CN103919810A - Method for preparing long-noded pit viper water-soluble total protein lyophilized powder - Google Patents
Method for preparing long-noded pit viper water-soluble total protein lyophilized powder Download PDFInfo
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Abstract
The invention discloses a method for preparing long-noded pit viper water-soluble total protein lyophilized powder. The method comprises the following steps: drying and crushing long-noded pit viper to prepare long-noded pit viper powder; mixing the long-noded pit viper powder and water in a mass ratio of 1:(5-20); decocting in a closed environment at 60-100 DEG C for 20-60 minutes; centrifuging the extract, and cooling the supernate to 0 DEG C; slowly adding solid ammonium sulfate until the saturation degree is 40-60 percent; standing at 4 DEG C for 2 hours, centrifuging, collecting precipitate, dissolving the precipitate with water, and dialyzing by using ultrapure water as dialyzate; replacing the dialyzate until the permeate does not contain ammonium sulfate; filtering a trapped fluid, lyophilizing the filter cake to obtain the long-noded pit viper water-soluble total protein lyophilized powder. The preparation process is simple, the condition is mild, prepared long-noded pit viper lyophilized powder is high in effective component content and can be better absorbed by a human body and play a better drug effect; and soluble crude protein with high purity can be obtained by precipitating the ammonium sulfate, and high protein activity can be maintained.
Description
(1) technical field
The present invention relates to a kind of preparation method of protein freeze-dried powder, particularly a kind of preparation method of Agkistrodon water solublity total protein lyophilized powder.
(2) background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of general autoimmune disease that aggressivity arthritis is main manifestations of take.Main clinical manifestation is symmetry, persistence arthroncus and pain, often stiff with morning, the joint of getting involved is with proximal interphalangeal joint, metacarpophalangeal joints, wrist, elbow and toes joint are the most common, and the extra-articular manifestations such as heating, anemia, subcutaneous nodule and lymphadenectasis can appear in some patients were.This disease is apt to occur in female middle-aged, and disability rate is higher, and the disability rate in 1 year of falling ill can be up to 20%, 10 year Nei Keda 60% left and right.In China, RA prevalence is 0.3-0.6%, and the sickness rate in the adult of Er developed country is up to 0.5%-1%.Modern medicine mainly comprises that for the Drug therapy of RA NSAID (non-steroidal anti-inflammatory drug) (NSAIDs), the state of an illness improve antirheumatic (DMARDs), glucocorticoid and biological preparation, but effect individual variation is obvious, toxic and side effects is larger, and this is the subject matter that clinical use antirheumatic faces.
Rheumatoid arthritis belongs to Chinese medical ' arthralgia syndrome ' category, how by positive QI-insufficiency, experiences due to the heresy of wind, cold, wet, heat, is often chronic progressive external process, and the course of disease is long, and the how obstinate difficulty of its card.The Qing Dynasty's leaf sky scholar point out: " wet three gas of wind and cold are combined into numbness, and for years, exopathogen stays work, and QI and blood is all hindered, and turn to and lose the solidifying expectorant of the stasis of blood, and mixed place meridians, must search and pick with insects, to move medicine, make blood without coagulating, and gas can be declared logical." Agkistrodon is conventional insect medicine, has another name called Agkistrodon, is the dry body of Viperidae animal Agkistrodon, sweet in the mouth, salty, property, temperature.Poisonous, enter liver spleen two warps, energy expel wind to dredge collateral, counteracting toxic substances arresting convulsion.Benefaction and nowhere less than, reach skin outward, interior the meridian dredging, and deep power of searching wind is especially strong, is called as cut-off wind key medicine, has remarkable result for treatment arthromyodynia.Modern study finds, Agkistrodon can promote the synthetic of antepituitary thyroliberin and discharge, and this hormone concentration in blood is raise, and produces antiinflammatory, Repercusion analgesia effect, and without the side effect of hormonelike.But Agkistrodon has now been listed in national secondary endangered watching for animals, medical material is famous and precious, and price is higher, and it is limited to originate, and artificial cultivation is more difficult, therefore, how at utmost to utilize the medical value of Agkistrodon, is clinical use Agkistrodon urgent problem.
