CN115487288B - Almond peptide oral liquid and preparation method thereof - Google Patents
Almond peptide oral liquid and preparation method thereof Download PDFInfo
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- CN115487288B CN115487288B CN202211191442.XA CN202211191442A CN115487288B CN 115487288 B CN115487288 B CN 115487288B CN 202211191442 A CN202211191442 A CN 202211191442A CN 115487288 B CN115487288 B CN 115487288B
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- 235000011437 Amygdalus communis Nutrition 0.000 title claims abstract description 93
- 241000220304 Prunus dulcis Species 0.000 title claims abstract description 93
- 235000020224 almond Nutrition 0.000 title claims abstract description 93
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 71
- 239000007788 liquid Substances 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000002105 nanoparticle Substances 0.000 claims abstract description 40
- 108010028144 alpha-Glucosidases Proteins 0.000 claims abstract description 38
- 230000000694 effects Effects 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 30
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 27
- 101800001442 Peptide pr Proteins 0.000 claims abstract description 23
- 238000004108 freeze drying Methods 0.000 claims abstract description 23
- 239000008213 purified water Substances 0.000 claims abstract description 20
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 15
- UDIPTWFVPPPURJ-UHFFFAOYSA-M Cyclamate Chemical compound [Na+].[O-]S(=O)(=O)NC1CCCCC1 UDIPTWFVPPPURJ-UHFFFAOYSA-M 0.000 claims abstract description 10
- 239000004386 Erythritol Substances 0.000 claims abstract description 10
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 claims abstract description 10
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000001768 carboxy methyl cellulose Substances 0.000 claims abstract description 10
- 239000000625 cyclamic acid and its Na and Ca salt Substances 0.000 claims abstract description 10
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 claims abstract description 10
- 235000019414 erythritol Nutrition 0.000 claims abstract description 10
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- 239000000243 solution Substances 0.000 claims description 18
- 239000002202 Polyethylene glycol Substances 0.000 claims description 17
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- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 description 2
- IFBHRQDFSNCLOZ-IIRVCBMXSA-N 4-nitrophenyl-α-d-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-IIRVCBMXSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FZNCGRZWXLXZSZ-CIQUZCHMSA-N Voglibose Chemical compound OCC(CO)N[C@H]1C[C@](O)(CO)[C@@H](O)[C@H](O)[C@H]1O FZNCGRZWXLXZSZ-CIQUZCHMSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
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- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- 102000004366 Glucosidases Human genes 0.000 description 1
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- 102000057297 Pepsin A Human genes 0.000 description 1
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- 101710184309 Probable sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 102400000472 Sucrase Human genes 0.000 description 1
- 101710112652 Sucrose-6-phosphate hydrolase Proteins 0.000 description 1
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- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/05—Dipeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Inorganic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- General Chemical & Material Sciences (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Immunology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The preparation method of the almond peptide oral liquid for inhibiting the activity of alpha-glucosidase comprises the steps of preparing almond peptide nanoparticles, erythritol, glycerol, pectin, citric acid, sodium carboxymethyl cellulose, sodium cyclamate and purified water, embedding the almond peptide nanoparticles with the almond peptide proline-arginine (PR), and then freeze-drying. The embedding rate is improved by embedding the almond peptide PR with the alpha glucosidase activity inhibition, the embedding rate reaches 88.09 percent, the particle size of the prepared almond peptide nano particles is as small as 105.3nm, the prepared almond peptide nano particles are stable in the neutral state of the oral liquid, the alpha glucosidase activity inhibition performance is effectively kept not to be reduced, the stability of the embedded shell is excellent, the phenomenon of falling off to form precipitation does not occur in pure 24 months, and the stable taste of the oral liquid is ensured. The inhibition rate of the final prepared almond peptide oral liquid on the alpha-glucosidase activity after gastrointestinal tract digestion is stabilized to be more than 64.51 percent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an almond peptide oral liquid and a preparation method thereof.
