CN105541967A - Leech small peptide chelate with coagulation preventing and blood activating effect and preparation method thereof - Google Patents
Leech small peptide chelate with coagulation preventing and blood activating effect and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
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- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a leech small peptide chelate with a coagulation preventing and blood activating effect and a preparation method thereof, and belongs to the field of biotechnology. The preparation method is characterized in that with leeches as a starting material, enzymolysis, ultrafiltration, resin column purification, a chelation reaction and drying are carried out to obtain the leech small peptide chelate, and preparation accessories can be added to prepare the leech small peptide chelate into a corresponding preparation through a conventional method. The leech small peptide chelate has a strong coagulation preventing and blood activating effect and has the advantages of being free of toxic and side effects and drug misadventures, low in cost and the like.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of small-peptide chelated thing of leech and preparation method thereof with the anti-freezing effect of invigorating blood circulation.
Background technology
Leech is the drying entirety of Hirudinidae animal whitmania WhitmaniapigraWhitman, leech HirudonipponicaWhitman, willow leaf leech WhitmaniaacranulataWhitman, is a kind of traditional Chinese medicine.Leech begins to be loaded in Shennong's Herbal: " (leech) is main by extravesated blood, hemostasis, and the moon closes, and broken blood lump in the abdomen is gathered, s.m.p, dredging water passages." Compendium of Material Medica: " salty flavor acting on blood, bitter victory blood.The salty hardship of leech, with except storing blood, is Liver Channel blood system medicine, therefore can lead to Liver Channel and gather blood." traditional Chinese medical science thinks that leech has broken blood, by the stasis of blood, effect of stimulating the menstrual flow, is mainly used in treatment lump in the abdomen disease, lump in the abdomen, blood stasis, amenorrhoea and wound.Clinical middle leech has been widely used in the diseases such as coronary heart diseases and angina pectoris, Acute Myocardial Infarction, ischemia apoplexy (as cerebral thrombosis, cerebral infarction and sequela thereof), hematencephalon, the sclerosis of artery congee and peripheral angiopathy.But directly take after the leech mode of being used as medicine adopting usually crude drug pulverize, or take after water extraction or alcohol extracting or water extract-alcohol precipitation, its DNA extration tradition taked, medicinal material utilization ratio is lower, and active substance there is no sufficient utilization.Through modern study, the definite curative effect of HIRULOG constituents becomes the chief active position of leech, peptide constituents exists with the form of albumen in leech body, after human oral, the stomach en-of albumen in gi tract and trypsin digestion become micromolecular peptide, absorbed into serum plays curative effect again, but albumen in vivo enzymolysis become the efficiency of small-molecular peptides extremely low, most directly got rid of external, cause a large amount of wastes of medicinal material, curative effect reduces greatly.Application enzymolysis process hydrolyzed animal protein, obtains the focus that the biologically active peptides with certain physiologically active is research both at home and abroad at present.
The little peptide of leech is the small-molecule peptide using biotechnology and zymolysis technique to extract from leech, its relative molecular mass at below 3KD, easy Transdermal absorption, solvability and digestive utilization ratio high.The preliminary test of pesticide effectiveness confirms, this project leech bionic enzymatic active small peptide efficient part has efficient anticoagulation, short fibrinolytic, confirm through the rat cerebral ischemia model test of pesticide effectiveness, leech bionic enzymatic active small peptide effectively can reduce brain water content, obviously alleviate the neurobehavioral symptom of rat neurocyte is had to provide protection, obviously suppresses cerebral ischemia scope; Toxic reaction is there is not in pilot acute toxicity test with maximum administration.Illustrate that leech bionic enzymatic active small peptide not only increases curative effect, and eliminate the immunogen reaction of taking animal M-band and causing, reduce adverse reaction rate, security is high.
