Background technology:
Rokitamycin is the Novel spiral adm derivative that utilizes the technique for gene engineering development, and former called after Biotechmycin, former name are shengjimycin [patent No.: ZL97104440.6].According to " Chinese adopted drug name nomenclature principle ", to examine and determine through Chinese Pharmacopoeia Commission's technology, the Universal Chinese character name change of Biotechmycin is rokitamycin, English name is Calimycin.
Rokitamycin is the genetic engineering bacterium tunning; its chemical constitution is take 4 "-the isovaleryl spiramycin is main constituent; comprise 4 "-isovaleryl Spiramycin I, II, III, secondly also contain and has an appointment 6 kind 4 "-spiramycin of position hydroxyl acyl group, therefore its chemical name is referred to as 4 " acidylated spiramycin.
The chemical constitution of rokitamycin main constituent is suc as formula shown in (1):
Wherein: R=H, COCH
3, COCH
2CH
3R '=COCH
2CH (CH
3)
2, COCH
2CH (CH
3)
2, COCH
2CH (CH
3)
2
Rokitamycin is 16 ring macrolide antibiotics, and its mechanism of action is to suppress its protein synthesis by being combined with bacterial ribosome.
The in vitro tests result shows, rokitamycin to gram positive bacteria, especially to some fastbacteria (as anti-β-lactams gold Portugal bacterium, anti-erythromycin gold Portugal bacterium etc.) effectively, with similar medicine without obvious cross resistance.It has good antibacterial activity to mycoplasma, chlamydia simultaneously, the part gram-negative bacteria is also had antibacterial activity, and toxoplasma, legionella etc. is had good antibacterial activity and tissue permeability, also has potential immunoregulation effect.Its antibacterial activity in vivo obviously is better than external [patent No.: ZL200310122420.9].Clinical research shows, takes rokitamycin tablet 0.2-0.4g every day 5-7 days, applicable to treatment micrococcus scarlatinae the acute bacterial pharyngitis, the acute suppurative tonsillitis that cause; Bacillary sinusitis, acute bronchitis that sensitive bacterial causes; Light disease pneumonia due to streptococcus pneumoniae, hemophilus influenza and mycoplasma pneumoniae; The non gonococcal urethritis that mycoplasma, chlamydia cause; The infectious disease such as the skin soft-tissue infection that sensitive bacterial causes, periodontitis, otitis media.Its total effective rate is 87.76%, and the untoward reaction rate of rokitamycin is low.
Clinical research proves, rokitamycin is an oral antibiotic safely and effectively.Yet because rokitamycin itself is multicomponent pharmaceutical, the product that obtains by fermentation in the plurality of impurities that sweat produces, brings certain difficulty for the component quantitative assay.The high performance liquid chromatography peak height computing method of setting up at present, can be with several materials that difficulty is separated such as isovaleryl spiramycin II in the rokitamycin sample and (different) butyryl spiramycin III, (different) butyryl spiramycin II separating degree with small component, propionylspiramycin II and the acetylspiramycin III of propionylspiramycin III, propionylspiramycin III and its front, reach that Chinese Pharmacopoeia stipulates more than 1.5; And the separating degree of the small component of acetylspiramycin III and its front is 1.2.The rokitamycin tablet quality control standard of setting up at present is to adopt high-efficient liquid phase analysis peak height computing method, measures 9 acidylated spiramycin components of rokitamycin, and wherein isovaleryl spiramycin (I+II+III) total content should be not less than 65%; The acidylated spiramycin total content should be not less than 80%.Although according to present tablet manufacturing technique, can obtain that the product component ratio is controlled, the reliable rokitamycin of steady quality, yet, in order to improve extraction and purification process, reduced mass testing process, to improve clinical therapeutic efficacy, be necessary to develop the good preparation of the main one-component of isovaleryl spiramycin.Especially for the patient of clinical critical patient or unsuitable oral medication, drug administration by injection takes effect rapidly, more easily accepts.Multicomponent antibiotic for the fermentation generation, be difficult to control its quality according to the chemical drugs quality control method, and any tiny variation, the variation of material base all might be caused, increase the difficulty of finished product quality control standard, cause uncertain bad kickback of using medicine.So still rare so far by the multicomponent injection of antibiotic agent that fermentation produces.
