CN105713942A - Broccoli polypeptide active ingredient, and preparation method and application thereof - Google Patents
Broccoli polypeptide active ingredient, and preparation method and application thereof Download PDFInfo
- Publication number
- CN105713942A CN105713942A CN201610060177.XA CN201610060177A CN105713942A CN 105713942 A CN105713942 A CN 105713942A CN 201610060177 A CN201610060177 A CN 201610060177A CN 105713942 A CN105713942 A CN 105713942A
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- China
- Prior art keywords
- caulis
- folium brassicae
- brassicae capitatae
- preparation
- active component
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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
- 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
- A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
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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
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Physiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Nutrition Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a broccoli polypeptide active ingredient, and a preparation method and application thereof. The preparation method comprises the following steps of taking broccoli stem and leaf juice; regulating the pH to 8 to 10; stirring the juice for 30 to 90 minutes at the temperature being 30 DEG C to 50 DEG C; then, performing centrifugation, sedimentation, washing and drying to obtain broccoli protein powder; adding water; under the conditions that the temperature is 45 to 60 DEG C and the pH is 4.5 to 7.5, adding alkaline protease for enzymolysis, wherein the enzymolysis time is 4.5 to 10h; performing enzyme deactivation, cooling, centrifugation, rotary steaming and concentration, and then performing freeze drying; obtaining a broccoli polypeptide zymolyte; performing separation by a sephadex column; performing elution by using a phosphate water solution as eluant; collecting the eluant; filtering and drying the eluant through steaming; and performing collection and drying to obtain the broccoli polypeptide active ingredient. The broccoli polypeptide active ingredient has an obvious auxiliary blood fat reducing effect, and can be used for preparing auxiliary blood fat reducing health care products and auxiliary blood fat reducing medicine.
Description
Technical field
The present invention relates to Caulis et Folium Brassicae capitatae field, be specifically related to a kind of Caulis et Folium Brassicae capitatae polypeptide active component and preparation method thereof and the application in preparation auxiliary blood fat reducing health products and auxiliary blood lipid-lowering medicine.
Background technology
Along with the change of the raising of living standards of the people and dietary structure, hyperlipemia sickness rate raises year by year, and in rejuvenation trend.Research data shows, hyperlipemia is the risk factor that the cardiovascular and cerebrovascular diseases such as hypertension, apoplexy, coronary heart disease and myocardial infarction occur, and is also one of risk factor promoting impaired glucose tolerance, diabetes and other important organ pathological changes simultaneously.Though having many chemicalses can reduce human serum cholesterol at present, but human body is often accompanied by obvious side reaction.Therefore, from natural plants, separation and Extraction has bioactive ingredients novel, efficient, low toxicity, for relevant disease prevention and treatment, it has also become the focus that countries in the world the world of medicine pays close attention to and studies in recent years.China's plant food resource is very abundant, Lipid-lowering activities composition effect in disease preventing and treating in further investigation plant, will to promoting that human health has immeasurable meaning.
Caulis et Folium Brassicae capitatae (BrassicaoleraceaL.var.botrytisL) has another name called broccoli, for Cruciferae Brassica genus one biennial plant, it is described as " vegetable Phaleria macrocarpa ", do not contain only protein, polysaccharide, vitamin and carotene, also having abundant Flavonoid substances, Flavonoid substances has the physiological active functionses such as antioxidation, antibacterial, defying age, scavenging free radicals, blood fat reducing, blood pressure lowering, blood sugar lowering, prevention cardiovascular and cerebrovascular disease.