The inventor thinks and should utilize modern advanced science and technology to excavate the magistery of conventional medicament, the dosage form of make easy easy clothes, easily promoting, make medicine better be absorbed by the body simultaneously, with less medical material, bring into play maximum effect, and then give full play to its therapeutical effect to rheumatoid arthritis.
(3) summary of the invention
Chinese medicine is used as medicine mainly with decoct, powder, exist effective ingredient absorb not good enough, be difficult for preserving and the curative effect deficiency such as built on the sand.The present invention is intended to overcome above deficiency, improves the processing technology of Agkistrodon water solublity total protein lyophilized powder, and making can good absorption, preserve for a long time and the lyophilized powder dosage form of stable curative effect, thereby has guaranteed effective utilization and the clinical expansion of Chinese medicine.Meanwhile, by the zoopery comparison of different dosage form, confirm that it is nontoxic, have no side effect, and the more traditional decoct of curative effect and powder more excellent.
The technical solution used in the present invention is:
The invention provides a kind of preparation method of Agkistrodon water solublity total protein lyophilized powder, described method is: Agkistrodon (preferably Agkistrodon decoction pieces) is dry, pulverize, make Agkistrodon powder, Agkistrodon powder is mixed with mass ratio 1:5～20 with water, airtight decoction 20～60 minutes (preferably 60 ℃ decoct 30min) under 60～100 ℃ of conditions, extracting solution is centrifugal, get supernatant and be cooled to 0 ℃, then slowly adding solid ammonium sulfate to saturation is 40%～60%, 4 ℃ standing 2 hours, centrifugal, collecting precipitation, the ultra-pure water (preferably MiliQ ultra-pure water) of take after precipitation is dissolved in water is dialysed as dialysis solution, change dialysis solution to liquid containing ammonium sulfate not in permeate, get trapped fluid, filter, filter cake lyophilization, obtain Agkistrodon water solublity total protein lyophilized powder.
Further, preferred described water is MiliQ ultra-pure water.
Further, the mass ratio of preferred described Agkistrodon powder and water is 1:15～20, more preferably 1:20.
Further, the cryodesiccated condition of preferred described filter cake is :-50～-70 ℃ of ℃ of lyophilization 24～48h, more preferably-60 ℃ of lyophilization 48h.
Further, the preparation method of Agkistrodon water solublity total protein lyophilized powder of the present invention recommends to carry out as follows: Agkistrodon is dry, being crushed to particle diameter is 60～100 orders, make Agkistrodon powder, Agkistrodon powder is mixed with mass ratio 1:20 with MiliQ ultra-pure water, at 60 ℃ of airtight decoction 30min, by decoction liquor at 4 ℃, centrifugal 10min under 1200 * g condition, get supernatant and be cooled to 0 ℃, then slowly adding solid ammonium sulfate to saturation is 60%, 4 ℃ standing 2 hours, centrifugal, collecting precipitation, precipitation is added and after MiliQ ultra-pure water dissolves, take MiliQ ultra-pure water and dialyse as dialysis solution, change dialysis solution to liquid containing ammonium sulfate not in permeate, get trapped fluid, with 0.45 μ m membrane filtration, filter cake is at-50～-70 ℃ of lyophilization 24～48h, obtain Agkistrodon water solublity total protein lyophilized powder, described dialysis is to carry out in the bag filter of molecular cut off 1000.
