Summary of the invention
The purpose of this invention is to provide a kind of antidotal medicine, it is by biological preparation and the compound antiaging agent oral liquid of making of Chinese medicine, and this oral liquid causes the factor of declining from the mechanism of aging at many-side, old and feeble process is checked in whole consideration from many aspects, at many levels.
Technical scheme of the present invention is: design a kind of antidotal medicine, it is characterized in that: it mainly comprises α-mannatide 15%-45%, melatonin 0.5%-5%, removes radical type Chinese medicine 50%-80%.
Described medicine comprises α-mannatide 20%-40%, melatonin 0.5%-3%, removes the Chinese medicine 5%-30% of radical type Chinese medicine 30%-60%, human body immunity improving power.
Described medicine comprises α-mannatide 20%-50%, melatonin 0.5%-3%, removes the Chinese medicine 5%-20% of radical type Chinese medicine 10%-50%, human body immunity improving power, regulates the Chinese medicine 10%-30% of neuroendocrine function.
Described medicine comprises the Chinese medicine 5%-30% of α-mannatide 20%-50%, melatonin 0.5%-3%, removing radical type Chinese medicine 10%-50%, the Chinese medicine 5%-20% of human body immunity improving power, the Chinese medicine 10%-30% that regulates neuroendocrine function, adjusting organism metabolism.
Described medicine comprises α-mannatide 20%40%, melatonin 0.5%-3%, removes the Chinese medicine 5%-20% of radical type Chinese medicine 10%-50%, human body immunity improving power, regulates Chinese medicine 10%-30%, the Chinese medicine 5%-30% of adjusting organism metabolism, the Chinese medicine 5%-20% of prolongation cell survival of neuroendocrine function.
Described medicine comprises α-mannatide 20%-40%, melatonin 0.5%-3%, removes the Chinese medicine 5%-20% of radical type Chinese medicine 10%-50%, human body immunity improving power, regulates Chinese medicine 10%-30%, the Chinese medicine 5%-30% of adjusting organism metabolism, Chinese medicine 5%-20%, the Chinese medicine 10%-20% of anti-gene mutation of prolongation cell survival of neuroendocrine function.
Described Chinese medicine with the effect of removing free radical is Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Cordyceps, green tea, Semen Ginkgo, Radix Salviae Miltiorrhizae, Cortex Magnoliae Officinalis, Fructus Jujubae, Radix Pseudostellariae, Semen Ziziphi Spinosae, Radix Rehmanniae; Chinese medicine with human body immunity improving power is Radix Ginseng, the Radix Astragali, Placenta Hominis, CORTEX ACANTHOPANACIS, Herba Epimedii, Herba Houttuyniae, Ramulus Mori; Having the Chinese medicine of regulating neuroendocrine function is Radix Et Caulis Acanthopanacis Senticosi, Rhizoma Polygonati, Radix Rehmanniae Preparata, Fructus Lycii, Fructus Mori, male Bombycis mori, Radix Codonopsis, the Rhizoma Pinelliae, Pericarpium Citri Reticulatae, Poria, Rhizoma Alismatis, Rhizoma Dioscoreae, Fructus Amomi, Cortex Cinnamomi; Having the Chinese medicine of regulating organism metabolism is Herba Lactucae formosanae, Ganoderma, Radix Ginseng, Folium Camelliae sinensis, Herba Ephedrae, Scolopendra, Fructus Arctii, Radix Angelicae Sinensis, Fructus Crataegi, Semen Juglandis, Semen Armeniacae Amarum, Cordyceps, Folium Camelliae sinensis, Radix Notoginseng, Radix Et Caulis Acanthopanacis Senticosi, Mel, the Radix Astragali, Radix Rehmanniae, Fructus Lycii, Rhizoma Polygonati, Fructus Ligustri Lucidi; Has the Chinese medicine that prolongs cell survival: Rhizoma Polygonati, the Radix Polygoni Multiflori, Margarita powder, Herb Gynostemmae Pentaphylli, the Radix Astragali, Radix Ginseng, Fructus Lycii, Radix Codonopsis, bee pollen, Radix Angelicae Sinensis, Semen Ziziphi Spinosae, Herba Epimedii, Poria; Chinese medicine with anti-gene mutation is Fructus Lycii, Radix Ginseng, the Radix Polygoni Multiflori, Herb Gynostemmae Pentaphylli, the Rhizoma Atractylodis Macrocephalae, Radix Asparagi, Radix Et Caulis Acanthopanacis Senticosi, Ganoderma.
Contain α-mannatide 300g, melatonin 20g, Fructus Schisandrae Chinensis 50g, Radix Angelicae Sinensis 50g, Cordyceps 50g, green tea 50g, Semen Ginkgo 50g, Radix Salviae Miltiorrhizae 50g, Cortex Magnoliae Officinalis 50g, Fructus Jujubae 80g, Radix Pseudostellariae 100g, Semen Ziziphi Spinosae 100g, Radix Rehmanniae 50g among the described medicine 1000g.