Background
Diabetes is a common metabolic disease, and the incidence rate of which is on the rise worldwide with the improvement of the living standard of people and the increase of the aged population. Diet therapy is the most important and fundamental therapeutic measure for various diabetes mellitus, and the active search for foods which are beneficial to control blood sugar from daily diet has become a current food science research hotspot.
Besides the function of reducing the blood sugar of insulin, an alpha-glucosidase inhibitor can be adopted, wherein the alpha-glucosidase inhibitor can delay the decomposition and digestion of complex carbohydrates and disaccharides by inhibiting the glucosidase activity of decomposing oligosaccharides into monosaccharides in villi of small intestine, delay and reduce the absorption of glucose by intestinal cavities, mainly reduce the function of postprandial blood sugar without influencing the utilization of glucose and the secretion of insulin, can be combined with insulin for application, and can improve the blood sugar reducing effect by mechanism complementation. Currently common alpha-glucosidase inhibitors are acarbose, voglibose, etc., e.g. acarbose mainly inhibits alpha-amylase, voglibose mainly inhibits maltase and sucrase. However, the long-term administration of the traditional Chinese medicine composition can cause nausea, vomiting, anorexia and other phenomena, and can cause abdominal distension, abdominal pain, diarrhea, gastrointestinal cramping pain, intractable constipation and the like. Therefore, many patients with other gastrointestinal diseases, liver and kidney functions and the like cannot use the alpha-glucosidase inhibitors, so that the use of the alpha-glucosidase inhibitors is greatly limited.
The almond has the biological activities of reducing blood sugar, reducing blood fat, reducing blood pressure, resisting cancer and the like. The almond can be eaten frequently to reduce cholesterol, promote cardiovascular health and control blood sugar level, and in the aspect of biologically active polypeptide auxiliary blood sugar reduction, research shows that the almond polypeptide with high dosage has the effect of inhibiting insulin reduction. However, there are many pathways that can cause blood glucose to drop, and the hypoglycemic activity of almond polypeptides has a significant relationship with the structure of the polypeptide. Therefore, the almond peptide with high-efficiency inhibition effect on the alpha-glucosidase is sought, and the almond peptide is prepared into oral liquid, so that the oral liquid is convenient to carry and take, and is beneficial to diabetics to take.
Disclosure of Invention
The invention aims to provide an almond peptide oral liquid capable of efficiently inhibiting the activity of alpha-glucosidase.
The invention also aims at providing a preparation method of the almond peptide oral liquid.
The invention aims at realizing the following technical scheme:
an almond peptide oral liquid for inhibiting alpha-glucosidase activity, which is characterized in that: the almond peptide nanoparticle is prepared from almond peptide proline-arginine (PR), bovine serum albumin, ferritin and chitosan by embedding treatment, and then freeze-drying.
Further, according to the mass percentage, the almond peptide nano-particles are 1-2%, erythritol is 0.03-0.04%, glycerin is 0.1-0.3%, pectin is 0.01-0.03%, citric acid is 0.02%, sodium carboxymethylcellulose is 0.02%, sodium cyclamate is 0.03%, and the balance is purified water.
Further, the embedding treatment is to sequentially dissolve bovine serum albumin, ferritin and chitosan in phosphate buffer solution with pH of 6 to prepare mixed solution, standing overnight at 4 ℃, freeze-drying to form freeze-dried powder, heating and preserving heat to form a compound, then dissolving the compound in purified water to form solution, adding silicon dioxide and polyethylene glycol, adjusting pH, heating and preserving heat again, cooling, adding almond peptide PR, performing ultrasonic treatment under ice bath condition, finally adjusting pH, centrifuging and freeze-drying.
Further, the mass ratio of the chitosan, the ferritin and the bovine serum albumin is 3-5:1:0.2-0.5.
Further, the mass ratio of the silicon dioxide to the polyethylene glycol is 2:0.3-0.6, wherein the silicon dioxide accounts for 2-3% of the mass of the compound.
Further, the heating and heat preservation is to heat the freeze-dried powder to 70-80 ℃ and preserve heat for 48 hours to form the compound.
Further, the concentration of the above compound dissolved in purified water is 2-4mg/mL, the pH is adjusted to 5-6, and the mixture is heated to 90-100 ℃ and kept for 30min.