The pharmacokinetic result shows, in rokitamycin, the active component of tool activity is mainly isovaleryl Spiramycin I, II, III.After rokitamycin enters in body, very fast metabolism is spiramycin, with the AUC of parent drug isovaleryl Spiramycin I, II, III and active metabolite Spiramycin I, II, III
0-tSummation is calculated, its oral absolute bioavailability average out to 91.6%.Bibliographical information, the oral absolute bioavailability of spiramycin human body are 30-40%[Frydman AM et al J Antimicrob Chemother.1988,22 (suppl B): 93-103].Illustrate that the structure of isovaleryl spiramycin obviously improved the bioavailability of active component spiramycin.The single medication rokitamycin is eliminated slower, T
1/2 βBetween 23-27 hour.
The discovery that the inventor is surprised, the key component isovaleryl spiramycin III of rokitamycin has better anti-infection activity, the invention provides the application of isovaleryl spiramycin III in the preparation anti-infectives for this reason.
Summary of the invention:
The purpose of this invention is to provide a kind of isovaleryl spiramycin III compound and pharmaceutical composition thereof, with and application in anti-infective therapy.The present invention is prepared into isovaleryl spiramycin III compound that a kind of production technology is simplified, quality standard is easily controlled, the one pack system antibiotic that effect of drugs is good.
Isovaleryl spiramycin III compound of the present invention has following structure:
Wherein: R=COCH
2CH
3R '=COCH
2CH (CH
3)
2
The structure that the invention provides the isovaleryl spiramycin III is determined;
The present invention also provides the preparation method of isovaleryl spiramycin III, it comprises the following steps: according to patent [ZL97104440.6] preparation rokitamycin, the sample crude separation, efficiently purifying, sample post processing etc., the preferred high performance liquid chromatography that adopts is separated the rokitamycin sample, obtains isovaleryl spiramycin III pure compounds.
The present invention carries isovaleryl spiramycin III antibacterium, anti-mycoplasma and chlamydia determination of activity also is provided.
The present invention also provides with the pharmaceutical composition of isovaleryl spiramycin III of the present invention as the pharmaceutically active substance preparation, isovaleryl spiramycin III shared percentage by weight in pharmaceutical composition can be 0.01-99.99%, and all the other are pharmaceutically acceptable carrier.Pharmaceutical composition of the present invention exists with unit dosage form, and described unit dosage form refers to the unit of preparation, as every of tablet, and every of capsule, every bottle of oral liquid, every of injection etc.
Pharmaceutical composition of the present invention can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, injection type preferably, as: liquid drugs injection, powder pin, the dosage forms such as transfusion.
Pharmaceutical composition of the present invention, the preparation of its oral administration can contain excipient commonly used, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet in case of necessity.
Applicable filler comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, for example sodium starch glycollate.Suitable lubricant comprises, for example magnesium stearate.The acceptable wetting agent of suitable medicine comprises sodium lauryl sulphate.
Can fill by mixing, the method that tabletting etc. are commonly used prepares solid oral composition.Repeatedly mix active substance is distributed in those compositionss of a large amount of filleies of whole use.
The form of oral liquid can be for example aqueous or oily suspensions, solution, Emulsion, syrup or elixir, can be perhaps a kind of available water before use or the composite dry products of other suitable carrier.This liquid preparation can contain conventional additive, such as suspending agent, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible fat, emulsifying agent, for example lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), for example almond oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerol; Antiseptic, for example para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if need, can contain conventional flavouring agent or coloring agent.