The report of the cholesterol levels of relevant Caulis et Folium Brassicae capitatae reduction at present is more.Broccoli stem-leaf is made feed meal by Hu Caihong et al., become the feed resource that China is novel, with the forage feed hen containing variable concentrations Caulis et Folium Brassicae capitatae stem, hen meat can be made to improve, and can reduce hen chest muscle and liver cholesterol levels (Hu Caihong, Qian Zhongcang, bang duckweed etc. broccoli stem-leaf powder is on the green impact [J] herding fast large-scale hen growth performance and meat. Animal nutrition journal, 2010,22 (1): 194-200).2010, Li Zhilan etc. reported that adding broccoli stem-leaf powder can improve Ovum Anas domestica quality, reduces yolk cholesterol level (Li Zhilan, Zuo Anyan, Hu Caihong. laying ducks is produced the impact of performance and Egg Quality by broccoli stem-leaf powder. herding and veterinary, 2010,42 (9): 37-39).2010, Zheng soldiers etc. report interpolation broccoli stem-leaf powder can improve fairy house triple-yolked egg egg quality, reduce yolk cholesterol level (Zheng soldier, Pan Hongying, Wang Degang, Qian Zhongcang, Zuo Anyan, Hu Caihong, Liu Jianxin. the broccoli stem-leaf powder impact on fairy house triple-yolked egg egg quality, fertility rate and hatchability. Practaculture Science, 2010,27 (3): 148-152).2009, Wang De has just waited and has found, add broccoli stem-leaf powder and can improve fairy house triple-yolked egg egg quality, reduce yolk cholesterol level (Wang Degang, Pan Hongying, Zheng soldier, Hu Caihong, Liu Jianxin. the broccoli stem-leaf powder impact on fairy house SANHUANG performance in layers and Egg Quality. feed industry, 2009,30 (21): 20-24).Finding from the graduate researcher of Milunovich bioscience by studying, edible Caulis et Folium Brassicae capitatae maybe can reduce low-density lipoprotein cholesterol in blood (LDL-Cholesterol) up to 6%.
But relevant Semen Camelliae (cake) first passes through organic solvent method (95% ethanol) and extracts Semen Camelliae (cake) albumen, then acid protease is adopted, neutral protease, alkaline protease, it is hydrolyzed by papain and compound enzyme respectively, in hydrolyzed solution, the content of bioactive peptide is target, Semen Camelliae (cake) protein hydrolyzate obtained can reduce the sickness rate of mortality rate and the hyperlipemia caused by coronary heart disease by reducing serum TC and TG concentration, and (king's layer flies, the preparation of oil tea bioactive peptide and antioxidation thereof, hypolipemic function evaluation study [D]. Changsha: Sino-South African Forestry University of Science and Technology: 2008.).
As far back as 20 beginnings of the century, just someone reports soybean protein and has the effect reducing blood fat and cholesterol, and its hydrolysate soybean polypeptide has this function equally, and effect is more preferably.Famous polypeptide expert Zou Yuandong points out that soybean polypeptide has reduction cholesterol effect, scalable blood viscosity, improves vascular permeability, it is prevented that arterial sclerosis, it is prevented that cerebral thrombosis, it is prevented that apoplexy.Sugano have studied the cholesterol level that the macromolecular gluco (HMF) that microbial protease enzymolysis protein obtains can reduce rapidly in mice serum, may also be combined with bile salts, increasing feces excretion, its neutrality adds 65-95% and 80-170% respectively with the excretion of acidic steroids compared with matched group.Many zooperies and clinical trial is also had to show: the molecule relative mass soybean polypeptide more than 5000Da has the effect significantly reducing cholesterol, and only the people that cholesterol value is high is had effect, on normal person's no impact.Liu Yimei etc. experiments show that with high (15mg/kg d), in (10mg/kg d) dosage soybean protein peptide gavage Mice Body in the concentration of low-density lipoprotein cholesterol reduce, atherogenic index (AI) value substantially reduces, and this illustrates that soybean polypeptide has the effect reducing serum cholesterol levels.
The research of Caulis et Folium Brassicae capitatae peptide separation auxiliary blood fat reducing aspect also seldom has bibliographical information.Caulis et Folium Brassicae capitatae is with Caulis et Folium Brassicae capitatae polypeptide and flavone for main target active component.
Though having many chemicalses can reduce human serum cholesterol at present, but human body is often accompanied by obvious side reaction.In plant food resource, extract hypolipidemic activity functional component report is multiple, but there is not yet the auxiliary significant extract of effect for reducing blood fat, also there are no closing the Caulis et Folium Brassicae capitatae report for auxiliary lipid-lowering function food, health food and medicine aspect.
In order to test and assess the application potential of the auxiliary blood fat reducing aspect of Caulis et Folium Brassicae capitatae polypeptide extract of the present invention, the method that those skilled in the art is familiar with has been used to test the biological activity of Caulis et Folium Brassicae capitatae polypeptide extract.These known method of testings include the lipid metabolism indexs such as TC, TG, HDL；The enzyme index determining experiments etc. such as ALT, AST, GGT, ALP, CYP7A1, ACAT2, CETP.Result shows that the Caulis et Folium Brassicae capitatae polypeptide extract that ad hoc approach of the present invention obtains has and assists effect for reducing blood fat preferably.
