump and a warm negative-pressure belt drying technology in patent CN 105440003A. Patent CN109111420A discloses a low-cost preparation method of oligomeric procyanidin, which comprises the steps of extracting, concentrating, adsorbing by macroporous resin, concentrating again, dissolving in water, reducing polymerization, and spray drying to obtain oligomeric procyanidin.
Although the related method can obtain the seabuckthorn seed procyanidin, the related method still has the following defects: (1) only the high polymer and the oligomer of the procyanidine in the seabuckthorn seeds are simply extracted by the means; (2) the macroporous resin has various varieties and specifications, complex operation and high technical requirements, and needs to be optimized when determining the process conditions; a resin is difficult to extract a plurality of chemical components under the same condition; (3) the above means involves degradation of procyanidin polymers, but the intermediate ring segments reduce the oligomeric procyanidin activity and extraction yield.
Disclosure of Invention
In order to solve the technical problems, the application provides a method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal, the process route utilizes separation technologies such as ultrahigh pressure equipment, molecular imprinting and molecular sieves to obtain three oligomeric proanthocyanidins with the average polymerization degree of less than 5, and the method has the characteristics of simple process, short reaction time, high extraction rate and the like, so that the effects of the oligomeric proanthocyanidins in the aspects of oxidation resistance and the like are expected to be further enhanced.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal comprises the following specific steps:
s1: drying and crushing: firstly, degreasing seabuckthorn seeds, drying, crushing and sieving to obtain seabuckthorn seed meal powder with 20-100 meshes;
s2: depolymerization and extraction of sea buckthorn procyanidin: pretreating the sea buckthorn seed meal by using ultrahigh pressure equipment (60-100 Mpa), then performing gradient extraction on the sea buckthorn seed meal by respectively adopting 50% -90% ethanol with the pH value of 1.00-6.00 under the condition of ultrasonic assistance, repeating the steps for 2-6 times, and mixing to obtain a procyanidine extracting solution;
s3: centrifugal separation: putting the extracting solution into a low-speed centrifuge, centrifuging for 5-30 min under the condition of 2000-10000 r/min, and separating to obtain filter residue and filtrate;
s4: and (3) concentrating under reduced pressure: putting the filtrate into a vacuum rotary evaporator, and recovering ethanol under reduced pressure to obtain a concentrated solution;
s5: molecular imprinting separation of procyanidine dimer: adding 1g of imprinting material into the concentrated solution in S4, oscillating and adsorbing in a water bath constant temperature oscillator, filtering after adsorption is balanced to obtain filtrate A and the adsorbed imprinting material, washing the adsorbed imprinting material with distilled water, placing the washed imprinting material in the water bath constant temperature oscillator for oscillating and desorbing, and obtaining procyanidine dimer solution after the desorption is finished, wherein the filtrate A is subjected to S6 operation and is mixed solution for adsorbing procyanidine dimer;
s6: molecular sieve separation of trimers: weighing 1-20 g of an adsorbent with a pore passage of 0.2-3.0 nm, adding the adsorbent into the filtrate A in S5, performing vortex for 1-10 min, and centrifuging for 5-20 min at 3000-10000 r/min to obtain a filtrate B and an adsorbed molecular sieve material; desorbing the adsorbed molecular sieve by using a desorption agent to obtain a procyanidine trimer solution, and performing S7 operation on the filtrate B, wherein the filtrate B is a mixed solution after the procyanidine trimer is adsorbed;
s7: molecular sieve separation of tetramer: weighing 1-20 g of adsorbent with pore channels of 3.0-10.0 nm, adding the filtrate B in S6, performing vortex for 1-10 min, and centrifuging for 5-20 min at 3000-10000 r/min to obtain filtrate C and an adsorbed molecular sieve material; desorbing the adsorbed molecular sieve by using a desorption agent to obtain a procyanidine tetramer solution, wherein the filtrate C is the procyanidine polymer solution, and the average polymerization degree is more than or equal to 5;
s8: and (3) freeze drying: prefreezing the procyanidine dimer, the procyanidine trimer and the procyanidine tetramer solution in S5-S7 to-80 ℃, putting the prefreezing solution into a freeze dryer, and freeze-drying the prefreezing solution for 2-12 hours at the temperature of-40 ℃ to-56 ℃ to obtain the procyanidine dimer, the procyanidine trimer and the procyanidine tetramer powder of the seabuckthorn seed meal;
s9: the sea-buckthorn seed meal procyanidine dimer, trimer and tetramer powder are compounded according to different proportions so as to improve the oxidation resistance of the sea-buckthorn seed procyanidine.
