Graded purification preparation method and application of a maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers

Abstract

The present invention discloses a graded purification preparation method for a maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers, and use thereof. Maritime pine bark is subjected to aqueous-ethanol extraction and low-temperature reduced-pressure concentration. The crude concentrate is purified by macroporous-resin gradient elution to remove small-molecule phenolic acids, catechin, and epicatechin and to obtain a DP2-DP6 oligomeric proanthocyanidin-enriched eluate. The eluate is subjected to two-stage membrane separation, wherein polymeric tannins are removed through a 3-10 kDa membrane and the permeate is concentrated through a 300-500 Da membrane to obtain an enriched liquid rich in oligomeric proanthocyanidins and low in monomers. Composite wall materials are added, followed by shearing, homogenization, and drying to obtain the microcapsule powder. The method regulates proanthocyanidin polymerization-degree distribution, reduces water activity, and improves storage stability and powder-processing compatibility for oral solid nutritional products.

Description

[0001] DESCRIPTION

[0002] Graded Purification Preparation Method and Application of a Maritime Pine Bark Extract Microcapsule Powder Rich in Oligomeric Proanthocyanidins and Low in Monomers

[0003] Technical Field

[0004] The present invention relates to the technical field of plant extract preparation and functional food raw materials, and in particular to a graded purification preparation method and application of a maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers.

[0005] Background Art

[0006] Maritime pine bark is a natural plant raw material rich in polyphenols, including proanthocyanidins, catechin, epicatechin and phenolic acids. The free-radical scavenging effect of proanthocyanidins has been widely recognized in the industry, and they are extensively used in oral nutritional supplements and functional foods. Existing production of maritime pine bark extracts generally uses an aqueous-ethanol extraction plus simple purification process. In the industry, total proanthocyanidin content is commonly used as a core quality-control indicator, while fine differentiation and targeted regulation of proanthocyanidin components with different degrees of polymerization are rarely carried out.

[0007] In actual production and application, it can be found that proanthocyanidins with different degrees of polymerization differ significantly in physicochemical properties, taste and processing compatibility. When the proportions of monomeric catechin, epicatechin and small-molecule phenolic acids are too high, the extract has a strong bitter and astringent taste, and such small-molecule components have poor chemical stability and are prone to oxidative degradation during storage, easily causing quality fluctuations among batches. When the proportion of highly polymerized condensed tannins is too high, the water solubility of the extract is reduced and the hygroscopicity of the powder is increased, causing caking and reduced flowability during storage and bringingDESCRIPTION

[0008] inconvenience to filling and tableting of oral solid preparations such as hard capsules and tablets. Current common improvement approaches mostly compound vitamins or other plant extracts to enhance product efficacy, or use a single wall material for simple embedding. None optimize the composition of the extract itself from the perspective of regulating the degree of polymerization of proanthocyanidins, and therefore cannot simultaneously improve compositional rationality and powder-processing compatibility.

[0009] Summary of the Invention

[0010] The object of the present invention is to remedy deficiencies in the prior art by providing a graded purification preparation method and application of a maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers. Through a graded purification process combining macroporous-resin gradient elution with two-stage membrane separation, together with a multi-component composite-wall-material microencapsulation technology, the maritime pine bark extract is directionally modified. Based on differences in component polarity and molecular weight, small-molecule phenolic acids, monomeric flavanols and polymeric tannins are removed stepwise, DP2-DP6 oligomeric proanthocyanidins are directionally enriched, and the polymerization-degree distribution of proanthocyanidins is precisely regulated; the microencapsulation treatment can reduce powder water activity, improve wet-heat storage stability and in-vitro digestion stability, and improve powder flowability and anti-caking properties. The obtained microcapsule powder has controllable composition and stable properties, and can be directly adapted to the processing and preparation of various oral solid nutritional products.

[0011] To solve the above technical problems, the present invention provides the following technical solutions: in one aspect, a graded purification preparation method for maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers comprises the following steps:DESCRIPTION

[0012] Raw-material pretreatment: taking maritime pine bark, drying and crushing it to 40-80 mesh to obtain maritime pine bark powder, thereby ensuring uniform raw-material particle size, facilitating sufficient wetting by the subsequent solvent and improving the extraction efficiency of active ingredients;

[0013] Aqueous-ethanol extraction: extracting the maritime pine bark powder with a 45%-65% ethanol aqueous solution by volume fraction at a solid-liquid ratio of 1:8-1:16, an extraction temperature of 45-65°C, an extraction time of 1.0-3.0 h each time and 1-2 extraction cycles, and combining the extracts;

[0014] Pre-concentration treatment: centrifuging and filtering the extract, and then concentrating it under reduced pressure at low temperature to obtain a crude concentrated maritime pine bark extract, removing medicinal residues and suspended particulate impurities, avoiding blockage of resin pores by solid particles, and ensuring stable operation of the subsequent purification process;

[0015] First-stage macroporous-resin fractionation: contacting the crude concentrate with a macroporous adsorption resin for adsorption, first eluting with a 20%-35% ethanol aqueous solution by volume fraction, and discarding the eluted fraction to remove small-molecule phenolic acids, catechin and epicatechin;

[0016] Enrichment of oligomeric fraction: eluting with a 45%-65% ethanol aqueous solution by volume fraction and collecting the eluate to obtain a DP2-DP6 oligomeric proanthocyanidin-enriched eluate, wherein gradient elution separates polyphenol components of different polarities and directionally enriches the target oligomeric proanthocyanidin fraction;