Trace element has four effects in vivo: 1. as launch vehicle; 2. composition or the metabolic process of enzyme is participated in; 3. participate in hormone form and play a role; 4. heredity and variation is affected.Stroke patient hair assay is pointed out, and trace element chromium, selenium obviously reduce; In cerebral infarction patient, micro-Fe content raises.Think after deliberation, scarce chromium can cause sugar, metabolism of fat change, produces adverse influence to the cerebrovascular; Scarce selenium can make prostaglandin I in body
2reduction, thromboxane A
2raise, thus accelerate vascular lesion; Manganese increases, and causes vascellum tunica interna incrassation, impels thrombosis.In addition, cerebral infarction patient, ratio and cadmium, aluminium, the rubidium of Copper in Serum, copper/zinc all raise, and zinc declines.And find that serum copper, copper/zinc ratio raises more obvious in acute phase, and cerebral infarction number of times is more, rises higher.Think after deliberation this with brain tissue impairment, downright bad relevant.Zinc participates in tissue repair, so content reduces.Therefore, determination of trace elements, as an auxiliary characteristics, has the certain significance to the understanding of the generation of apoplexy, development and prognosis.People also can supplement or control the object that some trace element reaches preventing apoplectic.
Trace element participates in composition and the Metabolic activity of living matter, wherein much participates in composition and the Metabolic activity of the koinomatter of cardiovascular and cerebrovascular.Polyphenoils is human body " street cleaner ", very important to the protection of cardiovascular and cerebrovascular.Wherein, the most important thing is superoxide-dismutase (SOD).The SOD participation competence exertion usefulness only had through trace element zinc, copper, manganese alive.Another kind of antioxidase one gsh proglumide enzyme, then after must being activated by trace element-selenium, with non-enzyme system as vitamins C, vitamin-E etc., could form anti-oxidant defence line together, remove the active oxygen radical in human body.
The synthesis of oxyphorase etc. participated in by iron, is distributed in red corpuscle, muscle, is responsible for transport and the exchange of oxygen and carbon dioxide.Iron can not cause anaemia completely, heart burden not only can be caused to increase, simultaneously due to the undersupply of oxygen, also directly have influence on the health of the heart, brain.If but iron too much also can cause a disease, especially bend I premature coronary heart disease.Finland's scholar's newly release achievement in research, serum ferritin often increases by 1%, and the danger of coronary heart disease will be made to increase by 4%, and serum ferritin has exceeded certain level, the incidence of coronary heart disease is the twice of normal people.Researchist also finds, easily suffers stroke when in the blood of people, iron level is too high.Iron can be combined with other chemical substances, forms oxidative pressure, thus damages cell.The mass storage of iron can increase the peroxidation of brain cell, cerebral microvascular, thus causes the generation of apoplexy.In general, the iron that the iron in food can not cause body accumulation too much, people are in fact only just passable otherwise abuse chalybeate.Certainly, under the condition of health license, strive for donating blood wise beyond doubt.
Copper can impel inorganic iron to become Organic Iron, promotes that iron enters people's bone, accelerates the synthesis of oxyphorase, and also contribute to the synthesis of s0D in histocyte etc., the cells such as the protection heart, liver, brain, marrow, from the evil of free radical.According to World Health Organization's document announcement, the whole world dies from first that cardiopathic people accounts for dead total population every year, and French deaths from heart disease rate is second from the bottom.Think after US Experts research, Frenchman's deaths from heart disease rate is low may be enriched relevant with copper content in their meals.If human body lacks copper for a long time, various physiological activity can be influenced, and cause obvious jf pupil cholesterol, blood fat and uric acid to raise, easily cause coronary atherosclerotic heart disease, severe patient also myocardial infarction can occur.The World Health Organization, according to the demand of human body to copper, recommends c to take the photograph people's 2 ~ 5 milligrams of copper everyone every day.Grownup takes the photograph copper amount every day and is often less than 2 milligrams, and heart function obstacle can occur.The a survey data of the U.S. shows, the U.S. has people's copper of 2/3 to take the photograph people's amount not reach this standard, and have further downward trend over nearly 30 years, heart disease rate of the same period adds 44%.In the age that current purified diet is in fashion, the mankind take the photograph copper amount and greatly decline.