Further, the seabuckthorn seed meal in the step S1 is prepared by extracting seabuckthorn seed oil in a supercritical manner, airing the seabuckthorn seed oil in a dark place or drying the seabuckthorn seed oil at a low temperature in a ventilated place, crushing the seabuckthorn seed meal, and sieving the seabuckthorn seed meal with a 40-80-mesh sieve.
Further, the sea buckthorn seed meal is pretreated by using ultrahigh pressure equipment in the step S2, wherein the ultrahigh pressure is 30-100 Mpa, the ultrahigh temperature is 20-80 ℃, and the ultrahigh time is 5-55 min. Then, performing gradient extraction by using an ultrasonic-assisted ethanol solvent, wherein the ultrasonic power is 100-500W, and the heating power is 100-600W; wherein the ethanol extraction is repeated for 2-6 times under the conditions that the ratio of the ethanol extraction material to the ethanol extraction liquid is 1: 5-1: 55 (g/mL), the temperature is 20-80 ℃, and the extraction time is 5-55 min. Adding acid into 50-90% ethanol as ethanol solvent to adjust pH to 1.00-6.00, wherein the acid can be inorganic acid such as hydrochloric acid, phosphoric acid, sulfurous acid and the like, and organic acid such as formic acid, acetic acid, lactic acid, citric acid, malic acid and the like; so as to achieve the purpose of extracting and preparing oligomeric procyanidin in depolymerization.
Further, in the step S4, the reduced pressure concentration condition is that the vacuum degree is 0.95MPa, the temperature is 28-40 ℃, and the residual volume is 0.25-0.5 of the original volume.
Further, the molecularly imprinted material in the step S5 is MIP-P4VP/SiO2, the desorption solution is 0.1-1 mol/L HCl, and the temperature of the thermostatic water bath is 20-30 ℃.
Further, in the step S6, the molecular sieve is SAPO-34, MCM-22, MCM-41, 5A, SAPO-11 and the like, the pore channel is 0.3-3.0 nm, and the desorbent is an acidic solution with the ratio of ethanol to acetic acid being 9:1 or 8:2 or 7:3 or 6:4 or 5: 5.
Furthermore, in the step S7, the molecular sieve is SBA-15, SBA-15-4.2, Al-SBA-15, KIT-6, 10X and the like, the pore channel is 3.0-9.5 nm, and the desorption agent is an acidic solution with the ratio of ethanol to acetic acid being 9:1 or 8:2 or 7:3 or 6:4 or 5: 5.
Further, the concentrated solution in the step S8 is pre-frozen at-80 ℃, and the freeze-drying vacuum degree is 5-20 Pa, and the temperature is-40 ℃ to-50 ℃.
Further, in the step S9, the antioxidant performance of the procyanidin is studied through different mixture ratios of the dimer, the trimer and the tetramer, the ratio is 8:1:1 or 7:2:1 or 6:2:2 or 6:3:1 or 5:3:2 or 5:4:1 or 4:3:3 or 4:4:2, and the procyanidin extracts with different average polymerization degrees are compounded to have a good promoting effect in some aspects.