Summary of the invention
The invention provides a kind of Caulis et Folium Brassicae capitatae polypeptide active component and preparation method thereof and the application in preparation auxiliary blood fat reducing health products and auxiliary blood lipid-lowering medicine.
In order to realize object above, the first aspect of the invention relates to Caulis et Folium Brassicae capitatae polypeptide active component and preparation method thereof, and it is to prepare by the following method:
The preparation method of a kind of Caulis et Folium Brassicae capitatae polypeptide active component, comprises the following steps:
(1) take broccoli stem-leaf juice, regulate pH to 8～10, in 30 DEG C～50 DEG C temperature, stir 30～90min, then through being centrifuged, precipitate, wash, obtaining Caulis et Folium Brassicae capitatae egg albumen powder after drying；
(2) the Caulis et Folium Brassicae capitatae egg albumen powder that above-mentioned steps (1) prepares is taken, water is added in Caulis et Folium Brassicae capitatae egg albumen powder, under temperature 45～60 DEG C, pH4.5～7.5 condition, the alkaline protease adding Caulis et Folium Brassicae capitatae egg albumen powder mass percent 1.0～5.0% carries out enzymolysis, and the time of enzymolysis is 4.5～10h, enzyme denaturing, cooling, centrifugal, rotary evaporation, concentration postlyophilization, obtains Caulis et Folium Brassicae capitatae polypeptide zymolyte；
(3) take the Caulis et Folium Brassicae capitatae polypeptide zymolyte that above-mentioned steps (2) prepares, adopt sephadex column to separate, with aqueous phosphatic as elution, collect eluent, filter, be evaporated, dry through collecting, obtain Caulis et Folium Brassicae capitatae polypeptide active component.
Following as the preferred technical solution of the present invention:
In step (1), the preparation of described broccoli stem-leaf juice, including: Caulis et Folium Brassicae capitatae stem and leaf are ground into the chip of 0.5-5cm length, and after making beating squeezing, the filter screen crossing aperture 1.0～5.0mm removes, and collects the juice squeezed and is broccoli stem-leaf juice.
Described adjustment pH to 8～10 adopts HCl/water solution and/or NaOH aqueous solution, it is preferred that, the concentration of described HCl/water solution is 0.5～2mol/L.The concentration of described NaOH aqueous solution is 0.5～2mol/L.It is preferred that, the concentration of described HCl/water solution is 1mol/L.The concentration of described NaOH aqueous solution is 1mol/L.
30 DEG C～50 DEG C temperature adopt water-bath to keep.It is preferred that, in 40 DEG C of temperature, stir 60min.
Described centrifugal, precipitation, washing, including: centrifugal 10～20min under 4000～6000r/min, regulate supernatant pH in isoelectric point, IP pI protein precipitation, abandoning supernatant, water washing and precipitating, and regulate pH to 6.5～7.5.It is preferred that, centrifugal 15min under 5000r/min, regulate supernatant pH in isoelectric point, IP pI protein precipitation, abandoning supernatant, water washing and precipitating three times, and regulate pH=7.0.
In step (2), the quality of described Caulis et Folium Brassicae capitatae egg albumen powder and the volume ratio of water are 0.5～2g:20mL.
It is the alkaline protease of Alcalase2.4L that described alkaline protease can specifically adopt letter (China) Bioisystech Co., Ltd of Novi to produce model.
In step (3), the pH of described aqueous phosphatic is 2～6.
The condition of described eluting is: in applied sample amount 100mg, flow velocity 10～20mL/h, it is preferred that, in applied sample amount 100mg, flow velocity 15mL/h.
Second aspect of the present invention relates to described Caulis et Folium Brassicae capitatae polypeptide active component for preparing auxiliary blood fat reducing health products and the purposes of auxiliary blood lipid-lowering medicine.
In order to detect the performance of the auxiliary effect for reducing blood fat of gained of the present invention, the Caulis et Folium Brassicae capitatae polypeptide active component of the auxiliary blood fat reducing of gained of the present invention is configured to 100～400mg/kg bw medicinal liquid, for lipid metabolism indexs such as TC (cholesterol), TG (triglyceride), HDL (HDL-C)；The enzyme index determining experiment etc. such as ALT (serum alanine aminotransferase (ALT) activity), AST (Mitochondria Isoenzyme), GGT (serum γ-glutamyl trans peptidase), ALP (alkali phosphatase), CYP7A1 (cholesterol 7α-hydroxylase), ACAT2 (S-acetyl-coenzyme-A Acetylase 2), CETP (cholesterol ester transfer protein).Result shows that Caulis et Folium Brassicae capitatae polypeptide active component has and assists effect for reducing blood fat significantly, can be used for preparing the functional food of auxiliary lipid-lowering function or health food.