First the present invention will extract Agkistrodon water solublity total protein, then be lyophilized into lyophilized powder; Set up the rat model of arthritis of II Collagen Type VI induction; Use respectively the Agkistrodon lyophilized powder high and low dose of extracting, Agkistrodon decoct high and low dose, Agkistrodon powder high and low dose is fed CIA model; Adopt toes volumetric measurement method to detect CIA rat model medication front and back biped ankle swelling degree, and carry out arthritis index scoring; Rat foot is carried out to roentgen radiation x, observe the variation of its tissue morphology; By ELISA method, detect the concentration of IL-1 β in rat blood serum; Make ankle joint pathological section, its pathological change is observed in HE dyeing.
The dosage of Agkistrodon water solublity total protein lyophilized powder of the present invention in preparation treatment rheumatic medicine is recommended as 1.72g/kg.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the invention provides a kind of preparation method of preparing Agkistrodon water solublity total protein lyophilized powder, this preparation process condition is simple, mild condition, gained Agkistrodon lyophilized powder active constituent content is high, can be absorbed by the body better, the larger drug action of performance simultaneously; The present invention can extract by ammonium sulfate precipitation the solubility crude protein that purity is higher, can keep its biological activity simultaneously, for the separated procedure of processing of follow-up purification provides reliable raw material, this method contrast trifluoroacetic acid/acetone precipitation extraction scheme, not only reduce total protein loss, keep higher protein active simultaneously.
(4) accompanying drawing explanation
Fig. 1 is the impact of Agkistrodon different dosage form on CIA rat arthritis index.
Fig. 2 takes the photograph sheet comparison for each group rat ankle joint x-ray, and A is blank group, and B is model group, and C is the low metering group of decoct, and D is decoct high dose group, and E is the low metering group of lyophilized powder, and F is lyophilized powder high dose group, and G is the low metering group of powder, and H is powder high dose group.
Fig. 3 is HE dyeing photo of each group rat ankle joint pathological tissue, and A is Normal group (HE dyeing, 20 *), B is model group (HE dyeing, 20 *), and C is (the HE dyeing of decoct low dose group, 20 *), D is decoct high dose group (HE dyeing, 20 *), E is lyophilized powder low dose group (HE dyeing, 20 *), and F is (the HE dyeing of lyophilized powder high dose group, 20 *), G is powder low dose group (HE dyeing, 20 *), H is powder high dose group (HE dyeing, 20 *).
Fig. 4 is the electrophoretogram of the Agkistrodon water solublity total protein that obtains under different temperatures, and a is molecular weight Mark, and b and c are the extracting solution that embodiment 1 obtains, and d and e are the extracting solution that embodiment 2 obtains, and f and g are the extracting solution that embodiment 3 obtains.
Fig. 5 is the electrophoretogram of the Agkistrodon water solublity total protein that obtains under different time, and a is 70 ℃ and extracts the extracting solution that obtain for 20 minutes; B is 70 ℃ and extracts the extracting solution obtaining for 40 minutes; C is 70 ℃ and extracts the extracting solution obtaining for 60 minutes.
(5) specific embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: Agkistrodon water solublity total protein lyophilized powder
Take Agkistrodon decoction pieces (purchased from decoction pieces factory of Zhejiang University of Traditional Chinese Medicine) 5g, pulverize, cross 100 orders, make Agkistrodon powder, dry 30 minutes for 60 ℃, actual weight is 4.976g, is placed in hermetic container, add 100mlMiliQ ultra-pure water, mix, 60 ℃ decoct 40 minutes, extracting solution are transferred to centrifuge tube (50ml), 4 ℃, the centrifugal 10min of 1200 * g, get supernatant and obtain crude protein; Crude protein is cooled to 0 ℃, progressively add solid ammonium sulfate to saturation 60%, 4 ℃ standing 2 hours, centrifugal 30 minutes of 1200 * g, collecting precipitation, MiliQ ultra-pure water is dissolution precipitation again, pack in bag filter (molecular cut off 1000), take MiliQ ultra-pure water as dialysis solution, 4 ℃ of dialysis desaltings, repeatedly change dialysis solution to permeate and do not detect ammonium sulfate, obtain protein extract (being trapped fluid), with 0.45 μ m membrane filtration degerming, filter cake is at-60 ℃ of lyophilizing 48h, obtain Agkistrodon water solublity total protein lyophilized powder 0.198g, standby.
