CN100368015C - Medicine for relieving drunk - Google Patents

Abstract

The present invention relates to a medicine for relieving alcoholism, which is characterized in that No. 33 alpha-hemolytic streptococcus strains are carried by a roundtrip deep space vehicle, DNA double strands in a microbial cell are broken under the special condition of the outer space, and the repair of impaired DNA is inhibited in a microgravity environment and a high vacuum environment, so that the hereditary properties of the NO. 33 alpha-hemolytic streptococcus strains are mutated, and strains with positive mutation and stable hereditary properties are preferably selected, bred and fermented to be produced into a biochemical preparation for relieving alcoholism. The strains are preserved in China General Microbiological Culture Collection Center and have a serial number of CGMCCNO. 1082. The biochemical preparation for relieving alcoholism is oral liquid, and 1000 ml of the biochemical preparation contains 0.8g to 15.0g of mannon peptide produced by using the strains mutated in the outer space. In the medicine for relieving alcoholism, a biochemical preparation is adopted to relieve and prevent alcoholism, and the present invention has the characteristics of no toxin, small side effect and high effective rate.

Description

A kind of oral formulations

Technical field

The present invention relates to a kind of medicine, particularly about being used for the medicine of relieving alcoholism and preventing drunkenness and treatment alcoholic liver injury.

Background technology

The hepatic injury that alcoholic liver injury that chronic alcoholism causes and viral hepatitis are caused is two big main causes of liver damage disease.The liver cirrhosis that causes thus sickness rate in recent years obviously raises.Internal medicine conventional treatment drug treatment reduces the triple therapy based on vitamins and hepatoprotective and gastric mucosa protectant at present.Alcoholic liver injury is longer through the above-mentioned medicine for treatment time, and onset is slow.Therefore seek and preparation to have a medicine of can relieving alcoholism and preventing drunkenness and promoting alcoholism that liver dysfunction is repaired very necessary.

Summary of the invention

The medicine that the purpose of this invention is to provide a kind of relieving alcoholism and preventing drunkenness, its adopts biochemical preparation relieving alcoholism and preventing drunkenness, has nontoxicly, and side effect is little, the characteristics that effective percentage is high.

Technical scheme of the present invention is: the medicine that designs a kind of relieving alcoholism and preventing drunkenness, it is characterized in that: it carries the reciprocation type space craft with No. 33 strains of Alpha-hemolytic streptococcus, under the specific condition of space, make the dna double chain interruption that causes in the microbial cell, at microgravity, the DNA that high vacuum environment suppresses damage down repairs, No. 33 strain hereditary properties of Alpha-hemolytic streptococcus are morphed, optimize the strain of plus variant stable hereditary property wherein, after cultivating fermentation, produce the antialcoholic biochemical preparation, this strain is numbered CGMCCNo.1082 through China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.

Described antialcoholic biochemical preparation is an oral liquid, contains the mannatide 0.8g-15.0g that the space flight strain is produced among the 1000ml.

Described oral liquid is to carry reciprocation type space craft, variation, strain after preferred with No. 33 strains of Alpha-hemolytic streptococcus, and it adds other adjuvant again through cultivating the stock solution of fermenting and producing.

Described oral liquid is to carry reciprocation type space craft, variation, strain after preferred with No. 33 strains of Alpha-hemolytic streptococcus, through cultivating the mannatide of fermenting and producing, adds the dissolving of adjuvant and purified water and stirs evenly.

Described adjuvant is essence and saccharin sodium, xylitol, sodium chloride.

The preparation method of described mannatide is; 1. preferred alpha-Hemolytic streptococcus 33 strains carry out space craft and carry, the variation bacterium pearl of the meticulous selection-breeding of process space flight is as producing strain, fermentation of preparation warp-level and second order fermentation, fermentation liquid is purified, final production goes out the mannatide that the space flight strain is produced. and 2. with the mannatide of space flight strain production, add adjuvant and make oral liquid.

Described strain preparation;

A. inclined-plane seed culture medium: Carnis Bovis seu Bubali cream 0.2-0.8%, yeast extract 0.2-0.8%, peptone 0.3-0.8%, glucose 0.1-0.8%, sodium chloride 0.2-0.6%, agar 1-5%, sheep blood 5-10%; PH 7.2-7.4;

105-125 ℃ of inclined-plane seed culture medium sterilising temp, 30 minutes time, steam pressure 0.11-0.14Mpa;

Unpacking strain cryovial, with aseptic broth bouillon dilution, access blood inclined-plane under aseptic condition, inoculate rearmounted 25-40 ℃ constant temperature culture 24-30 hour;

B. meat soup seed culture medium: Carnis Bovis seu Bubali cream 0.2-0.8%, yeast extract 0.2-0.8%, peptone 0.3-0.8%, glucose 0.1-0.8%, sodium chloride 0.2-0.6%; PH 7.0-7.4;

105-125 ℃ of meat soup seed culture medium sterilising temp, 30 minutes time, steam pressure 0.11-0.14Mpa;

Cultured slant strains is pressed the 1-9% inoculum concentration insert in the meat soup seed culture medium under aseptic condition, 25-40 ℃ constant temperature culture 10-30 hour.