[0017] Two-stage membrane separation and graded concentration: subjecting the DP2-DP6 oligomeric proanthocyanidin-enriched eluate first to membrane separation with a molecular-weight cutoff of 3-10 kDa, and collecting the permeate to remove DP>=10 polymeric tannins; then concentrating the permeate through a membrane with a molecular-weight cutoff of 300-500 Da, and collecting the retentate to obtain an enriched liquid rich in oligomeric proanthocyanidins and low in monomers;DESCRIPTION

[0018] Microencapsulation and drying: concentrating the enriched liquid at a temperature not higher than 45°C, adding composite wall materials and mixing, sequentially carrying out shearing and homogenization, and drying to obtain maritime pine bark extract microcapsule powder. A protective layer is formed by embedding with the wall materials to reduce the effects of external moisture and oxygen on the active ingredients while improving the processing compatibility of the powder.

[0019] Further, the macroporous adsorption resin is a nonionic styrene-divinylbenzene macroporous resin having a particle size of 0.3-1.25 mm and a specific surface area of 400-800 m2 / g. A resin within this parameter range has sufficient adsorption sites and an appropriate pore-size distribution and provides a good graded separation effect for polyphenol components of different degrees of polymerization; the amount of the macroporous adsorption resin is 0.5-2.0 times the mass of solids in the crude concentrate, and the contact adsorption time between the crude concentrate and the resin is 0.5-2.0 h, so that polyphenol components in the feed liquid are fully adsorbed on the resin while both separation efficiency and active-ingredient recovery are considered.

[0020] Further, in the two-stage membrane separation and graded concentration step, the operating temperature of membrane separation is 15-35°C and the transmembrane pressure is 0.1-0.4 MPa. Under these conditions, membrane flux is stable and oxidative degradation of oligomeric proanthocyanidins caused by excessively high temperature can be avoided; after concentration through a 300-500 Da molecular-weight cutoff membrane, the retention rate of the DP2-DP6 oligomeric proanthocyanidins is not less than 80%, ensuring controllable loss of core active components during concentration while balancing impurity removal and product yield.

[0021] Further, based on 100 parts by weight of the total mass of the final dry microcapsule powder, the system composition of the composite wall materials and active ingredients comprises: 70-84 parts of oligomericDESCRIPTION

[0022] proanthocyani din-enriched solids, 8-16 parts of resistant dextrin, 4-10 parts of gum arabic, 1-4 parts of hydroxypropyl methylcellulose, 0.2-1.2 parts of silicon dioxide, and the balance being maltodextrin; the amounts of the components are adjusted within the above ranges so that the total mass is 100 parts by weight. The multi-component composite wall-material system can form a dense embedding film layer while also providing film-forming properties, moisture resistance and powder flowability, and is adapted to the processing requirements of oral solid preparations.

[0023] Further, in the microencapsulation and drying step, the rotation speed of shearing is 6000-10000 r / min and the treatment time is 5-15 min. High-speed shearing enables the wall material and core material to be sufficiently dispersed and mixed, and preliminarily forms a uniform emulsion system; the pressure of homogenization is 20-50 MPa and homogenization is performed 1-3 times. High-pressure homogenization further refines the particle size of the emulsion to ensure uniform particle size and consistent embedding effect of the final microcapsule powder; the drying is spray drying or freeze drying, wherein the inlet air temperature of spray drying is 135-165°C and the outlet air temperature is 65-85°C. These drying parameters can rapidly remove water, shorten the heating time of the active ingredients and reduce component degradation.

[0024] In another aspect, a maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers is provided. In the microcapsule powder, the mass content of total proanthocyanidins is 60%-82%, the mass proportion of DP2-DP6 oligomeric proanthocyanidins relative to total proanthocyanidins is 45%-75%, the combined mass content of catechin and epicatechin is not higher than 10%, the mass proportion of DP>=10 polymeric tannins relative to total proanthocyanidins is not higher than 12%, and the water activity is not higher than 0.35. Under these compositional and physical-chemical parameters, the product combines a stable antioxidative activity basis with good physicochemical properties and meets the raw-material requirements of oral solidDESCRIPTION

[0025] nutritional products.

[0026] Further, the particle size D90 of the microcapsule powder is 80-220 micrometers, and the residual ethanol mass content is not higher than 0.5%, with uniform particle size and low solvent residue, meeting safety and processing requirements for food raw materials; after the powder is openly placed for 30 days at 40°C and 75% relative humidity under constant temperature and humidity, the retention rate of total proanthocyanidins is not less than 85%, so that the active ingredients can remain stable in a hot and humid environment and product quality stability during shelf life is ensured.

[0027] More further, the microcapsule powder is not exogenously added with niacin, nicotinamide, tocotrienol, grape seed extract, vitamin A, vitamin C or vitamin E as a skin-beautifying compounded active ingredient. The product-related effects are completely achieved by its own proanthocyanidin components and do not rely on exogenous compounded ingredients to enhance efficacy.

[0028] In still another aspect, the maritime pine bark extract microcapsule powder is used in the preparation of oral solid nutritional products. The microcapsule powder can be directly added and used as a functional raw material, and is suitable for production processes of various dosage forms such as hard capsules, tablets and granules.