For injection, the liquid unit dosage forms of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolving.Normally by active substance being dissolved in a kind of carrier, then filter-sterilized before it is packed into a kind of suitable bottle or ampoule seals in the preparation of solution.Adjuvant for example a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, can be after the bottle of packing into, that this compositions is freezing, and under vacuum, water is removed.
pharmaceutical composition of the present invention, optionally add suitable pharmaceutically acceptable carrier when being prepared into medicament, described pharmaceutically acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, the phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Pharmaceutical composition of the present invention is determined dosage form and usage and dosage according to patient's situation in use.
Advantage of the present invention and good effect are, for pharmacy corporation provides the easily isovaleryl spiramycin III one-component goods of control of production technology simplification, quality standard; For providing, clinical critical patient or patient that should not oral administration take effect rapidly, the probability of the drug formulation that is easy to accept.
Embodiment:
Below listed embodiment just in order to help those skilled in the art to understand better the present invention, but do not limit the present invention in any way.
<embodiment 1 〉, the separation of isovaleryl spiramycin III preparation
The rokitamycin raw material is according to " a kind of method of the utilizing technique for gene engineering MT mycin " patent (patent No.: ZL97104440.6) prepare.Adopt preparative high performance liquid chromatography that the rokitamycin sample is separated, obtain isovaleryl spiramycin III pure compounds.Specifically comprise the sample crude separation, efficiently purifying, sample post-treating and other steps.
1), crude separation: at first from the rokitamycin sample, isovaleryl spiramycin III component is carried out crude separation, adopt silica gel column chromatography method, with ethyl acetate and methanol as eluant, adopt respectively three times of column volume ethyl acetate: methanol (3: 1) and three to five times of column volume ethyl acetate: methanol (1: 1) carries out eluting, collect ethyl acetate: methanol (1: 1) part, to collect liquid is concentrated into dried, obtain isovaleryl Spiramycin I, II, III component, be used for being further purified separation.
2), efficiently purifying: adopt preparative high performance liquid chromatography to carry out purification to the crude separation sample, as chromatograph packing material, carry out gradient elution with acetonitrile and Ammonium Acetate buffer with ODS, pass through ultraviolet detection, the uv atlas that record separates, and isovaleryl spiramycin III target peak is collected.
Concrete chromatographic condition is as follows: instrument: technical grade preparative hplc.Critical piece comprises the binary gradient pump, UV-detector and chromatographic work station; Chromatographic column: ODS preparative hplc post (70i.d. * 380mm, 10 μ); Mobile phase: acetonitrile 25% (A), 100mM NH
4Ac aqueous solution 75% (B).Flow velocity: 260mL/min; Sample size: 10mL; Sample introduction concentration: 0.5g/mL; Detect wavelength: 231nm;
Collection mode: ultraviolet triggers collects; According to the retention time (RT 48.009) of isovaleryl spiramycin III, collect sample.Isovaleryl spiramycin III high pressure liquid chromatography figure sees (Fig. 1).
3), sample post processing: the isovaleryl spiramycin III sample of collection, the employing rotary evaporation is removed acetonitrile, then with 1 times of amount ethyl acetate extraction, removes ethyl acetate in extract with rotary evaporation, gets the paste sample.With the heavy molten gained sample of petroleum ether, then remove petroleum ether with rotary evaporation, obtain isovaleryl spiramycin III white powder solid.
<embodiment 2〉evaluation of isovaleryl spiramycin III
The molecular weight of the compound that separates by high resolution mass spectrum (Bruker APEXII, HR-SI-MS) is 982.Isovaleryl spiramycin II i high resolution mass spectrum figure is shown in (Fig. 2), through nuclear magnetic resonance
1H and
13C composes [BrukerAM500, solvent C DCl
3, interior mark TMS (tetramethylsilane)] etc. analyze conclusive evidence, the structure of the compound that separates is the isovaleryl spiramycin III.Isovaleryl spiramycin III nuclear magnetic resonance
1H and
13The C spectrum is shown in (Fig. 3,4), nuclear magnetic resonance
1H and
13C spectrum data are shown in (table 1,2).