The third aspect of the invention relates to the pharmaceutical composition providing Caulis et Folium Brassicae capitatae auxiliary hypolipidemic activity component with pharmaceutically acceptable adjuvant composition.
The following examples further description to the present invention, set forth below for embodiment be construed as limiting never in any form.
Caulis et Folium Brassicae capitatae polypeptide active component, through repeated multiple times auxiliary effect for reducing blood fat checking, confirms that Caulis et Folium Brassicae capitatae polypeptide active component has and significantly assists effect for reducing blood fat, carry out the determination experiment etc. of the indexs such as TC, TG, HDL-C.Show that Caulis et Folium Brassicae capitatae polypeptide active component has good auxiliary effect for reducing blood fat, there are the potentiality of exploitation.
Compared with prior art, present invention have the advantage that
The extraction process of Caulis et Folium Brassicae capitatae hypolipidemic activity component is optimized by the present invention, it is respectively adopted enzyme process and chromatography assists the active component of blood fat reducing to carry out pretreatment and refining Caulis et Folium Brassicae capitatae with after extracting before extraction, improving the yield of Caulis et Folium Brassicae capitatae auxiliary hypolipidemic activity component further, its technique is simple, yield is high；Equipment is simple, is suitable for industrialized production.
Accompanying drawing explanation
Fig. 1 is that in embodiment 1, SephadexG-25 separates little peptide design sketch.
Detailed description of the invention
The preparation of embodiment 1 Caulis et Folium Brassicae capitatae polypeptide active component
(1) Caulis et Folium Brassicae capitatae stem, leaf, feeder greens pulverizer is ground into the chip of 1-2cm length, pulls an oar with beater, squeezes through pressafiner, and the filter screen crossing aperture 2.0mm removes, and collects the juice that squeezes and is broccoli stem-leaf juice.Take broccoli stem-leaf juice, pH to 9 is regulated with 1M (mol/L) HCl/water solution or 1MNaOH aqueous solution, in 40 DEG C of stirred in water bath 60min, then through 15min centrifugal under 5000r/min, regulate supernatant pH in isoelectric point, IP pI protein precipitation, abandoning supernatant, water washing and precipitating three times, and regulate pH=7.0, dry to obtain Caulis et Folium Brassicae capitatae egg albumen powder.
(2) the Caulis et Folium Brassicae capitatae egg albumen powder that above-mentioned steps (1) prepares is taken, distilled water is added by solid-liquid ratio 1 20, namely the quality of Caulis et Folium Brassicae capitatae egg albumen powder is 1g:20mL with the volume ratio of distilled water, under temperature 50～55 DEG C, pH5.5～6.5 condition, add Aclase alkaline protease (letter (China) Bioisystech Co., Ltd of Novi of Caulis et Folium Brassicae capitatae egg albumen powder mass percent 3.0%, Alcalase2.4L), enzymolysis time is 7h, enzyme denaturing, cooling, centrifugal, rotary evaporation, concentration postlyophilization, obtains Caulis et Folium Brassicae capitatae polypeptide zymolyte standby.
Adopt the degree of hydrolysis DH=19 that pH-stat method records, gel chromatography is utilized to measure the molecular weight distribution of Caulis et Folium Brassicae capitatae polypeptide, record peptide in enzymatic hydrolysate and be mainly distributed on 310～2600Da, distribution is all had in each molecular weight ranges, relative molecular weight distribution is as shown in table 1, under this condition, little peptide content is 54.2%, need to separate purification further.