Embodiment 2
Change the decoction temperature in embodiment 1 into 80 ℃, other operate with embodiment 1, obtain Agkistrodon water solublity total protein lyophilized powder 0.207g.
Embodiment 3
Change the decoction temperature in embodiment 1 into 100 ℃, other operate with embodiment 1, obtain Agkistrodon water solublity total protein lyophilized powder 0.213g.
Utilize BCA protein concentration detection method, the extracting solution that embodiment 1-3 decoction is finished to rear acquisition carries out the detection (SDS-PAGE silver dyes) of protein concentration, it is 5mg/ml that different temperatures acquisition extracting solution is unified loading concentration, be specially: (1) is fixing: under room temperature, 10% glacial acetic acid fixative (glacial acetic acid: 150ml; Distilled water: 1350ml, concussion 30min left and right).(2) wash glue: the offset plate through fixing is placed in to the vinyl disc that fills 1.5L distilled water, cleans twice, each 2-4min.(3) dyeing: dyeing liquor (silver nitrate: 1.5g(0.1wt%), 37wt% methanol 2.25ml, add distilled water to 1.5L) concussion 30min left and right.(4) colour developing: nitrite ion (sodium carbonate: 45g (final concentration 0.03g/ml), volumetric concentration 37% formalin 2.25ml(final concentration 0.15%), sodium thiosulfate 300 μ l (final concentration 10mg/ml), add distilled water to 1.5L).(5) color development stopping: distilled water cleans.
Use instrument:
tetra Cell gel-electrophoretic apparatus, wherein b, d, f applied sample amount are 1 μ l, c, e and g applied sample amount are 2 μ l, the results are shown in Figure shown in 4.60 ℃ of extracting solution (b, c) have many obvious high molecular weight protein bands in 66-116KD interval; 80 ℃ of extracting solution (d, e) are higher at the protein content of 25kD left and right; 100 ℃ of extracting solution (f, g) are higher at the protein content of 25kD left and right, at 18.4kD place band content, increase.
Conclusion: rising temperature makes Agkistrodon water solublity total protein be decomposed into small molecular protein polypeptide.
Embodiment 4: the impact of extraction time on Agkistrodon water solublity total protein
Embodiment 1 is decocted to temperature and change 70 ℃ into, decocting time is followed successively by 20 minutes, 40 minutes and 60 minutes, obtains 3 parts of extracting solution, and extracting solution is carried out respectively to determination of protein concentration, the results are shown in Figure shown in 5.On the impact of total soluble protein in extracting solution, (silver dyes decocting time, applied sample amount 2 μ l): extend with extraction time, same position protein band color burn, show that content increases (contrast in 40 minutes 20 minutes), extraction time continues to increase, micromolecule band occurs, shows to continue to be heated to the decomposition of albumen (contrast in 60 minutes 40 minutes).
Embodiment 5: the intervention effect of Agkistrodon water solublity total protein lyophilized powder to rheumatoid arthritis rat
(1) materials and methods
1. laboratory animal
Wistar rat, female, body weight 160 ± 20g,, is provided credit number by totally 70 by Zhejiang University of Traditional Chinese Medicine's Experimental Animal Center: SYXK (Zhejiang) 2008-0115.Isolated rearing under totally-enclosed SPF state, 20 ℃ of room temperatures, relative humidity 40～60%, illumination every day 12h, freely ingests, drinks water.
2. experimental drug
Agkistrodon, purchased from Chinese Herbal Pieces Factory of Zhejiang Chinese Medical University, is dried medical material 250g.
3. experiment reagent
Cattle II Collagen Type VI is purchased from Chondrex company; Freund's complete adjuvant, not formula Freund's incomplete adjuvant is purchased from Sigma company; Rat il-1 β (IL-1 β) ELISA test kit is purchased from R & D company; Large mouse-anti cattle II Collagen Type VI antibody ELISA test kit is purchased from Chondrex company.