The nuclear magnetic resonance, NMR of table 1 isovaleryl spiramycin III
1H spectrum data (500MHz, CDCl
3)
The nuclear magnetic resonance, NMR of table 2 isovaleryl spiramycin III
13C spectrum data (125MHz, CDCl
3)
<embodiment 3〉Determination of Antibacterial Activity of isovaleryl spiramycin III
64) compound sample:
Compound 1 rokitamycin: lot number: 0812512, tire: 92.8%, Tonglian Group Co., Ltd., Shenyang provides;
Compound 2 isovaleryl spiramycin IIIs: lot number: 0811153, tire: 91.9%, Tonglian Group Co., Ltd., Shenyang provides
Erythromycin: lot number: 0706392, tire: 91.6%, Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
Azithromycin: lot number: 0421-9603, tire: 99.3%, Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
Clarithromycin: lot number: 0482-9901, tire: 90.4%, Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
2) test strain:
Reference culture: streptococcus pneumoniae ATCC49619;
Clinical separation gram positive bacteria;
Erythromycin-resistant streptococcus pneumoniae Streptococcus pneumoniae (7 strain);
Erythromycin-resistant micrococcus scarlatinae Streptococcus pyogenes (6 strain).
Every strain antibacterial all turns through flat board before test and lives minute purely, is used for test with new fresh thalli.Each experiment all uses reference culture as sensitive experiment Quality Control bacterium; With the plate that does not contain antibacterials as the test strain growth control.
3) culture medium and incubation conditions:
M-H (Mueller-Hinton) culture medium (g/L): acid hydrolysis casein 17.5, beef soak bone meal 2, starch 1.5, agar 12.Streptococcus pneumoniae on blood meida (adding 5% defiber Sanguis caprae seu ovis to make in the M-H culture medium), 35 ℃ of 5%CO
2Environment (CO
2Incubator) hatch 20-24h in.Micrococcus scarlatinae is hatched 20-24h for 35 ℃ on blood meida (adding 5% defiber Sanguis caprae seu ovis to make in the M-H culture medium).
4) minimum inhibitory concentration (MIC) is measured:
Employing standard plate doubling dilution.Tested bacteria suspension is inoculated with multiple spot inoculation instrument, and every some inoculum concentration is 10
4CFU.Measure each antibacterials to the minimum inhibitory concentration of various pathogenic bacterium.
Result:
In the erythromycin-resistant streptococcus of measuring, one pack system compound isovaleryl spiramycin III of the present invention is more superior to streptococcic rokitamycin, MIC
50And MIC
90Be respectively 0.5 and 2mg/L, rokitamycin is 2 and 4mg/L, obviously is better than contrast medicine erythromycin, azithromycin, clarithromycin (table 3) simultaneously.For the 7 high Antimicrobial Streptococcus Pneumoniaes of strain erythromycin (erythromycin MIC 64->256mg/L), azithromycin and clarithromycin show as the height drug resistance too, and the MIC value of the compounds of this invention is at 0.25-32mg/L, lower than rokitamycin 2-4 doubly, lower than contrast medicine 64-1024 doubly (table 4, Fig. 5).For the low drug resistance micrococcus scarlatinae (erythromycin MIC=1-8mg/L) of 6 strain erythromycin, the MIC of the compounds of this invention
50Value lower than rokitamycin, is better than contrasting medicine erythromycin, azithromycin, clamycin 2-32 times (table 3,5 Fig. 6) at 0.5mg/L.