The mensuration of table 1 relative molecular weight distribution
| Retention time (min) | Relative molecular mass distribution | Percentage ratio (%) |
| 12.767～16.483 | 13906～2644 | 3.79 |
| 16.483～17.817 | 2644～1457 | 12.10 |
| 17.817～18.183 | 1457～1237 | 6.16 |
| 18.183～18.933 | 1237～885 | 18.51 |
| 18.933～19.617 | 885～652 | 27.46 |
| 19.617～20.000 | 652～550 | 12.06 |
| 20.000～20.267 | 550～535 | 7.18 |
| 20.267～21.283 | 535～310 | 12.03 |
| 21.283～23.317 | 310～125 | 0.72 |
(3) the Caulis et Folium Brassicae capitatae polypeptide zymolyte that above-mentioned steps (2) prepares is taken, sephadex column (SephadexG-25) is adopted to separate, with aqueous phosphatic (buffer, pH is 4) as elution, flow velocity 15mL/h, applied sample amount 100mg, the little peptide effect of SephadexG-25 separation is best with this understanding, and the little peptide separation component I of three Caulis et Folium Brassicae capitataes of repeated collection, II and III, SephadexG-25 separates little peptide design sketch as shown in Figure 1, collect eluent, filter, it is evaporated, dry through repeatedly collecting, obtain Caulis et Folium Brassicae capitatae, it is Caulis et Folium Brassicae capitatae polypeptide active component, standby.
(4) take the Caulis et Folium Brassicae capitatae polypeptide active component that above-mentioned steps (3) prepares and carry out the zoopery of auxiliary lipid-lowering function.
The preparation of embodiment 2 Caulis et Folium Brassicae capitatae polypeptide active component
(1) Caulis et Folium Brassicae capitatae stem, leaf, feeder greens pulverizer or hay cutter are ground into the chip of 1-2cm length, pull an oar with beater, squeeze through pressafiner, and the filter screen crossing aperture 1.0mm removes, and collect the juice that squeezes and are broccoli stem-leaf juice.Take broccoli stem-leaf juice, pH to 8 is regulated with 1M (mol/L) HCl/water solution or 1MNaOH aqueous solution, in 30 DEG C of stirred in water bath 80min, then through 10min centrifugal under 6000r/min, regulate supernatant pH in isoelectric point, IP pI protein precipitation, abandoning supernatant, water washing and precipitating three times, and regulate pH=6.5, dry to obtain Caulis et Folium Brassicae capitatae egg albumen powder.
(2) the Caulis et Folium Brassicae capitatae egg albumen powder that above-mentioned steps (1) prepares is taken, distilled water is added by solid-liquid ratio 0.5 20, namely the quality of Caulis et Folium Brassicae capitatae egg albumen powder is 0.5g:20mL with the volume ratio of distilled water, under temperature 45～50 DEG C, pH4.5～5 condition, add Aclase alkaline protease (letter (China) Bioisystech Co., Ltd of Novi of Caulis et Folium Brassicae capitatae egg albumen powder mass percent 5.0%, Alcalase2.4L), enzymolysis time is 4h, enzyme denaturing, cooling, centrifugal, rotary evaporation, concentration postlyophilization, obtains Caulis et Folium Brassicae capitatae polypeptide zymolyte standby.
(3) the Caulis et Folium Brassicae capitatae polypeptide zymolyte that above-mentioned steps (2) prepares is taken, sephadex column (SephadexG-25) is adopted to separate, with aqueous phosphatic (buffer, pH is 3) as elution, flow velocity 20mL/h, applied sample amount 100mg, collects eluent, filters, it is evaporated, dry through repeatedly collecting, obtain Caulis et Folium Brassicae capitatae, be Caulis et Folium Brassicae capitatae polypeptide active component.
The preparation of embodiment 3 Caulis et Folium Brassicae capitatae polypeptide active component
(1) Caulis et Folium Brassicae capitatae stem, leaf, feeder greens pulverizer or hay cutter are ground into the chip of 1-2cm length, pull an oar with beater, squeeze through pressafiner, and the filter screen crossing aperture 4.0mm removes, and collect the juice that squeezes and are broccoli stem-leaf juice.Take broccoli stem-leaf juice, pH to 10 is regulated with 1M (mol/L) HCl/water solution or 1MNaOH aqueous solution, in 50 DEG C of stirred in water bath 40min, then through 20min centrifugal under 4000r/min, regulate supernatant pH in isoelectric point, IP pI protein precipitation, abandoning supernatant, water washing and precipitating three times, and regulate pH=7.5, dry to obtain Caulis et Folium Brassicae capitatae egg albumen powder.