Zinc is the important composition composition of human body more than 80 kind of enzyme.Zinc can improve immune function of human body, resists infection better, and thinks that the morbidity of some cardiovascular diseases is relevant with infection now.During scarce zinc, vitamin A normally can not play physiological action, and eyesight and dark adatpation are affected.The pancreas zinc content of diabetic subject only has the half of normal people, and diabetes are cardiovascular and cerebrovascular diseases " partners ".
The small-peptide chelated thing of leech further can not only improve effect of little peptide, can also improve the specific absorption of the trace element such as copper, zinc, make human body take in two class materials (little peptide and trace element), and the collaborative effect playing anti-freezing and invigorate blood circulation.
Summary of the invention
The first object of the present invention is to provide a kind of small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation, and the second object of the present invention is to provide the application of the small-peptide chelated thing of this leech in the protective foods of invigorating blood circulation for the preparation of anti-freezing, medicine and makeup.
The object of the invention first object is achieved in that
There is the small-peptide chelated thing of leech of the anti-freezing effect of invigorating blood circulation, it is characterized in that adopting following processing step to prepare:
(1) preparation of the little peptide of leech: leech was pulverized 80 mesh sieves, add 8-12 times of water gaging, heated and boiled 15-30 minute, wait to be chilled to about 35 ~ 45 DEG C, adjust ph 1.0 ~ 3.0, add 0.5 ~ 2% stomach en-, be incubated 1 ~ 3 hour, adjust ph 7.0 ~ 9.0, add 0.5 ~ 2% trypsinase, be incubated 3 ~ 5 hours, boil 10 ~ 15 minutes, centrifugal, supernatant liquor is the ultra-filtration membrane ultrafiltration of 3KD by relative molecular mass, permeate is added on DA201-C macroporous adsorptive resins, with water elution, discard water lotion, again with 60 ~ 90% ethanol elutions, collect ethanol eluate, decompression recycling ethanol also continues to be concentrated into the clear cream that 60 DEG C of relative densities are 1.10 ~ 1.15, (inlet temperature is 180 ~ 200 DEG C to spraying dry, air outlet temperature is 90 ~ 100 DEG C), obtain the little peptide extract of leech, detect through LC-MS, containing Thr-Leu-His-Glu-Arg-Trp-Asn-His-Thr-Val-Pro, Asn-Cys-Arg-Gly-Asn-Lys-Cys-Ser, Val-Asp-His-Glu-Arg-Leu-Leu-Gln-His, Arg-Val-Glu-Asp-Thr-Lys tetra-kinds of little peptides in the little peptide extract of leech, its total content is 50% ~ 90%.
The relative molecular mass of table 1 leech little peptide small molecular peptide and sequence composition table
(2) huge legendary turtle closes reaction: by copper sulfate (copper gluconate, or Cuprocitrol, or zinc chloride, or Zinc Gluconate, or zinc lactate) being dissolved in distilled water, to be made into concentration be 10 ~ 15mg/ml salts solution, with dilute hydrochloric acid adjust ph 2 ~ 4, the little peptide extract of leech is added in salts solution and leech little peptide extract ratio 8 ~ 10:1 ratio, stirring and dissolving, under temperature is 50 ~ 60 DEG C of conditions, chelatropic reaction 60 ~ 90 minutes, with nanofiltration membrane (retaining relative molecular mass is 150 ~ 300Da) nanofiltration desalination, obtains the small-peptide chelated thing solution of leech.
(3) spraying dry obtains inner complex: it is 1.10 ~ 1.15 that small-peptide chelated for leech thing solution decompression is concentrated into 60 DEG C of relative densities, and spraying dry (inlet temperature is 180 ~ 200 DEG C, and air outlet temperature is 90 ~ 100 DEG C), obtains the small-peptide chelated thing of leech.