Table 3. compound and contrast medicine MIC result
Table 4. compound and contrast medicine are to 7 strain erythromycin-resistant streptococcus pneumoniae MIC (mg/L)
Table 5 compound and contrast medicine are to 6 strain erythromycin-resistant micrococcus scarlatinae MIC (mg/L)
The anti-mycoplasma pneumoniae of B and Chlamydia pneumoniae are active
1. compound
Compound 1 rokitamycin: lot number: 0812512, tire: 92.8%, Tonglian Group Co., Ltd., Shenyang provides
Compound 2 isovaleryl spiramycin IIIs: lot number: 0811214, tire: 93.7%, Tonglian Group Co., Ltd., Shenyang provides
Erythromycin: lot number: 0706392, tire: 91.6%, Nat'l Pharmaceutical ﹠ Biological Products Control Institute
Azithromycin: lot number: 0421-9603, tire: 99.3%, Nat'l Pharmaceutical ﹠ Biological Products Control Institute
2. mycoplasma pneumoniae strain
Mycoplasma pneumoniae strain (ATCC-FH).
3. mycoplasma pneumoniae culture medium
1. mycoplasma agar culture medium: mycoplasma agar basal medium (Oxoid company, CM0401) 2.84g, add ultra-pure water 80ml, 121 ℃ of high pressure 15min sterilizations, be placed in the water-bath of 50 ℃, add 1 bottle of mycoplasma pneumoniae additive (Oxoid, SR0059C) (20ml), preparation agar culture dish.
2. mycoplasma PPLO broth bouillon: mycoplasma PPLO Mycoplasma Broth Base culture medium (BD company) 2.1g, deionization pure water 70ml, 121 ℃, 15min autoclaving.Be cooled to the phenol red 0.5ml that adds after room temperature the lyophilizing mycoplasma pneumoniae to cultivate additive (Oxoid additive) 1 bottle (dissolving of 20ml sterile deionized water), aseptic 50% glucose solution 2.0ml and 0.4%.
4. mycoplasma pneumoniae is cultivated
Mycoplasma pneumoniae FH strain is inoculated in and contains 20ml PPLO broth bouillon, and the 72h results scrape with bead, 12000rpm, and supernatant is abandoned in 10min centrifugation, and precipitation is outstanding again with the fresh PPLO broth bouillon of 8ml, and frozen-80 ℃ refrigerator is managed in packing 8.
The titration of mycoplasma pneumoniae FH inoculum
Above-mentioned preparation mycoplasma pneumoniae FH inoculum is coated the mycoplasma pneumoniae agar plate after cultivating and using the PPLO broth bouillon to carry out diluting in 1: 10,1: 100 and 1: 1000, and 37 ℃ of 5%CO2 cultivated 7 days, counted under * 40 power microscopes.Calculate the mycoplasma pneumoniae titre.
5. the mensuration of mycoplasma pneumoniae MIC
Compound is dissolved in dehydrated alcohol with 16mg/ml, uses mycoplasma PPLO broth bouillon with 10 doubling dilutions.The mycoplasma pneumoniae inoculum adds in the medication tube of above-mentioned serial dilution, and the final inoculum density of mycoplasma pneumoniae FH is 8.25 * 10
5CFU/ml, the variation of color is cultivated and observed every day to postvaccinal culture tube (comprising control tube) in 37 ℃.Test is set up:
(1) positive control: the mycoplasma pneumoniae FH inoculum (1.65 * 10 after dilution
6CFU/ml) 0.5ml+PPLO broth bouillon 0.5ml;
(2) negative control: PPLO broth bouillon 1ml;
(3) dehydrated alcohol is killed and is disturbed contrast: the mycoplasma pneumoniae FH inoculum (1.65 * 10 after dilution
6CFU/ml) 0.5ml, PPLO broth bouillon 0.5ml, ultimate density 0.05% dehydrated alcohol.
When obvious color change (by red flavescence color) occurs the positive control pipe, be the minimum antibacterial dense MIC of being concentration and this moment the antibiotic concentration of color change pipe does not occur.
7. Chlamydia pneumoniae strain
Chlamydia pneumoniae strain: CWL-029 (ATCC VR1310).