[0029] Further, the oral solid nutritional product is used for non-therapeutic antioxidation, non-therapeutic anti-photooxidation, improving skin dullness associated with daily light exposure, or improving skin-tone evenness, whereby oral intake exerts in-vivo antioxidative effects and thereby improves related skin-condition problems caused by daily light exposure.

[0030] Compared with the prior art, the graded purification preparation method and application solution of the maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers has the following beneficial effects:DESCRIPTION

[0031] First, the present invention uses a graded purification process combining macroporous-resin gradient elution with two-stage membrane separation. By utilizing differences in component polarity and molecular weight, it stepwise removes small-molecule phenolic acids, monomeric flavanols and highly polymerized condensed tannins, and directionally collects the DP2-DP6 oligomeric proanthocyanidin fraction. It can precisely control the polymerization-degree distribution of proanthocyanidins in the extract, increase the proportion of core active oligomers, and at the same time keep monomer impurities and polymeric tannins at low levels, thereby improving the activity basis and physicochemical properties of the extract at the compositional level.

[0032] Second, the present invention microencapsulates and embeds the purified extract with a multi-component composite wall-material system. After shearing, homogenization and drying, a complete microcapsule structure is formed, which can reduce powder water activity and reduce contact between external moisture, oxygen and active ingredients. This not only improves the retention rate of active ingredients in a hot and humid environment, but also improves the stability of oligomeric proanthocyanidins during digestion; meanwhile, it improves powder flowability and anti-caking ability and can be directly adapted to the processing and production of various oral solid nutritional products.

[0033] Other advantages, objectives and features of the present invention will be described to some extent in the following specification, and to some extent will be apparent to those skilled in the art based on review and study of the following text, or may be learned from practice of the present invention.

[0034] Brief Description of the Drawings

[0035] In order to more clearly explain the technical solutions in the embodiments of the present invention or in the prior art, the drawings required for describing the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention,DESCRIPTION

[0036] and a person of ordinary skill in the art may obtain other drawings based on these drawings without creative efforts.

[0037] FIG. 1 is a preparation process flowchart of the maritime pine bark extract microcapsule powder according to the present invention;

[0038] FIG. 2 is a graded flowchart of macroporous-resin gradient elution according to the present invention;

[0039] FIG. 3 is a flowchart of two-stage membrane separation and graded concentration according to the present invention.

[0040] Detailed Description of the Embodiments

[0041] The technical solutions of the present invention are further described below with reference to examples, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the following examples and comparative examples use the same batch of maritime pine bark raw material; the reagents used are food-grade or analytical-pure reagents; and the extraction, separation, drying and detection equipment are conventional equipment in the art. Unless otherwise specifically stated, the percentages refer to mass percentages.

[0042] In order to uniformly evaluate the component quality and performance of the samples of the examples and comparative examples, the following detection methods were used for determination:

[0043] Determination of total proanthocyanidin content: the DMAC colorimetric method was used, with proanthocyanidin B2 as the reference substance. Proanthocyanidin B2 standard solutions in a concentration range of 0.005-0.100 mg / mL were prepared, DMAC hydrochloric acid solution was added for reaction in the dark, and absorbance was measured at a wavelength of 640 nm to draw a standard curve, with a correlation coefficient R2of not less than 0.999 required. A test sample was measured by the same method and substituted into the standard curve to calculate the total proanthocyanidin content, and the result was expressed as proanthocyanidin B2 equivalents.DESCRIPTION

[0044] Determination of monomeric catechin and epicatechin content: high-performance liquid chromatography was used. A Cl 8 chromatographic column was used, with 0.1% formic acid aqueous solution and acetonitrile as mobile phases for gradient elution; the detection wavelength was 280 nm, the flow rate was 1.0 mL / min, and the column temperature was 30°C. Catechin and epicatechin standards were used for external-standard quantification, and the combined content of the two in the sample was calculated.

[0045] Determination of proanthocyanidin polymerization-degree distribution: normal-phase high-performance liquid chromatography with fluorescence detection was used. A diol silica-gel chromatographic column was used; mobile phase A was acetonitrile-acetic acid (volume ratio 98:2), and mobile phase B was methanol-water-acetic acid (volume ratio 95:3:2). The gradient elution program was: 0-10 min, 0%-10% B; 10-35 min, 10%-35% B; 35-50 min, 35%-60% B; 50-60 min, 60%-80% B. The fluorescence detector excitation wavelength was 276 nm and the emission wavelength was 316 nm.

[0046] Among them, the DP2 and DP3 components were quantified respectively by external-standard curves of proanthocyanidin B2 and proanthocyanidin Cl; the DP4-DP6 components were converted to proanthocyanidin B2 equivalents after correction according to the retention times and relative response factors of adjacent oligomers; and DP>=10 polymeric tannins were included in the corrected peak area of total proanthocyanidins after correction of the peak area in the corresponding retention interval with response factors. Finally, the percentage of each component's corrected peak area in the total proanthocyanidin corrected peak area was used to calculate the proportion of components with different degrees of polymerization.

[0047] Determination of moisture and water activity: moisture was determined by a constant- weight drying method at 105°C; water activity was determined with a water activity meter at a constant temperature of 25°C.

[0048] Particle size determination: a laser particle-size analyzer was used toDESCRIPTION

[0049] measure the particle-size distribution of the sample, and the volume cumulative distribution particle size D90 was used as the evaluation indicator.

[0050] Residual ethanol determination: headspace gas chromatography was used to determine the residual ethanol content in the sample.