Further, the mass ratio of PR to the freeze-dried powder is 1:3-7.
Further, the ultrasonic power of the ultrasonic is 200W, specifically 10s ultrasonic every 10s, and the total duration is 6-10min.
Further, after the above ultrasonic treatment, the pH was adjusted to 4-6, and after standing for 10min, the mixture was centrifuged at 3500rpm for 10min.
A preparation method of an almond peptide oral liquid for inhibiting alpha-glucosidase activity is characterized by comprising the following steps: the almond peptide nanoparticle is prepared from almond peptide proline-arginine (PR) through embedding treatment and freeze drying.
Further, according to the mass percentage, the almond peptide nano-particles are 1%, erythritol is 0.03-0.04%, glycerin is 0.1-0.3%, pectin is 0.01-0.03%, citric acid is 0.02%, sodium carboxymethylcellulose is 0.02%, sodium cyclamate is 0.03%, and the balance is purified water.
Further, the embedding treatment is to sequentially dissolve bovine serum albumin, ferritin and chitosan in phosphate buffer solution with pH of 6 to prepare mixed solution, standing overnight at 4 ℃, freeze-drying to form freeze-dried powder, heating and preserving heat to form a compound, then dissolving the compound in purified water to form solution, adding silicon dioxide and polyethylene glycol, adjusting pH to 5-6, heating and preserving heat again, cooling, adding almond peptide PR, carrying out ultrasonic treatment under ice bath condition, finally adjusting pH to 4-6, centrifuging and freeze-drying.
In the preparation process, the almond peptide PR is directly used as a raw material to prepare the oral liquid, the oral liquid can be gradually disabled after long-term storage, the inhibition effect on the alpha-glucosidase is gradually reduced, the PR directly enters the gastrointestinal tract, and the inhibition effect on the alpha-glucosidase is also reduced. Therefore, the almond peptide is firstly subjected to embedding treatment and then prepared into the oral liquid, but the chitosan and bovine serum albumin are compounded and embedded, and the fact that the shells of the embedded almond peptide gradually fall off in the long-term storage process of the oral liquid, sedimentation is formed in the oral liquid, the oral liquid becomes turbid, the almond peptide is exposed again, and the inhibition effect of the almond peptide on alpha-glucosidase cannot be ensured for a long time.
According to the invention, the novel ferritin is compounded with bovine serum albumin and then compounded with chitosan, and the free amino of the amino acid side chain in ferritin molecules reacts with the carbonyl of the reduction tail end of chitosan molecules through the hydrogen bond action formed between the amino of the bovine serum albumin and the hydroxyl of the chitosan, so that the compound of bovine serum albumin-chitosan-ferritin is formed, and the structural stability between polysaccharide and protein is further improved. And secondly, adding silicon dioxide and polyethylene glycol, adsorbing the silicon dioxide and chitosan through electrostatic attraction at a specific pH, and heating and preserving heat to form colloid, so that complete and stable embedding of the almond peptide PR is realized, and the embedding rate is remarkably improved. Finally, freeze drying is carried out to further stabilize the embedding structure, and the particle size of the prepared almond peptide nano particles is further controlled through the combination of multiple embedding and freeze drying, and the smaller particle size ensures that the almond peptide nano particles keep stable properties and are not easy to decompose in aqueous solution for a long time. After the nano particles are formed by embedding, the inhibition effect of the prepared almond peptide PR nano composite particles on alpha-glucosidase is obviously improved compared with PR without embedding. In addition, arginine in PR is basic amino acid, so that the antibacterial property of chitosan can be further enhanced, and other antibacterial components are not required to be added in the oral liquid.
Further, the mass ratio of the chitosan, the ferritin and the bovine serum albumin is 3-5:1:0.2-0.5, and the chitosan, the ferritin and the bovine serum albumin are dissolved in phosphate buffer solution to prepare mixed solution with the total mass concentration of 1 percent.
Further, the mass ratio of the silicon dioxide to the polyethylene glycol is 2:0.3-0.6, wherein the silicon dioxide accounts for 2-3% of the mass of the compound.