(2) the Caulis et Folium Brassicae capitatae egg albumen powder that above-mentioned steps (1) prepares is taken, distilled water is added by solid-liquid ratio 2 20, namely the quality of Caulis et Folium Brassicae capitatae egg albumen powder is 2g:20mL with the volume ratio of distilled water, under temperature 55～60 DEG C, pH7～7.5 condition, add Aclase alkaline protease (letter (China) Bioisystech Co., Ltd of Novi of Caulis et Folium Brassicae capitatae egg albumen powder mass percent 1.0%, Alcalase2.4L), enzymolysis time is 10h, enzyme denaturing, cooling, centrifugal, rotary evaporation, concentration postlyophilization, obtains Caulis et Folium Brassicae capitatae polypeptide zymolyte standby.
(3) the Caulis et Folium Brassicae capitatae polypeptide zymolyte that above-mentioned steps (2) prepares is taken, sephadex column (SephadexG-25) is adopted to separate, with aqueous phosphatic (buffer, pH is 5) as elution, flow velocity 10mL/h, applied sample amount 100mg, collects eluent, filters, it is evaporated, dry through repeatedly collecting, obtain Caulis et Folium Brassicae capitatae, be Caulis et Folium Brassicae capitatae polypeptide active component.
Embodiment 4 Caulis et Folium Brassicae capitatae polypeptide active component auxiliary lipid-lowering function zoopery
1, experiment material
1.1 test drugs
Caulis et Folium Brassicae capitatae polypeptide active component of the present invention (broccoli stem-leaf is provided by Taizhou Tian Lai Bioisystech Co., Ltd), adopts the Caulis et Folium Brassicae capitatae polypeptide active component of embodiment 1 preparation.
1.2 laboratory animals
Cleaning grade healthy SD rat, body weight 150-200g are selected in experiment, and single male, Zhejiang Academy of Medical Sciences Experimental Animal Center provides, and laboratory animal produces card credit number SCXK (Zhejiang) 2014-0001；Raising in Zhejiang Academy of Medical Sciences Experimental Animal Center SPF level Animal House, experiment condition keeps temperature to be 23 ± 2 DEG C, and relative humidity is 40%-70%, and fight-darkness cycle is 12h 12h, freely drinks water and ingests；
1.3 high lipid foods (weight percentage): normal feedstuff 78.8%, cholesterol 1%, yolk powder 10%, Adeps Sus domestica 10%, cholate 0.2%.
1.4 experimental techniques: feed rat with normal feedstuff, observe 7 days, take tail blood, measure serum total cholesterol (TC), triglyceride (TG), HDL-C (HDL-C) level, animal is randomly divided into high fat matched group, high, medium and low dosage group totally 4 groups, often 10 rats of group, totally 40.After formal experiment starts, each treated animal uses high lipid food instead.Administration group rat gives Caulis et Folium Brassicae capitatae polypeptide active component high dose group 400mg/kg bw, the middle dosage group 200mg/kg bw and low dose group 100mg/kg bw of embodiment 1 preparation, and gavage volume is in 1.0ml/100g bw.High fat matched group gavage gives the normal saline of suitable volume, the continuous gastric infusion 30d of laboratory animal, takes the every blood lipids index of tail hematometry.
1.5 key instruments and reagent
Animal balance, electronic balance, low speed centrifuge, Sysmex full automatic biochemical apparatus (CHEX-180), thermostat water bath, operating theater instruments.Measure TC, TG, HDL-C test kit and build up Bioengineering Research Institute's offer by Nanjing.
1.6 experimental data statistics:
Experiment the data obtained is numerical variable data.All data all carry out variance analysis through SPSS statistical software.Variance uses variance analysis together, and heterogeneity of variance is with being lost and checking.
2, experimental result
2.1 is as shown in table 2 on the impact of rat body weight:
Table 2 Caulis et Folium Brassicae capitatae polypeptide active component on the impact of rat body weight ()
| Group | Number of animals (only) | Original body mass (g) | Body weight in mid-term (g) | Body weight in latter stage (g) |
| High fat matched group | 10 | 165.12±10.17 | 261.33±26.18 | 357.28±29.76 |
| High dose group | 10 | 162.87±8.23 | 274.15±19.66 | 379.22±20.49 |
| Middle dosage group | 10 | 169.35±12.45 | 285.26±20.84 | 363.58±30.47 |
| Low dose group | 10 | 161.39±6.92 | 281.64±30.27 | 383.12±35.81 |
Note: compared with high fat matched group, * P < 0.01, * * P < 0.01
From table 2, in whole experimentation, each treated animal vegetative activity is normal, and each dosage treated animal body weight of experimental drug, compared with high fat matched group, there was no significant difference (P > 0.05).