[0051] Example 1

[0052] As shown in FIG. 1 , the maritime pine bark extract microcapsule powder of this example was prepared through seven steps in sequence. The specific operations were as follows:

[0053] Dried maritime pine bark was taken, and the moisture content of the raw material was controlled to be not higher than 12%. The total proanthocyanidin content in the raw material was detected to be 8%-18%. The bark was crushed and passed through a 60-mesh sieve to obtain maritime pine bark powder.

[0054] The maritime pine bark powder was weighed, and a 50% ethanol aqueous solution by volume fraction was added at a solid-liquid ratio of 1:12. After uniform stirring, the mixture was heated to 55 °C and extracted at constant temperature for 2 h, and the extract was discharged. The filter residue was again added with the same volume of ethanol aqueous solution at the same concentration and extracted once under the same conditions, and the two extracts were combined.

[0055] The obtained extract was first centrifuged to remove medicinal residues and then precisely filtered to remove suspended impurities. The filtrate was collected and concentrated under reduced pressure at a temperature not higher than 45 °C until the solids content was about 12%, thereby obtaining a crude concentrated maritime pine bark extract.

[0056] A nonionic styrene-divinylbenzene macroporous adsorption resin was taken and added to the crude concentrate at 1.2 times the mass of solids in the crude concentrate. After stirring contact adsorption for 1 h, the resin was packed into a chromatography column. As shown in FIG. 2, graded purification was carried out by gradient elution: first, the column was washed with 2 column volumes of ioDESCRIPTION

[0057] purified water; then it was eluted with 3 column volumes of a 25% ethanol aqueous solution by volume fraction, and this part of the eluate was discarded to remove small-molecule phenolic acids and monomeric flavanols. Thereafter, 4 column volumes of a 55% ethanol aqueous solution by volume fraction were used for elution, and this eluate fraction was collected to obtain a DP2-DP6 oligomeric proanthocyani din-enriched liquid.

[0058] The above oligomeric proanthocyanidin-enriched liquid was subjected to two- stage membrane separation and graded concentration treatment as shown in FIG. 3: first, it was introduced into an ultrafiltration membrane system with a molecular-weight cutoff of 5 kDa; the operating temperature was controlled at 25°C and the transmembrane pressure at 0.25 MPa, and the permeate was collected, while DP>=10 polymeric tannin components were retained and removed. The permeate was then introduced into a nanofiltration membrane system with a molecular- weight cutoff of 500 Da for concentration, and the retentate was collected to obtain a purified enriched liquid rich in oligomeric proanthocyanidins and low in monomers.

[0059] The purified enriched liquid was concentrated under reduced pressure at a temperature not higher than 45°C to an appropriate solids content. Based on 100 parts by weight of the total mass of the final dry powder, the feed ratio was: 78 parts of oligomeric proanthocyanidin-enriched solids, 10 parts of resistant dextrin, 6 parts of gum arabic, 3 parts of hydroxypropyl methylcellulose, 1 part of silicon dioxide, and 2 parts of maltodextrin. After all components were fully mixed with the concentrate, the mixture was first sheared at high speed at 8000 r / min for 8 min and then homogenized under high pressure at 35 MPa twice to obtain a uniform microcapsule emulsion.

[0060] The microcapsule emulsion was fed into a spray dryer for drying, with the inlet air temperature controlled at 150°C and the outlet air temperature at 75°C. The dried powder was collected to obtain the maritime pine bark extract microcapsule powder.

[0061] iiDESCRIPTION

[0062] Example 2

[0063] The preparation process of this example was basically the same as that of Example 1, except for the following:

[0064] In the extraction stage, a 45% ethanol aqueous solution by volume fraction was used, the solid-liquid ratio was 1:10, the extraction temperature was 50°C, and each extraction lasted 2.5 h; the amount of macroporous adsorption resin was 1.0 time the mass of solids in the crude concentrate; in the elution stage, a 30% ethanol aqueous solution by volume fraction was used to remove small-molecule components, a 60% ethanol aqueous solution by volume fraction was used to collect the oligomeric proanthocyanidin-enriched fraction, and an ultrafiltration membrane with a molecular-weight cutoff of 3 kDa was selected.

[0065] The microencapsulation system, based on 100 parts by weight of final dry powder, had the following ratio: 75 parts of oligomeric proanthocyanidin-enriched solids, 12 parts of resistant dextrin, 7 parts of gum arabic, 2.5 parts of hydroxypropyl methylcellulose, 0.8 part of silicon dioxide, and 2.7 parts of maltodextrin.

[0066] Shearing, homogenization and spray drying were completed according to the process of Example 1 to prepare maritime pine bark extract microcapsule powder.

[0067] Example 3

[0068] The preparation process of this example was basically the same as that of Example 1, except for the following:

[0069] In the extraction stage, a 60% ethanol aqueous solution by volume fraction was used, the solid-liquid ratio was 1:14, the extraction temperature was 60°C, and each extraction lasted 1.5 h; the amount of macroporous adsorption resin was 1.5 times the mass of solids in the crude concentrate; in the elution stage, a 25% ethanol aqueous solution by volume fraction was used to remove small-molecule components, a 50% ethanol aqueous solution by volume fraction was used to collect the oligomeric proanthocyanidin-enriched fraction, and an ultrafiltration membrane with a molecular-weight cutoff of 5 kDa was selected.DESCRIPTION

[0070] The microencapsulation system, based on 100 parts by weight of final dry powder, had the following ratio: 82 parts of oligomeric proanthocyanidin-enriched solids, 8 parts of resistant dextrin, 4 parts of gum arabic, 3 parts of hydroxypropyl methylcellulose, 1 part of silicon dioxide, and 2 parts of maltodextrin.