Further, the heating and heat preservation is to heat the freeze-dried powder to 70-80 ℃ and preserve heat for 48 hours to form the compound.
Further, the concentration of the above compound dissolved in purified water to form a solution is 2-4mg/mL, the pH is adjusted to 5-6, and the solution is heated to 90-100 ℃ and kept for 30min.
Further, the mass ratio of PR to the freeze-dried powder is 1:3-7.
Further, the ultrasonic power of the ultrasonic is 200W, specifically 10s ultrasonic every 10s, and the total duration is 6-10min.
Further, after the above ultrasonic treatment, the pH was adjusted to 4-6, and after standing for 10min, the mixture was centrifuged at 3500rpm for 10min.
The preparation method of the almond peptide oral liquid for inhibiting the activity of alpha-glucosidase is characterized by comprising the following steps:
step 1: dissolving chitosan, ferritin and bovine serum albumin in a mass ratio of 3-5:1:0.2-0.5, adding into phosphate buffer solution with pH of 6 to prepare a mixed solution with total mass concentration of 1%, standing overnight at 4 ℃, then freeze-drying to form freeze-dried powder, preserving heat of the freeze-dried powder at 70-80 ℃ for 48 hours to form a compound, dissolving the compound in purified water to form a solution with concentration of 2-4mg/mL, adding silicon dioxide and polyethylene glycol, adjusting pH to 4-6, heating to 90-100 ℃ to preserve heat for 30min, cooling, adding almond peptide PR, PR and the freeze-dried powder with mass ratio of 1:3-7, carrying out ultrasonic treatment under ice bath condition, specifically ultrasonic power of 200W, ultrasonic for 10s every interval, total time length of 10min, finally adjusting pH to 4-6, centrifuging at 3500rpm for 10min, and freeze-drying to form almond peptide nano particles, wherein the mass ratio of the silicon dioxide and the polyethylene glycol is 2:0.3-0.6, and the mass ratio of the silicon dioxide accounts for 2-3% of the compound;
step 2: the oral liquid is prepared from 1% of almond peptide nano particles, 0.03-0.04% of erythritol, 0.1-0.3% of glycerol, 0.01-0.03% of pectin, 0.02% of citric acid, 0.02% of sodium carboxymethyl cellulose, 0.03% of sodium cyclamate and the balance of purified water.
The invention has the following technical effects:
the embedding rate is improved by embedding the almond peptide PR with the alpha glucosidase activity inhibition, the embedding rate reaches 88.09 percent, the particle size of the prepared almond peptide nano particles is as small as 105.3nm, the prepared almond peptide nano particles are stable in the neutral state of the oral liquid, the alpha glucosidase activity inhibition performance is effectively kept not to be reduced, the stability of the embedded shell is excellent, the phenomenon of precipitation turbidity is avoided after the embedded almond peptide nano particles are purely and purely removed for 24 months, and the stable taste of the oral liquid is ensured. The inhibition rate of the final prepared almond peptide oral liquid on the alpha-glucosidase activity after gastrointestinal tract digestion is stabilized to be more than 64.51 percent.
Drawings
Fig. 1: the almond peptide nanoparticles prepared in example 3 and comparative example 1 mimic the inhibition of alpha-glucosidase activity following gastrointestinal digestion.
Detailed Description
The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will be to those skilled in the art in light of the foregoing disclosure.
The almond peptide PR (proline-arginine) used in the invention is synthesized by the biological technology company of Siananavi of China, and has the structural formula:the purity reaches more than 95 percent.
Experiments prove that the almond peptide PR has excellent effect of inhibiting the activity of alpha-glucosidase, and the specific steps are as follows:
120. Mu.L of phosphate buffer (pH 6.7,0.5M), 20. Mu.L of sample solution (concentration gradient of 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL, 10mg/mL and 12mg/mL in this order), 50. Mu.L of alpha-glucosidase (0.5. Mu.L), 50. Mu.L of substrate 4-nitrobenzene-beta-d-glucopyranoside (PNPG) were used, and after 1 hour of reaction at 37℃in a 96-well plate, na was added 2 CO 3 (0.67M) 50. Mu.L stopped the reaction and the absorbance of the solution was measured at 405 nm. Data from three independent experiments were collected. The inhibition activity was calculated as follows:
wherein Ai is absorbance of a blank portion of the sample replaced with an equal amount of buffer;
AB is the absorbance of the sample;
AC is the absorbance of the control when the sample and PNPG solution were replaced with equal amounts of buffer.