Through the small-peptide chelated thing of leech prepared by aforesaid method, for the preparation of the following preparation of one-tenth:
(1) by the small-peptide chelated thing of leech: Semen Maydis oil: beeswax: lecithin is 10:36:3:1 mixture, through colloidal mill grinding, soft capsule is prepared into;
(2) in the small-peptide chelated thing of leech: poly(oxyethylene glycol) 400: propylene glycol ratio is 10:38:2 mixture, through colloidal mill grinding, soft capsule is prepared into;
(3) the small-peptide chelated thing of leech, adds customary adjuvant, is prepared into oral liquid, capsule, tablet, granule;
(4) the small-peptide chelated thing of leech, add water, propylene glycol dissolve, using face paper infiltrate as cosmetic mask.
But the invention is not restricted to above-mentioned formulation.Moulding process conventional dose auxiliary material used can replace, as assistant formation pharmaceutical excipients such as Icing Sugar, honey, Xylo-Mucines with other kin auxiliary material; Also can share with other medicinal substances, jointly reach the effect that anti-freezing is invigorated blood circulation.
The second object of the present invention is to provide the purposes of the small-peptide chelated thing of above-mentioned leech, and it has the effect that anti-freezing is invigorated blood circulation, and it can apply in the protective foods of invigorating blood circulation for the preparation of anti-freezing, medicine and makeup.
The invention has the beneficial effects as follows: the small-peptide chelated thing of leech of the present invention, prepare easy, cost is low; The little peptide of leech and trace element work in coordination with the effect playing anti-freezing and invigorate blood circulation, have simultaneously toxic side effect little, have no drug resistance, the not easily advantage such as residual, reach the requirement of green food pure natural, can good economic benefit be obtained.
The test of pesticide effectiveness
For verifying the effect that the anti-freezing of the small-peptide chelated thing of leech of the present invention is invigorated blood circulation further, inventors performed the test of pesticide effectiveness investigate, test method and result as follows:
1. on the impact of clotting time of mice
(1) test medicine
The little peptide copper chelate of leech, self-control, containing the little peptide of leech (with total phthaleinometer) 81.5%, containing chromium 1.7%.Sterile purified water is adopted to be made into respective concentration liquid for gavage before use.
The little peptide chelates of zinc of leech, self-control, containing the little peptide of leech (with total phthaleinometer) 81.5%, containing zinc 1.7%.Sterile purified water is adopted to be made into respective concentration liquid for gavage before use.
The little peptide of leech, self-control, containing the little peptide of leech (with total phthaleinometer) 83.2%.Sterile purified water is adopted to be made into respective concentration liquid for gavage before use.
Copper gluconate, food grade, copper content is 14.1%.Sterile purified water is adopted to be made into respective concentration liquid for gavage before use.
Zinc Gluconate, food grade, Zn content is 14.3%.Sterile purified water is adopted to be made into respective concentration liquid for gavage before use.
(2) animal and raising
1. animal species, source
ICR mouse, SPF level, male and female half and half, body weight 18-22g, provides purchased from by Shandong University's experimental animal center, animal conformity certification number: SCXK(Shandong) 20130004.
2. rearing conditions
Mouse feeder in independent air-feeding cage (IVC), air purity 10000 grades, laboratory temperature 24 ± 2 DEG C; Relative humidity 60% ~ 80%; Air inerchange number of times per hour: 10-15 time/hour; Periodicity of illumination: 12(day)/12(night) hour.Male and female are separately raised, and every cage is no more than 5.
Feed: mouse full-valence pellet feed, works in coordination with medical bioengineering limited liability company purchased from Jiangsu Province, and its quality meets GB14924.1-2001 " laboratory animal mixed feed general-quality criteria ".
Bedding and padding: sterilizing particle bedding and padding, work in coordination with medical bioengineering limited liability company purchased from Jiangsu Province.
Drinking-water: drink purified water, freely drink after acidifying.