[0071] The emulsion after shearing and homogenization was treated by freeze drying to prepare maritime pine bark extract microcapsule powder.

[0072] Comparative Example 1

[0073] The same batch of maritime pine bark powder as in Example 1 was taken and extracted twice with a 50% ethanol aqueous solution by volume fraction at a solid-liquid ratio of 1:12 and 55°C for 2 h each time, and the extracts were combined. The extract was centrifuged, filtered and concentrated under reduced pressure, and then directly spray-dried to obtain maritime pine bark extract powder. In this comparative example, resin fractionation, membrane separation and composite-wall-material microencapsulation were not performed.

[0074] Comparative Example 2

[0075] The preparation process of this comparative example was basically the same as that of Example 1 , except that after resin adsorption and column packing, the step of eluting with a 25% ethanol aqueous solution by volume fraction to remove small-molecule components was not performed. Instead, elution was carried out directly with a 55% ethanol aqueous solution by volume fraction, and all eluted fractions were collected; the subsequent membrane separation and microencapsulation drying were completed according to the process of Example 1.

[0076] Comparative Example 3

[0077] The preparation process of this comparative example was basically the same as that of Example 1, except that after resin adsorption and column packing, graded elution collection was not performed. After water washing, elution was carried out directly with a 55% ethanol aqueous solution by volume fraction, andDESCRIPTION

[0078] all resin eluate was collected; the subsequent membrane separation and microencapsulation drying were completed according to the process of Example 1.

[0079] Comparative Example 4

[0080] The preparation process of this comparative example was basically the same as that of Example 1 , except that the oligomeric proanthocyanidin-enriched liquid obtained by resin elution was not subjected to ultrafiltration membrane separation to remove polymeric tannins, but was directly concentrated through a 500 Da nanofiltration membrane and then subjected to microencapsulation drying.

[0081] Comparative Example 5

[0082] The preparation process of this comparative example was basically the same as that of Example 1 , except that in the microencapsulation stage, maltodextrin alone was used as the wall material to make up the final total dry powder mass to 100 parts by weight. Resistant dextrin, gum arabic, hydroxypropyl methylcellulose and silicon dioxide were not added, and the remaining process parameters were the same as those in Example 1.

[0083] Effect Verification Tests

[0084] To verify the component quality, stability and processing compatibility of the maritime pine bark extract microcapsule powder prepared according to the present invention, the samples prepared in Examples 1-3 and Comparative Examples 1-5 were tested in parallel, and the results are as follows.

[0085] Test Example 1 Comparison of Product Quality Indicators

[0086] The core components and basic powder parameters of each group of samples were tested, and the results are shown in Table 1.DESCRIPTION

[0087] Table 1 Test results of quality indicators of each sample

[0088] WA PHE DP2-DP CATEC TER TOTAL DP>=I0 NOL

[0089] 6 HIN + ACT D90 / MIC SAM PROANTHO PROPO IC PROPO EPICAT IVIT ROMETE PLE CYANIDINS RTION / ACI

[0090] RTION / ECHIN / Y RS / % % DS /

[0091] % % (AW

[0092] %

[0093] )

[0094] Com

[0095] parati

[0096] 12.2± 0.48±

[0097] ve 67.8±2.3 34.6±1.5 15.4±0.7 21.8±1.1 232±10

[0098] 0.6 0.02

[0099] Exam

[0100] pie 1

[0101] Com

[0102] parati

[0103] 10.8± 0.39±

[0104] ve 72.6±2.1 50.8±1.7 13.6±0.6 10.9±0.7 178±8

[0105] 0.5 0.01

[0106] Exam

[0107] pie 2

[0108] Com

[0109] parati

[0110] 6.1± 0.37±

[0111] ve 70.4±1.9 38.9±1.5 6.4±0.4 20.7±1.0 166±7

[0112] 0.4 0.01

[0113] Exam

[0114] pie 3

[0115] Com

[0116] parati

[0117] 5.4± 0.36±

[0118] ve 75.8±2.0 42.2±1.6 5.8±0.3 23.5±1.2 171±8

[0119] 0.3 0.01

[0120] Exam

[0121] pie 4

[0122]

[0123] DESCRIPTION

[0124] Com

[0125] parati

[0126] 4.8± 0.46±

[0127] ve 72.1±1.8 60.3±1.8 5.5±0.3 8.2±0.6 238±12 0.3 0.02

[0128] Exam

[0129] pie 5

[0130] Exam 4.6± 0.31±

[0131] 74.8±2.1 62.4±1.8 5.1±0.4 7.3±0.6 142±6 pie 1 0.4 0.01

[0132] Exam 5.2± 0.33±

[0133] 70.2±1.9 58.6±1.7 6.2±0.4 8.8±0.7 153±7 pie 2 0.3 0.01

[0134] Exam 4.9± 0.34±

[0135] 72.6±2.0 60.1±1.6 5.8±0.3 9.4±0.7 165±8 pie 3 0.3 0.01

[0136]

[0137] As can be seen from Table 1, the proportions of DP2-DP6 oligomeric proanthocyanidins in the samples of Examples 1-3 all reached more than 58%, higher than that of the ordinary extract of Comparative Example 1 ; the combined content of monomeric catechin and epicatechin was lower than 6.5%, the proportion of polymeric tannins was lower than 10%, the water activity was not higher than 0.35, and the particle-size distribution was more uniform. The results show that the process combining resin fractionation and membrane separation can effectively adjust the polymerization-degree distribution of the extract, and, together with composite-wall-material microencapsulation, can further reduce powder water activity.