Compared with the prior art, the process has the following beneficial effects:
(1) pretreating semen Hippophae with ultrahigh pressure equipment, and gradient extracting to obtain oligomeric procyanidin (average degree of polymerization less than 5), with improved extraction rate and shortened reaction time; meanwhile, the reasonable selection of the factors such as the number of crushing meshes, the temperature, the time and the like can further promote the dissolution of specific components and the improvement of the extraction efficiency;
(2) the proanthocyanidins are adsorbed and separated by utilizing the molecular imprinting and molecular sieve technology, so that the interference of other substances on the analysis of the proanthocyanidins is effectively reduced, and the method has the advantages of short time consumption, solvent saving, simplicity and convenience in operation and the like; the separation effect of the procyanidin oligomer is further enhanced by improving the separation mode of the oligomeric procyanidin;
(3) the technological process is simple to operate, the activity of the oligomeric anthocyanin is not influenced, and the extraction efficiency is improved;
(4) through researching the combination of procyanidine with different polymerization degrees, the oxidation resistance of the obtained dimer is stronger than that of trimer and tetramer, and the lower the average polymerization degree is, the stronger the oxidation resistance is, and the better the effect is.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
A method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal comprises the following steps:
s1: drying and crushing: firstly, degreasing seabuckthorn seeds, drying, crushing and sieving to obtain seabuckthorn seed meal powder of 100 meshes;
s2: depolymerization and extraction preparation of sea-buckthorn procyanidine: pretreating the seabuckthorn seed meal by using ultrahigh pressure equipment (60-100 Mpa), then performing gradient extraction on the seabuckthorn seed meal by using 50-90% ethanol solution with the pH of about 2.50 under the assistance of ultrasonic waves, repeating the step for 3 times to obtain procyanidine extract, wherein the pH is adjusted by using lactic acid, 50g of seabuckthorn seed meal is taken, 750mL of ethanol solution with the pH of 2.50 is added according to the material-liquid ratio of 1:15(g/mL), the ultrasonic time is 12min, and the ultrasonic temperature is 45 ℃;
s3: centrifugal separation: placing the extract into a low-speed centrifuge, centrifuging for 15min at 4000r/min, and separating to obtain filter residue and filtrate;
s4: and (3) concentrating under reduced pressure: putting the filtrate into a vacuum rotary evaporator with the vacuum degree of 0.08MPa and the temperature of 35 ℃ for decompressing and recovering ethanol to obtain concentrated solution;
s5: molecular imprinting separation of procyanidine dimer: to the concentrated solution in S4, 1g of blotting material was added, and the mixture was adsorbed by shaking in a water bath constant temperature shaker. And after the adsorption is balanced, filtering to obtain filtrate A and the adsorbed imprinting material. Washing the adsorbed imprinting material with distilled water, placing the washed imprinting material in a water bath constant temperature oscillator for oscillation desorption, obtaining a procyanidine dimer solution after the desorption is finished, and performing S6 operation on filtrate A, wherein the filtrate A is a mixed solution for adsorbing procyanidine dimer;
s6: molecular sieve separation of trimers: weighing 1g of molecular sieve SAPO-11, adding the filtrate A in S5, carrying out vortex for 3min, and centrifuging for 10min at 5000r/min to obtain filtrate B and an adsorbed molecular sieve material; desorbing the adsorbed molecular sieve by using a desorption agent to obtain a procyanidine trimer solution, and performing S7 operation on the filtrate B, wherein the filtrate B is a mixed solution after the procyanidine trimer is adsorbed;
s7: molecular sieve separation of tetramer: weighing 1g of molecular sieve 10X, adding the filtrate B in S6, vortexing for 3min, and centrifuging for 10min at 5000r/min to obtain filtrate C and the adsorbed molecular sieve material; desorbing the adsorbed molecular sieve by using a desorption agent to obtain a procyanidine tetramer solution, wherein the filtrate C is the procyanidine polymer solution, and the average polymerization degree is more than or equal to 5;
s8: and (3) freeze drying: pre-freezing the procyanidine dimer, trimer and tetramer solutions obtained in the steps S5-S7 to-80 ℃, respectively, putting the pre-frozen procyanidine dimer, trimer and tetramer solutions into a freeze dryer, and freeze-drying the pre-frozen procyanidine dimer, trimer and tetramer solutions at the temperature of-40 ℃ for 8 hours to obtain seabuckthorn seed meal procyanidine dimer, trimer and tetramer powder;
s9: the sea buckthorn seed meal procyanidine dimer, trimer and tetramer powder are mixed according to the proportion of 6:3:1, and the oxidation resistance of DPPH, ABTS and FRAP is 2.078mmol of Trolox/g DW, 1.063mmol of Trolox/g DW and 1.173mmol of Trolox/g DW respectively.