Contain α-mannatide 300g, melatonin 20g, Fructus Schisandrae Chinensis 50g, Radix Angelicae Sinensis 50g, Cordyceps 50g, green tea 50g, Semen Ginkgo 50g, Radix Salviae Miltiorrhizae 50g, Cortex Magnoliae Officinalis 50g, Fructus Jujubae 80g, Radix Pseudostellariae 50g, Semen Ziziphi Spinosae 60g, Radix Rehmanniae 50g, Radix Ginseng 10g, Radix Astragali 10g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 15g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g among the described medicine 1000g.
Contain α-mannatide 250g among the described medicine 1000g, melatonin 20g, Fructus Schisandrae Chinensis 50g, Radix Angelicae Sinensis 50g, Cordyceps 30g, green tea 50g, Semen Ginkgo 50g, Radix Salviae Miltiorrhizae 50g, Cortex Magnoliae Officinalis 30g, Fructus Jujubae 30g, Radix Pseudostellariae 50g, Semen Ziziphi Spinosae 30g, Radix Rehmanniae 30g, Radix Ginseng 10g, Radix Astragali 10g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g Radix Et Caulis Acanthopanacis Senticosi 20g, Rhizoma Polygonati 20g, Radix Rehmanniae Preparata 20g, Fructus Lycii 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 15g, Pericarpium Citri Reticulatae 10g, Poria 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 15g, Cortex Cinnamomi 15g.
Contain α-mannatide 250g, melatonin 15g, Fructus Schisandrae Chinensis 30g, Cordyceps 30g, green tea 40g, Semen Ginkgo 40g, Radix Salviae Miltiorrhizae 30g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 30g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 30g, Radix Rehmanniae 30g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g among the described medicine 1000g.Radix Et Caulis Acanthopanacis Senticosi 10g, Rhizoma Polygonati 10g, Radix Rehmanniae Preparata 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 5g, Pericarpium Citri Reticulatae 10g, Poria 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 5g, Cortex Cinnamomi 5g, Herba Lactucae formosanae 20g, Ganoderma 10g, Radix Ginseng 10g, Folium Camelliae sinensis 15g, Herba Ephedrae 10g, Scolopendra 5g, Fructus Arctii 10g, Radix Angelicae Sinensis 10g, Fructus Crataegi 20g, Semen Juglandis 10g, Semen Armeniacae Amarum 10g, Cordyceps 5g, Folium Camelliae sinensis 10g, Radix Notoginseng 20g, Radix Et Caulis Acanthopanacis Senticosi 10g, Mel 10g, Radix Astragali 10g, Radix Rehmanniae 10g, Fructus Lycii 10g, Rhizoma Polygonati 10g, Fructus Ligustri Lucidi 10g.
Contain α-mannatide 200g among the described medicine 1000g, melatonin 15g, Fructus Schisandrae Chinensis 30g, Cordyceps 30g, green tea 30g, Semen Ginkgo 20g, Radix Salviae Miltiorrhizae 30g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 30g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 30g, Radix Rehmanniae 30g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g, Radix Et Caulis Acanthopanacis Senticosi 10g, Rhizoma Polygonati 10g, Radix Rehmanniae Preparata 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 5g, Pericarpium Citri Reticulatae 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 5g, Cortex Cinnamomi 5g, Herba Lactucae formosanae 20g, Ganoderma 10g, Radix Ginseng 10g, Folium Camelliae sinensis 15g, Herba Ephedrae 10g, Scolopendra 5g, Fructus Arctii 10g, Radix Angelicae Sinensis 10g, Fructus Crataegi 20g, Semen Juglandis 10g, Semen Armeniacae Amarum 10g, Cordyceps 5g, Folium Camelliae sinensis 10g, Radix Notoginseng 20g, Radix Et Caulis Acanthopanacis Senticosi 10g, Mel 10g, Radix Astragali 10g, Radix Rehmanniae 10g, Fructus Lycii 10g, Fructus Ligustri Lucidi 10g, Rhizoma Polygonati 5g, Radix Polygoni Multiflori 5g, Margarita powder 10g, Herb Gynostemmae Pentaphylli 10g, Radix Astragali 5g, Radix Ginseng 5g, Fructus Lycii 10g, Radix Codonopsis 5g, bee pollen 5g, Radix Angelicae Sinensis 5g, Semen Ziziphi Spinosae 10g, Herba Epimedii 10g, Poria 15g.
