CN105267247A - Application of bee pollen extract in preparation of blood pressure-lowering or liver-protecting medicines and health-care products - Google Patents
Application of bee pollen extract in preparation of blood pressure-lowering or liver-protecting medicines and health-care products Download PDFInfo
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Abstract
The invention relates to a bee pollen extract, specifically relates to an application of the bee pollen extract in preparation of blood pressure-lowering and liver-protecting medicines and health-care products; the bee pollen extract as an active ingredient has the blood pressure-lowering and liver-protecting functions, can be made into various oral and externally-applied medicines or injections, and has a good application prospect in the fields of medicines and health-care products.
Description
Technical field
The present invention relates to bee pollen extract, bee pollen extract prepared by the present invention has blood pressure lowering and protects the liver function.This technology belongs to biological technical field.
Background technology
Bee pollen is the pollen grain of honey collection flowering plant, secretions, saliva and nectar on the gland adding Mel self in gatherer process and formed.Bee pollen is one of Major Nutrient source of bee colony growth and procreation, has very high nutritive value.Bee pollen comprises the multiple nutritional components such as protein, carbohydrate, flavone compound, trace element, enzyme, and therefore bee pollen usually just has good reputations such as " natural miniature nutrition libraries ".Bioactivity research about bee pollen shows, bee pollen has effects such as improving cardiovascular disease, enhancing immunity, blood sugar lowering, antioxidation, defying age, suppression prostatosis.
Document 1 (Chinese patent 200610052876.6) discloses by alcohol extraction broker wall bee pollen active component, and this active component has the function of blood fat reducing.Document 2 (Chinese patent 200810134816.8) discloses and extracts bee pollen protein and prepare peptide with enzymatic isolation method, and bee pollen peptide has angiotensin transferase to be suppressed.Document 3 (Chinese patent 201010295791.7) discloses from Brassica campestris L pollen, to obtain a kind of method of extract with antiinflammatory, analgesia, bacteriostatic activity.Document 4 (Chinese patent 201010169477.4) discloses a kind of method extracting oil compounds from bee pollen.Document 5 (Chinese patent 201110364321.6) discloses a kind of method extracting coenzyme Q10 from bee pollen.Document 6 (Chinese patent 200410073514.6) discloses the method that preparation from Pollen Brassicae campestris has the effective site for the treatment of tumor of prostate.The open effective site extracting Brassica campestris L pollen by different organic solvents of document 7 (Chinese patent CN200810007853.2), effective site is at the novelty teabag of preventing alcoholic liver injury.Although the method extracted about bee pollen activity component at present has a lot, existing method is all for target product with certain one matter in bee pollen.Although extract the component that obtains there is good activity, in leaching process, but to have lost in bee pollen other active component, make bee pollen only become the raw material of certain composition, and the feature in bee pollen " multipotential nutrition storehouse " cannot be embodied.Crude bee pollen is larger as reaching desirable effect required dosage when using when functional food or medicine.
Hypertension is the common cardiovascular diseases that China's sickness rate is very high, is the significant risk factor causing the various complication such as the heart, brain, kidney and Ocular Vessels and cause apoplexy, promotion atherosclerosis, coronary heart disease.Hypertension does not generally have malaise symptoms in early days, until there is clinical picture-heart attacks, rupture of blood vessel in brain, therefore has the early stage health care of family history of hypertension and light symptoms person just most important.
The initiation position of chemical liver injury is plasma membrane, and then under the induction of Cytochrome P450, hepatotoxicant matter produces active oxygen (ROS) and a series of mesostate, cause biomembranous lipid peroxidation, cell membrane integrity is destroyed, organelle function is impaired, finally cause hepatocellular apoptosis or necrosis, cause liver dysfunction.
Summary of the invention
The object of the invention is to be that bee pollen extract prepared by raw material with bee pollen, blood pressure lowering should be had concurrently and the function protected the liver by thing in advance, and can be applicable in medicine or functional food.
For achieving the above object, the technical solution used in the present invention is as follows:
Concrete grammar: dry bee pollen is according to raw material: the ratio of solvent=1kg:5 ~ 50L adds solvent, 20-70 DEG C is extracted 1 ~ 10 hour, and extraction terminates rear filtration or centrifugal separation with solid content by extracting solution processes respectively:
(1) processing method of extracting solution: extracting solution is through vacuum concentration, concentrating sample macroporous resin column upper after volumetric concentration 40-70% dissolve with ethanol, first use the deionized water drip washing of 2 ~ 10 times of column volumes, and then wash pillar with volumetric concentration 40 ~ 90% ethanol and collect flow point, the flow point vacuum concentration containing ethanol elution agent eluting is obtained component 1.