4 experimental apparatus
YLS-7A toes capacity measurer (An Hemeng development in science and technology company limited), multi-functional microplate reader (Thermo Scientific company), small animal imaging instrument, pathological section, microscope.
5. experimental technique
5.1 modeling method
Choose 70 of SPF level Healthy female wistar rats, adaptability is fed one week.Modeling method with reference to CIA rat in < < Current Protocols in Immunology > >, specific as follows: cattle II Collagen Type VI 8ml, be dissolved in 0.05mol/L, pH3.2 glacial acetic acid aqueous solution, modeling preparation the previous day, 4 ℃ of refrigerator shaken overnight.Second day, mixes with isopyknic complete Freund's adjuvant, with the emulsifying repeatedly of syringe method, makes the emulsion (being collagen Emulsion) containing cattle II Collagen Type VI 1.0mg/mL.Choose at random 8 as blank group, remaining 62 rats all carry out initial immunity, and minute multiple spot is in rat root of the tail portion subcutaneous injection collagen Emulsion, and 0.2mL/ only.After initial immunity 1 week, again configure collagen Emulsion, change complete Freund's adjuvant into incomplete Freund's adjuvant, all the other methods are the same, and the collagen Emulsion that then contains cattle II Collagen Type VI 1.0mg/mL in the difference injection of rat root of the tail portion carries out booster immunization, and 0.15mL/ only.During first and booster immunization, the normal saline of 8 rat skin lower injection equivalent of blank group.
5.2 medicine preparations
Agkistrodon decoct: 43g Agkistrodon decoction pieces is placed in to 300ml distilled water, and 100 ℃ of decocting and concentrating are to 100ml, and obtaining concentration is the Agkistrodon water decoction of 0.43g/ml; 86g Agkistrodon decoction pieces is placed in to 300ml distilled water, and 100 ℃ of decocting and concentrating are to 100ml, and obtaining concentration is the Agkistrodon water decoction of 0.86g/ml.
Agkistrodon powder: Agkistrodon decoction pieces is worn into powder, and 100 orders sieve, and are dissolved in distilled water, prepares respectively the Agkistrodon suspension of 0.145g/ml and 0.29g/ml.
5.3 grouping and administrations
After initial immunity 20 days, 56 experimental rat arthritis index surpass 6 minutes (comprising 6 minutes), it is modeling success, then the successful rat of modeling is divided into totally 7 groups of Agkistrodon decoct low dose group, Agkistrodon decoct high dose group, Agkistrodon powder low dose group, Agkistrodon powder high dose group, Agkistrodon lyophilized powder low dose group, Agkistrodon lyophilized powder high dose group, model group, 8 every group according to foot swelling degree after two with randomized blocks.
After grouping, start gastric infusion and observe, gavage dosage is 2ml/, and every day 1 time, administration is 24 days altogether.According to laboratory animal drug dose, converse drug level: Agkistrodon decoct low dose group 0.86g/kg, Agkistrodon decoct high dose group 1.72g/kg, Agkistrodon lyophilized powder low dose group 0.25g/kg, Agkistrodon lyophilized powder high dose group 0.50g/kg, the loose low dose group 0.29g/kg of Agkistrodon, the loose high dose group 0.58g/kg of Agkistrodon.Equivalent distilled water gavage for blank group and model group.
6. observation index and method
6.1 ordinary circumstance
Modeling, the variation of administration fore-and-aft observing rat body weight, pattern of activity, chaeta color and luster, changes in diet, excretion situation etc.
6.2 toes swellings
With toes capacity measurer, measure the swelling of the two metapedes of rat, using the sufficient cubical content of the two metapedes of rat as the index of foot swelling.Initial immunity pre-test 1 time, measures weekly 1 time afterwards.