Described sweat is to use fermentation medium, Carnis Bovis seu Bubali cream 0.2-0.8%, yeast extract 0.2-0.8%, peptone 0.3-0.8%, glucose 0.1-0.9%, sodium chloride 0.2-0.6%;

105-125 ℃ of fermentation medium sterilising temp, 30 minutes time, steam pressure 0.11-0.14Mpa;

A. first class seed pot: good meat soup strain is pressed the 1-10% inoculum concentration insert first class seed pot under aseptic condition, at 25-40 ℃ of constant temperature culture 10-50 hour.Tank pressure is no more than 0.02Mpa, and ventilation is advisable can stir culture fluid, fully stirs continuously;

B. second order fermentation jar: the one grade fermemtation culture fluid is pressed the 2-20% inoculum concentration insert fermentation tank under aseptic condition, at 25-40 ℃ of constant temperature culture 20-100 hour, tank pressure was no more than 0.02Mpa; Jar is put in deactivation, and deactivation is adopted to heat and made the fermentation liquid temperature reach 70-120 ℃, is incubated 20-60 minute, and cooling is left standstill.

Described leaching process;

A, fermentation liquid concentrate, and make concentrated solution volume and fermentating liquid volume ratio be controlled at 1: 15-1: 20 or be determined by circumstances;

The concentrated solution of b, fermentation liquid adds 80-99% ethanol, and the volume ratio is controlled at 1: 1.5-1: 5.5 or be determined by circumstances; Fully stir leave standstill after, centrifugal removal supernatant, the precipitation dissolved in distilled water, accent pH1.0-5.0 obtains lysate and lysate is left standstill;

C, the more centrifugal removal impurity of lysate is obtained supernatant.Accurately measure the supernatant volume, calculate required amount of alcohol, ethanol is slowly joined in the supernatant, and fully stir, leave standstill the centrifugal removal supernatant in back and obtain precipitate by the supernatant stereometer;

D, precipitate reuse dissolved in distilled water are transferred pH, and be centrifugal.Supernatant adds ethanol precipitation again.Such technology repeatable operation to content detection qualified till, the precipitate that obtains is the mannatide crude product that the space flight strain is produced.Promptly got the mannan peptide product that the space flight strain is produced in vacuum drying 3-8 hour.

Characteristics of the present invention are: because the present invention is carried recoverable space craft (spacecraft or retrievable satellite) with the Alpha-hemolytic streptococcus strain, utilize the comprehensive function of factors such as little in the space (zero) gravity, cosmic ray, alternating magnetic field, fine vacuum, hyperpyrexia deep cooling, make the dna double chain structure fracture of bacterial strain, genome is reset, thereby produces new bacterial strain.After returning ground, bacterial strain through space treatment is cultivated and screened, by studying contents such as its morphological characteristic, cultural characteristic, biochemical reactions, metabolite, hereditary character and protein expression, hereditary stability, pilot scale production target, optimize the stable strain of plus variant, inherited character wherein, the medicine of the ulcerative colitis that after cultivation and fermentation, obtains medical treatment.This strain in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, is numbered CGMCCNo.1082.

Particularly Alpha-hemolytic streptococcus D33 bacterial strain behind ground screening, separation and the purification, selects higher, the secreted mannan peptide content of fermentation unit higher high yield, quality strains T33 strain through space treatment mutation.By Institute of Microorganism, Academia Sinica whole-cell protein SDS-polyacrylamide gel (SDS-PAGE) analysis of T33 bacterial strain and D33 bacterial strain and the genomic DNA restricted enzyme cutting analysis (RFLP/PFGE) of bacterial strain are compared, can prove that its gene of T33 bacterial strain that space mutagenesis and ground filter out has variation.This mutant strain is stable in heredity, stronger production adaptability is arranged, the mannan peptide content that is produced by its fermentation improves, content of peptides improves 3 to 5 times than national standard (80%), institute for drug control, Xi'an sample presentation is measured and is reached 271.6%, and fermentation content on average exceeds more than three times than matched group product content.The mannatide of producing behind the space induction mutation of bacterium has the removing ability of relieving alcoholism and preventing drunkenness drug effect and animal interior free yl, reduces its lipid peroxidation speed.The mannatide of producing behind the space induction mutation of bacterium has the effect that promotes that alcoholism is repaired liver dysfunction.(see Appendix: adnexa material 1: science and technology bureau Shaanxi Province, Shaanxi Province scientific and technological achievement assay certificate material

Adnexa material 2: the mannatide that the space flight strain is produced is produced bacterial strain and is carried notarization through " No. three, divine boat " airship.