Example 2
A method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal comprises the following steps:
s1: drying and crushing: firstly, degreasing seabuckthorn seeds, drying, crushing and sieving to obtain 60-mesh seabuckthorn seed meal powder;
s2: depolymerization and extraction preparation of sea-buckthorn procyanidine: pretreating the seabuckthorn seed meal by using ultrahigh pressure equipment (60-100 Mpa), then performing gradient extraction on the seabuckthorn seed meal by using 50-90% ethanol solution with the pH value of 3.55 under the assistance of ultrasonic waves, repeating the steps for 3 times to obtain procyanidine extract, wherein the pH value is adjusted by using citric acid, 25g of seabuckthorn seed meal is taken, 375mL of ethanol solution with the pH value of 3.55 is added according to the material-to-liquid ratio of 1:15(g/mL), and the ultrasonic time is 28min and the ultrasonic temperature is 56 ℃;
s3: centrifugal separation: placing the extract into a low-speed centrifuge, centrifuging for 15min at 4000r/min, and separating to obtain filter residue and filtrate;
s4: and (3) concentrating under reduced pressure: putting the filtrate into a vacuum rotary evaporator with the vacuum degree of 0.08MPa and the temperature of 35 ℃ for decompressing and recovering ethanol to obtain concentrated solution;
s5: molecular imprinting separation of procyanidine dimer: to the concentrated solution in S4, 1g of blotting material was added, and the mixture was adsorbed by shaking in a water bath constant temperature shaker. And after the adsorption is balanced, filtering to obtain filtrate A and the adsorbed imprinting material. Washing the adsorbed imprinting material with distilled water, placing the washed imprinting material in a water bath constant temperature oscillator for oscillation desorption, obtaining a procyanidine dimer solution after the desorption is finished, and performing S6 operation on filtrate A, wherein the filtrate A is a mixed solution for adsorbing procyanidine dimer;
s6: molecular sieve separation of trimers: weighing 5g of molecular sieve SAPO-34, adding the filtrate A in S5, performing vortex for 6min, and centrifuging at 8000r/min for 10min to obtain filtrate B and the adsorbed molecular sieve material; desorbing the adsorbed molecular sieve by using a desorption agent to obtain a procyanidine trimer solution, and performing S7 operation on the filtrate B, wherein the filtrate B is a mixed solution after the procyanidine trimer is adsorbed;
s7: molecular sieve separation of tetramer: weighing 5g of molecular sieve SBA-15, adding the filtrate B in S6, whirling for 8min, and centrifuging at 3000r/min for 18 min to obtain filtrate C and the adsorbed molecular sieve material; desorbing the adsorbed molecular sieve by using a desorption agent to obtain a procyanidine tetramer solution, wherein the filtrate C is the procyanidine polymer solution, and the average polymerization degree is more than or equal to 5;
s8: and (3) freeze drying: pre-freezing procyanidine dimer, trimer and tetramer solutions in S5-S7 to-80 ℃, putting the pre-frozen procyanidine dimer, trimer and tetramer solutions into a freeze dryer, and freeze-drying the pre-frozen procyanidine dimer, trimer and tetramer solutions for 6 hours at-45 ℃ to obtain seabuckthorn seed meal procyanidine dimer, trimer and tetramer powder;
s9: the sea buckthorn seed meal procyanidine dimer, trimer and tetramer powder are proportioned according to the ratio of 4:4:2, and the oxidation resistance of DPPH, ABTS and FRAP is respectively 1.460mmol of Trolox/g DW, 0.604mmol of Trolox/g DW and 0.799mmol of Trolox/g DW.