2.2 impacts on rat fat content:
2.2.1 rat fat contents level before experiment: Analysis of variance, each group rat blood TC, TG, HDL-C content difference not statistically significant (P > 0.05) before experiment, result is in Table 3.
Table 3 each group rat blood TC, TG, HDL-C comparision contents before testing ()
| Group | Number of animals (only) | TC(mmol/L) | TG(mmol/L) | HDL-C(mmol/L) |
| High fat matched group | 10 | 0.72±0.19 | 0.89±0.52 | 1.34±0.53 |
| High dose group | 10 | 0.61±0.21 | 0.92±0.47 | 1.17±0.39 |
| Middle dosage group | 10 | 0.76±0.24 | 0.79±0.56 | 1.25±0.42 |
| Low dose group | 10 | 0.69±0.11 | 0.90±0.42 | 1.29±0.48 |
Note: compared with high fat matched group, * P < 0.01, * * P < 0.01
2.2.2 rat fat contents level after experiment: compared with high fat matched group, the TC content of dosage group high, middle is all remarkably decreased (P < 0.01)；The TG content of dosage group high, middle is all remarkably decreased (P < 0.01)；High dose group HDL-C content significance rises (P < 0.05), in Table 4.
After table 4 administration each organize rat blood TC, TG, HDL-C content comparison ()
| Group | Number of animals (only) | TC(mmol/L) | TG(mmol/L) | HDL-C(mmol/L) |
| High fat matched group | 10 | 1.72±0.23 | 3.26±0.62 | 1.59±0.84 |
| High dose group | 10 | 0.81±0.14** | 1.37±0.37** | 2.97±1.35* |
| Middle dosage group | 10 | 1.13±0.24** | 1.63±0.46** | 2.26±1.42 |
| Low dose group | 10 | 1.61±0.54 | 2.90±0.32 | 1.79±1.28 |
Note: compared with high fat matched group, * P < 0.01, * * P < 0.01
3, experiment conclusion:
Originally experiments show that, per os gives the Caulis et Folium Brassicae capitatae polypeptide active component of rat various dose, dosage group high, middle can significantly reduce rat blood TC and TG contents level (P < 0.01), high dose group can significantly improve HDL-C contents level (P < 0.05) simultaneously, compared with high fat matched group, all statistically significant.It is shown that Caulis et Folium Brassicae capitatae polypeptide active component has significantly assists effect for reducing blood fat.
The preparation of embodiment 5 Caulis et Folium Brassicae capitatae tablets of reducing blood fat pressed candy
Caulis et Folium Brassicae capitatae tablets of reducing blood fat pressed candy, adopts the raw material of following weight percentage: 20% Caulis et Folium Brassicae capitatae polypeptide active component, 10% Monas cuspurpureus Went, 55% pregelatinized Starch, 10% hypromellose, 5% calcium hydrogen phosphate；By the prescription of science, adopt existing technique to prepare, obtain Caulis et Folium Brassicae capitatae tablets of reducing blood fat pressed candy.
The model adopting embodiment 4 is tested, it was shown that Caulis et Folium Brassicae capitatae enhancing immunity pressed candy has significantly assists effect for reducing blood fat.
The preparation of embodiment 6 Caulis et Folium Brassicae capitatae lipid-lowering health food
Caulis et Folium Brassicae capitatae lipid-lowering health food, adopts the raw material of following weight content: Caulis et Folium Brassicae capitatae polypeptide active component 325.5g, extract of Radix Ginseng stem and leaf 14g, microcrystalline Cellulose 10.5g, 350g gram altogether, makes 1000,350mg/ grain.
The model adopting embodiment 4 is tested, it was shown that Caulis et Folium Brassicae capitatae enhancing immunity pressed candy has significantly assists effect for reducing blood fat.