(3) test method and result
Dosage arranges and selects 18-22 gram of ICR mouse 60 with grouping, male and female half and half, 6 groups are divided at random by body weight, often organize 10: leech little peptide copper chelate group (30mg/kg), leech little peptide chelates of zinc group (30mg/kg), the little peptide group (30mg/kg) of leech, copper gluconate group (5.0mg/kg), Zinc Gluconate group (5.0mg/kg), blank group, gastric infusion every day each trial-product 0.2m1/20g body weight.Blank group to same volume tap water, continuous 7 days.
Test method respectively organizes fasting 12h before the administration of mouse last, after last administration 1h, plucks side eyeball, in the drop of blood of each 1 diameter 5mm in slide glass two ends, record from blood sampling to provoking the trace of blood, institute's elapsed-time standards is the clotting time, records the clotting time of each mouse.
Each group of test-results the results are shown in Table 2 to the impact of clotting time of mice.
Table 2 is on the impact (N=10, X ± s) of clotting time of mice
#p<0.05,
##p<0.01, compares with blank group.
Above experimental result shows, compared with control group, leech little peptide copper chelate group, leech little peptide chelates of zinc group be energy pole significant prolongation clotting time of mice (P<0.01) all; Leech little peptide group energy significant prolongation clotting time of mice (P<0.05); Chromium picolinate group, Zinc Gluconate group there was no significant difference (P > 0.05).
2. on the impact of Mouse Whole Brain ischemic
(1) test medicine
The same
(2) animal and raising
The same
(3) test method and result
Dosage is arranged and grouping ICR mouse 60, male and female half and half, body weight 18-22g, be divided into 6 groups at random, often organize 10, be respectively: leech little peptide copper chelate group (30mg/kg), leech little peptide chelates of zinc group (30mg/kg), the little peptide group (30mg/kg) of leech, copper gluconate group (5.0mg/kg), Zinc Gluconate group (5.0mg/kg), blank group.
Test method mouse is pressed such scheme grouping and prevents administration 7 days continuously, and after last 1 administration 30min, break end after ear under waking state, 5min gets brain, prepares the brain homogenate of 10% with ice-cold physiological saline.Mda (MDA) and serum lactic dehydrogenase (LDH) level in cerebral tissue is measured by test kit specification sheets.
Each group of test-results the results are shown in Table 3 to the impact of LDH and MDA level in Mouse Whole Brain Neurons Against Cerebral Ischemia tissue.
Table 3 is on the impact (N=10, X ± s) of LDH and MDA level in Mouse Whole Brain Neurons Against Cerebral Ischemia tissue
#p<0.05,
##p<0.01, compares with control group.
Above experimental result shows, compared with control group, leech little peptide copper chelate group, leech little peptide chelates of zinc group all extremely significantly can reduce LDH level (P<0.01) in Mouse Whole Brain Neurons Against Cerebral Ischemia tissue, also obviously can reduce MDA level (P<0.01) in cerebral tissue simultaneously; The little peptide group of leech significantly can reduce LDH level (P<0.05) in Mouse Whole Brain Neurons Against Cerebral Ischemia tissue, also obviously can reduce MDA level (P<0.05) in cerebral tissue simultaneously; Chromium picolinate group, Zinc Gluconate group there was no significant difference (P > 0.05).
Embodiment
Enumerate embodiment below, further illustrate the present invention, each embodiment, only for illustration of the present invention, does not limit the present invention:
Embodiment 1
Leech was pulverized 80 mesh sieves, add 12 times amount water boils, wait to be chilled to about 40 DEG C, adjust ph 2.0, add 1% stomach en-, be incubated 1 hour, adjust ph 8.0, add 1% trypsinase, be incubated 3 hours, boil 15 minutes, centrifugal, supernatant liquor is the ultra-filtration membrane ultrafiltration of 3KD by relative molecular mass, permeate is added on DA201-C macroporous adsorptive resins, with water elution, discard water lotion, again with 90% ethanol elution, collect ethanol eluate, decompression recycling ethanol also continues to be concentrated into the clear cream that 60 DEG C of relative densities are 1.10 ~ 1.15, (inlet temperature is 180 ~ 200 DEG C to spraying dry, air outlet temperature is 90 ~ 100 DEG C), obtain the little peptide extract of leech.
