CN102251005B - Preparation method of royal jelly polypeptide - Google Patents
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Abstract
The invention relates to the technical field of biomedicines, and in particular relates to a preparation method of a royal jelly polypeptide. 1-3kDa of the royal jelly polypeptide is obtained by extracting intestinal enzymes from the bodies of queen bee larva and simulating the enzymolysis environment in the queen bee bodies to carry out enzymolysis on royal jelly. The prepared royal jelly polypeptide can be used for preparing Abeta inhibitors.
Description
Technical field
The present invention relates to the biological medicine technology field, particularly a kind of preparation method of royal jelly polypeptide.
Background technology
In recent years, along with the increase of mankind's mean lifetime, the ratio of elderly population constantly rises, China's aging population becomes clear day by day, alzheimer's disease (Alzheimer ' s disease, AD) as the modal type of senile dementia, caused the extensive concern of the whole society.Alzheimer's disease is a kind of multifactor chronic progressive external mental deterioration disease of take the complexity that hypomnesis and cognition dysfunction be feature, clinical manifestation for carrying out property memory and cognition ability goes down, the symptom such as aphasis, psychomotor be abnormal.The patient drops to the master closely to remember in early days, the disease later stage, memory far away was also involved, daily life is affected, and shows as studying new knowledge and knows difficulty, and the work initiative descends, bearing new task can't be competent at, and pass in time and increase the weight of, with aphasis, dyscalculia, the visual space obstacle, lose daily technical ability, lose cognitive ability etc.The onset of Alzheimer disease concealment, carrying out property increases the weight of, and brings huge misery can to patient and household thereof, seriously hampers socioeconomic healthy and stable development.Nowadays, alzheimer's disease has become the fourth-largest killer after heart trouble, tumour and apoplexy, and the international and domestic treatment for alzheimer's disease does not also have specific medicament, and its complex therapy course for the treatment of is long, produce effects is slow.
International and domestic scholar shows the result of study of alzheimer's disease, onset of Alzheimer disease mechanism is very complicated, and its definite interpretation of the cause, onset and process of an illness it be not immediately clear, the same with other chronicity diseases of great majority, alzheimer's disease is caused by multifactor, has formed thus multiple hypothesis.But numerous studies have shown that, A β cascade hypothesis is the main flow theory of current onset of Alzheimer disease mechanism, and this hypothesis points out that the excessive accumulation of A β is the basis of AD morbidity, and A β produces and the unbalance of removing is the reason that causes its excessive accumulation.
Royal jelly (Royal Jelly, RJ) has another name called Lac regis apis, is commonly called as the queen bee breast, is the secreted particular matters of body of gland such as the hypopharynx cephalic gland of the young worker bee of 6-18 age in days and mandibular gland.Royal jelly is creamy white or is faint yellow, translucent, micro-sticky, peat-reek is arranged, distinguish the flavor of sour, puckery, hot, micro-sweet, be mainly first three day food after the lifelong food of queen bee and worker bee larvae hatching, have immunomodulatory, antibiotic, anti-inflammatory, antitumor, regulate blood pressure, reduce the functions such as blood sugar and Promote cell's growth.
Research is found, royal jelly has good improvement effect to the nervous system disease that comprises alzheimer's disease, Cucumber in royal jelly has the differentiation that improves neural propagation function, strengthens Neural Stem Cells, stellate cell and spongiocyte in the trophic function to brain, the expression amount that increases specific neurotrophic factor, the nerve growth that promotes brain cell, promotion brain, improve the cognition dysfunction of mouse, improve the effects such as learning and memory index of rat.Therefore, royal jelly can become the potential drug source of the generation that improves the neurological disorder diseases such as alzheimer's disease and development.
China is maximum in the world royal jelly producing country, output accounts for more than 90% of the world, but it is mainly with the products export of former material property, lack deep processed product, thereby cause product price lower, profit gained is less, therefore, royal jelly is carried out to deep processing, can effectively improve the quality of China's royal jelly, increase foreign exchange earnings from exports.
Fresh royal jelly contains various bioactivators, but store, improperly very easily rots, and loses original biological activity and nutritive value.Research shows, the rotten of royal jelly is mainly that its contained protein changes, thereby causes a series of chemical reaction to impel it rotten, so the protein in royal jelly plays a part extremely crucial to royal jelly performance overall efficacy.Protein contained in royal jelly is very responsive to temperature, and in storage process, partial protein is easily degraded, and, under different temperature condition, the degradation rate of different proteins is different.Storage temperature is higher, the time is longer, and Maillard reaction (Maillard reaction) more easily occurs royal jelly, and sex change speed is faster.Studies have found that at present, play the water soluble protein of very important effect in the health-care effect of royal jelly, polymerization can occur in degraded product, thereby water-insoluble protein is increased.The investigator finds, the royal jelly polypeptide obtained by enzymic hydrolysis not only storage requirement reduces, and the protein after enzymolysis has not perishablely, and the metabolism be more conducive in body absorbs, ingredient has more preferably pharmacological function, more contributes to illustrate the mechanism of action of royal jelly.But up to now, research to polypeptide after royal jelly enzymolysis is less, and the method great majority of enzymolysis are undertaken by exogenous enzyme such as stomach en-, trypsinase, papoids, are not suitable for the hydrolysis of royal jelly albumen, more difficult screening obtains the real effectively biologically active polypeptides of human body.
Summary of the invention
In view of this, the invention provides a kind of royal jelly polypeptide and uses thereof.This royal jelly polypeptide is by extract the enteron aisle enzyme in the queen bee nit body, and simulation queen bee body endoenzyme solution environment carries out enzymolysis to royal jelly, and the royal jelly polypeptide biological activity obtained is better, can be used in preparation A beta inhibitor.
In order to realize the foregoing invention purpose, the invention provides following technical scheme:
A kind of preparation method of royal jelly polypeptide comprises:
Step 1: by the queen bee nit of 2～3 ages in days, with after 0.9% physiological saline washing, take out the enteron aisle of described queen bee nit, add phosphate buffer soln or the Tris-HCl buffered soln that the pH value is 7.0～7.2 to grind to form homogenate, at 18000～25000g, centrifugal 20～30min under 4 ℃ of conditions, collect the middle level liquid after centrifugal for the first time, then at 18000～25000g, centrifugal 20～30min under 4 ℃ of conditions, collect the middle level liquid after centrifugal for the second time, obtain enteron aisle enzyme liquid;
Step 2: in royal jelly, add phosphate buffered saline buffer or Tris-HCl buffered soln that the pH value is 7.0～7.2, at 18000～25000g, centrifugal 20～30min under 4 ℃ of conditions, (removing insoluble composition) collects supernatant liquor, ice bath dialysis 42～72h in the phosphate buffered saline buffer that the dialysis tubing that is 8000～14000 through molecular weight cut-off is 7.0～7.2 in the pH value or Tris-HCl buffered soln, (every 12h, changing a dialyzate) collects dialyzate in dialysis tubing, obtains the water soluble royal jelly protein liquid;
Step 3: in mg/mL, by the enteron aisle enzyme liquid of step 1 gained, with the water soluble royal jelly protein liquid of step 2 gained, according to the protein concentration ratio, be 30～35: 80～90 mix, in the pH value, be 8.3～8.7, enzymolysis 22～26h under the condition that temperature is 34～39 ℃, obtain the water soluble royal jelly protein enzymatic hydrolyzate;
Step 4: after the described water soluble royal jelly protein enzymatic hydrolyzate of step 3 ice bath is stopped to enzyme digestion reaction, at 8000～15000g, centrifugal 10～20min under 4 ℃ of conditions, collect supernatant liquor, with after 10 μ m filtering with microporous membrane, collect filtrate and obtain the first filtrate, ultrafiltration membrance filter by described the first filtrate through 3kDa, collect filtrate and obtain the second filtrate, the ultrafiltration membrance filter by described the second filtrate through 1kDa, collect the royal jelly polypeptide that trapped substance obtains 1～3kDa.