Adnexa material 3: the mannatide that the space flight strain is produced is produced bacterial strain and is returned ground screening report through the lift-launch of " No. three, divine boat " airship

Adnexa material 4: Microbe Inst., Chinese Academy of Sciences produces the form Physiology and biochemistry probation report book of bacterial strain and ground contrast bacterium about the mannatide that " No. three, divine boat " airship carries the production of space flight strain

Adnexa material 5: Microbe Inst., Chinese Academy of Sciences produces SDS-PAGE and the RFLP/PFGE Analysis and Identification statement of bacterial strain and ground contrast bacterium about the mannatide that " No. three, divine boat " airship carries the production of space flight strain

Adnexa material 6: the mannatide oral liquid examining report that institute for drug control, Shaanxi Province " No. three, divine boat " space flight strain is produced

Adnexa material 7: scientific and technological novelty assessment report copy

Adnexa material 8: the notice copy is accepted in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation culture presevation)

New pharmacological action analysis of experiments is verified: the new pharmacological action of treatment alcoholic liver injury mannatide is relieving alcoholism and preventing drunkenness and promotes the effect that alcoholism is repaired liver dysfunction, verifies by following zoopery:

1.1 material animal: Kunming kind white mice, body weight (20 ± 2) g, the male and female dual-purpose, The Fourth Military Medical University's Experimental Animal Center provides. medicament: relieving alcoholism and preventing drunkenness agent A (vitamin C), B (No. three mannatide oral liquids of divine boat) and C (taurine).Wine use in experiment: 53 ° of Luzhou Old Cellar two fermented wine (commercially available, Luzhou Old Cellar company limited product, ig during to wine distilled water diluting become 40 °).

1.2 method: relieving alcoholism and preventing drunkenness effect experiment (with thiobarbituricacid colorimetric method for determining serum lipid peroxide): in the experimentation that carries out animal alcoholic liver injury pharmacodynamics, the 2nd, 5,10 and 20d statistical observation mice right the anti-disappearance situation of thanking, calculate its drunk rate.

The morphologic observation of when dissected mice internal organs: great little with reference to Mus, from gallbladder assist, comprehensive test is carried out in 4 aspects of fatty number under the enlargement situation of pancreas and liver and the abdomen.

Mouse liver even slurry LPO value adopts the TBA colorimetric method for determining: mice is behind the pharmacodynamic experiment of continuous 28d, fasting 12h, the cervical vertebra dislocation is put to death next day. and dissect and get the Hepar Mus 0.18～0.20g at right lobules of liver position, with adding cold 1.15%KCl solution behind the normal saline flush away blood stains, in the full glass grinding device of 10mL, make 10% liver homogenate. measure the LOP value according to literature method.

Mouse Liver section microexamination: mice is after the experiment of continuous 28d, fasting 12h, the cervical vertebra dislocation is put to death next day. and dissect and get right lobules of liver, core is cut into the piece of tissue of 1cm2 size. use formaldehyde fixed according to a conventional method, paraffin embedding, make common section, the hepatic cell fattydegeneration degree is examined under a microscope in haematoxylin-Yihong (HE) dyeing.

1.3 the tissue of experiment: Kunming kind white mice, body weight (20 ± 2) g, through after the environmental adaptation of 3d, 80 mices are divided into 8 groups of (model control group at random by body weight, the normal control group, the A low dosage, the A high dose, the B low dosage, the B high dose, C low dosage and C high dose) after, every group is each 5 of male and female. except that the normal raising of normal control group, all the other each experimental grouies wait to try the mice overnight fasting, the all first ig administration of each experimental group next day, behind the 20min each experimental group again ig give wine. by every 20g Mus re-computation, model control group ig dosage is the 0.60mL normal saline, the dosage of low dose group ig is the agent of 0.30mL relieving alcoholism and preventing drunkenness, the dosage of high dose group ig is the agent of 0.60mL relieving alcoholism and preventing drunkenness, each experimental group ig is 0.37mL to capacity for liquor, pharmacodynamic experiment through continuous 28d, behind the fasting 12h, dissect, carry out internal organs and observe and liver homogenate LPO pH-value determination pH and liver slice microexamination hepatocellular degeneration situation.

2 results: the relieving alcoholism and preventing drunkenness drug effect of table 1 relieving alcoholism and preventing drunkenness agent A, B and C

The drunk number of project/only drunk rate/%

Model contrast 2255.0

A low 1025.0

A is high by 717.5

B low 37.5

B is high by 410.0

C low 512.5

C is high by 410.0

Annotate: each is organized effective number of animals and is 40, under the same conditions relieving alcoholism and preventing drunkenness agent A, B and C is carried out the Comprehensive Experiment of relieving alcoholism and preventing drunkenness drug effect, liver homogenate LPO pH-value determination pH, the observation of when dissected internal organs and liver slice electron microscopic observation.

2.1 relieving alcoholism and preventing drunkenness drug effect situation

Experimental result is shown in table 1 (" A is low " expression low dosage in the table, down together). compare with model control group, each administration group mice drunk rate after drinking of giving obviously reduces, and relieving alcoholism and preventing drunkenness agent A, B and C have tangible relieving alcoholism and preventing drunkenness drug effect, wherein the effects of relieving alcoholism and preventing drunkenness of B and C is more remarkable

2.2 when dissected mice internal organs morphologic observation

Experimental result (table 2) shows, normal control group mice its gallbladder behind normal raising 28d is assisted, pancreas, liver is all normal, fat is seldom under its abdomen. and model control group mice visible its gallbladder of naked eyes after ig gives wine 28d is continuously assisted, pancreas, the plump accumulation of fat deposit under enlargement phenomenon and the abdomen appears in liver. each administration group mice internal organs size appropriateness, do not observe abnormal phenomena, fat is less than the model control group mice under the interior abdomen of visible each the administration group mice body of naked eyes simultaneously, A height wherein, B is low, B is high and that C is high, and effect is better. and experimental result shows, long-term a large amount of ethanol of taking in, internal organs to animal can cause certain damage, gallbladder is assisted, pancreas and liver enlargement, relieving alcoholism and preventing drunkenness agent A, B and C have certain effect that alleviates animal internal organs damage after drinking; The long-term ethanol of taking in easily causes the animal shape obesity, the phenomenon of athero under the abdomen, and relieving alcoholism and preventing drunkenness agent A, B and C have and necessarily prevent to cause because of drinking the sedimentary effect of body fat.