Swelling (%)=(modeling metapedes volume-modeling front foot volume)/modeling front foot volume
6.3 arthritis index (AI)
After initial immunity, start to observe and record rat extremities joint lesion degree, every 7d1 time.5 grades of scoring system in post-therapeutic evaluations of reference: 0 minute: without arthritis; 1 minute: little toe joint mild swelling; 2 minutes: little toe joint and pedal swelling; 3 minutes: the sufficient pawl swelling below ankle joint; 4 minutes: comprise whole arthroncuss of ankle joint.Accumulation score 6 minutes and above person's modeling success, be up to 16 minutes.
6.4 serum il-1 β, the antibody test of anti-cattle II Collagen Type VI
After administration 35 days, 10% chloral hydrate intraperitoneal injection of anesthesia, through abdominal aortic blood, 3000r/ minute centrifugal rear collection serum, subpackage is preserved.In strict accordance with test kit description, detect IL-1 β, anti-cattle II Collagen Type VI antibody content.
6.5 small animal imaging systems
After administration 35 days, rat is put to death in anesthesia, gets bilateral ankle joint, is placed on small animal imaging instrument, sets parameter, and x-ray is taken, and observes its change.
6.6 ankle joint pathological observations
X-ray is put into joint 10% formalin after taking in time, fixes one week, specimens paraffin embedding slices, HE dyeing, micro-Microscopic observation pathological change.
6.7 statistical procedures
Measurement data represents by average ± standard deviation; Between many groups, relatively adopt method of analysis of variance, during homogeneity of variance, between group, relatively adopt between two LSD-t method, during heterogeneity of variance, adopt Kruskal-wallis H check.All adopt two-sided test, P < thinks statistical significance, adopts SPSS17.0 statistical software to carry out statistical analysis.
(2) result
1. respectively organize rat treatment front and back bilateral toes swelling testing result
After modeling, model group is compared with Normal group, and left back paw swelling obviously increases (P<0.01); After administration two weeks, compare with model group, each group is all on a declining curve, and wherein low, the left back paw swelling of high dose group of decoct low dose group, lyophilized powder high dose group, powder declines and has statistical significance (P<0.05, P<0.01); After administration three weeks, compare with model group, each is organized left back paw swelling and continues to decline, and wherein low, the high dose group of low, the high dose group of decoct, lyophilized powder high dose group, powder declines and has notable difference (P<0.05, P<0.01).(in Table 1)
Table 1 Agkistrodon different dosage form on the impact of CIA rat left hind paw swelling (
%, n=8)
Note: with Normal group comparison, △ △ P<0.01; With model group comparison, * P<0.05, * * P<0.01.
After modeling, model group is compared with Normal group, and right back paw swelling obviously increases (P<0.01); After administration one week, compare with model group, decoct low dose group, the right back paw swelling of freeze dried powder high dose group decline and have statistical significance (P<0.05); After administration two weeks, compare with model group, each group is all on a declining curve, but only have the right back paw swelling of lyophilized powder high dose group to decline, has statistical significance (P<0.01); After administration three weeks, compare with model group, each is organized right back paw swelling and continues to decline, and wherein low, the high dose group of low, the high dose group of decoct low dose group, lyophilized powder, powder declines and has notable difference (P<0.05, P<0.01).(in Table 2)
Table 2 Agkistrodon different dosage form on the impact of CIA rat right hind leg paw swelling (
%, n=8)
Note: with Normal group comparison, △ △ P<0.01; With model group comparison, * P<0.05, * * P<0.01.