As preferably, in step 1 or step 2, the concentration of phosphate buffered saline buffer or Tris-HCl buffered soln is 50mmol/L.
As preferably, the mass volume ratio of the enteron aisle of queen bee nit described in step 1 and phosphate buffered saline buffer or Tris-HCl buffered soln with g/mL count 1: 1～1: 2.
As preferably, in step 1, the middle level liquid of homogenate or the first suspension is centrifugal under the condition of 20000g.
As preferably, in step 1, the middle level centrifugal 20min of homogenate or the first suspension.
As preferably, in step 2, the volume ratio of phosphate buffered saline buffer or Tris-HCl buffered soln and described royal jelly is 2: 1～4: 1.
As preferably, in step 2, centrifugal force is centrifugal 20min under the condition of 20000g.
As preferably, in step 2, dialysis time is 48h.
As preferably, in mg/mL, in step 3, described enteron aisle enzyme liquid is 32.5: 85 with the protein concentration ratio of described water soluble royal jelly protein liquid.
Preferably, in step 3, the protein concentration of described enteron aisle enzyme liquid is 20～35mg/mL, and the protein concentration of described water soluble royal jelly protein liquid is 25～45mg/mL, and the mass ratio of described enteron aisle enzyme liquid and described water soluble royal jelly protein liquid is 1: 2.3～3.0.
As preferably, in step 3, hydrolysis temperature is 37 ℃.
As preferably, in step 3, enzymolysis pH value is 8.5.
As preferably, in step 3, enzymolysis time is 24h.
As preferably, in step 4, centrifugal condition is at 10000g, centrifugal 10min under 4 ℃ of conditions.
The invention provides a kind of preparation method of royal jelly polypeptide.The method is carried out enzymolysis by extraction enteron aisle enzyme in the queen bee nit body to royal jelly and is obtained, and can simulate queen bee body endoenzyme solution environment, obtains the better royal jelly polypeptide of biological activity, can be used in preparation A beta inhibitor.
Test shows, the royal jelly protein polypeptide of 1～3kDa can effectively suppress 25 μ M A β
25-35to the toxic action of SH-SY5Y cell, to A β
25-35the SH-SY5Y cell injury caused has significant protective effect (P<0.01).
With A β
25-35model group is compared, the royal jelly protein polypeptide of 1～3kDa of three kinds of different concns all can make the LDH burst size in cell culture fluid significantly reduce (P<0.01), and, along with the increase of royal jelly protein polypeptide concentration, the LDH burst size reduces gradually, show significant concentration dependent relation.With A β
25-35model group is compared, and in the experiment of this group, the royal jelly protein polypeptide of 1～3kDa of 92 μ g/ml can farthest suppress the release of LDH, and inhibiting rate is in 66% left and right.
The A β of 25 μ M
25-35after the royal jelly protein polypeptide acting in conjunction 24h of 1～3kDa of different concns, the overall apoptosis rate of SH-SY5Y cell descends to some extent, and along with the increase of royal jelly protein polypeptide concentration, the early apoptosis rate and late period apoptosis rate reduce gradually, be certain concentration dependent relation.When royal jelly protein polypeptide concentration is 92 μ g/ml, the early apoptosis rate and late period apoptosis rate drop to 6.97 ± 1.01% (P<0.001) from 26.39 ± 2.24%, approach the level of blank group.Explanation thus, the royal jelly protein polypeptide of 1～3kDa is to A β
25-35the SH-SY5Y apoptosis of inducing has certain restraining effect.
The A β of 25 μ M
25-35after the royal jelly protein polypeptide co-treatment SH-SY5Y cell 24h of 1～3kDa of different concns, the generation of ROS all has decline, and along with the increase of concentration separately, and the generation of ROS is and first reduces the trend increased afterwards.For the royal jelly protein polypeptide treatment group of 1～3kDa, it is minimum that the dosage of 46 μ g/ml can make the generation of ROS drop to, and from 19.99 ± 0.59, is reduced to 14.06 ± 0.34 (P<0.001).The royal jelly protein polypeptide that shows 1～3kDa can stop A β
25-35the generation of the SH-SY5Y intracellular reactive oxyradical (ROS) of inducing.Dose concentration is within the specific limits the time, and along with the increase of royal jelly protein polypeptide concentration, the ROS growing amount is the trend reduced; When royal jelly protein polypeptide concentration continues to increase, the ROS growing amount rises again gradually.
The A β of 25 μ M
25-35all can reduce the value of Bax/Bcl-2 with the royal jelly protein polypeptide acting in conjunction 24h of 1～3kDa of different concns, and dose concentration is between 11.5～92 μ g/ml the time, along with the increase of concentration has the trend reduced gradually, is certain dose-dependently; When concentration is 46 μ g/ml, reducing to minimum level, is 0.65 ± 0.05 times (P<0.001) of control group.When concentration is 92 μ g/ml, the value of Bax/Bcl-2 has again the trend of slight increase.The reason that the royal jelly protein polypeptide of 1～3kDa reduces the Bax/Bcl-2 value is mainly the royal jelly protein polypeptide and can suppresses significantly A β
25-35induce the rising of Bax expression amount in the SH-SY5Y cell.
Comprehensive above-mentioned test-results, the preparation method of the royal jelly protein polypeptide of 1～3kDa provided by the invention is to A β
25-35induce the SH-SY5Y cell to have and suppress active, the royal jelly protein polypeptide of the 1～3kDa therefore made can, for the preparation of the A beta inhibitor, reach preparation treatment alzheimer's disease related drugs.
The accompanying drawing explanation
Fig. 1 shows enteron aisle enzyme liquid that the present invention makes and the SDS-PAGE electrophorogram of water soluble royal jelly protein liquid, and wherein, swimming lane 1 and swimming lane 2 are enteron aisle enzyme liquid, and swimming lane 3 and swimming lane 4 are the water soluble royal jelly protein liquid, and swimming lane 5 is marker.
Fig. 2 shows the impact of enzymolysis time on hydrolysis result, and wherein, swimming lane 1 is the water soluble royal jelly protein liquid, and swimming lane 2 to 9 is respectively enzymolysis time 2h, 4h, 6h, 8h, 10h, 15h, 20h, 25h, and swimming lane 10 is marker.
Fig. 4 shows the impact of enzymolysis pH value on hydrolysis result, and wherein, swimming lane 1 is the water soluble royal jelly protein liquid, and swimming lane 2 to 7 is respectively pH value 5,6,6.5,7,7.5,8, and swimming lane 8 is marker.