Calculating the in vitro alpha-glucosidase inhibition capability IC of the almond peptide PR according to the formula 50 The value was 19.79.+ -. 0.006. Mu.M.
Example 1
The preparation method of the almond peptide oral liquid for inhibiting the activity of alpha-glucosidase comprises the following steps:
step 1: dissolving chitosan, ferritin and bovine serum albumin in a mass ratio of 3:1:0.2, adding the mixture into phosphate buffer solution at pH of 6 to prepare a mixed solution with a total mass concentration of 1%, standing overnight at 4 ℃, then freeze-drying to form freeze-dried powder, insulating the freeze-dried powder at 80 ℃ for 48 hours to form a compound, dissolving the compound in purified water to form a solution with a concentration of 2mg/mL, adding silicon dioxide and polyethylene glycol, adjusting the pH to 4, heating to 100 ℃ for 30min, cooling, adding almond peptide PR, PR and the freeze-dried powder with a mass ratio of 1:3, performing ultrasonic treatment under ice bath conditions, specifically ultrasonic power of 200W, 10s per interval, total time length of 10min, finally adjusting the pH to 6, centrifuging at 3500rpm for 10min, and freeze-drying to form almond peptide nano-particles, wherein the mass ratio of the silicon dioxide and the polyethylene glycol is 2:0.6, and the silicon dioxide accounts for 2% of the mass of the compound;
step 2: the oral liquid is prepared from 1% of almond peptide nano particles, 0.03% of erythritol, 0.3% of glycerol, 0.01% of pectin, 0.02% of citric acid, 0.02% of sodium carboxymethylcellulose, 0.03% of sodium cyclamate and the balance of purified water.
The oral liquid prepared by the embodiment is stored for 24 months under the normal temperature environment, has no change in properties, is uniform and transparent in color and luster and stable in taste, and does not have any precipitation or layering phenomenon.
Example 2
The preparation method of the almond peptide oral liquid for inhibiting the activity of alpha-glucosidase comprises the following steps:
step 1: dissolving chitosan, ferritin and bovine serum albumin in a mass ratio of 5:1:0.5, adding the mixture into phosphate buffer solution with pH of 6 to prepare a mixed solution with total mass concentration of 1%, standing overnight at 4 ℃, then freeze-drying to form freeze-dried powder, preserving heat of the freeze-dried powder at 70 ℃ for 48 hours to form a compound, dissolving the compound in purified water to form a solution with concentration of 3mg/mL, adding silicon dioxide and polyethylene glycol, adjusting pH to 6, heating to 95 ℃ to preserve heat for 30min, cooling, adding almond peptide PR, PR and the freeze-dried powder with mass ratio of 1:7, carrying out ultrasonic treatment under ice bath conditions, specifically ultrasonic power of 200W, carrying out ultrasonic treatment for 10s at intervals of 10s at the total time of 10min, finally adjusting pH to 4-6, centrifuging at 3500rpm for 10min, and then freeze-drying to form almond peptide nano-particles, wherein the mass ratio of the silicon dioxide and the polyethylene glycol is 2:0.6, and the silicon dioxide accounts for 3% of the mass of the compound;
step 2: the oral liquid is prepared from 1% of almond peptide nano particles, 0.04% of erythritol, 0.3% of glycerol, 0.03% of pectin, 0.02% of citric acid, 0.02% of sodium carboxymethylcellulose, 0.03% of sodium cyclamate and the balance of purified water.
The oral liquid prepared by the embodiment is stored for 24 months under the normal temperature environment, has no change in properties, is uniform and transparent in color and luster and stable in taste, and does not have any precipitation or layering phenomenon.