Contain α-mannatide 200g among the described medicine 1000g, melatonin 15g, Fructus Schisandrae Chinensis 20g, Cordyceps 20g, green tea 10g, Semen Ginkgo 10g, Radix Salviae Miltiorrhizae 20g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 20g, Radix Pseudostellariae 20g, Semen Ziziphi Spinosae 15g, Radix Rehmanniae 15g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g, Rhizoma Polygonati 10g, Radix Rehmanniae Preparata 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 15g, Pericarpium Citri Reticulatae 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 5g, Cortex Cinnamomi 5g, Herba Lactucae formosanae 20g, Folium Camelliae sinensis 25g, Herba Ephedrae 10g, Scolopendra 15g, Fructus Arctii 10g, Radix Angelicae Sinensis 10g, Fructus Crataegi 20g, Semen Juglandis 10g, Semen Armeniacae Amarum 10g, Cordyceps 5g, Folium Camelliae sinensis 10g, Radix Notoginseng 20g, Radix Et Caulis Acanthopanacis Senticosi 10g, Mel 10g, Radix Astragali 10g, Radix Rehmanniae 15g, Fructus Ligustri Lucidi 15g, Rhizoma Polygonati 5g, Radix Polygoni Multiflori 5g, Margarita powder 10g, Herb Gynostemmae Pentaphylli 10g, Radix Astragali 5g, Radix Codonopsis 5g, bee pollen 5g, Radix Angelicae Sinensis 5g, Semen Ziziphi Spinosae 10g, Herba Epimedii 10g, Poria 15g, Fructus Lycii 25g, Radix Ginseng 20g, Radix Polygoni Multiflori 10g, Herb Gynostemmae Pentaphylli 10g, Rhizoma Atractylodis Macrocephalae 10g, Radix Asparagi 10g, Radix Et Caulis Acanthopanacis Senticosi 20g, Ganoderma 20g.
Characteristics of the present invention are: because Chinese medicines such as Fructus Schisandrae Chinensis, Radix Angelicae Sinensis can effectively be removed interior free yl, activate free radical scavenger superoxide dismutase (SOD); Medicine such as Rhizoma Polygonati, the Radix Polygoni Multiflori can effectively prolong the brain cell life-span, prevents the minimizing of neurocyte quantity, improves audition and vision, improves intelligence and memory, thereby reaches antidotal purpose; Herba Epimedii etc., α-mannatide have the effect of regulating immunity of organisms; Chinese medicines such as Cortex Cinnamomi have regulating action to hypothalamus-hypophysis-target gland axle; Chinese medicines such as Ganoderma have the raising metabolism, effects such as blood sugar regulation, blood fat reducing; Chinese medicine such as Fructus Lycii, Radix Ginseng has repair ability or the synthesis capability that promotes DNA damage, also has the effect of anti-gene mutation; Melatonin has the free radical of removing, raise immunity, the neuroendocrine effect of adjusting.In addition, also contain abundant vitamin and trace element in these natural drugs, they are that the normal physiological activity of body is indispensable, the same defense reaction that has to aging.
The specific embodiment
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.This strain is through the spaceship-carried space flight of Shenzhou series of spacecraft, and further screening and culturing breeding forms stable hereditary property, the tangible high-yield highly-effective strain of variation features to strain by BeiJing ZhongKe Institute of Micro-biology of institute and biology department of Northwest University.The mannan peptide content of α in the every fermentation unit of this strain-space flight strain production has improved more than several times.(the material 1 of seeing Appendix: 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: notice is accepted in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation culture presevation)
Strain improvement of the present invention: 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%; PH7.4.
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%; PH7.4.
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 10mg 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 VII of Chinese Pharmacopoeia version in 2000 D, second method). 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 in the 100ml measuring bottle, is dissolved in water and is diluted to scale. shake up; Precision is measured 5ml, puts in the measuring bottle of 100ml, adds water to scale, shakes 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 l% 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 and O.2ml 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.
Embodiment 1 gets above-mentioned α-mannatide 300g, melatonin 20g, Fructus Schisandrae Chinensis 50g, Radix Angelicae Sinensis 50g, Cordyceps 50g, green tea 50g, Semen Ginkgo 50g, Radix Salviae Miltiorrhizae 50g, Cortex Magnoliae Officinalis 50g, Fructus Jujubae 80g, Radix Pseudostellariae 100g, Semen Ziziphi Spinosae 100g, Radix Rehmanniae 50g, 1000g makes 10, every 10ml altogether.
During preparation with above-mentioned Chinese medicine dense fry in shallow oil three times → collecting decoction → filter and remove residue → with α-mannatide and melatonin mixing → filtration → concentrate → fill → seal → sterilize → lamp inspection → packing.
Concrete production process and equipment are pressed preparation technology's production (Chinese Pharmacopoeia Commission compiles, and Chemical Industry Press publishes) of 2000 editions oral liquids of Pharmacopoeia of People's Republic of China.