Fig. 6 shows in enzymolysis solution that albumen is with pH value changing conditions, wherein, swimming lane 1 is the water soluble royal jelly protein liquid, swimming lane 2 is enteron aisle enzyme liquid provided by the invention, enzymolysis solution after enzymolysis under the condition that swimming lane 3 to 7 is is 8,8.1,8.3,8.5,8.7 in the pH value respectively by water soluble royal jelly protein liquid and enteron aisle enzyme liquid, swimming lane 8 is marker.
Fig. 8 shows the best proportioning of enteron aisle enzyme liquid provided by the invention and water soluble royal jelly protein liquid, wherein, swimming lane 1,8 is enteron aisle enzyme liquid provided by the invention, swimming lane 2,9 is the water soluble royal jelly protein liquid, swimming lane 3 to 6 is respectively enteron aisle enzyme liquid and water soluble royal jelly protein liquid volume ratio is 1: 1,1: 2,1: 3,1: 4, swimming lane 10 to 13 is respectively enteron aisle enzyme liquid and water soluble royal jelly protein liquid volume ratio is 1: 5,1: 6,1: 7,1: 8, and swimming lane 7,14 is marker.
Fig. 9 is shown under optimum enzymolysis condition, comparison diagram before and after enzyme digestion reaction, and wherein, the reaction system cumulative volume is 10mL, swimming lane 1 to 8 is collection of illustrative plates after reaction for the front collection of illustrative plates of reaction, swimming lane 10 to 17; Swimming lane 1,10 is respectively containing 0.5mL enteron aisle enzyme liquid and 0.5mL water soluble royal jelly protein liquid; Swimming lane 2,11 is respectively containing 0.5mL enteron aisle enzyme liquid and 1mL water soluble royal jelly protein liquid; Swimming lane 3,12 is respectively containing 1mL enteron aisle enzyme liquid and 1mL water soluble royal jelly protein liquid; Swimming lane 4,13 is respectively containing 1mL enteron aisle enzyme liquid and 2mL water soluble royal jelly protein liquid; Swimming lane 5,14 is respectively containing 1.5mL enteron aisle enzyme liquid and 1.5mL water soluble royal jelly protein liquid; Swimming lane 6,15 is respectively containing 1.5mL enteron aisle enzyme liquid and 3mL water soluble royal jelly protein liquid; Swimming lane 7,16 is respectively containing 2mL enteron aisle enzyme liquid and 2mL water soluble royal jelly protein liquid; Swimming lane 8,17 is marker containing 2mL enteron aisle enzyme liquid and 4mL water soluble royal jelly protein liquid swimming lane 9,18 respectively.
Figure 10 shows the royal jelly polypeptide liquid phase analysis color atlas of 1～3kDa, and wherein, solid line is the color atlas under 280nm, and dotted line is the color atlas under 214nm.
Figure 11 shows that the royal jelly polypeptide of 1～3kDa is to A β
25-35the provide protection of the SH-SY5Y cell injury of inducing, wherein, Figure 11 (a) is the Control group, Figure 11 (b) is 25 μ mol/L A β
25-35group, Figure 11 (c) is 25 μ mol/LA β
25-35+ 9.2 μ g/mL royal jelly polypeptide groups, Figure 11 (d) is 25 μ mol/LA β
25-35+ 23 μ g/mL royal jelly polypeptide groups, Figure 11 (e) is 25 μ mol/L A β
25-35+ 46 μ g/mL royal jelly polypeptide groups, Figure 11 (f) is 25 μ mol/LA β
25-35+ 92 μ g/mL royal jelly polypeptide groups.
Figure 12 shows that the royal jelly polypeptide of 1～3kDa is to A β
25-35the apoptotic restraining effect of the SH-SY5Y induced, wherein, ordinate zou is cytoactive (take the control group as 100%), X-coordinate is A β
25-35with the addition of royal jelly polypeptide, the first cylindricality is the control group, and the second cylindricality is A β
25-35model group, the 3rd to the 6th cylindricality is the royal jelly polypeptide treatment group; The second cylindricality and the first cylindricality have utmost point significant difference (P<0.001), have utmost point significant difference (P<0.01) between the 3rd to the 6th cylindricality and the second cylindricality.
Figure 13 shows that the royal jelly polypeptide of 1～3kDa is to A β
25-35the impact of the SH-SY5Y cell LDH burst size of inducing.
Figure 14 shows that the two royal jelly polypeptides of methods detection 1～3kDa that dye of Annexin V/PI are to A β
25-35the impact of the SH-SY5Y cell of inducing, wherein, Figure 14 (a) is the control group, Figure 14 (b) is A β model group, the royal jelly polypeptide that Figure 14 (c) is A β+23 μ g/mL 1～3kDa, the royal jelly polypeptide that Figure 14 (d) is A β+46 μ g/mL 1～3kDa, the royal jelly polypeptide that Figure 14 (e) is A β+92 μ g/mL 1～3kDa.
Figure 15 shows that the two royal jelly polypeptides of methods detection 1～3kDa that dye of Annexin V/PI are to A β
25-35the result that affects of the SH-SY5Y cell of inducing is summed up.
Figure 16 shows the impact of the royal jelly polypeptide of 1～3kDa on active oxygen radical.
Figure 17 shows that western blotting detects the impact of the royal jelly polypeptide of 1～3kDa on Bax in the SH-SY5Y cell and Bcl-2 protein expression level.
Figure 18 shows the impact of the royal jelly polypeptide of 1～3kDa on Bax/Bcl-2 ratio in the SH-SY5Y cell, and wherein, the oblique line cylindricality represents the value of Bax/Bcl-2, and white cylindricality represents the expression amount of Bax, and the black cylindricality represents the expression amount of Bcl-2.
Embodiment
The invention discloses a kind of preparation method of royal jelly polypeptide, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they all are deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can be changed methods and applications as herein described or suitably change and combination within not breaking away from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
In royal jelly polypeptide provided by the invention, every reagent all can be buied by market.Royal jelly and queen bee nit all, purchased from Meng Wu village, Jun Zhuan town, Mentougou, Beijing City district bee-keeping specialist, show through 50 tests, in royal jelly protein content without significant difference, from the protein content of the enteron aisle enzyme that extracts in the queen bee nit body also without significant difference.SH-SY5Y cell (Beijing Normal University's Resource Institute resource ecology and natural resources of Chinese medicinal materials institute cell bank).Prestained Protein Ladder (U.S. Fermentas), D-MEM/F12 substratum (U.S. Gibco company), foetal calf serum (FBS, U.S. Gibco company), pancreatin (U.S. R& D), penicillin/streptomycin (U.S. Gibco company), A β
25-35(purity 98.4%, Zhongtai Bio-Chem. Co., Ltd., Hangzhou), 3-(4, 5-dimethylthiazole-2)-2, 5-phenylbenzene tetrazole bromine salt (MTT, U.S. Amresco company), Cellstar Tissue Culture Plate (German Greiner company), serum lactic dehydrogenase (LDH) is measured test kit (bio-engineering research institute is built up in Nanjing), Annexin V-FITC apoptosis detection kit (Beijing Bao Sai Bioisystech Co., Ltd), 2 ', 7 '-dichlorofluorescein diacetate (DCFH-DA, U.S. Sigma company), Polyvinylidene Fluoride Membrane (pvdf membrane) (U.S. Roche), mouse anti human Bcl-2 multispecific antibody, the anti-human Bax polyclonal antibody of rabbit, mouse anti human β-actin polyclonal antibody, HRP mark goat anti-mouse igg polyclonal antibody, the anti-rabbit igg polyclonal antibody of HRP mark mouse, ECL colouring reagents (Santa Cruz company), other reagent are domestic analytical pure.
