CN111454323A - Preparation method and application of rosa roxburghii tratt natural polypeptide - Google Patents

Preparation method and application of rosa roxburghii tratt natural polypeptide Download PDF

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CN111454323A
CN111454323A CN201910051097.1A CN201910051097A CN111454323A CN 111454323 A CN111454323 A CN 111454323A CN 201910051097 A CN201910051097 A CN 201910051097A CN 111454323 A CN111454323 A CN 111454323A
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rosa roxburghii
roxburghii tratt
natural
polypeptide
natural polypeptide
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菅晓勇
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Beijing Zhongke Weixin Biomedical Research Institute Co ltd
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Beijing Zhongke Weixin Biomedical Research Institute Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography

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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Nutrition Science (AREA)
  • Mycology (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention provides a preparation method of natural polypeptide of rosa roxburghii tratt, which comprises the following steps: cleaning and cutting the rosa roxburghii tratt into pieces, homogenizing at low temperature, standing for 12 hours at low temperature, separating and purifying the centrifuged supernatant extracting solution through gel chromatography, collecting the part with the molecular weight of 500 plus 12000 daltons, namely the rosa roxburghii tratt natural polypeptide, and carrying out vacuum freeze drying on the collected liquid of the part of the natural polypeptide to obtain the rosa roxburghii tratt natural polypeptide powder. The golden roxburgh rose natural polypeptide prepared by the preparation method provided by the invention has lower molecular weight, retains higher natural activity, and can be used for preparing health-care food and food.