Table 2 when dissected mice internal organs situation

Project gallbladder pancreas liver abdomen is fat down

Normal control is normally less

The obvious enlargement of model contrast enlargement enlargement is piled up

A is low normally medium

The A height is normally less

B is low normally less

The B height is normally less

C is low normally medium

The C height is normally less

Table 3 mouse liver even slurry LPO value (± S)

Project LPO value/nmol.L-1

Normal control 0.725+0.313

Model contrast 9.682+1.002

A hangs down 7.165+1.1901)

The high 9.358+0.901 of A

B hangs down 3.718+1.0341)

The high 6.201+0.8491 of B)

C hangs down 8.567+0.6571)

The high 7.430+1.0371 of C)

1) compares P＜0.01 through the t check with the model contrast

2.3 the agent of medicine relieving alcoholism and preventing drunkenness is to the influence of chronic alcoholism mouse liver even slurry LPO value, the a large amount of ethanol of long-term absorption can cause fatty liver, lipid peroxide value in the animal livers significantly raises, can detect the rising of malonaldehyde (MDA) content during animal alcoholism. lipid peroxide (Lipidperoxides LPO) the class peroxide that to be free radical unsaturated fatty acid such as active oxygen generate through enzymatic or non-enzymatic route, LPO can be metabolized to malonic acid again. can reflect alcoholism mouse liver free radical level indirectly by the LPO content that detects liver homogenate, be the snperoxiaized index of animal body inner lipid. experimental result (table 3) shows, the liver homogenate LPO value of normal control group mice is lower, it is higher to show that its interior free yl is removed ability, level of lipid peroxidation is lighter. compares with the normal control group, no matter whether ig gives the relieving alcoholism and preventing drunkenness agent, long-term a large amount of the absorption under the ethanol condition, the LPO value of mouse liver even slurry all significantly raises, although show that B is low, the B height, the A height, the C height has certain effect for reducing LPO, but long-term a large amount of ethanol of taking in will cause alcoholic liver injury.

Compare with model control group, each administration group mouse liver even slurry LPO all obviously reduces, show that relieving alcoholism and preventing drunkenness agent A, B and C all have preferably antioxidation and protect the liver function, except that A is low, each administration group all has utmost point significance, especially with the low best results of B. these No. three mannatides of explanation divine boat have the removing ability that improves the animal interior free yl, reduce its lipid peroxidation speed, thereby alleviate the alcoholic liver injury that long-term heavy drinking causes.

2.4 murine liver tissue section microexamination, long-term heavy drinking can induce in the hepatic mitochondria cytochrome P 450 enzymes to cause that the hepatic mitochondria electron transport system is activated and lipid peroxidation is hyperfunction, reduced glutathion in the liver (GSH) concentration reduces, free radical generates in a large number, cause hepatic injury. simultaneously, because hepatic mitochondria malfunction, the ability that acetaldehyde resolves into acetic acid is reduced, acetaldehyde and hepatocyte interior participation secretion of protein transport and combining of tubulin, cause that albumen is accumulated in the liver, the hepatocyte that stores with water be the swelling of balloon sample=, because liver cell mitochondria respiratory chain hyperfunctioning, cause the residing district of alcoholdehydrogenase (ADH) band be in; Group Mouse Liver section microexamination project: liver cirrhosis is soaked in cloudy swelling, hydropic degeneration, spotty necrosis, hyperemia, area under control

Normal control------

The model contrast ++ ++ +++±+

A is low ++ ±+--

The A height ++ ±----

B is low ± and-----

The B height ++-±--

C is low ++ ±---

The C height ++----

Model control group Mouse Liver section microexamination is found, tangible cloudy swelling and hydropic degeneration phenomenon all occur around the nuclear, and the portal area is soaked into, and fat drop is a lot of in the born of the same parents, and the congested and swelling of iuntercellular causes perisinusoidal space to blur or disappearance, and hepatic sinusoid joins together; The central vein edge is loose, and hepatocyte is arranged disorderly and unsystematic, and hepatic cords is fuzzy, and cell boundaries disappears, and the kernel enlargement is loose, and is painted darker, and spotty necrosis and local liver cirrhosis phenomenon appear in hepatocyte.

Compare with model control group, each administration group lobules of liver hydropic degeneration and cloudy swelling phenomenon are all obviously lighter, and spotty necrosis regulating liver-QI hardening phenomenon does not also appear in hepatocyte; The central vein edge is comparatively tight, hepatocyte is radial arrangement more, hepatic cords is clearer basically, the cell boundary is clearer, kernel is slightly seen enlargement and loose, and is painted deep mixed, and the hepatic sinusoid gap is slightly obvious, fat drop has also lacked much in the born of the same parents, and lighter away from the local hepatic injury situation of central vein.Show that relieving alcoholism and preventing drunkenness agent A, B and C have the certain protection effect for alleviating the alcoholic liver injury that long-term heavy drinking causes, especially with B low to protect the liver function best. the Comprehensive Experiment result as can be seen, the drug effect that B is low is better.Through No. three mannatide optimal doses of further experiment measuring and calculating divine boat are 0.5mg/ml/kg.