Below in conjunction with embodiment, further set forth the present invention:
Get the queen bee nit of fresh 2～3 ages in days, filter after waiting 0.9% ice-cold physiological saline washes clean of oozing, dissect one by one and collect whole enteron aisles of polypide.After the phosphate buffered saline buffer dilution of the 50mmol/L that the pH value that to add with polypide enteron aisle mass volume ratio (g/mL) be 1: 1 is 7, with electric homogenizer, under condition of ice bath, grind to form homogenate.At 20000 * g, centrifugal 20min under 4 ℃, centrifugal rear suspension liquid is divided into three layers, and floating matter upper strata (lipid component), aaerosol solution middle level, precipitation lower floor, reclaim middle level.By middle level, again at 20000 * g, centrifugal 20min under 4 ℃, get the enteron aisle enzyme liquid that middle level obtains yellow transparent thus, is kept in-20 ℃ of refrigerators.
Get partial enteral enzyme liquid, by the BCA method, detect protein concentration, the protein concentration that obtains enteron aisle enzyme liquid is 31.9mg/mL.SDS-PAGE electrophoresis detection result, be shown in Fig. 1.
The phosphate buffered saline buffer of the 50mmol/L that is 7 by the pH value of the royal jelly of 1 times of volume and 2 times of volumes mixes, and prepares the royal jelly diluting soln.By above-mentioned royal jelly diluting soln, at 20000 * g, centrifugal 20min under 4 ℃, remove insoluble composition, collects supernatant liquor.The 48h that dialyses under ice bath in the phosphate buffered saline buffer of the 50mmol/L that the dialysis tubing (molecular weight cut-off 8000-14000) that uses 70mm is 7 in pH value, every a dialyzate of 12h replacing.Get liquid in dialysis tubing, be highly purified water soluble royal jelly albumen, be kept in-20 ℃ of refrigerators.Get part water soluble royal jelly albumen, by the BCA method, detect protein concentration, the protein concentration that obtains water soluble royal jelly albumen is 42.7mg/mL.SDS-PAGE electrophoresis detection result, be shown in Fig. 1.
Determining of enteron aisle enzyme and royal jelly enzymolysis condition: pH value, enzymolysis time to enzymolysis are groped, and the results are shown in Figure 2 to Fig. 9.Determine that optimum enzymolysis condition is: in the pH value, be 8.3～8.7, enzymolysis 22～26h under the condition that temperature is 34～39 ℃.
According to adding water soluble royal jelly albumen that the 4mL protein concentration is 42.7mg/mL, enteron aisle enzyme liquid that the 2mL protein concentration is 31.9mg/mL and the proportionlity of 4mL damping fluid, the reaction system of preparation 2000mL in the 10mL reaction system.And the pH value of system is adjusted under 8.5,37 ℃ to the reaction through row 24h.After reaction 24h, ice bath, stopped reaction.Reaction solution is at 10000 * g, and centrifugal 10min under 4 ℃, remove insoluble composition, obtains the 1900mL supernatant liquor.With 10 μ m filtering with microporous membrane, collect filtrate, then the membrane filtration system that is 3kDa and 1kDa with molecular size range successively filtration, obtain the filtrate of 200mL 1-3kDa.By the filtrate lyophilize, then dissolve with distilled water, the membrane filtration of 0.22 μ m, obtain the royal jelly polypeptide of 1-3kDa after enzymolysis.After the BCA method detects, obtaining the protein concentration of royal jelly protein polypeptide after enzymolysis is 2.30mg/mL.
Get the queen bee nit of fresh 2～3 ages in days, filter after waiting 0.9% ice-cold physiological saline washes clean of oozing, dissect one by one and collect whole enteron aisles of polypide.After the phosphate buffered saline buffer of the 50mmol/L that the pH value that to add with polypide enteron aisle mass volume ratio (g/mL) be 1: 1 is 7, with electric homogenizer, under condition of ice bath, grind to form homogenate.At 18000 * g, centrifugal 30min under 4 ℃, centrifugal rear suspension liquid is divided into three layers, and floating matter upper strata (lipid component), aaerosol solution middle level, precipitation lower floor, reclaim middle level.By middle level, again at 18000 * g, centrifugal 30min under 4 ℃, get the enteron aisle enzyme liquid that middle level obtains yellow transparent thus, is kept in-20 ℃ of refrigerators.
Get partial enteral enzyme liquid, by the BCA method, detect protein concentration, the protein concentration that obtains enteron aisle enzyme liquid is 35mg/mL.
The royal jelly of 1 times of volume is mixed with the phosphate buffered saline buffer of the 50mmol/L of the pH=7 of 4 times of volumes, prepare the royal jelly diluting soln.By above-mentioned royal jelly diluting soln, at 18000 * g, centrifugal 30min under 4 ℃, remove insoluble composition, collects supernatant liquor.The 42h that dialyses under ice bath in the phosphate buffered saline buffer of the 50mmol/L that the dialysis tubing (molecular weight cut-off 8000-14000) that uses 70mm is 7 in pH value, every a dialyzate of 12h replacing.Get liquid in dialysis tubing, be highly purified water soluble royal jelly albumen, be kept in-20 ℃ of refrigerators.Get part water soluble royal jelly albumen, by the BCA method, detect protein concentration, the protein concentration that obtains water soluble royal jelly albumen is 26.8mg/mL.
Determining of enteron aisle enzyme and royal jelly enzymolysis condition: pH value, enzymolysis time to enzymolysis are groped, and the results are shown in Figure 2 to Fig. 9.Determine that optimum enzymolysis condition is: in the pH value, be 8.3～8.7, enzymolysis 22～26h under the condition that temperature is 34～39 ℃.
According to adding water soluble royal jelly albumen that the 6mL protein concentration is 26.8mg/mL, enteron aisle enzyme liquid that the 2mL protein concentration is 35mg/mL and the proportionlity of 2mL damping fluid, the reaction system of preparation 2000mL in the 10mL reaction system.And the pH value of system is adjusted under 8.3,39 ℃ to the reaction through row 26h.After reaction 26h, ice bath, stopped reaction.Reaction solution is at 8000 * g, and centrifugal 20min under 4 ℃, remove insoluble composition, obtains the 1860mL supernatant liquor.With 10 μ m filtering with microporous membrane, collect filtrate, then the membrane filtration system that is 3kDa and 1kDa with molecular size range successively filtration, obtain the filtrate of 180mL 1-3kDa.By the filtrate lyophilize, then dissolve with distilled water, the membrane filtration of 0.22 μ m, obtain the royal jelly polypeptide of 1-3kDa after enzymolysis.After the BCA method detects, obtaining the protein concentration of royal jelly protein polypeptide after enzymolysis is 2.10mg/mL.