8. Chlamydia pneumoniae is cultivated
(1) cell culture fluid: 10% hyclone (HyClone company) Dulbecco ' s MEM (Sigma company) culture fluid.
(2) Chlamydia pneumoniae culture fluid: 10% hyclone Dulbecco ' s MEM culture fluid contains the cycloheximide (Sigma company) of 2 μ g/ml.
(3) BGMK (green monkey kidney cell, Diagnostic HYBRIDS company) is planted in 96 porocyte culture plates, and 37 ℃, 5%CO2 cultivates became cell monolayer in 48 hours.
9. the mensuration of Chlamydia pneumoniae MIC
Compound is dissolved in dehydrated alcohol with 16mg/ml, at first uses the chlamydia culture fluid with 10 doubling dilutions, is 3.3 * 10 with concentration
7Cfu (inclusion body formation unit)/1ml Chlamydia pneumoniae inoculum 1: 200 dilution, ultimate density is: 1.65 * 10
5Cfu/1ml sucks the cell culture fluid in 96 well culture plates, then inoculates by the 0.1ml/ hole.Strain is inoculated complete centrifugal 96 porocyte culture plates, uses the J-6MC centrifuge of Beckman-Coulter company, centrifugal force * 1500g, 35 ℃ of centrifuging temperatures, centrifugation time 60min.
Set up contrast
(1) positive control: BGMK cell, the Chlamydia pneumoniae CWL-029 strain of dilution.
(2) negative control: BGMK cell, Chlamydia pneumoniae culture fluid.
(3) medicine contrast: BGMK cell, the compound of highly diluted concentration (8 μ g/ml), ultimate density 0.05% dehydrated alcohol.
Centrifugal complete after, suck the Chlamydia pneumoniae inoculum, add respectively the compound 0.1ml/ hole of serial dilution.37 ℃, 5%CO2 cultivates 72min.Cultivate completely, draw antibiotic medicine solution, PBS (0.01M, pH 7.4) washing 2 times, 100% dehydrated alcohol is 15min fixedly.
Indirect IF staining is identified: monoclonal antibody to Chlamydia pneumoniae, 50 μ l/ holes, incubation 30min in 37 ℃ of wet boxes, then wash plate machine washing plate 4 times, add again the anti-Mus fluorescent antibody of rabbit (Sigma company), 50 μ l/ holes, same method and condition incubation and wash plate.Add mounting glycerol, 100 μ l/ holes are observed under the OlympusX71 inverted fluorescence microscope
The definition of MIC: the minimum antibiotic diluted concentration in the complete suppressed hole of Chlamydia pneumoniae inclusion body growth (inclusion body of fluorescence staining is not found in full hole) in 96 hole bread boards.
10. result:
Isovaleryl spiramycin III activity in the anti-mycoplasma pneumoniae activity of measuring obviously is better than rokitamycin, and obviously is better than contrasting medicine.To the activity of Chlamydia pneumoniae, isovaleryl spiramycin III and rokitamycin are suitable, are better than erythromycin.
Table 6 compound is to mycoplasma pneumoniae/chlamydia active (μ g/ml)
<embodiment four〉preparation of isovaleryl spiramycin III pharmaceutical preparation
The present invention can be with isovaleryl spiramycin III one pack system as active component, is prepared into useful clinically various dosage form with receivable carrier pharmaceutically.As injection: will be dissolved in 1-5ml water after isovaleryl spiramycin III (abbreviation raw material) 100-500mg and solvent adipic acid 18-90mg or mole mix homogeneously such as citric acid 26-130mg or maleic acid 14-70mg respectively, can obtain faint yellow clear and bright solution, pH is between 4.6-5.6.Add mannitol 30-150mg as the lyophilizing proppant, after cryogenic quick freezing 9h, lyophilization obtains faint yellow loose block again, dissolves with the 2-10ml sterilized water before using.