(2) solid content processing method: according to raw material: the ratio of solution=1kg:2 ~ 10L adds water or buffer mixing, pH adjusts mixed liquor in scope 2 ~ 10, add the proteolytic enzyme of 0.001 ~ 3% of raw materials quality again, in 20 ~ 60 DEG C of enzymolysis 1 ~ 48 hour; After enzymolysis terminates, temperature of reaction system is risen to 80-100 DEG C and keep 10-60 minute, make enzyme deactivation, then centrifugal or filtration, collect filtrate concentrate drying and obtain component 2.
Component 1 is mixed again with component 2, obtains bee pollen extract.
Dry pollen of the present invention extracts solvent used: the solvent mixture of one or two or more kinds in water, methanol, ethanol, ethyl acetate, dichloromethane, chloroform, cyclohexane extraction, normal hexane, petroleum ether
Proteolytic enzyme used in solid content enzymolysis of the present invention is: proteolytic enzyme is one or two or more kinds in pepsin, trypsin, flavor protease, bromelain, papain, alkaline protease or neutral protease.
Buffer of the present invention is: the one in phosphate buffer, Tris-HCI buffer, acetate buffer solution, deionized water.
Medicine containing extract of the present invention, functional food, food can be prepared into any form, such as peroral dosage form: powder, tablet, capsule, soft capsule, aqueous pharmaceutical, syrup, tincture, pill, powder, parcel, granule; Topical formulations: emulsifiable paste, ointment, emulsion.Gel.Semi-solid cream, patch paste.Spray.Aerosol etc.; Injection: solution, outstanding agent, Emulsion.
Feature of the present invention is as follows:
1. bee pollen has long edible history as miniature nutrition library in China, the extraction of the flavone compound in bee pollen separates with the preparation of biologically active peptide and carries out by the present invention, not only concentratedly remain not only two kinds of main components simultaneously but also increase new active function by the method for enzymolysis, improve the bioavailability of bee pollen.
2. the effect that by the bee pollen extract prepared by the present invention, there is blood pressure lowering simultaneously and protect the liver.
3. have a good application prospect.The function that the bee pollen extract prepared by the present invention is had blood pressure lowering and protects the liver, likely becomes medicine or health functional food, therefore has a good application prospect.
Detailed description of the invention
Embodiment 1
One, bee pollen extract preparation method
Take Pollen Brassicae campestris as raw material, prepare bee pollen extract according to following technique:
Get dry bee pollen 1000 grams, add volumetric concentration 70% ethanol 5L, 20 DEG C are extracted 10 hours, extract to terminate rear filtration and be separated with solid content by extracting solution and process respectively:
(1) processing method of extracting solution: extracting solution is dry through vacuum concentration, concentrating sample macroporous resin column upper after 20 ml volumes concentration 70% dissolve with ethanols, first use the deionized water drip washing of 5 times of column volumes, and then wash pillar with 3 times of column volume volumetric concentration 70% ethanol and collect flow point, the component vacuum concentration drying of being washed by 70% ethanol obtains component 1.
(2) solid content processing method: solid content is according to raw material: the ratio of solution=1kg:10L adds water, it is 2 that 1MHCI adjusts pH of mixed, then add raw materials quality 0.2% pepsin, in 40 DEG C of enzymolysis 48 hours; After enzymolysis terminates, temperature of reaction system is risen to 90 DEG C to keep 20 minutes, make enzyme deactivation, then centrifugal or filtration, collect filtrate concentrate drying and obtain component 2.
Component 1 is mixed again with component 2, obtains Brassica campestris L pollen extract.
Two, the antihypertensive function evaluation of bee pollen:
(1) antihypertensive function evaluation
1. experimental section
1.1 laboratory animal
Spontaneous hypertensive rat SHR (Shanghai Slac Experimental Animal Co., Ltd.), body weight 200 ~ 220g; Normal arterial pressure SD rat (Dalian Medical Univ SPF animal experimental center), body weight 200 ~ 250g.
1.2 assay methods and instrument
The mensuration of blood pressure, heart rate adopts method of indirectly measuring blood pressure-tail pulses method.Arteria caudalis blood pressure measuring instrument (Alcott bio tech ltd, Shanghai).