Get the queen bee nit of fresh 2～3 ages in days, filter after waiting 0.9% ice-cold physiological saline washes clean of oozing, dissect one by one and collect whole enteron aisles of polypide.After the phosphate buffered saline buffer dilution of the 50mmol/L that the pH value that to add with polypide enteron aisle mass volume ratio (g/mL) be 1: 1.5 is 7.2, with electric homogenizer, under condition of ice bath, grind to form homogenate.At 25000 * g, centrifugal 24min under 4 ℃, centrifugal rear suspension liquid is divided into three layers, and floating matter upper strata (lipid component), aaerosol solution middle level, precipitation lower floor, reclaim middle level.By middle level, again at 25000 * g, centrifugal 24min under 4 ℃, get the enteron aisle enzyme liquid that middle level obtains yellow transparent thus, is kept in-20 ℃ of refrigerators.
Get partial enteral enzyme liquid, by the BCA method, detect protein concentration, the protein concentration that obtains enteron aisle enzyme liquid is 25.5mg/mL.
The royal jelly of 1 times of volume is mixed with the phosphate buffered saline buffer of the 50mmol/L of the pH=7 of 3 times of volumes, prepare the royal jelly diluting soln.By above-mentioned royal jelly diluting soln, at 25000 * g, centrifugal 24min under 4 ℃, remove insoluble composition, collects supernatant liquor.Use the dialysis tubing (molecular weight cut-off 8000-14000) of the 70mm 72h that dialyses under ice bath in the phosphate buffered saline buffer of the 50mmol/L of pH=7, every 12h, change a dialyzate.Get liquid in dialysis tubing, be highly purified water soluble royal jelly albumen, be kept in-20 ℃ of refrigerators.Get part water soluble royal jelly albumen, by the BCA method, detect protein concentration, the protein concentration that obtains water soluble royal jelly albumen is 32.5mg/mL.
Determining of enteron aisle enzyme and royal jelly enzymolysis condition: pH value, enzymolysis time to enzymolysis are groped, and the results are shown in Figure 2 to Fig. 9.Determine that optimum enzymolysis condition is: in the pH value, be 8.3～8.7, enzymolysis 22～26h under the condition that temperature is 34～39 ℃.
According to adding water soluble royal jelly albumen that the 4mL protein concentration is 32.5mg/mL, enteron aisle enzyme liquid that the 1.7mL protein concentration is 25.5mg/mL and the proportionlity of 4.3mL damping fluid, the reaction system of preparation 2000mL in the 10mL reaction system.And the pH value of system is adjusted under 8.7,34 ℃ to the reaction through row 22h.After reaction 22h, ice bath, stopped reaction.Reaction solution is at 15000 * g, and centrifugal 14min under 4 ℃, remove insoluble composition, obtains the 1930mL supernatant liquor.With 10 μ m filtering with microporous membrane, collect filtrate, then the membrane filtration system that is 3kDa and 1kDa with molecular size range successively filtration, obtain the filtrate of 220mL 1-3kDa.By the filtrate lyophilize, then dissolve with distilled water, the membrane filtration of 0.22 μ m, obtain the royal jelly polypeptide of 1-3kDa after enzymolysis.After the BCA method detects, obtaining the protein concentration of royal jelly protein polypeptide after enzymolysis is 2.56mg/mL.
Get the queen bee nit of fresh 2～3 ages in days, filter after waiting 0.9% ice-cold physiological saline washes clean of oozing, dissect one by one and collect whole enteron aisles of polypide.After the phosphate buffered saline buffer dilution of the 50mmol/L that the pH value that to add with polypide enteron aisle mass volume ratio (g/mL) be 1: 2 is 7.2, with electric homogenizer, under condition of ice bath, grind to form homogenate.At 25000 * g, centrifugal 24min under 4 ℃, centrifugal rear suspension liquid is divided into three layers, and floating matter upper strata (lipid component), aaerosol solution middle level, precipitation lower floor, reclaim middle level.By middle level, again at 25000 * g, centrifugal 24min under 4 ℃, get the enteron aisle enzyme liquid that middle level obtains yellow transparent thus, is kept in-20 ℃ of refrigerators.
Get partial enteral enzyme liquid, by the BCA method, detect protein concentration, the protein concentration that obtains enteron aisle enzyme liquid is 20.2mg/mL.
The royal jelly of 1 times of volume is mixed with the phosphate buffered saline buffer of the 50mmol/L of the pH=7 of 3 times of volumes, prepare the royal jelly diluting soln.By above-mentioned royal jelly diluting soln, at 25000 * g, centrifugal 24min under 4 ℃, remove insoluble composition, collects supernatant liquor.Use the dialysis tubing (molecular weight cut-off 8000-14000) of the 70mm 56h that dialyses under ice bath in the phosphate buffered saline buffer of the 50mmol/L of pH=7, every 12h, change a dialyzate.Get liquid in dialysis tubing, be highly purified water soluble royal jelly albumen, be kept in-20 ℃ of refrigerators.Get part water soluble royal jelly albumen, by the BCA method, detect protein concentration, the protein concentration that obtains water soluble royal jelly albumen is 32.5mg/mL.
Determining of enteron aisle enzyme and royal jelly enzymolysis condition: pH value, enzymolysis time to enzymolysis are groped, and the results are shown in Figure 2 to Fig. 9.Determine that optimum enzymolysis condition is: in the pH value, be 8.3～8.7, enzymolysis 22～26h under the condition that temperature is 34～39 ℃.
According to adding water soluble royal jelly albumen that the 4mL protein concentration is 32.5mg/mL, enteron aisle enzyme liquid that the 2.5mL protein concentration is 20.2mg/mL and the proportionlity of 3.5mL damping fluid, the reaction system of preparation 2000mL in the 10mL reaction system.And the pH value of system is adjusted under 8.7,34 ℃ to the reaction through row 22h.After reaction 22h, ice bath, stopped reaction.Reaction solution is at 15000 * g, and centrifugal 14min under 4 ℃, remove insoluble composition, obtains the 1930mL supernatant liquor.With 10 μ m filtering with microporous membrane, collect filtrate, then the membrane filtration system that is 3kDa and 1kDa with molecular size range successively filtration, obtain the filtrate of 220mL 1-3kDa.By the filtrate lyophilize, then dissolve with distilled water, the membrane filtration of 0.22 μ m, obtain the royal jelly polypeptide of 1-3kDa after enzymolysis.After the BCA method detects, obtaining the protein concentration of royal jelly protein polypeptide after enzymolysis is 2.42mg/mL.
The royal jelly protein polypeptide rotation of respectively getting the 1-3kDa that 1mL embodiment 1 to 4 makes volatilizes, and by the water dissolution of 0.06%TFA, to 1mL, the centrifugal 10min of 12000rpm, get supernatant.Condition of gradient elution is as follows: level pad (A): 0.06%TFA, elution buffer (B): the acetonitrile of 0.05%TFA; Gradient: 0～100%B over 6column volumes (CV); Flow velocity: 1mL/min; Sampling volume: 500 μ L; Detect wavelength: 280nm/214nm.Detected result as shown in figure 10.