Example 3
The preparation method of the almond peptide oral liquid for inhibiting the activity of alpha-glucosidase comprises the following steps:
step 1: dissolving chitosan, ferritin and bovine serum albumin in a mass ratio of 4:1:0.3, adding the mixture into phosphate buffer solution with pH of 6 to prepare a mixed solution with a total mass concentration of 1%, standing overnight at 4 ℃, then freeze-drying to form freeze-dried powder, preserving the heat of the freeze-dried powder at 75 ℃ for 48 hours to form a compound, dissolving the compound in purified water to form a solution with a concentration of 4mg/mL, adding silicon dioxide and polyethylene glycol, adjusting the pH to 5.5, heating to 95 ℃ to preserve heat for 30 minutes, cooling, adding almond peptide PR, PR and the freeze-dried powder with a mass ratio of 1:6, carrying out ultrasonic treatment under ice bath conditions, specifically ultrasonic power of 200W for 10 seconds every 10 seconds, adjusting the pH to 5 at last, centrifuging at 3500rpm for 10 minutes, and then freeze-drying to form almond peptide nano-particles, wherein the mass ratio of the silicon dioxide and the polyethylene glycol is 2:0.4, and the silicon dioxide accounts for 2-3% of the mass of the compound;
step 2: the oral liquid is prepared from 1% of almond peptide nano particles, 0.04% of erythritol, 0.2% of glycerol, 0.02% of pectin, 0.02% of citric acid, 0.02% of sodium carboxymethylcellulose, 0.03% of sodium cyclamate and the balance of purified water.
The oral liquid prepared by the embodiment is stored for 24 months under the normal temperature environment, has no change in properties, is uniform and transparent in color and luster and stable in taste, and does not have any precipitation or layering phenomenon.
Comparative example 1:
in this protocol, as compared to example 3, the compound was prepared using bovine serum albumin instead of ferritin, the mass ratio of chitosan to bovine serum albumin was 4:1.3, and the remaining steps were consistent with example 3.
The oral liquid prepared in the embodiment has the characteristics changed when being stored for 12 months under the normal temperature environment, the oral liquid starts to be turbid, and fine precipitates appear on the flat bottom, so that the test is not continued.
Comparative example 2:
in this protocol, in comparison to example 3, where the compound was prepared using ferritin instead of bovine serum albumin, the mass ratio of chitosan to ferritin was 4:1.3, and the remaining steps were consistent with example 3.
The oral liquid prepared in the embodiment has changed properties when stored for 9 months under normal temperature environment, the oral liquid has turbidity and fine sediment on the flat bottom, and the test is not continued.
Comparative example 3:
in this case, the procedure was the same as in example 3 except that the procedure was conducted in example 3 without adjusting pH or freeze-drying after the ultrasonic treatment in step 1.
The oral liquid prepared by the scheme is stored for 12 months under the normal temperature environment, the properties of the oral liquid change, the oral liquid is turbid, fine precipitates appear on the flat bottom, and the test is not continued.
In the preparation process of each scheme, 100 mu L of filtrate obtained by centrifuging after ultrasonic treatment is diluted by 10 times is taken, and an ultraviolet spectrophotometer is used for measuring the embedding rate. The absorbance of the almond peptide at 214nm was maximized by ultraviolet scanning, so that the absorbance was measured at 214nm, and the concentration of the free almond peptide was calculated to obtain the entrapment rate, and the results are shown in table 1.
| Embedding ratio (%) | Nanoparticle particle size (nm) | Absolute potential (mV) | |
| Example 1 | 86±2.09 | 117.4±2.15 | 11.21±0.97 |
| Example 2 | 84±1.73 | 124.9±3.21 | 10.72±1.05 |
| Example 3 | 87±1.94 | 105.3±5.46 | 11.54±1.34 |
| Comparative example 1 | 70±2.12 | 260.4±6.25 | 6.28±2.23 |
| Comparative example 2 | 68±1.85 | 239.5±4.67 | 4.06±1.19 |
| Comparative example 3 | 76±3.34 | 177.1±4.49 | 8.07±1.26 |
The potential reflects the stability of the nano-particles to a certain extent, the positive and negative properties of the nano-particles represent the positive and negative properties of the charged particles, and the larger the absolute value of the potential is, the higher the stability of the nano-particles is, and the better the properties and performance stability of the prepared oral liquid are.