2. respectively organize rat treatment front and back arthritis index appraisal result
This experiment CIA rat arthritis sign starts to occur, before and after drug treatment, peaks for 7 days after booster immunization.After administration 8 days, 12 days and 16 days, though each group has decline in various degree, not statistically significant; After administration 20 days, compare with model group, decoct is low, high dose group, lyophilized powder high dose group, powder high dose group arthritis index decline all statistical significance (P<0.05, P<0.01); After administration 24 days, compare with model group, decoct high dose group, lyophilized powder high dose group, powder high dose group arthritis index decline and have statistical significance (P<0.05, P<0.01).(see Fig. 1, (
n=8), with model group comparison, * P<0.05, * * P<0.01)
3. respectively organize rat blood serum IL-1 β, anti-cattle II Collagen Type VI antibody test result
Experimental result shows, compares with Normal group, and each content of organizing serum il-1 β significantly increases (P<0.01); Compare with model group, each content of organizing serum il-1 β is all lower, and wherein low, the high dose group of low, the high dose group of decoct, lyophilized powder declines and has statistical significance (P<0.05, P<0.01).And the content of respectively organizing serumanti-typeⅡcollagenantibody is compared all lower with model group, but only have, decoct high dose group, lyophilized powder high dose group, powder are low, high dose group declines has statistical significance (P<0.05, P<0.01).(in Table 3)
Table 3 respectively organize serum il-1 β, anti-II Collagen Type VI antibody (
n=8)
Note: with Normal group comparison, △ △ P<0.01; With model group comparison, * P<0.05, * * P<0.01.
4. respectively organize rat ankle joint x-ray and take the photograph sheet observed result
X-ray is taken the photograph sheet result and is shown, Normal group: ankle joint complete form, and sclerotin is not destroyed; Model group: ankle joint swollen tissue is obvious, and bone destruction is serious, has many places cortical bone interrupted; Each administration group: the ankle joint swollen tissue of decoct high dose group, lyophilized powder high dose group is lighter, bone destruction is also not serious, and other medication group swelling are all obvious, and bone destruction is serious, and cortical bone many places are not connected.(see figure 2).
5. respectively organize rat ankle joint pathology section examination result
Experimental result shows, Normal group: the surface-coated thin layer hyaline cartilage of ankle joint cell, and cellular morphology is complete, and top layer synovial cell arranges evenly, without obvious hypertrophy, without lymphocyte, inflammatory cell infiltration; Model group: ankle joint pathological change is obvious, and cartilage layers is destroyed, during synovial cell has, degeneration and the hypertrophy of severe, visible massive inflammatory cells infiltrated; Each administration group: ankle joint all has pathological change in various degree, wherein lyophilized powder high dose group cartilage destruction is lighter, hypertrophy that synovial cell is slight, visible a small amount of inflammatory cell infiltration.Compare with lyophilized powder low dose group, powder high dose group, decoct is low, high dose group, powder low dose group cartilage destruction are obvious, synovial cell's moderate hypertrophy and degeneration, and inflammatory cell infiltration is more.(see figure 3).
(3) conclusion
This experiment by and model, medication group between comparison, from paw swelling, arthritis index, serum il-1 β, anti-II Collagen Type VI antibody horizontal, x-ray, take the photograph the aspects such as sheet and pathological section, comparatively system has compared the curative effect of Agkistrodon lyophilized powder various dose to CIA rat, the Agkistrodon lyophilized powder that has confirmed high dose is rapid recovery RA symptom comparatively, stop aggravation, in early stage the controlling inflammation and prevent of RA, aspect destruction of joint, have better curative effect.Therefore,, for saving rare medicinal herbs, bring into play to greatest extent the medical value of Agkistrodon, the Agkistrodon lyophilized powder of this experimental result recommendation high dose (1.72g/kg).
Claims (5)
1. the preparation method of an Agkistrodon water solublity total protein lyophilized powder, it is characterized in that described method is: Agkistrodon is dry, pulverize, make Agkistrodon powder, Agkistrodon powder is mixed with mass ratio 1:5～20 with water, 60～100 ℃ of airtight decoctions 20～60 minutes, extracting solution is centrifugal, get supernatant and be cooled to 0 ℃, then slowly adding solid ammonium sulfate to saturation is 40%～60%, 4 ℃ standing 2 hours, centrifugal, collecting precipitation, the ultra-pure water of take after precipitation is dissolved in water is dialysed as dialysis solution, change dialysis solution to liquid containing ammonium sulfate not in permeate, get trapped fluid, filter, filter cake lyophilization, obtain Agkistrodon water solublity total protein lyophilized powder.