3 conclusions:

1) mannatide of producing behind the space induction mutation of bacterium has the removing ability of relieving alcoholism and preventing drunkenness drug effect and animal interior free yl, reduces its lipid peroxidation speed.

2) the long-term a large amount of ethanol of taking in can cause certain damage to the internal organs of animal, and relieving alcoholism and preventing drunkenness agent A, B and C have certain effect that alleviates animal internal organs damage after drinking and prevent to cause because of drinking the sedimentary effect of body fat.

The mannatide effect that B produces behind the space induction mutation of bacterium is the most obvious.

3) alcoholism can cause liver to damage, and the mannatide of producing behind the space induction mutation of bacterium has the effect that promotes that alcoholism is repaired liver dysfunction, and optimal dose is 0.5mg/ml/kg.

(4) new purposes checking: find that through 1,000 examples being used the mannatide oral liquid patient's who behind the space induction mutation of bacterium, produces investigation 75 examples drink for a long time, alcoholic liver injury, Patients with Fatty Liver, the uncomfortable n or V of no upper abdominal pain after the 10-20ml that takes medicine before drinking drinks by common custom amount, control goes down, and speech increases, the emotion excitement, action increases, and behavior is frivolous, and is sleepy.Instability of gait, face and whole skin flushing, symptom such as drowsiness.Three months persons that adhere to taking medicine pay a return visit 39 example check liver B ultrasonic fatty liver indications and disappear.Total effective rate 98%

(5) new curative effect checking:

Li Jian, man, 32 years old.The north pass, the Xi'an Hua Lu that shakes.Cause work in 2 years dinner party is drunk more, drinks Chinese liquor 3-4 two average every day, finds the chronic gastritis medical history is previously arranged fatty liver when hospital's health check-up at the end of the year.The normal sense headache in back of drinking is weak, and nausea and vomiting is drowsiness.Long-term inappetence, the difficulty falling asleep at night of not drinking then.JIUYUE in 2003 began to take mannatide oral liquid to 17 a day drug withdrawal above-mentioned symptom of producing on the 5th and all disappears behind the space induction mutation of bacterium.December return visit in the same year symptom does not have repeatedly, checks liver B ultrasonic fatty liver indication November and disappears.

Specific embodiments

Strain improvement: on the basis of Alpha-hemolytic streptococcus growth rhythm, selected than the proper growth strain in period, adopt the plate growth bacterium colony, immerse methods such as the bacteria suspension in other material, germ-carrying sand, carry " No. three, divine boat " spacecraft on March 25th, 2002.Carried out 6 days 0 18 hours flying at space track (200 kilometers of perigee altitudes, 350 kilometers of altitude of the apogees) at rail.The specific condition of space mainly is presented as special environments such as microgravity, fine vacuum, high radiation, alternating magnetic field.The strain of outer-space flight be placed in one with the diverse environment of tellurian ecological environment in, the electronics, proton and the mental retardation heavy particle that mainly comprise coming self-magnetic field to capture; Cosmic ray such as proton, particle and heavier high energy heavy particle from the milky way galaxy; From the proton of sun magnetic storm and heavy particle etc.These particles act on the dna double chain structure in the microbial cell nuclear, can cause double-strand break.Microgravity, high vacuum environment can make the base sequence of DNA recombinate, promptly genomic reorganization and variation.The new bacterial strain that the variation back produces, variation in various degree all can take place in its morphological characteristic, cultural characteristic, biochemical reactions, metabolite, hereditary character and protein expression, pilot scale production target etc.After ground is returned in spacecraft, through further screening, only optimize wherein 0.2% plus variant and the stable bacterial strain of inherited character, make the production strain through cultivation.This strain that after spaceship-carried mutation, selects December in 2003 29 days by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, be numbered CGMCCNo.1082.

Experiment and middle trial production result show, stable on inherited character through the novel space strain behind the space mutagenesis, stronger production adaptability is arranged, the mannan peptide content that is produced by its fermentation improves, content of peptides improves 3 to 5 times than national standard (80%), institute for drug control, Xi'an sample presentation is measured and is reached 271.6%, and fermentation content on average exceeds more than three times than matched group product content, and fermentation period shortens greatly.

Preparation method:

The space flight strain is produced the preparation process of mannatide:

The mannatide of space flight strain of the present invention production is by following method preparation: the space strain preparation--purify, and final production goes out mannatide by one grade fermemtation--second order fermentation-----fermentation liquid.

1. space strain preparation:

With through the Alpha-hemolytic streptococcus of space mutagenesis breeding meticulous selection-breeding as producing strain.

A. inclined-plane seed culture medium: Carnis Bovis seu Bubali cream 0.5%, yeast extract 0.6%, peptone 0.5%, glucose 0.4%, sodium chloride 0.5%, agar 3%, sheep blood 8%; PH 7.4.

121 ℃ of inclined-plane seed culture medium sterilising temps, 30 minutes time, steam pressure 0.12Mpa.