The almond peptide PR without any embedding treatment, the almond peptide nanoparticles prepared in example 3 of the invention and the almond peptide nanoparticles prepared in comparative example 1 were formulated into solutions of the same concentration, and after simulated in vitro gastrointestinal digestion, the change of the product on the alpha-glucosidase inhibitory activity was determined:
the first step: simulating the gastrointestinal tract
Regulating pH value of the almond peptide nanoparticle solution to 2.0 with 1mol/L hydrochloric acid, heating to 37deg.C in water bath, adding pepsin (the addition amount is 5% of the almond peptide nanoparticle content), stirring, and performing enzymolysis at constant temperature for 2 hr. In the process, samples are taken at 0, 0.5, 1.0, 1.5 and 2.0 hours respectively, and are immediately put into a boiling water bath to inactivate enzymes for 10 minutes, and after cooling, the alpha-glucosidase inhibitory activity of the samples after being digested by gastric digestive enzymes is measured.
And a second step of: simulated intestinal digestive tract
After the sample was digested by the gastric digestive tract enzyme system for 2 hours, the pH value of the system was adjusted to 7.0 with a 5mol/L NaOH solution. Heating to 37deg.C, adding trypsin (5% of polypeptide content), stirring, and performing enzymolysis at constant temperature for 2 hr. In the process, samples are taken at 3.0 h, 4.0 h, 5.0 h and 6.0h respectively, the samples are immediately placed in a boiling water bath for enzyme deactivation for 10min, and after cooling, the alpha-glucosidase inhibition activity of the samples after digestion by gastric digestive enzymes is measured.
The inhibitory activity on alpha-glucosidase after the three samples mimic the gastrointestinal digestive enzyme digestion process is shown in figure 1. After three samples respectively enter the stomach, the independent PR enters the intestinal tract after being digested by the stomach tract, the alpha-glucosidase inhibition rate is obviously reduced, and the free PR is digested by intestinal digestive enzymes, so that the absorption rate is reduced, and the alpha-glucosidase inhibition activity is influenced. The entrapped PR is released after the PR nanoparticle solution enters the gastric tract, and the inhibition rate of alpha-glucosidase in the gastric tract is lower than that of PR alone due to the entrapment loss. However, after entering the gastric tract, the embedded shell on the surface of the almond peptide PR plays a role in slow-release protection, and gastric digestive enzymes cannot hydrolyze PR, so that the alpha-glucosidase inhibition rate of the nanoparticle solution in the gastric tract is obviously improved compared with PR. After entering the intestinal tract, the outer shell of the nanoparticle is used as a barrier, PR is still not degraded by intestinal digestive enzymes, so that the alpha-glucosidase inhibition rate is in a gentle state, the alpha-glucosidase inhibition activity in the intestinal tract is obviously enhanced compared with PR, and after the almond peptide nanoparticle prepared in the embodiment 3 is digested in the gastrointestinal tract, the alpha-glucosidase inhibition rate is stabilized to be more than 64.51%. However, as can be seen from comparative example 1, the single bovine serum albumin and chitosan are compounded to form a shell, compared with the shell formed by compounding ferritin and bovine serum albumin and chitosan, the slow release effect is poorer, the dissolution rate of the shell is faster, so that the almond peptide PR is exposed faster, the inhibition tendency of alpha-glucosidase tends to be reduced within 6 hours to 41.19%, and the inhibition rate of alpha-glucosidase of the almond peptide PR which is not subjected to any embedding is reduced to 39.62% after 6 hours.