Comparative example 1
A method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal comprises the following steps:
s1: drying and crushing: firstly, degreasing seabuckthorn seeds, drying, crushing and sieving to obtain seabuckthorn seed meal powder of 100 meshes;
s2: depolymerization and extraction preparation of sea-buckthorn procyanidine: pretreating the seabuckthorn seed meal by using ultrahigh pressure equipment (60-100 Mpa), then performing gradient extraction on the seabuckthorn seed meal by using 50-90% ethanol solution with the pH of about 2.50 under the assistance of ultrasonic waves, repeating the step for 3 times to obtain procyanidine extract, wherein the pH is adjusted by using lactic acid, 50g of seabuckthorn seed meal is taken, 750mL of ethanol solution with the pH of 2.50 is added according to the material-liquid ratio of 1:15(g/mL), the ultrasonic time is 12min, and the ultrasonic temperature is 45 ℃;
s3: centrifugal separation: placing the extract into a low-speed centrifuge, centrifuging for 15min at 4000r/min, and separating to obtain filter residue and filtrate;
s4: and (3) concentrating under reduced pressure: putting the filtrate into a vacuum rotary evaporator with the vacuum degree of 0.08MPa and the temperature of 35 ℃ for decompressing and recovering ethanol to obtain concentrated solution;
s5: adsorption separation: adding macroporous resin AB-8 into the concentrated solution in S4, oscillating and adsorbing in a water bath constant temperature oscillator, selecting a gradient elution mode after adsorption is balanced, eluting with 5-20% ethanol solution, eluting with 25-45% ethanol solution, and finally eluting with 50-70% ethanol solution, wherein the use amount of the eluent is 10 times of the column volume, and the flow rate during elution is 2 BV/h;
s6: pre-freezing the eluates with three gradients to-80 deg.C, respectively, placing into a freeze-drying machine, and freeze-drying at-45 deg.C for 6 hr to obtain procyanidin powder with three gradients; the antioxidant properties of DPPH, ABTS and FRAP are respectively 0.884mmol of Trolox/g DW, 0.501mmol of Trolox/g DW and 0.561mmol of Trolox/g DW according to the ratio of 6:3: 1.
The difference from the embodiment 1 is that macroporous resin AB-8 is adopted to carry out adsorption separation on the concentrated solution in the step S4, and a gradient elution mode is selected, wherein 5-20% ethanol solution is used for elution at first, 25-45% ethanol solution is used for elution, and 50-70% ethanol solution is used for elution at last; the dosage of the eluent is 10 times of the column volume, and the flow rate during elution is 2 BV/h; the oxidation resistance of DPPH, ABTS and FRAP is obviously reduced.
Comparative example 2
A method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal comprises the following steps:
s1: drying and crushing: firstly, degreasing seabuckthorn seeds, drying, crushing and sieving to obtain 60-mesh seabuckthorn seed meal powder;
s2: depolymerization and extraction preparation of sea-buckthorn procyanidine: pretreating the seabuckthorn seed meal by using ultrahigh pressure equipment (60-100 Mpa), then performing gradient extraction on the seabuckthorn seed meal by using 50-90% ethanol solution with the pH value of 3.55 under the assistance of ultrasonic waves, repeating the steps for 3 times to obtain procyanidine extract, wherein the pH value is adjusted by using citric acid, 25g of seabuckthorn seed meal is taken, 375mL of ethanol solution with the pH value of 3.55 is added according to the material-to-liquid ratio of 1:15(g/mL), and the ultrasonic time is 28min and the ultrasonic temperature is 56 ℃;
s3: centrifugal separation: placing the extract into a low-speed centrifuge, centrifuging for 15min at 4000r/min, and separating to obtain filter residue and filtrate;
s4: and (3) concentrating under reduced pressure: putting the filtrate into a vacuum rotary evaporator with the vacuum degree of 0.08MPa and the temperature of 35 ℃ for decompressing and recovering ethanol to obtain concentrated solution;
s5: adsorption separation of procyanidine: and (3) adding macroporous resin D101 into the concentrated solution in the S4, oscillating and adsorbing in a water bath constant temperature oscillator, and after adsorption is balanced, selecting a gradient elution mode, eluting with 5-20% ethanol solution, eluting with 25-45% ethanol solution, and eluting with 50-70% ethanol solution. The dosage of the eluent is 10 times of the column volume, and the flow rate during elution is 2 BV/h;
s6: pre-freezing the eluates with three gradients to-80 deg.C, respectively, placing into a freeze-drying machine, and freeze-drying at-45 deg.C for 6 hr to obtain procyanidin powder with three gradients. The antioxidant properties of DPPH, ABTS and FRAP are respectively 0.608mmol of Trolox/g DW, 0.233mmol of Trolox/g DW and 0.435mmol of Trolox/g DW according to the ratio of 4:4: 2.