Unpacking strain cryovial with aseptic broth bouillon dilution, inserts the blood inclined-plane under aseptic condition, the rearmounted 38 ℃ of constant temperature culture of inoculation 30 hours.

B. meat soup seed culture medium: Carnis Bovis seu Bubali cream 0.5%, yeast extract 0.6%, peptone 0.5%, glucose 0.4%, sodium chloride 0.5%; PH 7.4.

121 ℃ of meat soup seed culture medium sterilising temps, 30 minutes time, steam pressure 0.12Mpa.

With cultured slant strains under aseptic condition by 9% inoculum concentration, insert in the meat soup seed culture medium 38 ℃ of constant temperature culture 30 hours.

2. sweat:

Fermentation medium: Carnis Bovis seu Bubali cream 0.4%, yeast extract 0.5%, peptone 0.5%, glucose 0.4%, sodium chloride 0.5%.

121 ℃ of fermentation medium sterilising temps, 30 minutes time, steam pressure 0.12Mpa.

A. one grade fermemtation jar: with good meat soup strain under aseptic condition by 10% inoculum concentration, insert first class seed pot, 29 ℃ of constant temperature culture 30 hours, tank pressure was no more than 0.02Mpa, ventilation is advisable can stir culture fluid, fully stirs continuously.

B. second order fermentation jar: with the one grade fermemtation culture fluid under aseptic condition by 20% inoculum concentration, insert fermentation tank, 29 ℃ of constant temperature culture 70 hours, tank pressure is 0.02Mpa not, jar is put in deactivation.Deactivation is adopted to heat and is made the fermentation liquid temperature reach 100 ℃, is incubated 60 minutes, and cooling is left standstill.

3. leaching process:

A, fermentation liquid concentrate, and make concentrated solution volume and fermentating liquid volume ratio be controlled at 1: 15-1: 20 or be determined by circumstances.

The concentrated solution of b, fermentation liquid adds 80-99% ethanol, and the volume ratio is controlled at 1: 1.5-1: 5.5 or be determined by circumstances.Fully stir leave standstill after, centrifugal removal supernatant, the precipitation dissolved in distilled water, accent pH5.0 obtains lysate and lysate is left standstill.

C, the more centrifugal removal impurity of lysate is obtained supernatant, accurately measure the clear liquid volume, calculate required amount of alcohol by the supernatant stereometer, adjust pH slowly joins ethanol in the supernatant, and fully stirs, and leaves standstill the centrifugal removal supernatant in back and obtains precipitate.

D, precipitate reuse dissolved in distilled water are transferred pH, and centrifugal, supernatant adds ethanol precipitation again.Such technology repeatable operation to intermediate detection qualified till, the precipitate that obtains is the mannatide crude product that the space flight strain is produced.Vacuum drying 3-8 hour, promptly obtain the mannan peptide product that the space flight strain is produced.

The check of the product that said method obtains:

[character] this product is white or little yellow amorphous powder; Odorless, tasteless.

This product is easily molten in water, and is insoluble in ethanol, chloroform and acetone.

Specific optical rotation: get this product, accurate claim surely, be dissolved in water and be diluted to the solution that contains 10mg among every 1ml approximately, measure (two appendix vI of Chinese Pharmacopoeia version in 2000 E) in accordance with the law, specific optical rotation is+70 ℃ to+80 ℃.

[discriminating] 1, get this product 10mg, add water 0.5ml and make dissolving, add a-naphthols alcoholic solution (5-100) 1ml, shake up, slowly add sulphuric acid 0.5ml along tube wall, after several minutes, the interface is aubergine.

2, get this product, add water and make the solution that contains 1mg among every 1ml, get about 10ul point on filter paper, dry, fix, put high salpeter solution and (get periodic acid 1.2g with dehydrated alcohol, after adding water 30ml dissolving. add 0.2mol/L sodium acetate solution 1.5ml and ethanol 100ml, mixing is promptly.Put the dark place and preserve, can use the several months) the middle immersion 5 minutes, take out, wash with 70% alcoholic solution, (get potassium iodide 5g, sodium thiosulfate 5g adds water 100ml and makes dissolving to put reducing solution, add ethanol 150ml, 2mol/L hydrochloric acid solution 2.5ml again, with adding, face the time spent and join with stirring) the middle immersion 5 minutes, take out, wash with 70% alcoholic solution, put in the pinkish red industry sulphuric acid test solution and soaked about 30 minutes, take out, (get sodium pyrosulfite 0.4g with sodium metabisulfite solution, add hydrochloric acid 1ml, being dissolved in water makes into 100ml, promptly), and flushing, dry, the place should be aubergine at the filter paper point sample.

3, get test sample and reference substance is an amount of, add respectively the chlorination sodium injection make contain 1mg among every 1ml solution as need testing solution and reference substance solution, press the test of mannatide immunogenicity determining method, test sample and contrast QC should all not have haemolysis to be taken place.

[inspection] 1, acidity: get this product, add water and make the solution that contains IOmg among every 1ml, measure (two appendix VI of Chinese Pharmacopoeia version in 2000 H) in accordance with the law, pH value should be 3.0-5.0.

2, trap: get this product, add water and make the solution that contains 0.4mg among every 1ml, according to spectrophotography (two appendix VIA of Chinese Pharmacopoeia version in 2000), wavelength place at 260nm, its trap must not be greater than 0.25, and at the wavelength place of 280nm, its trap must not be greater than 0.20.