[0138] Supplementary testing showed that the residual ethanol contents of Examples 1-3 were respectively 0.12%, 0.15% and 0.18%, all not higher than 0.5%; based on the DP2-DP6 oligomeric proanthocyanidin content in the feed liquid, the oligomeric proanthocyanidin retention rates of Examples 1-3 during the nanofiltration concentration stage were respectively 88.6%, 86.9% and 85.8%, all not less than 80%, indicating that the loss of core components during membrane concentration was relatively small.DESCRIPTION

[0139] Test Example 2 Investigation of Wet-Heat Storage Stability

[0140] Each sample was spread in an open glass weighing bottle at a thickness of about 2 mm and placed in an accelerated storage environment at 40°C and 75% relative humidity under constant temperature and humidity. Samples were taken at 30 days, 60 days and 90 days, and the retention rates of total proanthocyanidins and DP2-DP6 oligomeric proanthocyanidins were measured. The results are shown in Table 2.

[0141] Table 2 Wet-heat storage stability results of each sample

[0142] 30-DA 90-DA 30-DAY 60-DAY 90-DAY

[0143] Y Y TOTAL TOTAL TOTAL DP2-DP DP2-DP SAMP PROANTHOC PROANTHOC PROANTHOC

[0144] 6 6 LE YANIDIN YANIDIN YANIDIN RETEN RETEN RETENTION RETENTION RETENTION TION TION RATE RATE RATE RATE RATE

[0145] Compa

[0146] rative 66.1± 39.2±

[0147] 69.4±2.6% 55.7±2.1% 43.6±1.8%

[0148] Exampl 2.4% 1.7% e 1

[0149] Compa

[0150] rative 73.4± 46.5±

[0151] 76.8±2.5% 63.5±2.2% 50.1±1.9%

[0152] Exampl 2.3% 1.8% e 5

[0153] Exampl 92.7± 83.6±

[0154] 93.2±2.4% 89.4±2.1% 85.1±1.8%

[0155] e 1 2.3% 1.9% Exampl 90.4± 80.8±

[0156] 91.5±2.3% 86.7±2.0% 82.2±1.7%

[0157] e 2 2.1% 1.8% Exampl 89.8±2.2% 84.1±1.9% 79.4±1.7% 88.9± 77.1±

[0158]

[0159] DESCRIPTION

[0160] e 3 2.0% 1.8%

[0161]

[0162] As can be seen from Table 2, the active ingredients in the ordinary extract degraded quickly under hot and humid conditions, and embedding with maltodextrin alone only slightly delayed degradation. After being placed for 30 days, the samples of the examples of the present invention all had total proanthocyanidin retention rates higher than 85%; after 90 days, they still maintained more than 79%, and the oligomeric proanthocyanidins also remained at a relatively high level.

[0163] Test Example 3 Investigation of In-Vitro Digestion Stability

[0164] A simulated gastrointestinal fluid system was used to evaluate the in-vitro digestion stability of the samples: the samples were first incubated in simulated gastric fluid for 60 min, then transferred to simulated intestinal fluid and incubated for 120 min, and the retention rates of DP2-DP6 oligomeric proanthocyanidins and total proanthocyanidins at different stages were determined. The results are shown in Table 3.

[0165] Table 3 In-vitro digestion stability results of each sample

[0166] DP2-DP6 DP2-DP6

[0167] TOTAL RETENTION RETENTION PROANTHOCYANIDIN RATE IN RATE IN SAMPLE RETENTION RATE IN GASTRIC INTESTINAL INTESTINAL FLUID FLUID AT 60 FLUID AT 120

[0168] AT 120 MIN MIN MIN

[0169] Comparative

[0170] 61.2±2.4% 48.5±2.0% 50.6±2.2% Example 1

[0171] Comparative

[0172] 70.4±2.3% 58.2±2.1% 60.1 ±2.0% Example 5

[0173] Example 1 86.8±2.5% 78.6±2.2% 80.4±2.3% Example 2 84.1 ±2.4% 75.2±2.1% 77.5±2.2%

[0174]

[0175] DESCRIPTION

[0176] Example 3 82.5±2.3% 72.8±2.0% 74.6±2.1%

[0177]

[0178] The results show that, in simulated gastric and intestinal fluids, the retention rates of oligomeric proanthocyanidins and total proanthocyanidins in the microcapsule powders of the examples were higher than those of the comparative examples, and the microcapsule embedding structure can reduce destruction of active ingredients by digestive fluids.

[0179] Test Example 4 Investigation of Powder Compatibility

[0180] The hygroscopicity, flowability and caking of each sample were investigated. The hygroscopic weight gain rate and angle of repose after open placement for 24 h at 25°C and 75% relative humidity were measured, and the appearance state was observed. The results are shown in Table 4.