Just the medication composition is different with embodiment 1 for other embodiment, and other production process is identical.
Contain α-mannatide 300g, melatonin 20g, Fructus Schisandrae Chinensis 50g, Radix Angelicae Sinensis 50g, Cordyceps 50g, green tea 50g, Semen Ginkgo 50g, Radix Salviae Miltiorrhizae 50g, Cortex Magnoliae Officinalis 50g, Fructus Jujubae 80g, Radix Pseudostellariae 50g, Semen Ziziphi Spinosae 60g, Radix Rehmanniae 50g, Radix Ginseng 10g, Radix Astragali 10g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 15g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g among the medicine 1000g of embodiment 2.
Contain α-mannatide 250g among the medicine 1000g of embodiment 3, melatonin 20g, Fructus Schisandrae Chinensis 50g, Radix Angelicae Sinensis 50g, Cordyceps 30g, green tea 50g, Semen Ginkgo 50g, Radix Salviae Miltiorrhizae 50g, Cortex Magnoliae Officinalis 30g, Fructus Jujubae 30g, Radix Pseudostellariae 50g, Semen Ziziphi Spinosae 30g, Radix Rehmanniae 30g, Radix Ginseng 10g, Radix Astragali 10g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g Radix Et Caulis Acanthopanacis Senticosi 20g, Rhizoma Polygonati 20g, Radix Rehmanniae Preparata 20g, Fructus Lycii 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 15g, Pericarpium Citri Reticulatae 10g, Poria 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 15g, Cortex Cinnamomi 15g.
Contain α-mannatide 250g, melatonin 15g, Fructus Schisandrae Chinensis 30g, Cordyceps 30g, green tea 40g, Semen Ginkgo 40g, Radix Salviae Miltiorrhizae 30g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 30g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 30g, Radix Rehmanniae 30g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g among the medicine 1000g of embodiment 4.Radix Et Caulis Acanthopanacis Senticosi 10g, Rhizoma Polygonati 10g, Radix Rehmanniae Preparata 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 5g, Pericarpium Citri Reticulatae 10g, Poria 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 5g, Cortex Cinnamomi 5g, Herba Lactucae formosanae 20g, Ganoderma 10g, Radix Ginseng 10g, Folium Camelliae sinensis 15g, Herba Ephedrae 10g, Scolopendra 5g, Fructus Arctii 10g, Radix Angelicae Sinensis 10g, Fructus Crataegi 20g, Semen Juglandis 10g, Semen Armeniacae Amarum 10g, Cordyceps 5g, Folium Camelliae sinensis 10g, Radix Notoginseng 20g, Radix Et Caulis Acanthopanacis Senticosi 10g, Mel 10g, Radix Astragali 10g, Radix Rehmanniae 10g, Fructus Lycii 10g, Rhizoma Polygonati 10g, Fructus Ligustri Lucidi 10g.
Contain α-mannatide 200g among the medicine 1000g of embodiment 5, melatonin 15g, Fructus Schisandrae Chinensis 30g, Cordyceps 30g, green tea 30g, Semen Ginkgo 20g, Radix Salviae Miltiorrhizae 30g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 30g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 30g, Radix Rehmanniae 30g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g, Radix Et Caulis Acanthopanacis Senticosi 10g, Rhizoma Polygonati 10g, Radix Rehmanniae Preparata 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 5g, Pericarpium Citri Reticulatae 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 5g, Cortex Cinnamomi 5g, Herba Lactucae formosanae 20g, Ganoderma 10g, Radix Ginseng 10g, Folium Camelliae sinensis 15g, Herba Ephedrae 10g, Scolopendra 5g, Fructus Arctii 10g, Radix Angelicae Sinensis 10g, Fructus Crataegi 20g, Semen Juglandis 10g, Semen Armeniacae Amarum 10g, Cordyceps 5g, Folium Camelliae sinensis 10g, Radix Notoginseng 20g, Radix Et Caulis Acanthopanacis Senticosi 10g, Mel 10g, Radix Astragali 10g, Radix Rehmanniae 10g, Fructus Lycii 10g, Fructus Ligustri Lucidi 10g, Rhizoma Polygonati 5g, Radix Polygoni Multiflori 5g, Margarita powder 10g, Herb Gynostemmae Pentaphylli 10g, Radix Astragali 5g, Radix Ginseng 5g, structure Qi 10g, Radix Codonopsis 5g, bee pollen 5g, Radix Angelicae Sinensis 5g, Semen Ziziphi Spinosae 10g, Herba Epimedii 10g, Poria 15g.