Copper gluconate being dissolved in distilled water, to be made into concentration be 15mg/ml salts solution, with dilute hydrochloric acid adjust ph 3, the little peptide extract of leech is added in salts solution and leech little peptide extract ratio 8:1 ratio, stirring and dissolving, under temperature is 60 DEG C of conditions, chelatropic reaction 90 minutes, with retaining the nanofiltration membrane nanofiltration desalination that relative molecular mass is 150 ~ 300Da, obtain the small-peptide chelated thing solution of leech, being evaporated to 60 DEG C of relative densities is 1.10 ~ 1.15, (inlet temperature is 180 ~ 200 DEG C to spraying dry, air outlet temperature is 90 ~ 100 DEG C), obtain the small-peptide chelated thing of leech.
Embodiment 2
Leech was pulverized 80 mesh sieves, add 10 times amount water boils, wait to be chilled to about 45 DEG C, adjust ph 1.5, add 2% stomach en-, be incubated 2 hours, adjust ph 9.0, add 2% trypsinase, be incubated 4 hours, boil 15 minutes, centrifugal, supernatant liquor is the ultra-filtration membrane ultrafiltration of 3KD by relative molecular mass, permeate is added on DA201-C macroporous adsorptive resins, with water elution, discard water lotion, again with 75% ethanol elution, collect ethanol eluate, decompression recycling ethanol also continues to be concentrated into the clear cream that 60 DEG C of relative densities are 1.10 ~ 1.15, (inlet temperature is 180 ~ 200 DEG C to spraying dry, air outlet temperature is 90 ~ 100 DEG C), obtain the little peptide extract of leech.
Zinc Gluconate being dissolved in distilled water, to be made into concentration be 10mg/ml salts solution, with dilute hydrochloric acid adjust ph 4, the little peptide extract of leech is added in salts solution and leech little peptide extract ratio 10:1 ratio, stirring and dissolving, under temperature is 60 DEG C of conditions, chelatropic reaction 60 minutes, with retaining the nanofiltration membrane nanofiltration desalination that relative molecular mass is 150 ~ 300Da, obtain the small-peptide chelated thing solution of leech, being evaporated to 60 DEG C of relative densities is 1.10 ~ 1.15, (inlet temperature is 180 ~ 200 DEG C to spraying dry, air outlet temperature is 90 ~ 100 DEG C), obtain the small-peptide chelated thing of leech.
Embodiment 3
The small-peptide chelated thing 100g of leech of Example 1 method gained, by the small-peptide chelated thing of leech: Semen Maydis oil: beeswax: lecithin is 10:36:3:1 mixture, 30 minutes are ground through colloidal mill, with gelatin, glycerine, purified water, iron oxide black makes soft capsule hide glue gelatin in 100:40:100:1 ratio, control glue box temperature 55 ± 5 DEG C, heat insulation tank 40 ± 5 DEG C, sprinkler body temperature 36 ± 5 DEG C, with pellet press compacting soft capsule, the tolerant heavy 0.50g of every intragranular, 18 DEG C ~ 26 DEG C cold air drying 4h(relative humidity are 30% ~ 40%) sizing, wash ball, dry, select ball, packaging, be prepared into soft capsule finished product.