[0181] Table 4 Powder compatibility results of each sample

[0182] 24-H ANGLE OF

[0183] INITIAL HYGROSCOPIC REPOSE APPEARANCE SAMPLE ANGLE OF WEIGHT AFTER 24 STATE REPOSE / 0

[0184] GAIN / % H / °

[0185] Comparative

[0186] 13.2±0.6 45.8±1.2 53.4±1.5 Obvious caking Example 1

[0187] Comparative

[0188] 9.6±0.5 42.7±1.0 49.2±1.3 Slight caking Example 5

[0189] No obvious Example 1 4.3±0.3 34.6±0.8 38.4±0.9

[0190] caking No obvious Example 2 4.8±0.3 35.7±0.9 39.8±1.0

[0191] caking Slight moisture Example 3 5.4±0.3 37.2±0.9 41.5±1.0

[0192] absorption

[0193]

[0194] As can be seen from Table 4, the ordinary extract had strong hygroscopicity, and its flowability significantly decreased after placement with caking. TheDESCRIPTION

[0195] microcapsule powders of the examples of the present invention had lower hygroscopic weight gain rates and smaller angles of repose, and showed no obvious caking after placement, and therefore can be directly used in production operations of oral solid preparations such as hard-capsule filling, tablet compression and granule filling.

[0196] Test Example 5 Determination of In-Vitro Antioxidant Capacity

[0197] The DPPH free-radical scavenging method, ABTS free-radical scavenging method and ORAC method were respectively used to evaluate the in-vitro antioxidant capacity of each sample. The results are shown in Table 5.

[0198] Table 5 In-vitro antioxidant capacity results of each sample DPPH ABTS ORAC / (MICROMOL SAMPLE SCAVENGING SCAVENGING TE / G) RATE / % RATE / %

[0199] Comparative

[0200] 54.8±2.0 62.3±2.1 2450±96 Example 1

[0201] Comparative

[0202] 63.7±2.2 70.4±2.3 3180±110 Example 5

[0203] Example 1 76.8±2.5 82.H2.6 4I20±I35 Example 2 73.6±2.4 79.5±2.5 3890±I28 Example 3 71.9±2.3 77.8±2.4 37I0±I20

[0204]

[0205] The results show that the antioxidant indicators of the samples of the examples were all higher than those of the comparative examples. After graded purification increased the proportion of oligomeric proanthocyanidins, the in-vitro antioxidant activity of the samples was improved.

[0206] Test Example 6 HaCaT Intracellular ROS Scavenging Test

[0207] An oxidative damage model was established by inducing human immortalized epidermal cells with H2O2, and the scavenging effect of the samples on intracellular reactive oxygen species was investigated. Cell viabilityDESCRIPTION

[0208] was also detected to evaluate sample safety. The results are shown in Table 6.

[0209] Table 6 Results of HaCaT cell ROS test

[0210] ROS LEVEL CELL GROUP CONCENTRATION (MODEL VIABILITY / %

[0211] GROUP=100%) Blank control

[0212] 0 100.0±3.0 51.8±2.3 group

[0213] H2O2 model

[0214] 0 79.2±2.7 100.0±3.5 group

[0215] Comparative

[0216] lOOpg / mL 86.H2.8 82.6±3.0 Example 1

[0217] Example 1 , low

[0218] 25pg / mL 93.5±3.0 78.4±2.8 dose

[0219] Example 1,

[0220] 50pg / mL 94.H3.1 68.5±2.6 medium dose

[0221] Example 1 , high

[0222] lOOpg / mL 93.6±2.9 60.7±2.4 dose

[0223]

[0224] The results show that Example 1 had no obvious toxicity to epidermal cells in the concentration range of 25-100 micro grams / mL, and could reduce H2O2 -induced intracellular reactive oxygen species levels in a dose-dependent manner, with an effect better than that of the ordinary extract at the same concentration.

[0225] Test Example 7 B16-F10 Melanin Generation Inhibition Test

[0226] A melanin generation model was established by inducing mouse melanoma B16-F10 cells with alpha-melanocyte-stimulating hormone, and the effects of the samples on melanin generation and tyrosinase activity were investigated. The results are shown in Table 7.

[0227] Table 7 Results of B16-F10 melanin generation testDESCRIPTION

[0228] MELANIN TYROSINAS CELL CONTENT E ACTIVITY CONCENTRATI GROUP VIABILITY / (MODEL (MODEL ON

[0229] % GROUP=IOO GROUP=IOO %) %) Blank

[0230] control 0 100.0±3.1 59.4±2.4 63.7±2.6 group

[0231] alpha-MSH

[0232] model 0 I00.0±3.4 I00.0±3.5 I00.0±3.4 group

[0233] Comparati

[0234] ve lOOpg / mL 94.6±2.9 90.8±3.1 92.4±3.0 Example 1

[0235] Example 1,

[0236] 25pg / mL 97.8±3.0 91.6±3.0 93.5±3.1 low dose

[0237] Example 1,

[0238] medium 50pg / mL 95.9±2.9 84.7±2.8 88.6±2.7 dose

[0239] Example 1,

[0240] lOOpg / mL 93.8±2.8 78.9±2.6 82.5±2.5 high dose

[0241] Arbutin

[0242] positive 200pM 91.5±2.7 64.2±2.3 68.4±2.4 control

[0243]

[0244] The results show that, within a concentration range that did not significantly affect cell viability, Example 1 could inhibit melanin generation and tyrosinase activity in a dose-dependent manner, with an effect better than that of the ordinary extract at the same concentration.DESCRIPTION

[0245] The above descriptions are merely preferred embodiments of the present invention and do not limit the present invention in any form. Although the present invention has been disclosed above by preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art may make some changes or modifications to the technical content disclosed above into equivalent embodiments without departing from the scope of the technical solutions of the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the scope of the technical solutions of the present invention.