Contain α-mannatide 200g among the medicine 1000g of embodiment 6, melatonin 15g, Fructus Schisandrae Chinensis 20g, Cordyceps 20g, green tea 10g, Semen Ginkgo 10g, Radix Salviae Miltiorrhizae 20g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 20g, Radix Pseudostellariae 20g, Semen Ziziphi Spinosae 15g, Radix Rehmanniae 15g, Placenta Hominis 20g, CORTEX ACANTHOPANACIS 10g, Herba Epimedii 15g, Herba Houttuyniae 10g, Ramulus Mori 10g, Rhizoma Polygonati 10g, Radix Rehmanniae Preparata 10g, Fructus Mori 10g, male Bombycis mori 15g, Radix Codonopsis 10g, Rhizoma Pinelliae 15g, Pericarpium Citri Reticulatae 10g, Rhizoma Alismatis 10g, Rhizoma Dioscoreae 15g, Fructus Amomi 5g, Cortex Cinnamomi 5g, Herba Lactucae formosanae 20g, Folium Camelliae sinensis 25g, Herba Ephedrae 10g, Scolopendra 15g, Fructus Arctii 10g, Radix Angelicae Sinensis 10g, Fructus Crataegi 20g, Semen Juglandis 10g, Semen Armeniacae Amarum 10g, Cordyceps 5g, Folium Camelliae sinensis 10g, Radix Notoginseng 20g, Radix Et Caulis Acanthopanacis Senticosi 10g, Mel 10g, Radix Astragali 10g, Radix Rehmanniae 15g, Fructus Ligustri Lucidi 15g, Rhizoma Polygonati 5g, Radix Polygoni Multiflori 5g, Margarita powder 10g, Herb Gynostemmae Pentaphylli 10g, Radix Astragali 5g, Radix Codonopsis 5g, bee pollen 5g, Radix Angelicae Sinensis 5g, Semen Ziziphi Spinosae 10g, Herba Epimedii 10g, Poria 15g, Fructus Lycii 25g, Radix Ginseng 20g, Radix Polygoni Multiflori 10g, Herb Gynostemmae Pentaphylli 10g, Rhizoma Atractylodis Macrocephalae 10g, Radix Asparagi 10g, Radix Et Caulis Acanthopanacis Senticosi 20g, Ganoderma 20g.
Composition principle: the Fructus Schisandrae Chinensis in the prescription, Radix Angelicae Sinensis can effectively be removed interior free yl, activate free radical scavenger superoxide dismutase (SOD); Rhizoma Polygonati, the Radix Polygoni Multiflori can effectively prolong the brain cell life-span, prevent the minimizing of neurocyte quantity, improve audition and vision, improve intelligence and memory, thereby reach antidotal purpose; Herba Epimedii, α-mannatide have the effect of regulating immunity of organisms; Cortex Cinnamomi has regulating action to hypothalamus-hypophysis-target gland axle; Ganoderma has raising metabolism, effects such as blood sugar regulation, blood fat reducing; Fructus Lycii, Radix Ginseng have repair ability or the synthesis capability that promotes DNA damage, also have the effect of anti-gene mutation; Melatonin has the free radical of removing, raise immunity, the neuroendocrine effect of adjusting.In addition, also contain abundant vitamin and trace element in these natural drugs, they are that the normal physiological activity of body is indispensable, the same defense reaction that has to aging.
The toxicological study of anti-senility oral liquid of the present invention: 1. acute toxicity test: the disposable filling stomach of Canis familiaris L. 300ml, do not see that dead and any untoward reaction takes place; 2. subacute toxicity test: the each oral 50ml of Canis familiaris L., every day three times, hepatic and renal function, hemogram and all no abnormal variation of each organs and tissues are checked in logotype one month; 3. the Cavia porcellus hypersensitive test is negative; 4. the deadly test of teratogenesis is all negative.
The animal pharmacodynamic study
1 test material
1.1 experimental animal: the Km mice, the Wistar rat, it is qualified to be one-level, is provided by The Fourth Military Medical University's Experimental Animal Center.Drosophila melanogaster (Drosophila malanogaster) is provided by Xi'an Jiaotong University Medical College's bioengineering dept.
1.2 medicine: α-mannatide oral liquid, seven kinds of oral liquids of the present invention provide by our company, Radix Polygoni Multiflori tablet, and the 0.25g/ sheet is produced by Tong Junge pharmaceutical factory, Chongqing.