Claims (9)
1. A first partA preparation method of an almond peptide oral liquid for inhibiting alpha-glucosidase activity is characterized by comprising the following steps: is prepared from almond peptide nanoparticles, erythritol, glycerin, pectin, citric acid, sodium carboxymethyl cellulose, sodium cyclamate and purified water, wherein the almond peptide nanoparticles are prepared from almond peptide PR through embedding treatment, and freeze drying, the embedding treatment comprises the steps of sequentially dissolving bovine serum albumin, ferritin and chitosan in phosphate buffer solution with pH of 6 to prepare mixed solution, standing overnight at 4 ℃, freeze drying to form freeze-dried powder, heating the freeze-dried powder to form a compound, dissolving the compound in the purified water to form solution, adding silicon dioxide and polyethylene glycol, regulating the pH to 5-6, heating again to preserve heat, cooling, adding the almond peptide PR, carrying out ultrasonic treatment under ice bath condition, finally regulating the pH to 4-6, centrifuging, and freeze drying, wherein the structural formula of the almond peptide PR is that。
2. The method for preparing the almond peptide oral liquid according to claim 1, which is characterized in that: according to the mass percentage, the almond peptide nano-particles are 1 percent, erythritol is 0.03-0.04 percent, glycerol is 0.1-0.3 percent, pectin is 0.01-0.03 percent, citric acid is 0.02 percent, sodium carboxymethylcellulose is 0.02 percent, sodium cyclamate is 0.03 percent, and the balance is purified water.
3. The method for preparing the almond peptide oral liquid according to claim 2, which is characterized in that: the mass ratio of the chitosan to the ferritin to the bovine serum albumin is 3-5:1:0.2-0.5, and the chitosan, the ferritin and the bovine serum albumin are dissolved in phosphate buffer solution to prepare mixed solution with the total mass concentration of 1 percent.
4. The method for preparing the almond peptide oral liquid according to claim 3, which is characterized in that: the mass ratio of the silicon dioxide to the polyethylene glycol is 2:0.3-0.6, wherein the silicon dioxide accounts for 2-3% of the mass of the compound.
5. The method for preparing the almond peptide oral liquid according to claim 4, which is characterized in that: the heating and heat preservation is to heat the freeze-dried powder to 70-80 ℃ and preserve heat for 48 hours to form the compound.
6. The method for preparing the almond peptide oral liquid according to claim 5, which is characterized in that: the mass ratio of the almond peptide PR to the freeze-dried powder is 1:3-7.
7. The method for preparing the almond peptide oral liquid according to claim 6, which is characterized in that: the ultrasonic power of the ultrasonic is 200W, specifically 10s of ultrasonic every 10s, and the total duration is 6-10min.
8. The method for preparing the almond peptide oral liquid according to claim 7, which is characterized in that: after the ultrasonic treatment, the pH is adjusted to 4-6, and after standing for 10min, the solution is centrifuged at 3500rpm for 10min.
9. The preparation method of the almond peptide oral liquid is characterized by comprising the following steps:
step 1: dissolving chitosan, ferritin and bovine serum albumin with a mass ratio of 3-5:1:0.2-0.5 in a phosphate buffer solution with a pH of 6 to prepare a mixed solution with a total mass concentration of 1%, standing overnight at 4 ℃, then freeze-drying to form freeze-dried powder, preserving heat for 48 hours at 70-80 ℃ to form a compound, dissolving the compound in purified water to form a solution, adding silicon dioxide and polyethylene glycol, adjusting the pH to 4-6, heating and preserving heat, cooling, and then adding almond peptide PR with a structural formula ofPerforming ultrasonic treatment under ice bath condition with specific ultrasonic power of 200W and ultrasonic power of 10s every 10s, total time length of 10min, regulating pH to 4-6, centrifuging at 3500rpm for 10min, and freeze drying to obtain almond peptide nanoparticle, wherein the mass ratio of silicon dioxide to polyethylene glycol is 2:0.3-0.6, and silicon dioxide accounts for 2-3% of the mass of the compound;
step 2: the oral liquid is prepared from 1% of almond peptide nano particles, 0.03-0.04% of erythritol, 0.1-0.3% of glycerol, 0.01-0.03% of pectin, 0.02% of citric acid, 0.02% of sodium carboxymethyl cellulose, 0.03% of sodium cyclamate and the balance of purified water.
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