2. the preparation method of Agkistrodon water solublity total protein lyophilized powder as claimed in claim, is characterized in that described water is MiliQ ultra-pure water.
3. the preparation method of Agkistrodon water solublity total protein lyophilized powder as claimed in claim, the mass ratio that it is characterized in that described Agkistrodon powder and water is 1:20.
4. the preparation method of Agkistrodon water solublity total protein lyophilized powder as claimed in claim, is characterized in that the cryodesiccated condition of described filtrate is :-50～-70 ℃ of lyophilization 24～48h.
5. the preparation method of Agkistrodon water solublity total protein lyophilized powder as claimed in claim, it is characterized in that described method carries out as follows: Agkistrodon is dry, being crushed to particle diameter is 60～100 orders, make Agkistrodon powder, Agkistrodon powder is mixed with mass ratio 1:20 with MiliQ ultra-pure water, 60 ℃ of airtight decoctions 30 minutes, by extracting solution at 4 ℃, centrifugal 10min under 1200 * g condition, get supernatant and be cooled to 0 ℃, then slowly adding solid ammonium sulfate to saturation is 60%, 4 ℃ standing 2 hours, centrifugal, collecting precipitation, precipitation is added and after MiliQ ultra-pure water dissolves, take MiliQ ultra-pure water and dialyse as dialysis solution, change dialysis solution to liquid containing ammonium sulfate not in permeate, get trapped fluid, with 0.45 μ m membrane filtration, filter cake is at-50～-70 ℃ of lyophilization 24～48h, obtain Agkistrodon water solublity total protein lyophilized powder, described dialysis is to carry out in the bag filter of molecular cut off 1000.
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Cited By (3)
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|---|---|---|---|---|
| CN107854731A (en) * | 2017-09-30 | 2018-03-30 | 山东省药学科学院 | A kind of preparation method of medical polyester polymers superfines |
| CN109758480A (en) * | 2019-03-27 | 2019-05-17 | 景德镇陈锋特种野生动物科技开发有限公司 | A kind of production method of the long-noded pit viper freeze-dried powder for rheumatoid arthritis |
| CN114191453A (en) * | 2021-12-15 | 2022-03-18 | 湖南永州异蛇生物制药有限公司 | Preparation method of agkistrodon Chinese medicine raw powder |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088096A (en) * | 2012-12-30 | 2013-05-08 | 浙江中医药大学 | Method for extracting collagen type II by taking agkistrodon as raw material |
-
2014
- 2014-03-27 CN CN201410119382.XA patent/CN103919810A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088096A (en) * | 2012-12-30 | 2013-05-08 | 浙江中医药大学 | Method for extracting collagen type II by taking agkistrodon as raw material |
Non-Patent Citations (3)
| Title |
|---|
| 汪世华: "《蛋白质工程》", 29 February 2008, 科学出版社 * |
| 谷恒存 等: "《蕲蛇Ⅱ型胶原蛋白对CIA大鼠的治疗作用》", 《中华中医药学刊》 * |
| 陈振江: "《金钱白花蛇可溶性蛋白凝胶电泳图谱的研究》", 《中草药》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107854731A (en) * | 2017-09-30 | 2018-03-30 | 山东省药学科学院 | A kind of preparation method of medical polyester polymers superfines |
| CN109758480A (en) * | 2019-03-27 | 2019-05-17 | 景德镇陈锋特种野生动物科技开发有限公司 | A kind of production method of the long-noded pit viper freeze-dried powder for rheumatoid arthritis |
| CN114191453A (en) * | 2021-12-15 | 2022-03-18 | 湖南永州异蛇生物制药有限公司 | Preparation method of agkistrodon Chinese medicine raw powder |
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