1.3 reagent: india ink, 25ml/ bottle, the daily jointing material of Shanghai Changjiang River factory.Natrium carbonicum calcinatum, AR, 500g/ bottle, Beijing Chemical Plant's product.Acetylphenylhydrazine, CP, specification 100g/ bottle, Shanghai reagent three factories.The D-galactose, biochemical reagents, 25g/ bottle, Shanghai reagent two factories.Thiobarbituricacid (TBA), biochemical reagents, Shanghai reagent two factories.Tetraethoxypropane (TEP), standard substance, 10nmol/L is provided by Third Military Medical University's Regional research centers.Trichloroacetic acid, AR, Shanghai chemical reagent head factory.N-butyl alcohol, AR, Chongqing Dongfanghong chemical reagent work.Pyrogallol, AR, second chemical plant, zunyi, guizhou city.Chloroform, AR, the safe fine chemicals company limited in sky, Tianjin.Cyanmethemoglobin conversional solution (Drabkin liquid), serum alanine aminotransferase, aspartate amino transferase, total protein, measure test kit from albumen, blood glucose, blood urea nitrogen and inosine and provide by Chongqing medicinal agents institute.
1.4 instrument: day island proper Tianjin EB-3200D-AW electronic balance (sensibility reciprocal 10mg), the Shanghai second accurate torsion balance of the JN-B of balance factory (scale division value 0.5mg), day island proper Tianjin UV-730 automatic biochemical colour comparatour, optical instrument factory, Chongqing ordinary optical microscope, mice diving tower instrument, stopwatch, thermostatic water tank.
2 statistical procedures
Measurement data represents with X ± SD, and contrast in twos between organizing with the t check.Enumeration data x2
(2 * 2) contrast in twos between check or definite probabilistic method are organized.Contrast in twos between grade type data is organized with rank test.Inspection level a=0.05.All statistical procedures are all carried out on computers with the Sigmastat statistical software.
3 methods and result
3.1 influence to the mouse immune organ weight
The Km mice, body weight 11-15g, male and female half and half are divided into 10 groups at random by body weight, and promptly blank group, seven kinds of oral liquids of the present invention form seven groups, α-mannatide oral liquid group, Radix Polygoni Multiflori tablet group.Make solution with 0.5ml distilled water, 0.5ml seven kinds of oral liquids of the present invention, 0.5ml α-mannatide oral liquid, 0.5g Radix Polygoni Multiflori tablet respectively and irritate stomach, once a day, continuous 10 days.After the last administration 24 hours, putting to death animal with the cervical vertebra displacement method, weigh and the weight of thymus, spleen, is thymus or spleen coefficient (the results are shown in Table 1) with thymus or spleen weight (mg) with likening to of body weight (g).
The various medicines of table 1 are to mouse immune organ weight's influence
Group |
Number of animals |
Thymus coefficient (mg/g) |
Spleen coefficient (mg/g) |
Blank group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
10 10 10 10 10 10 10 10 10 10 |
3.0±0.9 4.8±0.6** 5.0±1.0** 5.2±0.6** 5.4±1.0** 5.6±0.5** 6.0±0.9** 6.5±0.8** 4.0±0.3* 3.9±0.5 |
3.0±0.4 5.2±0.8** 5.3±0.7** 5.5±0.4** 5.6±0.3** 5.8±0.7** 6.2±0.8** 6.3±1.1** 4.3±0.5* 3.8±0.7 |
Annotate: compare * P<0.05, * * P<0.01 with the blank group.
From the experimental data of table 1 as can be seen: seven kinds of oral liquids of the present invention can significantly increase the weight of mouse thymus childhood, spleen, and along with oral liquid prescription is constantly strengthened, the weightening finish of thymus and spleen is also more and more obvious, with the blank group utmost point significant difference is arranged relatively; Single also have tangible effect of gain with α-mannatide oral liquid to thymus and spleen, but effect is lower than seven kinds of oral liquids of the present invention; Radix Polygoni Multiflori tablet also has slight effect of gain to thymus and spleen, and gaining effect is starkly lower than seven kinds of oral liquids of the present invention and α-mannatide oral liquid.
3.2 influence to the effect of mice carbon clearance
The Km mice, body weight 18-22g, male and female half and half are divided into 10 groups at random by body weight, and promptly blank group, seven kinds of oral liquids of the present invention form seven groups, α-mannatide oral liquid group, Radix Polygoni Multiflori tablet group.Make solution with 0.5ml distilled water, 0.5ml seven kinds of oral liquids of the present invention, 0.5ml α-mannatide oral liquid, 0.5g Radix Polygoni Multiflori tablet respectively and irritate stomach, once a day, continuous 7 days.After the last administration 1 hour, tail vein injection dilution in 1: 2 india ink 0.1ml/10gBW is in annotating back 1 minute (t
1), 5 minutes (t
5), get blood 20 μ l with suction pipe through the eye socket rear vein beard respectively, measure the carbon granules phagocytic index K of every animal and proofread and correct phagocytic index α (the results are shown in Table 2).