D-MEM/F12 minimum medium: the D-MEM/F12 substratum is dissolved in tri-distilled water, mixes, use NaHC0
3the pH value of substratum is adjusted to 7.2, through the filtering with microporous membrane of 0.22 μ M, after packing, 4 ℃ of preservations.
The D-MEM/F12 perfect medium: add 10% foetal calf serum in the D-MEM/F12 minimum medium, the penicillin/streptomycin of each 100,000 μ/L of 1%, mix 4 ℃ of preservations.
On super clean bench, by 1mg A β
25-35be dissolved in 928 μ l sterilizing tri-distilled waters fully, be mixed with the mother liquor of 1mmol/L ,-20 ℃ of preservations, with front 37 ℃ of aging 72h, after packing ,-20 ℃ of preservations are stand-by.
The FBS that is 10% containing volume fraction for the SH-SY5Y cell, the DMEM/F12 substratum of 1% penicillin/streptomycin, in 37 ℃, 5% CO
2cultivate in incubator, within every 2 days, carry out 1 time and go down to posterity.Take the logarithm the vegetative period cell through row experiment.Cell is divided into to 3 groups.Normal group: perfect medium is changed to serum-free DMEM/F12 substratum after cultivating 24h; A β
25-35model group: the A β that adds 25 μ mol/L
25-35; After royal jelly protein polypeptide (different concns) the preincubate 4h of the 1-3kDa that royal jelly protein polypeptide protection group: embodiment 1 to 4 makes, add the A β of 25 μ mol/L
25-35.
MTT measures cell survival rate: the SH-SY5Y cell in vegetative period of taking the logarithm, and with 3 * 10
4the density of cells/ml, 200 μ l/well are inoculated into 96 well culture plates, and 37 ℃, 5% CO
2cultivate in incubator, after 24h, inhale and abandon old substratum, Normal group and A β
25-35model group adds respectively 100 μ l serum-free DMEM/F12 substratum, the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide (different concns) of the 1-3kDa that royal jelly protein polypeptide protection group adds 100 μ l to make containing embodiment 1 to 4.After preincubate 4h, Normal group changes and adds 100 μ l serum-free DMEM/F12 substratum, A β
25-35model group is changed the A β that adds 100 μ l 25 μ mol/L
25-35serum-free DMEM/F12 substratum, royal jelly protein polypeptide protection group is changed and is added 100 μ l simultaneously containing 25 μ mol/LA β
25-35the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa made with different concns embodiment 1 to 4.6 multiple holes are set respectively.After drug effect 24h, MTT (pH=7.0PBS preparation) the 11 μ l that every hole adds 5mg/ml, after 4h, abandon substratum, adds DMSO 150 μ l/well, and dark lower light shaking 10min, read absorbancy (OD) value under microplate reader 492nm and 630nm.
Cell survival rate (%)=(experimental group OD
492-630-blank organizes OD
492-630)/(control organizes OD
492-630-blank organizes OD
492-630) * 100%.The inverted microscope observations is shown in Figure 11.The MTT colorimetry the results are shown in Figure 12.
Result by inverted microscope shows: the SH-SY5Y cell of control group is uniformly distributed growth, well-grown, and cell space is plentiful, and transmittance is good, and cynapse is obvious and stretching, extension is good, and most cells is fusiformis, trilateral or Polygons, the cell culture fluid clear.25 μ M A β
25-35after the 24h processed, the SH-SY5Y cell distribution is inhomogeneous, the clustering growth, and the cellular form heterogeneity, cell space shrinks, and transmittance descends, decrease of synapses or disappearance, the cell rounding is serious; Cell has suspension and cell culture fluid that turbid phenomenon is arranged.The RJP treatment group of the 1-3kDa of different concns gradient and 25 μ M A β
25-35model group is compared, increase gradually along with RJP concentration, the phenomenon of cell aggregation cluster weakens gradually, growth is uniformly distributed gradually, and cell space is full gradually, and transmittance strengthens gradually, cynapse becomes changeable length gradually, cell rounding phenomenon weakens, and the cell culture fluid turbid phenomenon also dies down, and the general cell state has clear improvement.
Result by the MTT colorimetric determination shows: 25 μ M A β
25-35after treatment S H-SY5Y cell 24h, can cause the MTT metabolic rate of cell obviously to descend.25 μ MA β are described
25-35can reduce significantly the vigor of cell mitochondrial, thereby cell is caused to damage significantly, form the obvious toxicity of cell.The royal jelly protein polypeptide of the 1-3kDa of four kinds of concentration and 25 μ MA β
25-35after acting in conjunction 24h, can make the metabolic rate of cell MTT significantly improve, with A β model group obvious difference (P<0.01), the royal jelly protein polypeptide effect of the 1-3kDa of 92 μ g/ml is best.Explanation thus, along with the increase of the concentration of 1-3kDa royal jelly protein polypeptide, the metabolic rate of MTT improves gradually, presents significant concentration dependent relation; The royal jelly protein polypeptide of 1-3kDa can effectively suppress 25 μ M A β
25-35to the toxic action of SH-SY5Y cell, to A β
25-35the SH-SY5Y cell injury caused has significant protective effect (P<0.01).
The detection that serum lactic dehydrogenase (Lactate dehydrogenase, LDH) discharges: the SH-SY5Y cell in the vegetative period of taking the logarithm, adjusting cell density is 3 * 10
4cells/ml, 200 μ l/well are inoculated into 96 well culture plates, and 37 ℃, 5% CO
2cultivate in incubator, after 24h, inhale and abandon old substratum, Normal group and A β
25-35the every hole of model group adds respectively 100 μ l serum-free DMEM/F12 substratum, the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa that royal jelly protein polypeptide protection group adds 100 μ l to make containing different concns embodiment 1 to 4.After 4h, Normal group changes and adds 100 μ l serum-free DMEM/F12 substratum, A β
25-35model group is changed the A β that adds 100 μ l 25 μ mol/L
25-35serum-free DMEM/F12 substratum, royal jelly protein polypeptide protection group is changed and is added 100 μ l simultaneously containing 25 μ mol/LA β
25-35the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa made with different concns embodiment 1 to 4.4 multiple holes are set respectively.Draw supernatant after drug effect 24h, every hole is got 0.02ml and is detected through row LDH concentration immediately.Do blank tube, standard pipe, mensuration pipe and control tube according to LDH test kit operation steps, and add respectively corresponding solution and mix, room temperature is placed 3min, the zeroing of 440nm distilled water, and the 1cm optical path is measured the absorbancy of each pipe.Calculate LDH vigor in nutrient solution according to following formula:
(2mmol/L) extension rate before * sample test * 1000.The results are shown in Figure 13.
As seen from the figure: the LDH amount discharged in the cell culture fluid of control group is very low, and in two groups of experiments, the LDH activity is respectively 22.87 ± 3.76U/L and 38.17 ± 6.52U/L.25 μ M A β
25-35after treatment S H-SY5Y cell 24h, the LDH amount discharged in nutrient solution significantly increases (P<0.001), and 25 μ M A β are described
25-35membrane structure to the SH-SY5Y cell has caused certain damage, and the part LDH in tenuigenin is discharged in nutrient solution through the cytolemma damaged.With A β
25-35model group is compared, the royal jelly protein polypeptide of the 1-3kDa of three kinds of different concns all can make the LDH burst size in cell culture fluid significantly reduce (P<0.01), and, along with the increase of royal jelly protein polypeptide concentration, the LDH burst size reduces gradually, show significant concentration dependent relation.With A β
25-35model group is compared, and in the experiment of this group, the royal jelly protein polypeptide of the 1-3kDa of 92 μ g/ml can farthest suppress the release of LDH, and inhibiting rate is in 66% left and right.