3, total nitrogen: get this product, measure according to N2 method (two appendix VIID second methods of Chinese Pharmacopoeia version in 2000). press dry product and calculate, contain total nitrogen and should be 0.8-2.0%.

4, immunogenicity: get test sample and reference substance is an amount of, add the chlorination sodium injection respectively and make the solution that contains 10mg among every 1ml, make 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64,1: 128,1: 256 diluent as need testing solution and reference substance solution with phosphate buffer respectively again, check (attached mannatide immunogenicity determining method) in accordance with the law, the least concentration of the insoluble blood vessel of test sample should be higher than the reference substance respective concentration-doubly more than.

5, loss on drying is got this product, is dried to constant weight at 105 ℃, subtracts weight loss and must not cross 5.0% (two appendix VIII of Chinese Pharmacopoeia version in 2000 L).

6, heavy metal is got this product, at 1.0g, checks that in accordance with the law (Chinese Pharmacopoeia version VIII in 2000 H) contains heavy metal and must not cross 20/1000000ths.

7, the undue toxicity gets this product, adds the chlorination sodium injection and makes the solution that contains 0.5mg among every 1ml, checks (Chinese Pharmacopoeia version appendix in 2000 XI C) in accordance with the law. press the intravenous injection administration, and should (injection) up to specification.

[assay]

1. the preparation of reference substance solution

Precision takes by weighing through 105 ℃ of D-mannose reference substance 0.1g that are dried to constant weight and puts 100ml

In the measuring bottle, be dissolved in water and be diluted to scale. shake up; Precision is measured 5ml, puts 100ml's

In the measuring bottle, add water to scale, shake up. contain mannose 50ug among every 1ml.

2. need testing solution is equipped with

It is an amount of to get this product, and accurate the title decides, and is dissolved in water and makes the solution that contains 40ug among every 1ml.

3. the preparation of standard curve

Precision takes by weighing reference substance solution 0,0.2,0.4,0.6,0.8,1.0ml, put respectively in the tool plug test tube, respectively add water to 1.0ml, add 3% phenol solution 1.0ml again, shake up, pour sulphuric acid 4.5ml, shake up, being positioned over room temperature, is blank with 0 pipe. measure trap according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A) at the wavelength place of 490nm.To corresponding trap, calculate regression equation with mannose ug number.

4. algoscopy

Precision is measured need testing solution 1.0ml, and from " adding 3% phenol solution 1.0ml again ", operation is in accordance with the law measured trap, by the content of regression equation calculation mannose under the sighting target directrix curve preparation.

Mannatide immunogenicity determining method (complement combined techniques);

1, test solution

A. phthalate buffer (pH7.2)

Get sodium chloride 8.5g, sodium hydrogen phosphate 0.565g and potassium dihydrogen phosphate 0.135g, add water to 1000ml and make its dissolving, add 10% Adlerika 1ml, shake up.

B.1% sheep erythrocyte suspension

The preparation of sheeps blood erythrocyte: (get glucose 20.5g, sodium citrate 8.0g, sodium chloride 4.2g in filling equivalent A Shi liquid by sheep jugular vein sterile blood sampling, citric acid 5.5g, add water to 1000mI and make dissolving, 100 ℃ the sterilization 30 minutes) sterile chamber in, 4 ℃ of preservations.

The preparation of 1% sheeps blood erythrocyte suspension: it is an amount of to get above-mentioned sheeps blood erythrocyte, and with sodium chloride injection washing three times, each centrifugal 5 minutes (2000 rev/mins), sheeps blood erythrocyte is amassed in pressure, makes 1% sheeps blood erythrocyte suspension with sodium chloride injection.Get suspension, make need testing solution for 20 times with the sodium chloride injection dilution, other gets 20 times of equivalent suspension thin ups as blank solution, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A), wavelength place at 541nm measures, its trap should be 0.65-0.70, should regulate the concentration of 1% sheep erythrocyte suspension as overrun.

C. hemolysin and sensitization sheeps blood erythrocyte

The preparation of hemolysin: get above-mentioned hematocrit sheeps blood erythrocyte, make 25% sheeps blood erythrocyte suspension with sodium chloride injection. get 1 of rabbit, the above-mentioned sheeps blood erythrocyte suspension of intravenous injection, once a day, totally seven times, first three time 3ml, three 5ml in back, last is injected blood sampling in back seven days.Separation of serum, 56 ℃ of deactivations in .30 minute, packing is preserved below 0 ℃.

The mensuration of amboceptor unit: it is an amount of to get hemolysin, add phosphate buffer and make 1: 1000,1: 2000,1: 3000,1: 4000,1: 5000,1: 6000,1: 7000,1: 8000,1: 9000,1: 10000 diluent respectively, respectively getting 0.1ml puts in the test tube, add 1% Sanguis caprae seu ovis cell suspension 0.1ml, shake up, add dilution factor and be 1: 30 guinea pig serum (complement) 0.2ml and phosphate buffer 0.2ml, shake up, 37 ℃ of insulations 30 minutes, the high dilution of complete hemolysis pipe is 1 unit hemolysin.