The various medicines of table 2 are to the influence of mice carbon clearance effect
Group |
Number of animals |
Phagocytic index K (* 10
-2)
|
Proofread and correct phagocytic index α |
Blank group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
10 10 10 10 10 10 10 10 10 10 |
7.33±1.87 10.07±1.01** 10.27±0.94** 10.86±0.73** 11.25±1.05** 12.86±1.16** 13.07±0.97** 13.63±0.88** 9.25±0.73* 7.47±1.52 |
7.06±1.14 9.52±0.84** 9.73±1.13** 10.01±0.43** 10.25±0.62** 10.89±0.75** 11.34±0.89** 11.58±0.68** 9.01±0.51* 7.12±0.89 |
Annotate: compare * P<0.05, * * P<0.01 with the blank group.
From the experimental data of table 2 as can be seen: seven kinds of oral liquids of the present invention can significantly increase engulf the clean up ability of reticuloendothelial system to inertia carbon granules the blood, and along with oral liquid prescription is constantly strengthened, reticuloendothelial system to inertia carbon granules in the blood to engulf the ability of cleaning up also more and more stronger, with the blank group utmost point significant difference (P<0.01) is arranged relatively; Single also have tangible increase with reticuloendothelial system behind α-mannatide oral liquid to the ability of cleaning up of engulfing of inertia carbon granules in the blood, but effect is lower than seven kinds of oral liquids of the present invention; The reticuloendothelial system of Radix Polygoni Multiflori tablet group mice does not have obvious change to the ability of cleaning up of engulfing of inertia carbon granules in the blood.Show that seven kinds of oral liquids of the present invention have enhancing body nonspecific immunity function, α-mannatide oral liquid also has enhancing body nonspecific immunity function, but effect significantly is lower than the former.
3.3 influence to the generation of mice serum hemolytic antibody
The Km mice, body weight 18-22g, male and female half and half are divided into 11 groups at random by body weight, and promptly blank group, immune matched group, seven kinds of oral liquids of the present invention form seven groups, α-mannatide oral liquid group, Radix Polygoni Multiflori tablet group.Except that the blank group, the equal lumbar injection 2%RBC of all the other animals 0.2ml/ only carries out immunity, began to make solution with 0.5ml distilled water, 0.5ml distilled water, 0.5ml seven kinds of oral liquids of the present invention, 0.5ml α-mannatide oral liquid, 0.5g Radix Polygoni Multiflori tablet respectively on the immune same day and irritate stomach, once a day, continuous 6 days.1 hour eye socket, the 20 μ l that take a blood sample measure every mice sample half hemolysis value HC after the last administration
50(the results are shown in Table 3).
The influence that the various medicines of table 3 generate the mice hemolytic antibody
Group |
Number of animals |
HC
50 |
Blank group immunity control group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
10 10 10 10 10 10 10 10 10 10 10 |
4.59±1.32 14.67±5.89## 19.54±3.92* 22.25±8.86* 26.96±9.98** 28.57±7.23** 35.47±6.89** 40.97±7.68** 43.03±10.23** 20.14±5.13* 13.05±6.47 |
Annotate: compare ##P<0.01 with the blank group, compare * P<0.05, * * P<0.01 with immune matched group.
From the experimental data of table 3 as can be seen: after with 2%RBC mice being carried out immunity, its HC
50Significantly raise than the blank group.After using seven kinds of oral liquids of the present invention, visible HC
50All more immune matched group height, and along with the reinforcement of oral liquid prescription, to HC
50The rising effect more remarkable, show that seven kinds of oral liquids of the present invention have the effect that improves the humoral immunity of organism function.Single α-mannatide oral liquid of using is equally to HC
50Rising effect is to a certain degree arranged, but effect shows that inferior to seven kinds of oral liquids of the present invention seven kinds of oral liquids of the present invention are better than single with α-mannatide oral liquid to the raising effect of humoral immunity of organism function.Radix Polygoni Multiflori tablet does not have tangible rising effect to the humoral immunity of organism function.
3.4 acetylphenylhydrazine is caused the influence of mice model of blood dificiency
The Km mice, body weight 18-22g, male and female half and half are divided into 11 groups at random by body weight, and promptly blank group, model control group, seven kinds of oral liquids of the present invention form seven groups, α-mannatide oral liquid group, Radix Polygoni Multiflori tablet group.Tested the 1st, 4,7 day, to mice difference subcutaneous injection 2% acetylphenylhydrazine 0.1,0.05,0.05ml/10g, (normal control injection isometric(al) normal saline).From testing the 1st day beginning gastric infusion, normal group and model group are given distilled water 0.5ml, all the other each groups make 0.5ml solution for respectively 0.5ml seven kinds of oral liquids of the present invention, 0.5ml α-mannatide oral liquid, 0.5 g Radix Polygoni Multiflori tablet, once a day, continuous 8 days, eye socket blood sampling in the 9th day is with cyaniding high ferro spectrophotometry hemoglobin (Hb), with turbidimetry for Determination erythrocyte (RBC) number (the results are shown in Table 4).