The difference from the embodiment 2 is that macroporous resin D101 is adopted to perform adsorption separation on the concentrated solution in the step S4, and a gradient elution mode is selected, wherein 5-20% ethanol solution is used for elution at first, 25-45% ethanol solution is used for elution, and 50-70% ethanol solution is used for elution at last. The dosage of the eluent is 10 times of the column volume, the flow rate during elution is 2BV/h, and the oxidation resistance of DPPH, ABTS and FRAP is obviously reduced.
Claims (9)
1. A method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal is characterized by comprising the following steps:
s1: drying and crushing: firstly, degreasing seabuckthorn seeds, drying, crushing and sieving to obtain seabuckthorn seed meal powder with 20-100 meshes;
s2: depolymerization and extraction preparation of sea-buckthorn procyanidine: pretreating the sea buckthorn seed meal by using ultrahigh pressure equipment (60-100 Mpa), performing gradient extraction on the sea buckthorn seed meal by respectively adopting 50% -90% ethanol with the pH value of 1.00-6.00 under the condition of ultrasonic assistance, repeating the steps for 2-6 times, and mixing to obtain a procyanidine extracting solution;
s3: centrifugal separation: putting the extracting solution into a low-speed centrifuge, centrifuging for 5-30 min under the condition of 2000-10000 r/min, and separating to obtain filter residue and filtrate;
s4: and (3) concentrating under reduced pressure: putting the filtrate into a vacuum rotary evaporator, and recovering ethanol under reduced pressure to obtain concentrated aqueous solution of procyanidine;
s5: molecular imprinting separation of procyanidine dimer: adding the imprinting material into the concentrated solution in S4, and oscillating and adsorbing in a water bath constant temperature oscillator; after the adsorption is balanced, filtering to obtain filtrate A and the adsorbed imprinting material; washing the adsorbed imprinting material with distilled water, placing the imprinting material in a water bath constant temperature oscillator for oscillation desorption, obtaining a procyanidine dimer solution after the desorption is finished, and performing S6 operation on the filtrate A, wherein the filtrate A is a mixed solution for adsorbing procyanidine dimer;
s6: molecular sieve separation of trimers: adding a molecular sieve into the filtrate A of S5 for adsorption, and centrifuging to obtain filtrate B and an adsorbed molecular sieve material; desorbing the adsorbed molecular sieve material by using a desorption agent to obtain a procyanidine trimer solution, and performing S7 operation on a filtrate B, wherein the filtrate B is a mixed solution after the procyanidine trimer is adsorbed;
s7: molecular sieve separation of tetramer: adding a molecular sieve into the filtrate B of S6 for adsorption, and centrifuging to obtain filtrate C and an adsorbed molecular sieve material; desorbing the adsorbed molecular sieve by using a desorption agent to obtain a procyanidine tetramer solution, wherein the filtrate C is the procyanidine polymer solution;
s8: and (3) freeze drying: prefreezing the procyanidine dimer, the procyanidine trimer and the procyanidine tetramer solution in S5-S7 to-80 ℃, putting the prefreezing solution into a freeze dryer, and freeze-drying the prefreezing solution for 2-12 hours at the temperature of-40 ℃ to-56 ℃ to obtain the procyanidine dimer, the procyanidine trimer and the procyanidine tetramer powder of the seabuckthorn seed meal;
s9: the sea-buckthorn seed meal procyanidine dimer, trimer and tetramer powder are compounded according to different proportions so as to improve the oxidation resistance of the sea-buckthorn seed procyanidine.