Description

Preparation method and application of rosa roxburghii tratt natural polypeptide
Technical Field
The invention belongs to the field of natural product extraction, and particularly relates to a rosa roxburghii tratt natural polypeptide, and a preparation method and application thereof.
Background
The Rosa roxburghii is named as smooth branch Rosa roxburghii, etc. The rosa roxburghii tratt is not only a fruit, but also a medicine, is suitable for both fresh eating and processing, is rich in SOD (superoxide dismutase), carotene, vitamin E, vitamin C, more than 20 amino acids and more than 10 trace elements beneficial to human bodies, has the effects of removing freckles, beautifying, resisting aging, helping sleep, preventing and resisting cancers, expelling toxin and removing toxicity, discharging lead and manganese, resisting radiation, resisting fatigue, enhancing immunity, invigorating stomach, promoting digestion, nourishing and strengthening body and the like, is a 'nutritional precious fruit' for preventing diseases and preserving health, and is beneficial to 'a new mountain precious' and 'a life-prolonging fruit'. Because of the limitation of strong seasonality, great preservation difficulty and other factors, the golden roxburgh rose is processed into products such as roxburgh rose juice, roxburgh rose fruit wine, roxburgh rose cake and the like after being dried at high temperature, but the golden roxburgh rose processed by the products at high temperature loses the original color and luster and nutritive value.
Peptides are classified into oligopeptides and polypeptides, which are bioactive substances in which two or more amino acids are condensed and linked by peptide bonds. The peptides are characterized by high biological activity and diversity compared with other substances in organisms. In the organism, many bioactive substances exist in the form of the molecular structure and the natural conformation of the polypeptide, and play an important role in the organism in the mode of the natural bioactivity. Polypeptides play a crucial role in the growth and development and metabolism of organisms. The active biological polypeptide can regulate and control the vital activities of cells to ensure normal cell division and normal protein synthesis, thereby playing an important role in cell division, differentiation and repair. Therefore, the active biological polypeptide is closely related to human health, anti-aging, life prolonging and the like and has great significance. Recent scientific studies have demonstrated that the major forms of protein absorbed by the human body are not all amino acids, but active biological peptides, and thus can condition health, fight disease and aging by supplementing active biological polypeptides.
At present, a plurality of methods for extracting and preparing the polypeptide comprise an acid hydrolysis method, an alkali hydrolysis method, an enzyme hydrolysis method, a combination of a plurality of methods and the like. However, extraction of polypeptides that retain their native activity, i.e., trying to separate impurities from the extract without altering the structure and properties of these compounds, requires milder extraction conditions. If the selected solution system is too acidic or alkaline, the extracted substance may be irreversibly denatured or excessively hydrolyzed and the molecular conformation may be significantly changed, so that the biological activity and function of the polypeptide are gradually lost. Furthermore, in order to prevent the hydrolysis of the protease added in the enzymatic method after the extraction of the polypeptide is completed, it is generally necessary to boil for inactivating the enzyme, which may cause a decrease or destruction of the biological activity of the polypeptide to some extent.
Importantly, the polypeptide is obtained by decomposing protein in the methods, the operation steps are complicated on the whole, more media are introduced, and the biological activity of the obtained polypeptide has more problems. The method provides a simple and convenient polypeptide preparation method capable of directly extracting natural polypeptide from biological tissues, the method has mild extraction conditions, physiological conditions are adopted as separation conditions, and the obtained polypeptide can keep the natural activity of the polypeptide.
Disclosure of Invention
The invention provides a simple polypeptide preparation method for directly extracting natural polypeptide from biological tissues and retaining the natural activity of the polypeptide, which at least solves the problems of complex technology of a polypeptide extraction method and natural activity retention in the prior art.
The invention provides a preparation method of natural polypeptide of rosa roxburghii tratt, which is characterized by comprising the following steps:
(1) cleaning fresh rosa roxburghii tratt, dicing, homogenizing at 2-8 ℃ in ice bath, adding 0.02-0.2 mol/l (mol/L) of phosphate buffer solution with pH of 6.0-7.2 which is 1-3 times of the weight of the precooled rosa roxburghii tratt, soaking in a refrigerator at 2-8 ℃ for 12 hours, freezing and centrifuging at 2-8 ℃, and obtaining supernatant which is crude extract of natural polypeptides containing rosa roxburghii tratt;
(2) adding eluent into the crude extract containing the natural polypeptide of the rosa roxburghii tratt obtained in the step (1) to dilute the crude extract into a solution containing 30-100 mg/ml (mg/m L) of the polypeptide, separating the solution by using sephadex chromatography, and collecting the component with the molecular weight of 500-12000 dalton (D);
(3) and (3) freezing and drying the golden roxburgh rose natural polypeptide collecting liquid in the step (2) to obtain the golden roxburgh rose natural polypeptide extract freeze-dried powder.
Preferably, the concentration of the phosphate buffer is 0.05 mol/L, the pH of the phosphate buffer is 7.0, and the dosage of the phosphate buffer is 2 times of that of the rosa roxburghii tratt.
Preferably, in the step (2), the crude extract of natural polypeptides of sweet potato leaves collected in the step (1) is diluted by an eluent to a solution containing 50mg/m L of polypeptides.
Preferably, the separation conditions of the Sephadex G-25 gel chromatography column, the inner diameter of 0.5-2 cm (cm), the column length of 30-120cm, the pH7.0 phosphate buffer solution with 0.05 mol/L eluent, the sodium chloride solution containing 0.15 mol/L, the flow rate of 0.4-7 ml/min (m L/min), the sample amount of 0.5-1m L and the detection wavelength of 280nm are adopted as the separation conditions of the Sephadex G-25 gel chromatography column.
The Rosa roxburghii natural polypeptide extract obtained by the preparation method retains high natural biological activity, and can be used for preparing health food and food.