The two method flow cytometers that dye of Annexin V-FITC/PI detect apoptosis rate: the SH-SY5Y cell in the vegetative period of taking the logarithm, adjusting cell density is 8.5 * 10
4cells/ml, 2ml/well is inoculated into 6 well culture plates, and 37 ℃, 5% CO
2cultivate 24h in incubator, after 24h, inhale and abandon old substratum, Normal group and A β
25-35the every hole of model group adds respectively 2ml serum-free DMEM/F12 substratum, and royal jelly protein polypeptide protection group adds 2ml to contain the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa that different concns embodiment 1 to 4 makes.After 4h, Normal group changes and adds 2ml serum-free DMEM/F12 substratum, A β
25-35model group is changed the A β that adds 2ml 25 μ mol/L
25-35serum-free DMEM/F12 substratum, royal jelly protein polypeptide protection group is changed and is added 2ml simultaneously containing 25 μ mol/L A β
25-35the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa made with different concns embodiment 1 to 4.After drug effect 24h, sucking-off is cultivated based on preserving in the EP pipe, with containing the trysinization 30s of EDTA, with 1ml, containing the cold PBS of 2% serum, blows and beats gently afterwards, then with the substratum of previous step sucking-off, mixes, and 4 ℃ of centrifugal 10min of lower 1000r/min, abandon supernatant.Add 1ml to contain the cold PBS of 2% serum, light shaking makes cell resuspended, and 4 ℃ of centrifugal 10min of lower 1000r/m, abandon supernatant.Repeat previous step once, then cell is resuspended in 1 * binding buffer that 500 μ l have diluted, transfer to streaming loading pipe, add 25 μ l Annexin V-FITC (final concentration is 0.5 μ g/ml).Normal temperature lucifuge 15min or 4 ℃ of lucifuge 30min, add 5 μ l propidium iodide (Propidium iodide, PI, final concentration 0.5-1.0 μ g/ml), mix, with FACS Calibar flow cytometer (U.S. Becton Dickinson), selective exitation wavelength 488nm, emission wavelength 530nm detects.Adopt the Cell-Quest data processing software, obtain 10000 cell analysis.The results are shown in Figure 14, result is summed up and is seen Figure 15.
As seen from the figure: the early apoptosis rate of control group cell and late period apoptosis rate less, only account for 5.32 ± 0.98%-6.88 of total cellular score ± 0.37%.The A β of 25 μ M
25-35after processing 24h, obvious apoptosis appears in the SH-SY5Y cell, the early apoptosis rate and late period apoptosis rate account for 26.39 ± 2.24%-36.71 of cell count ± 1.47%, show on streaming figure to be fourth quadrant and first quartile cell count showed increased (P<0.001).The A β of 25 μ M
25-35after the royal jelly protein polypeptide acting in conjunction 24h of the 1-3kDa of different concns, the overall apoptosis rate of SH-SY5Y cell descends to some extent, and along with the increase of royal jelly protein polypeptide concentration, the early apoptosis rate and late period apoptosis rate reduce gradually, be certain concentration dependent relation.When royal jelly protein polypeptide concentration is 92 μ g/ml, the early apoptosis rate and late period apoptosis rate drop to 6.97 ± 1.01% (P<0.001) from 26.39 ± 2.24%, approach the level of blank group.Explanation thus, the royal jelly protein polypeptide of 1-3kDa is to A β
25-35the SH-SY5Y apoptosis of inducing has certain restraining effect.
The generation of active oxygen radical (ROS) is measured with the DCFH-DA detecting probe method: the SH-SY5Y cell in the vegetative period of taking the logarithm, adjusting cell density is 8.5 * 10
4cells/ml, 2ml/well is inoculated into 6 well culture plates, and 37 ℃, 5% CO
2cultivate in incubator, after 24h, inhale and abandon old substratum, Normal group and A β
25-35the every hole of model group adds respectively 2ml serum-free DMEM/F12 substratum, and royal jelly protein polypeptide protection group adds 2ml to contain the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa that different concns embodiment 1 to 4 makes.After 4h, Normal group changes and adds 2ml serum-free DMEM/F12 substratum, A β
25-35model group is changed the A β that adds 2ml 25 μ mol/L
25-35serum-free DMEM/F12 substratum, royal jelly protein polypeptide protection group is changed and is added 2ml simultaneously containing 25 μ mol/L A β
25-35the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa made with different concns embodiment 1 to 4.After drug treating 24h, inhale and abandon old substratum, wash one time with serum free medium, every hole adds the serum free medium containing DCFH-DA (final concentration 20 μ mol/L) of 2ml preparation in 1: 1000,37 ℃, 5% CO
220min in incubator, jiggle every 5min.After serum free medium is washed three times, trysinization 30s, resuspended with the piping and druming of the PBS containing 2% serum cell.Use FACS Vantage SE flow cytometer (U.S. Becton Dickinson), selective exitation wavelength 488nm, emission wavelength 525nm detects.The results are shown in Figure 16.
As seen from the figure, the A β of 25 μ M
25-35process 24h and can make the ROS in the SH-SY5Y cell significantly raise (P<0.001), fluorescence intensity is elevated to 19.99 ± 0.59 from 6.27 ± 0.60.The A β of 25 μ M
25-35after the royal jelly protein polypeptide co-treatment SH-SY5Y cell 24h of the 1-3kDa of different concns, the generation of ROS all has decline, and along with the increase of concentration separately, and the generation of ROS is and first reduces the trend increased afterwards.For the royal jelly protein polypeptide treatment group of 1-3kDa, it is minimum that the dosage of 46 μ g/ml can make the generation of ROS drop to, and from 19.99 ± 0.59, is reduced to 14.06 ± 0.34 (P<0.001).The royal jelly protein polypeptide that shows 1-3kDa can stop A β
25-35the generation of the SH-SY5Y intracellular reactive oxyradical (ROS) of inducing.Dose concentration is within the specific limits the time, and along with the increase of royal jelly protein polypeptide concentration, the ROS growing amount is the trend reduced; When royal jelly protein polypeptide concentration continues to increase, the ROS growing amount rises again gradually.