Claims (9)
1. the preparation method of a Caulis et Folium Brassicae capitatae polypeptide active component, it is characterised in that comprise the following steps:
(1) take broccoli stem-leaf juice, regulate pH to 8～10, in 30 DEG C～50 DEG C temperature, stir 30～90min, then through being centrifuged, precipitate, wash, obtaining Caulis et Folium Brassicae capitatae egg albumen powder after drying；
(2) the Caulis et Folium Brassicae capitatae egg albumen powder that above-mentioned steps (1) prepares is taken, water is added in Caulis et Folium Brassicae capitatae egg albumen powder, under temperature 45～60 DEG C, pH4.5～7.5 condition, the alkaline protease adding Caulis et Folium Brassicae capitatae egg albumen powder mass percent 1.0～5.0% carries out enzymolysis, and the time of enzymolysis is 4.5～10h, enzyme denaturing, cooling, centrifugal, rotary evaporation, concentration postlyophilization, obtains Caulis et Folium Brassicae capitatae polypeptide zymolyte；
(3) take the Caulis et Folium Brassicae capitatae polypeptide zymolyte that above-mentioned steps (2) prepares, adopt sephadex column to separate, with aqueous phosphatic as elution, collect eluent, filter, be evaporated, dry through collecting, obtain Caulis et Folium Brassicae capitatae polypeptide active component.
2. the preparation method of Caulis et Folium Brassicae capitatae polypeptide active component according to claim 1, it is characterized in that, in step (1), the preparation of described broccoli stem-leaf juice, including: Caulis et Folium Brassicae capitatae stem and leaf are ground into the chip of 0.5～5cm length, after making beating squeezing, the filter screen crossing aperture 1.0～5.0mm removes, and collects the juice squeezed and is broccoli stem-leaf juice.
3. the preparation method of Caulis et Folium Brassicae capitatae polypeptide active component according to claim 1, it is characterized in that, in step (1), described centrifugal, precipitation, washing, including: centrifugal 10～20min under 4000～6000r/min, regulate supernatant pH in isoelectric point precipitates albumen, abandoning supernatant, water washing and precipitating, and regulate pH to 6.5～7.5.
4. the preparation method of Caulis et Folium Brassicae capitatae polypeptide active component according to claim 1, it is characterised in that in step (2), the quality of described Caulis et Folium Brassicae capitatae egg albumen powder and the volume ratio of water are 0.5～2g:20mL.
5. the preparation method of Caulis et Folium Brassicae capitatae polypeptide active component according to claim 1, it is characterized in that, in step (2), it is the alkaline protease of Alcalase2.4L that described alkaline protease specifically adopts letter Bioisystech Co., Ltd of Novi to produce model.
6. the preparation method of Caulis et Folium Brassicae capitatae polypeptide active component according to claim 1, it is characterised in that in step (3), the pH of described aqueous phosphatic is 2～6.
7. the preparation method of Caulis et Folium Brassicae capitatae polypeptide active component according to claim 1, it is characterised in that in step (3), the condition of described eluting is: flow velocity 10～20mL/h.
8. the Caulis et Folium Brassicae capitatae polypeptide active component that prepared by the preparation method according to any one of claim 1～7.
9. the Caulis et Folium Brassicae capitatae polypeptide active component according to claim 8 application in preparation auxiliary blood fat reducing health products and auxiliary blood lipid-lowering medicine.
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| CN109517035A (en) * | 2018-11-29 | 2019-03-26 | 宁波大学 | A kind of ace inhibitory peptide of broccoli albumen source, ace inhibitory peptide digestion metabolite and its preparation method and application |
| CN111011589A (en) * | 2019-12-30 | 2020-04-17 | 中国农业科学院饲料研究所 | Method for hydrolyzing forage broccoli leaf protein and hydrolysate thereof |
| CN111280443A (en) * | 2020-03-24 | 2020-06-16 | 浙江劢康生物科技有限公司 | Health food with functions of regulating fat and losing weight and preparation method thereof |
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| CN109517035A (en) * | 2018-11-29 | 2019-03-26 | 宁波大学 | A kind of ace inhibitory peptide of broccoli albumen source, ace inhibitory peptide digestion metabolite and its preparation method and application |
| CN109517035B (en) * | 2018-11-29 | 2021-10-19 | 宁波大学 | ACE inhibitory peptide derived from broccoli protein, ACE inhibitory peptide enzyme digestion metabolite, and preparation method and application thereof |
| CN111011589A (en) * | 2019-12-30 | 2020-04-17 | 中国农业科学院饲料研究所 | Method for hydrolyzing forage broccoli leaf protein and hydrolysate thereof |
| CN111280443A (en) * | 2020-03-24 | 2020-06-16 | 浙江劢康生物科技有限公司 | Health food with functions of regulating fat and losing weight and preparation method thereof |
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