Claims

CLAIMS1. A graded purification preparation method for maritime pine bark extract microcapsule powder rich in oligomeric proanthocyanidins and low in monomers, characterized in that the preparation method comprises the following steps:Raw-material pretreatment: taking maritime pine bark, drying and crushing it to 40-80 mesh to obtain maritime pine bark powder;Aqueous-ethanol extraction: extracting the maritime pine bark powder with a 45%-65% ethanol aqueous solution by volume fraction at a solid-liquid ratio of 1:8-1:16, an extraction temperature of 45-65°C, an extraction time of 1.0-3.0 h each time, and 1 -2 extraction cycles, and combining the extracts;Pre-concentration treatment: centrifuging and filtering the extract, and then concentrating it under reduced pressure at low temperature to obtain a crude concentrated maritime pine bark extract;First-stage macroporous-resin fractionation: contacting the crude concentrate with a macroporous adsorption resin for adsorption, first eluting with a 20%-35% ethanol aqueous solution by volume fraction, and discarding the eluted fraction to remove small-molecule phenolic acids, catechin and epicatechin;Enrichment of oligomeric fraction: eluting with a 45%-65% ethanol aqueous solution by volume fraction and collecting the eluate to obtain a DP2-DP6 oligomeric proanthocyanidin-enriched eluate;Two- stage membrane separation and graded concentration: subjecting the DP2-DP6 oligomeric proanthocyanidin-enriched eluate first to membrane separation with a molecular- weight cutoff of 3-10 kDa, and collecting the permeate to remove DP>=10 polymeric tannins; then concentrating the permeate through a membrane with a molecular- weight cutoff of 300-500 Da, and collecting the retentate to obtain an enriched liquid rich in oligomeric proanthocyanidins and low in monomers;Microencapsulation and drying: concentrating the enriched liquid at a temperature not higher than 45°C, adding composite wall materials and mixing, sequentially carrying out shearing and homogenization, and drying to obtainCLAIMSmaritime pine bark extract microcapsule powder.

2. The preparation method according to claim 1, characterized in that the macroporous adsorption resin is a nonionic styrene-divinylbenzene macroporous resin having a particle size of 0.3-1.25 mm and a specific surface area of 400-800 m2 / g; the amount of the macroporous adsorption resin is 0.5-2.0 times the mass of solids in the crude concentrate, and the contact adsorption time between the crude concentrate and the resin is 0.5-2.0 h.

3. The preparation method according to claim 1 , characterized in that, in the two-stage membrane separation and graded concentration step, the operating temperature of the membrane separation is 15-35°C and the transmembrane pressure is 0.1-0.4 MPa; after concentration through a 300-500 Da molecular-weight cutoff membrane, the retention rate of the DP2-DP6 oligomeric proanthocyanidins is not less than 80%.

4. The preparation method according to claim 1, characterized in that, based on 100 parts by weight of the total mass of the final dry microcapsule powder, the system composition of the composite wall materials and active ingredients comprises: 70-84 parts of oligomeric proanthocyanidin-enriched solids, 8-16 parts of resistant dextrin, 4-10 parts of gum arabic, 1-4 parts of hydroxypropyl methylcellulose, 0.2-1.2 parts of silicon dioxide, and the balance being maltodextrin; the amounts of the components are adjusted within the above ranges so that the total mass is 100 parts by weight.

5. The preparation method according to claim 1 , characterized in that, in the microencapsulation and drying step, the rotation speed of shearing is 6000-10000 r / min and the treatment time is 5-15 min; the pressure of homogenization is 20-50 MPa and homogenization is performed 1-3 times; the drying is spray drying or freeze drying, wherein the inlet air temperature of spray drying is 135-165°C and the outlet air temperature is 65-85°C.

6. The maritime pine bark extract microcapsule powder prepared by the preparation method according to any one of claims 1-5, characterized in that theCLAIMSmass content of total proanthocyanidins in the microcapsule powder is 60%-82%, the mass proportion of DP2-DP6 oligomeric proanthocyanidins relative to total proanthocyanidins is 45%-75%, the combined mass content of catechin and epicatechin is not higher than 10%, the mass proportion of DP>=10 polymeric tannins relative to total proanthocyanidins is not higher than 12%, and the water activity is not higher than 0.35.

7. The maritime pine bark extract microcapsule powder according to claim 6, characterized in that the particle size D90 of the microcapsule powder is 80-220 micrometers, and the residual ethanol mass content is not higher than 0.5%; after the powder is openly placed for 30 days at 40°C and 75% relative humidity under constant temperature and humidity, the retention rate of total proanthocyanidins is not less than 85%.

8. The maritime pine bark extract microcapsule powder according to claim 6, characterized in that the microcapsule powder is not exogenously added with niacin, nicotinamide, tocotrienol, grape seed extract, vitamin A, vitamin C or vitamin E as a skin-beautifying compounded active ingredient.

9. Use of the maritime pine bark extract microcapsule powder according to any one of claims 6-8 in the preparation of oral solid nutritional products.

10. The use according to claim 9, characterized in that the oral solid nutritional product is used for non-therapeutic antioxidation, non-therapeutic anti-photooxidation, improving skin dullness associated with daily light exposure, or improving skin-tone evenness.

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