Embodiment 4
The small-peptide chelated thing 100g of leech of Example 1 method gained, in the small-peptide chelated thing of leech: poly(oxyethylene glycol) 400: propylene glycol ratio is 10:38:2 mixture, grind through colloidal mill, with gelatin, glycerine, purified water, iron oxide black makes soft capsule hide glue gelatin in 100:40:100:1 ratio, control glue box temperature 55 ± 5 DEG C, heat insulation tank 40 ± 5 DEG C, sprinkler body temperature 36 ± 5 DEG C, with pellet press compacting soft capsule, the tolerant heavy 0.50g of every intragranular, 18 DEG C ~ 26 DEG C cold air drying 4h(relative humidity are 30% ~ 40%) sizing, wash ball, dry, select ball, packaging, be prepared into soft capsule finished product.
Embodiment 5
The small-peptide chelated thing 200g of leech of Example 2 method gained, adds starch to total amount 250g, and granulation, dry, whole grain, load capsule, packaging, is prepared into capsule finished product.
Embodiment 6
The small-peptide chelated thing 50g of leech of Example 2 method gained, adds water, propylene glycol is dissolved to 1000ml, infiltrates as cosmetic mask using face paper.
Claims (9)
1. there is the small-peptide chelated thing of leech of the anti-freezing effect of invigorating blood circulation, it is characterized in that adopting following processing step to prepare:
(1) preparation of the little peptide of leech: leech was pulverized 80 mesh sieves, add 8-12 times of water gaging, heated and boiled 15-30 minute, wait to be chilled to about 35 ~ 45 DEG C, adjust ph 1.0 ~ 3.0, add 0.5 ~ 2% stomach en-, be incubated 1 ~ 3 hour, adjust ph 7.0 ~ 9.0, add 0.5 ~ 2% trypsinase, be incubated 3 ~ 5 hours, boil 10 ~ 15 minutes, centrifugal, supernatant liquor is the ultra-filtration membrane ultrafiltration of 3KD by relative molecular mass, permeate is added on DA201-C macroporous adsorptive resins, with water elution, discard water lotion, again with 60 ~ 90% ethanol elutions, collect ethanol eluate, decompression recycling ethanol also continues to be concentrated into the clear cream that 60 DEG C of relative densities are 1.10 ~ 1.15, spraying dry, obtain the little peptide extract of leech,
(2) huge legendary turtle closes reaction: copper sulfate is dissolved in distilled water and is made into salts solution, with dilute hydrochloric acid adjust ph 2 ~ 4, add the little peptide extract of above-mentioned leech, stirring and dissolving, under temperature is 50 ~ 60 DEG C of conditions, chelatropic reaction 60 ~ 90 minutes, with nanofiltration membrane nanofiltration desalination, obtains the small-peptide chelated thing solution of leech;
(3) spraying dry obtains inner complex: it is 1.10 ~ 1.15 that small-peptide chelated for leech thing solution decompression is concentrated into 60 DEG C of relative densities, and spraying dry obtains the small-peptide chelated thing of leech.
2. the small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation according to claim 1, it is characterized in that in the little peptide extract of step (1) described leech containing Val-Asp-His-Glu-Pro-Gly-Asn-His, Arg-Val-Glu-Asp-Thr-Lys, Val-Asp-His-Glu-Arg-Leu-Leu-Gln-His, Thr-Leu-His-Glu-Arg-Trp-Asn-His-Thr-Val-Pro, Lys-Leu-Tyr-Pro-Ala-Pro-Tyr-Met-Thr-Val-Thr, Val-Gly-Ser-Ala-Gly-Ala-Gly-Val-Thr, Cys-Cys-Gly-Phe-Val-Gln-Trp-Cys-Pro-Lys, Asn-Cys-Arg-Gly-Asn-Lys-Cys-Ser eight kinds of little peptides, its total content is 50% ~ 90%.
3. the small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation according to claim 1, is characterized in that step (2) described copper sulfate can use copper gluconate, Cuprocitrol, zinc chloride, Zinc Gluconate, zinc lactate to replace.
4. the small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation according to claim 1, is characterized in that the concentration of step (2) described salts solution is 10 ~ 15mg/ml.
5. the small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation according to claim 1, is characterized in that step (2) described salts solution and the little peptide extract ratio of silkworm chrysalis are 8 ~ 10:1.