1.3 experimental technique
All rats divide cage to feed, and 4, every cage, freely drinks water, freely take food.For experiment after one week situation without exception of feeding.Random packet, often organizes 8.Hypertensive Rats is grouped into: blank group, positive controls, bee pollen extract group.Altace Ramipril Captopril is as positive control.Dosage is respectively: Captopril medicine 2mg/kg, bee pollen extract 600mg/kg.
Experimental rat is according to grouping dosage gastric infusion, and every day is administered once, successive administration 4 weeks,
Table 1 bee pollen extract is on the impact of original hypertensive rat blood pressure
Measuring blood pressure and pulse are once weekly.
1.4 date processing and result judge
Test group of animals blood pressure is starkly lower than matched group, and difference has significance, and on the blood pressure of test group of animals heart rate and intact animal and heart rate without impact, can judge that auxiliary function for lowering blood pressure results of animal is positive.
2. experimental result
Table 1 is the impact of bee pollen extract on original hypertensive rat blood pressure, visible from the 7th day dosage be the bee pollen extract of 600mg/kg, diastolic pressure and the systolic pressure of SHR rat all obviously decline, having significant difference (P<0.05) compared with blank group, is that the positive group of contrast also had significant difference with blank group from the 7th day with captopril.Illustrate that bee pollen extract has the function of blood pressure lowering to original hypertensive rat.
Table 1 bee pollen extract affects (Continued) to original hypertensive rat blood pressure
# has significant difference (P<0.05) compared with blank group
(2) bee pollen liver-protecting function is evaluated
1. experimental section
1.1 experiment material
60, SPF level Kunming kind adult male mice (18-22g) purchased from Dalian Medical Univ's animal experimental center, the quality certification number: SCXX (the Liao Dynasty) 2013-0003.
1.2 experimental technique
1.2.1 the foundation of the subacute alcoholic liver injury model of mice
1.2.1.1 dosage choice and grouping
Kunming mice is divided into five groups at random, is normal group, model group, bee pollen extract group: low dose group 1.34g/kg, middle high dose 2.68g/kg, high dose group 4.02g/kg respectively.
1.2.1.2 animal modeling
Kunming mice adaptability is raised one week, each group according to setting dosage gavage 30 days afterwards, be specially normal group and the normal saline of model group gavage 0.2% methylcellulose in the morning, the extract (normal saline with 0.2% methylcellulose) of administration group gavage respective concentration, after the 4h of interval except normal group gives normal saline, other groups give 20% ethanol according to 12mL/kg dosage, and (ethanol density is 0.8g/mL, the dosage amounting to ethanol is 1.92g/kg) 15d, within 15 days afterwards, only 20% ethanol is adjusted to 30% ethanol (dosage amounting to ethanol is 2.88g/kg), gavage volume is 0.2-0.5mL, within 2 days, weigh once, adjustment gavage volume.
After last gavage, water 4h is can't help in fasting, carries out injecting anesthetic with 10% chloral hydrate by 0.003mL/g body weight, plucks eyeball and gets blood, blood 37 DEG C of water-bath 30min, the centrifugal 15min of 3000r/min, gets supernatant-80 DEG C and preserves to be measured.
1.2.2 Biochemical Indices In Serum detects
The mensuration of low-density lipoprotein cholesterol (LDL), serum mesobilirubin (TBIL), mice very low density lipoprotein (VLDL) (VLDL), murine tumor necrosis factor (TNF-α), aspartate aminotransferase (AST), alanine aminotransferase (ALT), superoxide dismutase (SOD) in body weight, cholesterol in serum (CHOL), serum, uses corresponding reagent box to measure.
1.2.3 date processing
Data acquisition variance analysis, but first need carry out homogeneity test of variance by the program of variance analysis.Carry out the significance of difference with SPSS software and calculate P>0.05, there is no significant difference between two groups, between P<0.05 two groups, have significant difference.Add up with the comparative approach between two of mean between multiple experimental group and a matched group.Suitable variable transitions is carried out to the data of abnormal or heterogeneity of variance, after meeting normal state or variance and requiring together, adds up by the data after conversion; If do not reach normal state or the neat object of variance after variable transitions yet, use rank test instead and add up.