The preparation method of the rosa roxburghii tratt natural polypeptide provided by the invention is simple to operate, can directly extract natural active polypeptide from biological tissues, has mild extraction conditions, adopts physiological conditions for separation, and can keep the natural biological activity of the obtained polypeptide. The natural active polypeptide of rosa roxburghii tratt prepared by the method provided by the invention has lower osmotic pressure than free amino acid, is easier to absorb, and does not cause adverse reactions such as diarrhea and the like after eating; meanwhile, the natural bioactive peptide of rosa roxburghii tratt has lower antigenicity and cannot cause anaphylactic reaction; the active polypeptide can participate in the synthesis of in vivo protein in human body, promote mitosis and proliferation differentiation of related cells, and enhance metabolism of organism tissues and cells, so that the active polypeptide has the effects of regulating and controlling cell metabolism at molecular level and repairing abnormal damaged cells. In addition, the natural active polypeptide also has the potential functions of reducing blood pressure, blood fat, oxidation resistance, virus resistance, bacteria resistance, cholesterol, mineral substance absorption, substance metabolism improvement, body immunity enhancement and the like, so that the rosa roxburghii tratt natural active polypeptide can effectively regulate and control the health of a human body on a cellular level, prevent degenerative diseases and cardiovascular and cerebrovascular diseases, improve the immunity, delay aging and the like.
Drawings
FIG. 1 gel chromatogram of native polypeptide of Rosa roxburghii Tratt, i.e. the graph of example 1.
FIG. 2 is a schematic diagram showing the effect of natural polypeptides of Rosa roxburghii Tratt on the maintenance of mouse swimming time under load.
The following detailed description is intended to further illustrate but not limit the invention, the following example being only one preferred embodiment of the invention.
Detailed Description
Example 1 a method for preparing a rosa roxburghii tratt natural active polypeptide extract, comprising the following steps:
(1) taking rosa roxburghii tratt, dicing, homogenizing at 4 ℃ in ice bath, then adding 0.05 mol/L pH7.0 phosphate buffer solution which is 2 times of the precooled rosa roxburghii tratt by weight, soaking, placing in a refrigerator at 4 ℃ for 12 hours, freezing and centrifuging at 4 ℃, and obtaining supernatant which is crude extract containing rosa roxburghii tratt natural polypeptide;
(2) slowly adding eluent into the crude extract containing the gold roxburgh rose polypeptide obtained in the step (1) to dilute the crude extract into a solution containing 50mg/m L of the polypeptide, separating the solution by Sephadex G-25 (the inner diameter is 1cm, the column length is 30cm, the eluent is 0.05 mol/L, phosphate buffer solution with the pH value of 7.0 and 0.15 mol/L sodium chloride solution at the flow rate of 2m L/min, the sample injection amount is 1m L and the detection wavelength is 280nm), and collecting components with the molecular weight of 500-12000D (the peak between 8ml and 17ml in figure 1 is the target polypeptide to be collected);
(3) and (3) freezing and drying the natural polypeptide collecting solution of the rosa roxburghii tratt in the step (2) to obtain the rosa roxburghii tratt active polypeptide extract freeze-dried powder.
Embodiment 2 a method for preparing a rosa roxburghii tratt natural active polypeptide extract, comprising the following steps:
(1) taking rosa roxburghii tratt, dicing, homogenizing at 4 ℃ in ice bath, then adding a phosphate buffer solution with the weight of 3 times of that of the precooled rosa roxburghii tratt and the pH value of 0.2 mol/L being 6.0 for soaking, placing the rosa roxburghii tratt in a refrigerator with the temperature of 2-8 ℃ for 12 hours, freezing and centrifuging at the temperature of 2-8 ℃, and obtaining a supernatant, namely a crude extract containing natural polypeptides of rosa roxburghii tratt;
(2) slowly adding eluent into the crude extract containing the gold roxburgh rose polypeptide obtained in the step (1) to dilute the crude extract into a solution containing the polypeptide of 100mg/m L, separating the solution by Sephadex G-25 (the inner diameter is 1cm, the column length is 30cm, the eluent is 0.05 mol/L, phosphate buffer solution with the pH value of 7.0 and sodium chloride solution containing 0.15 mol/L at the flow rate of 1m L/min, the sample injection amount is 1m L and the detection wavelength is 280nm), and collecting components with the molecular weight of 500-;
(3) and (3) freezing and drying the natural polypeptide collecting solution of the rosa roxburghii tratt in the step (2) to obtain the rosa roxburghii tratt active polypeptide extract freeze-dried powder.
Embodiment 3 a method for preparing a rosa roxburghii tratt natural active polypeptide extract, comprising the following steps:
(1) taking rosa roxburghii tratt, dicing, homogenizing at 2-8 ℃ in ice bath, then adding 0.02 mol/L pH7.2 phosphate buffer solution with the weight 1 time that of the precooled rosa roxburghii tratt to soak, placing in a refrigerator with the temperature of 2-8 ℃ for 12 hours, freezing and centrifuging at the temperature of 2-8 ℃, and obtaining supernatant, namely the crude extract containing the rosa roxburghii tratt natural polypeptide;
(2) slowly adding eluent into the crude extract containing the gold roxburgh rose polypeptide obtained in the step (1) to dilute the crude extract into a solution containing the polypeptide of 30mg/m L, separating by Sephadex G-25 (inner diameter is 1cm, column length is 30cm, eluent is 0.05 mol/L, phosphate buffer solution with pH of 7.0 and sodium chloride solution of 0.15 mol/L at the flow rate of 0.8m L/min, sample introduction amount is 1m L and detection wavelength is 280nm), and collecting components with the molecular weight of 500-12000D;
(3) and (3) freezing and drying the natural polypeptide collecting solution of the rosa roxburghii tratt in the step (2) to obtain the rosa roxburghii tratt active polypeptide extract freeze-dried powder.
The natural active polypeptide extract of rosa roxburghii tratt prepared in the above example 1 is used for verifying the improvement of the body movement capacity: the mice are divided into a control group (filled with physiological saline), an experiment 1 group (filled with golden roxburgh rose natural active polypeptide 5mg/kg every day), an experiment 2 group (filled with golden roxburgh rose natural active polypeptide 10mg/kg every day) and an experiment 3 group (filled with golden roxburgh rose natural active polypeptide 15mg/kg every day). Each group of animals was kept for 30 days with 10 animals. The animals in each group were respectively bound with lead wire (calculated by 5% of the weight) at their tails, and the weight-bearing swimming test was performed to observe the time that the animals in each group swim without drowning, and the results are shown in fig. 2, where the experimental group and the control group have significant differences (P < 0.001). As shown in figure 2, compared with the control group, the mice taking the natural active polypeptide of rosa roxburghii tratt have longer time for swimming, and the larger the dosage, the longer the time for swimming, which proves that the natural active polypeptide of rosa roxburghii tratt can effectively improve the anti-fatigue capability of the body.