2. The method for efficiently preparing high-purity oligomeric procyanidin from seabuckthorn seed meal as claimed in claim 1, wherein the seabuckthorn seed meal in the step S1 is prepared by performing supercritical extraction on seabuckthorn seed oil, then airing the seabuckthorn seed oil in a dark place or drying the seabuckthorn seed oil at a low temperature, crushing the seabuckthorn seed meal and sieving the seabuckthorn seed meal with a 40-80-mesh sieve.
3. The method for efficiently preparing high-purity oligomeric procyanidins from seabuckthorn seed meal as claimed in claim 1, wherein the seabuckthorn seed meal is subjected to pretreatment by using ultrahigh pressure equipment in step S2, wherein the ultrahigh pressure is 30-100 MPa, the ultrahigh pressure temperature is 20-80 ℃, and the ultrahigh pressure time is 5-55 min; then, performing gradient extraction by using an ultrasonic-assisted ethanol solvent, wherein the ultrasonic power is 100-500W, and the heating power is 100-600W; wherein the ethanol extraction material-liquid ratio is 1: 5-1: 55 (g/mL), the extraction is repeated for 2-6 times under the conditions that the temperature is 20-80 ℃, the extraction time is 5-55 min, and acid is added into ethanol with 50-90% of ethanol solvent to adjust the pH value to be 1.00-6.00, wherein the acid can be hydrochloric acid, phosphoric acid, sulfurous acid inorganic acid, formic acid, acetic acid, lactic acid, citric acid and malic acid organic acid.
4. The method for efficiently preparing high-purity oligomeric procyanidin from seabuckthorn seed meal as claimed in claim 1, wherein the reduced pressure concentration condition in step S4 is that the vacuum degree is 0.95MPa, the temperature is 28-40 ℃, and the residual volume is 0.25-0.5 of the original volume.
5. The method for efficiently preparing high-purity oligomeric procyanidins from seabuckthorn seed meal as claimed in claim 1, wherein the molecularly imprinted material in step S5 is MIP-P4VP/SiO2The composite material is a self-synthesized material in a laboratory, desorption solution is 0.1-1 mol/L HCl, and constant-temperature water bath is 20-30 ℃.
6. The method for efficiently preparing high-purity oligomeric procyanidin from seabuckthorn seed meal according to claim 1, wherein the molecular sieve in step S6 is SAPO-34, MCM-22, MCM-41, 5 a, SAPO-11, the pore channel is 0.3-3.0 nm, and the desorption agent is an acidic solution with a ratio of ethanol to acetic acid of 9:1 or 8:2 or 7:3 or 6:4 or 5: 5.
7. The method for efficiently preparing high-purity oligomeric procyanidin from seabuckthorn seed meal as claimed in claim 1, wherein the molecular sieve in step S7 is SBA-15, SBA-15-4.2, Al-SBA-15, KIT-6, 10X, the pore channel is 3.0-9.5 nm, and the desorbent is an acidic solution prepared from ethanol and acetic acid in a ratio of 9:1, 8:2, 7:3, 6:4, or 5: 5.
8. The method for efficiently preparing high-purity oligomeric procyanidin from seabuckthorn seed meal as claimed in claim 1, wherein the concentrated solution in the step S8 is pre-frozen at-80 ℃, the vacuum degree of freeze drying is 5-20 Pa, and the temperature is-40 ℃ to-50 ℃.
9. The method for efficiently preparing high-purity oligomeric procyanidins from seabuckthorn seed meal as claimed in claim 1, wherein the ratio of dimer, trimer and tetramer in step S9 is 8:1:1 or 7:2:1 or 6:2:2 or 6:3:1 or 5:3:2 or 5:4:1 or 4:3:3 or 4:4: 2.
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