2 experimental results
2.1 zoopery Biochemical Indices In Serum testing results
Table 2 result shows: the alcohol damaged rising causing Serum ALT, AST, TNF-a level of model group, the decline of SOD in serum level, and there is significant difference with normal group, and modeling success is described.Compared with model group, for SOD in serum level, the equal significance of dosage group rises, and Serum ALT levels, the equal significance of dosage group declines, and for serum AST, TNF-a level, dosage group declines all to some extent, but only has high dose group to have significant difference.Illustrate that the mixture of Brassica campestris L pollen flavone and Peptides has certain protective effect for alcoholic liver injury, especially high dose group successful.
Table 2 respectively group mice serum SOD, ALT, AST, TNF-a level compares (n=12)
Note: * represents and compares P<0.05 with normal group, and * * represents and compares P<0.01 with normal group
Δrepresent and compare P<0.05 with model group,
Δ Δrepresent and compare P<0.01 with model group
Table 3 respectively group mice serum CHOL, LDL, TBIL, VLDL level compares (n=12)
Note: * represents and compares P<0.05 with normal group, and * * represents and compares P<0.01 with normal group
Δrepresent and compare P<0.05 with model group,
Δ Δrepresent and compare P<0.01 with model group
Table 3 result shows: the alcohol damaged rising causing change of serum C HOL, LDL, TBIL, VLDL level of model group, and there is significant difference with normal group, and modeling success is described.For serum VLDL level, dosage group declines relative to the equal significance of model group, and change of serum C HOL, LDL, TBIL content decline all to some extent relative to model group, but low dose group does not have significant difference, and middle dosage group, high dose group exist significant difference.Illustrate that the mixture of Brassica campestris L pollen flavone and Peptides has certain protective effect for alcoholic liver injury, dosage group, high dose group successful especially.
Above result shows, bee pollen extract has protective effect to alcoholic liver injury.
To sum up, Brassica campestris L pollen extract not only has antihypertensive function but also have protection hepatic injury function.
Embodiment 2
With tea bee pollen for raw material, prepare bee pollen extract according to following technique:
Get dry tea bee pollen 1000 grams, add ethyl acetate 50L, 50 DEG C are extracted 2 hours, extract to terminate rear filtration and be separated with solid content by extracting solution and process respectively:
(1) processing method of extracting solution: extracting solution is dry through vacuum concentration, concentrating sample macroporous resin column upper after 50 milliliter of 70% dissolve with ethanol, first use the deionized water drip washing of 5 times of column volumes, and then wash pillar with 5 times of column volume 70% ethanol and collect flow point, the component vacuum concentration drying of being washed by 70% ethanol obtains component 1.
(2) solid content processing method: solid content is according to raw material: the ratio of solution=1kg:2L adds the Tris-HCI buffer of 0.01M, adds the bromelain of 3% of raw materials quality, in 50 DEG C of enzymolysis 1 hour; After enzymolysis terminates, temperature of reaction system is risen to 90 DEG C to keep 20 minutes, make enzyme deactivation, then centrifugal or filtration, collect filtrate concentrate drying and obtain component 2.
Component 1 is mixed again with component 2, obtains tea bee pollen extract.
Tea bee pollen measures its content and biological activity according to the method for embodiment 1, and tea bee pollen and Brassica campestris L pollen had not only had antihypertensive function but also had protects hepatic injury function.
Embodiment 3
Take bee pollen of maize as raw material, prepare bee pollen of maize extract according to following technique:
Get dry bee pollen of maize 1000 grams, add normal hexane 20L, 30 DEG C are extracted 5 hours, extract to terminate rear filtration and be separated with solid content by extracting solution and process respectively:
(1) processing method of extracting solution: extracting solution is dry through vacuum concentration, concentrating sample macroporous resin column upper after 30 milliliter of 70% dissolve with ethanol, first use the deionized water drip washing of 10 times of column volumes, and then wash pillar with 2 times of column volume 50% ethanol and collect flow point, the component of being washed by 50% ethanol merges final vacuum concentrate drying and obtains component 1.
(2) solid content processing method: solid content is according to raw material: the ratio of solution=1kg:5L adds 0.05M phosphate buffer, then add raw materials quality 0.1% trypsin, in 40 DEG C of enzymolysis 10 hours; After enzymolysis terminates, temperature of reaction system is risen to 80 DEG C to keep 60 minutes, make enzyme deactivation, then centrifugal or filtration, collect filtrate concentrate drying and obtain component 2.