Claims (4)

1. A preparation method of natural polypeptide of rosa roxburghii tratt is characterized by comprising the following steps:
(1) cleaning fresh rosa roxburghii tratt, dicing, homogenizing at 2-8 ℃ in ice bath, adding 0.02-0.2 mol/L of 0.0-7.2 phosphate buffer solution with the pH value of 6.0-7.2 which is 1-3 times of the weight of the precooled rosa roxburghii tratt, soaking in a refrigerator with the temperature of 2-8 ℃ for 12 hours, and then freezing and centrifuging at 2-8 ℃, wherein the supernatant is crude extract containing natural polypeptides of rosa roxburghii tratt;
(2) adding an eluent into the crude extract containing the natural polypeptide of the rosa roxburghii tratt obtained in the step (1) to dilute the crude extract into a solution containing 30-100 mg/ml of polypeptide, separating the solution by using sephadex chromatography, and collecting components with the molecular weight of 500-;
(3) and (3) carrying out vacuum freeze drying on the golden roxburgh rose natural polypeptide collecting solution in the step (2) to obtain the golden roxburgh rose natural polypeptide extract freeze-dried powder.
2. The method according to claim 1, wherein the concentration of the phosphate buffer solution in step (1) is 0.05 mol/liter, the pH of the phosphate buffer solution is 7.0, and the amount of the phosphate buffer solution is 2 times that of the Rosa roxburghii.
3. The method of claim 1, wherein: in the step (2), the crude extract of the natural polypeptides of the rosa roxburghii tratt collected in the step (1) is diluted by eluent to be a solution containing 50 mg/ml of polypeptides; the separation conditions of the sephadex chromatography are as follows: the gel chromatography column was Sephadex G-25, and the eluent was 0.05 mol/l of pH7.0 phosphate buffer containing 0.15 mol/l of sodium chloride solution.
4. The Rosa roxburghii natural polypeptide extract obtained by the preparation method of any one of claims 1-3 has high natural activity, and can be used for preparing health food and food.
CN201910051097.1A 2019-01-21 2019-01-21 Preparation method and application of rosa roxburghii tratt natural polypeptide Pending CN111454323A (en)

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Application publication date: 20200728