The preparation of sensitization sheeps blood erythrocyte: before facing usefulness, the sheeps blood erythrocyte suspension with 2% mixes with 2 unit haemolysis rope equal-volumes, and 37 ℃ are incubated 15 minutes promptly.

Complement: get the Cavia porcellus more than three, the heart blood sampling, centrifugalize serum is preserved below 0 ℃.

The mensuration of complement unit: it is an amount of to get complement, add phosphate buffer, make 1: 40,1: 60,1: 80,1: 100,1: 120,1: 140,1: 160,1: 180 diluent respectively, respectively get 0.2ml and put in the test tube, add the 0.2ml phosphate buffer, shake up, 37 ℃ of insulations are after 30 minutes, add sensitization sheeps blood erythrocyte 0.2ml respectively, shake up, 37 ℃ are incubated 30 minutes again. and the high dilution of complete hemolysis pipe is the complement of 1 unit.

Antibody: it is an amount of to get the mannatide reference substance, add the chlorination sodium injection and make the reference substance solution immunizing rabbit that contains 10mg among every 1ml, the next day adopt ear vein injection reference substance solution once.Inject for the first time 0.2ml, for the second time respectively inject 0.5ml to the 5th time, respectively inject 1.0ml the 6th time to the tenth time, the tenth once respectively injects 2.0ml to the 15 time, and last is injected blood sampling in back three days, centrifugalize serum, 56 ℃ of deactivations in following 30 minutes, (before facing usefulness, needing 56 ℃ of deactivations once more in 30 minutes) preserved in packing below 0 ℃.

The mensuration of antibody unit: it is an amount of to get antibody, add phosphate buffer and make 1: 2,1: 4,1: 8,1: 16,1: 32.1: 64,1: 128 diluent respectively, respectively getting 0.1ml puts in the test tube, add mannatide reference substance solution 0.1ml and the 2 complement 0.2m of unit], shake up, place more than 4 hours in 4-8 ℃, put 37 ℃ of insulations 30 minutes, the high dilution of insoluble blood vessel is 1 unit antibody.Establish antigen simultaneously and (do not add antibody, replace with the 0.1ml phosphate buffer), antibody (do not add antigen, replace with the 0.1ml phosphate buffer), complement (do not add antigen, antibody, replace with the 0.2ml phosphate buffer) control tube, more than three kinds of control tube haemolysis entirely; The sensitization sheeps blood erythrocyte that other establishes (do not add antigen, antibody and complement, replace with the 0.4ml phosphate buffer) control tube is haemolysis not.

2, immunogenicity determining method

It is an amount of to get rapid glycopeptide reference substance of manna and test sample, make the reference substance solution and the need testing solution of variable concentrations according to the regulation under the medicine item, respectively getting 0.1ml puts in the test tube, add the 2 antibody 0.1ml of unit and the 2 complement 0.2ml. of unit shake up, more than 4 hours, put 37 ℃ of insulations 30 minutes in 4-8 ℃ of placement, add sensitization sheeps blood erythrocyte 0.2ml, shake up, 37 ℃ are incubated 30 minutes again.Observe the haemolysis situation of each pipe, the least concentration of insoluble blood vessel is represented the immunogenicity of mannatide.Establish antigen simultaneously and (do not add antibody, replace with the 0.1ml phosphate buffer), antibody (do not add antigen, replace with the 0.1ml phosphate buffer), complement (do not add antigen, antibody, replace) control tube with the 0.2ml phosphate buffer, more than three kinds of control tube haemolysis entirely: the sensitization sheeps blood erythrocyte that other establishes (do not add antigen, antibody and complement, replace with the 0.4ml phosphate buffer) control tube is haemolysis not.

The space flight strain is produced the preparation method of " No. three, divine boat " mannatide oral liquid:

Get the mannatide that the space flight strain is produced, add an amount of essence, correctives dissolving, add purified water, the after-filtration that stirs, fill, pressure sterilizing 105-121 ℃, 20-30 minute, packing warehouse-in after lamp inspection is qualified to full dose.

Embodiment 1:

Get mannatide 1 gram that the space flight strain is produced; Sweeting agent is an amount of and an amount of sodium chloride of essence is an amount of; Make 1000ml.

The mannatide 1g that the space flight strain is produced, saccharin sodium 0.15g, sodium chloride 1.0g dissolve with a small amount of purified water, adding essence 0.1ml, add purified water and stir evenly to 1000ml, filtration, chemically examine qualified after, embedding, 121 ℃ of steam sterilizations, 30 minutes.

Embodiment 2:

The mannatide material liquid 1000ml that the space flight strain is produced, active carbon 0.25g, xylitol 1.0g, sodium chloride 1.0g, essence 0.1ml makes 1000ml.

Get the mannatide raw material 1000ml that the space flight strain is produced, add active carbon 0.25g, stir evenly, filter, add xylitol 1.0g, sodium chloride 1.0g adds essence 0.1ml, stirs evenly, chemically examine qualified after, embedding, 121 ℃ of steam sterilizations, 30 minutes.

Claims (1)

1. antialcoholic oral liquid is characterized in that: contain among the medicine 1000ml that relieves the effect of alcohol and be numbered mannatide 1 gram that CGMCCNo.1082 produces, essence 0.1ml, saccharin sodium 0.15g, sodium chloride 1.0g make 1000ml.