Component 1 is mixed again with component 2, obtains bee pollen of maize extract.
Bee pollen of maize measures its content and biological activity according to the method for embodiment 1, has not only had antihypertensive function but also has had protect hepatic injury function with Brassica campestris L pollen.
Claims (8)
1. bee pollen extract is preparing the application in blood pressure lowering or hepatic or health product.
2. according to application according to claim 1, it is characterized in that: described bee pollen extract is that the proteolytic enzyme enzymolysis bee pollen activity peptide of bee pollen flavone compound and the bee pollen solvent extraction residual residue extracted from bee pollen by solvent mixes.
3. according to application according to claim 2, it is characterized in that: bee pollen extract is prepared in accordance with the following methods: dry bee pollen is according to raw material: the ratio of solvent=1kg:5 ~ 50L adds solvent, 20-70 DEG C is extracted 1 ~ 10 hour, and extraction terminates rear filtration or centrifugal separation with solid content by extracting solution processes respectively:
(1) processing method of extracting solution: extracting solution is dry through vacuum concentration, drying sample macroporous resin column upper after volumetric concentration 40-70% dissolve with ethanol, first use the deionized water drip washing of 2 ~ 10 times of column volumes, and then wash pillar with volumetric concentration 40 ~ 90% ethanol of 1 ~ 5 times of column volume and collect flow point, the flow point vacuum concentration containing ethanol elution agent eluting is obtained component 1;
(2) solid content processing method: according to raw material: the ratio of solution=1kg:2 ~ 10L adds water or buffer mixing, the pH of mixed liquor is adjusted in scope 2 ~ 10 with 0.1 ~ 1MHCI or 0.1 ~ 1MNaOH, add the proteolytic enzyme of 0.001 ~ 3% of raw materials quality again, in 20 ~ 60 DEG C of enzymolysis 1 ~ 48 hour; After enzymolysis terminates, temperature of reaction system is risen to 80-100 DEG C and keep 10-60 minute, make enzyme deactivation, then centrifugal or filtration, collect filtrate concentrate drying and obtain component 2;
Component 1 is mixed again with component 2, obtains bee pollen extract.
4. according to application according to claim 3, it is characterized in that: bee pollen extracts solvent used and is: the solvent mixture of one or two or more kinds in water, methanol, ethanol, ethyl acetate, dichloromethane, chloroform, cyclohexane extraction, normal hexane, petroleum ether.
5. according to the application described in Claims 2 or 3, it is characterized in that: proteolytic enzyme used in solid content enzymolysis is: one or two or more kinds in pepsin, trypsin, flavor protease, bromelain, papain, alkaline protease or neutral protease.
6. according to application according to claim 3, it is characterized in that: described buffer is: the one in phosphate buffer, Tris-HCI buffer or acetate buffer solution.
7. according to application according to claim 3, it is characterized in that: described bee pollen is one or two or more kinds the different pollen mixture in Pollen Brassicae campestris, Pollen Pini, Pollen Maydis, Flos Camelliae Japonicae powder, Semen Castaneae pollen, Flos Nelumbinis pollen, pollen of Semen Fagopyri Esculenti, Flos Pruni part, peach flower powder, Pollen Helianthi.
8. according to application according to claim 1, it is characterized in that: described medicine with preparation bee pollen extract for active constituent, with the preparation process of routine, be used alone or prepare into powder, pill, capsule, tablet, microcapsule, soft capsule, suppository, unguentum, tincture or electuary with other drug.
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| CN112022881A (en) * | 2020-09-08 | 2020-12-04 | 中国农业科学院蜜蜂研究所 | Bee pollen fat extract and extraction method and application thereof |
| CN112385811A (en) * | 2020-11-02 | 2021-02-23 | 杭州蜂之语蜂业股份有限公司 | Method for extracting nutrient components of bee pollen |
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| CN103182001A (en) * | 2011-12-29 | 2013-07-03 | 新昌县冠阳技术开发有限公司 | Microencapsulated high-ester type catechin-tea flavonoid crystal and its making method |
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| CN112022881A (en) * | 2020-09-08 | 2020-12-04 | 中国农业科学院蜜蜂研究所 | Bee pollen fat extract and extraction method and application thereof |
| CN112385811A (en) * | 2020-11-02 | 2021-02-23 | 杭州蜂之语蜂业股份有限公司 | Method for extracting nutrient components of bee pollen |
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