Western blotting detects the expression amount of Bcl-2 and Bax: select Bcl-2 and Bax antibody that primary antibodie is the mouse source, with the expression amount of Bcl-2 and Bax in the method detection SH-SY5Y different treatment group of western blotting.The SH-SY5Y cell of taking the logarithm vegetative period, adjusting cell density is 8.5 * 10
4cells/ml, 2ml/well is inoculated into 6 well culture plates, and 37 ℃, 5% CO
2cultivate in incubator, after 24h, inhale and abandon old substratum, Normal group and A β
25-35the every hole of model group adds respectively 2ml serum-free DMEM/F12 substratum, and royal jelly protein polypeptide protection group adds 2ml to contain the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa that different concns embodiment 1 to 4 makes.After 4h, Normal group changes and adds 2ml serum-free DMEM/F12 substratum, A β
25-35model group is changed the A β that adds 2ml25 μ mol/L
25-35serum-free DMEM/F12 substratum, royal jelly protein polypeptide protection group is changed and is added 2ml simultaneously containing 25 μ mol/L A β
25-35the serum-free DMEM/F12 substratum of the royal jelly protein polypeptide of the 1-3kDa made with different concns embodiment 1 to 4.After drug treating 24h, cold PBS washes twice, collect cell, and with lysis buffer (10% glycerine, the Tris-HCl of 50mM pH=6.8,2% beta-mercaptoethanol, 0.02% tetrabromophenol sulfonphthalein, 2% or 5% SDS) at 100 ℃ of lower cracking 10min, ice bath 5min, after vortex mixes and gently gets rid of, loading or-80 ℃ frozen standby.
Select 15% SDS-PAGE separation gel, 10 μ l/ hole loadings, press 100V 20min, the method electrophoretic separation of 160V 1.5h.0.5% skim- milk sealing 2h, 4 ℃ of reactions of the primary antibodie of respective concentration are spent the night, and under two anti-normal temperature, react 1.5h.ECL sent out the expression amount that detection system detects Bcl-2 and Bax, UMAX1120 scanner scanning, the quantitative analysis of ImageJ software.The results are shown in Figure 17, Figure 18.
As seen from the figure, the A β of 25 μ M
25-35after processing 24h, in the SH-SY5Y cell, the value of Bax/Bcl-2 significantly improves, and becomes 2.24 ± 0.18 times (P<0.001) of control group, and the A β of 25 μ M
25-35all can reduce the value of Bax/Bcl-2 with the royal jelly protein polypeptide acting in conjunction 24h of the 1-3kDa of different concns, and dose concentration is between 11.5-92 μ g/ml the time, along with the increase of concentration has the trend reduced gradually, is certain dose-dependently; When concentration is 46 μ g/ml, reducing to minimum level, is 0.65 ± 0.05 times (P<0.001) of control group.When concentration is 92 μ g/ml, the value of Bax/Bcl-2 has again the trend of slight increase.
The A β of 25 μ M
25-35after processing 24h, in the SH-SY5Y cell, the expression amount of Bax albumen increases significantly, becomes 1.69 ± 0.14 times (P<0.001) of control group, and the A β of 25 μ M
25-35all can reduce the expression amount of Bax with the royal jelly protein polypeptide acting in conjunction 24h of the 1-3kDa of different concns, and dose concentration is between 11.5-92 μ g/ml the time, along with the expression amount of the increase Bax of concentration has the trend reduced gradually, is certain dose-dependently; When concentration is 46 μ g/ml, reducing to minimum level, is 0.38 ± 0.04 times (P<0.001) of control group.When concentration is 92 μ g/ml, the expression amount of Bax has again the trend of slight increase.The expression amount of Bcl-2 albumen is irregular variation, and control group and A β
25-35model group, A β
25-35difference all not significantly (P>0.05) between the royal jelly protein polypeptide treatment group of the 1-3kDa of model group and different concns.The royal jelly protein polypeptide that shows 1-3kDa can stop A β
25-35in the SH-SY5Y cell of inducing, the rising of Bax/Bcl-2 value all is certain concentration dependent relation in a certain concentration range.The optimal concentration of the RJP of 1-3kDa is 46 μ g/ml.The reason that the royal jelly protein polypeptide of 1-3kDa reduces the Bax/Bcl-2 value is mainly the royal jelly protein polypeptide and can suppresses significantly A β
25-35induce the rising of Bax expression amount in the SH-SY5Y cell.
Above-mentioned all data adopts the SPSS13.0 statistical software through the row statistical study, and data are used
mean, relatively use one-way analysis of variance (One-way ANOVA) check between group, significant difference is thought in P<0.05 o'clock.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. the preparation method of a royal jelly polypeptide, is characterized in that, comprising:
Step 1: by the queen bee nit enteron aisle of 2～3 ages in days, add phosphate buffer soln or the Tris-HCl buffered soln that the pH value is 7.0～7.2 to grind to form homogenate, at 18000～25000g, centrifugal 20～30min under 4 ℃ of conditions, collect the middle level liquid after centrifugal for the first time, then at 18000～25000g, centrifugal 20～30min under 4 ℃ of conditions, collect the middle level liquid after centrifugal for the second time, obtain enteron aisle enzyme liquid;
Step 2: in royal jelly, add phosphate buffered saline buffer or Tris-HCl buffered soln that the pH value is 7.0～7.2, at 18000～25000g, centrifugal 20～30min under 4 ℃ of conditions, collect supernatant liquor, in the phosphate buffered saline buffer that the dialysis tubing that is 8000～14000Da through molecular weight cut-off is 7.0～7.2 in the pH value or Tris-HCl buffered soln, ice bath dialysis 42～72h, collect dialyzate in dialysis tubing, obtains the water soluble royal jelly protein liquid;
Step 3: in mg/mL, by the enteron aisle enzyme liquid of step 1 gained, with the water soluble royal jelly protein liquid of step 2 gained, according to the protein concentration ratio, be that mix 30～35:80～90, in the pH value, be 8.3～8.7, enzymolysis 22～26h under the condition that temperature is 34～39 ℃, obtain the water soluble royal jelly protein enzymatic hydrolyzate;
Step 4: after the described water soluble royal jelly protein enzymatic hydrolyzate of step 3 ice bath is stopped to enzyme digestion reaction, at 8000～15000g, centrifugal 10～20min under 4 ℃ of conditions, collect supernatant liquor, with after 10 μ m filtering with microporous membrane, collect filtrate and obtain the first filtrate, ultrafiltration membrance filter by described the first filtrate through 3kDa, collect filtrate and obtain the second filtrate, the ultrafiltration membrance filter by described the second filtrate through 1kDa, collect the royal jelly polypeptide that trapped substance obtains 1～3kDa.
2. preparation method as claimed in claim 1, is characterized in that, in step 1 or step 2, the concentration of phosphate buffered saline buffer or Tris-HCl buffered soln is 50mmol/L.
3. preparation method as claimed in claim 1, is characterized in that, the mass volume ratio of the enteron aisle of queen bee nit described in step 1 and phosphate buffered saline buffer or Tris-HCl buffered soln is counted 1:1～1:2 with g/mL.
4. preparation method as claimed in claim 1, is characterized in that, in step 2, the volume ratio of phosphate buffered saline buffer or Tris-HCl buffered soln and described royal jelly is 2:1～4:1.
5. preparation method as claimed in claim 1, is characterized in that, in mg/mL, in step 3, described enteron aisle enzyme liquid is 32.5:85 with the protein concentration ratio of described water soluble royal jelly protein liquid.
6. preparation method as claimed in claim 1, is characterized in that, in step 3, hydrolysis temperature is 37 ℃.
7. preparation method as claimed in claim 1, is characterized in that, in step 3, enzymolysis pH value is 8.5.
8. preparation method as claimed in claim 1, is characterized in that, in step 3, enzymolysis time is 24h.
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| CN102251005A (en) | 2011-11-23 |
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