6. the small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation according to claim 1, is characterized in that the relative molecular mass that retains of step (2) described nanofiltration membrane is 150 ~ 300Da.
7. the small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation according to claim 1, it is characterized in that step (1) and (3) described spraying dry inlet temperature are 180 ~ 200 DEG C, air outlet temperature is 90 ~ 100 DEG C.
8. the small-peptide chelated thing of leech with the anti-freezing effect of invigorating blood circulation according to claim 1, is characterized in that being prepared into following preparation:
(1) by the small-peptide chelated thing of leech: Semen Maydis oil: beeswax: lecithin is 10:36:3:1 mixture, through colloidal mill grinding, soft capsule is prepared into;
(2) in the small-peptide chelated thing of leech: poly(oxyethylene glycol) 400: propylene glycol ratio is 10:38:2 mixture, through colloidal mill grinding, soft capsule is prepared into;
(3) the small-peptide chelated thing of leech, adds customary adjuvant, is prepared into oral liquid, capsule, tablet, granule;
(4) the small-peptide chelated thing of leech, add water, propylene glycol dissolve, using face paper infiltrate as cosmetic mask.
9. the small-peptide chelated thing of leech according to claim 1 is invigorated blood circulation for anti-freezing.
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| CN201610021721.XA CN105541967A (en) | 2016-01-14 | 2016-01-14 | Leech small peptide chelate with coagulation preventing and blood activating effect and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106035983A (en) * | 2016-06-24 | 2016-10-26 | 河南广安生物科技股份有限公司 | Preparation method of feed additive nano small peptide chelated copper |
| CN106173185A (en) * | 2016-07-15 | 2016-12-07 | 安徽精准医疗产业研究院有限公司 | A kind of preparation method of the little peptide of Pupa bombycis |
| CN107279458A (en) * | 2017-07-13 | 2017-10-24 | 安徽生物肽产业研究院有限公司 | It is a kind of that there is zinc-rich pea small peptide of enhancing immunity of organisms and preparation method thereof |
| CN110066318A (en) * | 2019-05-13 | 2019-07-30 | 山东大学 | A kind of hirudin and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1483340A (en) * | 2002-09-17 | 2004-03-24 | 祝光伟 | Protein single fodder containing microelement chelate and preparation process thereof |
-
2016
- 2016-01-14 CN CN201610021721.XA patent/CN105541967A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1483340A (en) * | 2002-09-17 | 2004-03-24 | 祝光伟 | Protein single fodder containing microelement chelate and preparation process thereof |
Non-Patent Citations (2)
| Title |
|---|
| 王艳等: "水蛭提取方法及生理活性的研究", 《中国生化药物杂志》 * |
| 苏纯阳等: "微量元素氨基酸（小肽）螯合物的研究应用进展", 《饲料工业》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106035983A (en) * | 2016-06-24 | 2016-10-26 | 河南广安生物科技股份有限公司 | Preparation method of feed additive nano small peptide chelated copper |
| CN106035983B (en) * | 2016-06-24 | 2019-08-20 | 河南广安生物科技股份有限公司 | A kind of preparation method of the small-peptide chelated copper of feed addictive nanometer |
| CN106173185A (en) * | 2016-07-15 | 2016-12-07 | 安徽精准医疗产业研究院有限公司 | A kind of preparation method of the little peptide of Pupa bombycis |
| CN107279458A (en) * | 2017-07-13 | 2017-10-24 | 安徽生物肽产业研究院有限公司 | It is a kind of that there is zinc-rich pea small peptide of enhancing immunity of organisms and preparation method thereof |
| CN110066318A (en) * | 2019-05-13 | 2019-07-30 | 山东大学 | A kind of hirudin and its application |
| CN110066318B (en) * | 2019-05-13 | 2022-02-15 | 山东大学 | Leech peptide and application thereof |
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Application publication date: 20160504 |