The various medicines of table 4 are to the influence of blood deficiency mice peripheral blood Hb, RBC
Group |
Number of animals |
Hb(g/L) |
RBC(/L) |
Blank group model control group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
10 10 10 10 10 10 10 10 10 10 10 |
161.8±4.1 89.0±14.4## 106.07±12.5* 110.9±21.8* 112.8±17.8** 115.78±15.07** 120.34±11.16** 133.47±10.97** 160.63±11.36** 90.57±10.73 91.49±11.52 |
6.19±1.98 1.70±0.72## 2.21±0.54* 2.40±0.13* 2.69±0.58** 2.74±0.62** 3.89±0.75** 4.99±0.89** 6.04±0.38** 1.81±0.51 1.69±0.89 |
Annotate: compare ##P<0.01 with the blank group, compare * P<0.05, * * P<0.01 with model control group.
From the experimental data of table 4 as can be seen: model control group mice Hb and RBC all significantly are lower than the normal control group, show that model of blood dificiency makes successfully.After using seven kinds of oral liquids of the present invention the blood deficiency animal being treated, Hb and RBC are significantly increased in the animal blood, and along with the reinforcement of oral liquid prescription, curative effect is more remarkable, and Hb and RBC have returned to normal level in the part animal blood.Hb and RBC in α-mannatide oral liquid group fleece-flower root sheet group blood deficiency animal blood do not have obvious improvement.Show that seven kinds of oral liquids of the present invention have blood tonification effect preferably, its list α-mannatide oral liquid fleece-flower root sheet that is better than evident in efficacy.
3.5 the weary source of biochemistry is caused the influence of deficiency of vital energy mouse model
The Km mice, body weight 18-22g, male and female half and half are divided into 11 groups at random by body weight, and promptly blank group, model control group, seven kinds of oral liquids of the present invention form seven groups, α-mannatide oral liquid group, Radix Polygoni Multiflori tablet group.Except that the normal control group is given capacity feedstuff (2.5g/10g/d), all the other animal limitations of food intake (forage volume 1g/10g/d).The limit food began to make solution with 0.5ml distilled water, 0.5ml distilled water, 0.5ml seven kinds of oral liquids of the present invention, 0.5ml α-mannatide oral liquid, 0.5g Radix Polygoni Multiflori tablet respectively on the 5th day and irritates stomach, once a day, continuous 7 days, after the last administration 1 hour, by mice body weight 10% additional load, then mice is put into thermostatic water tank (depth of water 30cm, water temperature 14-16 ℃), observing 10 seconds of mouse head submerged can not the person of emerging be muscle power exhaustion, is mice swimming time (the results are shown in Table 5).
The various medicines of table 5 are to the influence of deficiency of vital energy mice low temperature load swimming time
Group |
Number of animals |
Low temperature load swimming time |
Blank group model control group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
10 10 10 10 10 10 10 10 10 10 10 |
169.0±54.1 119.0±30.9# 145.0±38.7* 146.0±36.8* 148.0±39.3* 150.0±37.2* 152.0±36.6* 155.0±35.6** 156.0±36.8** 120.0±35.1 123.0±36.4 |
Annotate: compare #P<0.05 with the blank group, compare * P<0.05, * * P<0.01 with immune matched group.
From the experimental data shown in the table 5 as can be seen: model control group animal low temperature load swimming time significantly shortens, and shows the obvious deficiency of vital energy of animal.After using seven kinds of oral liquids of the present invention deficiency of vital energy animal being treated, the equal significant prolongation of animal low temperature load swimming time, and along with the reinforcement of oral liquid prescription, curative effect is more remarkable, and part animal low temperature load swimming time returns to normal level.α-mannatide oral liquid group fleece-flower root sheet group deficiency of vital energy animal low temperature load swimming time does not have obvious improvement.Show that seven kinds of oral liquids of the present invention have QI invigorating effect preferably, its list α-mannatide oral liquid fleece-flower root sheet that is better than evident in efficacy.
3.6 influence to life span of drosophila melanogaster
Preparation fruit bat basal medium, the ovum of fruit bat, larva, pupa, adult are all cultivated in the cylindricality glass cell of 25 ± 1 ℃ and relative humidity 60%-70%, the about 60cm of every chamber volume
2Press different sexes, the raising of group locellus, 25-30 is only raised in every chamber.Collect the adult that sprouts wings in 10 hours, in basal medium, raised one month, make it to become aging fruit bat, continue to raise in random packet, establish blank group (0.5ml distilled water), seven kinds of oral liquids of the present invention form seven groups (each oral liquid of 0.5ml), α-mannatide oral liquid group (0.5ml), Radix Polygoni Multiflori tablet group (0.5g).Above drug level is the amount of contained seven kinds of oral liquids, α-mannatide oral liquid and Radix Polygoni Multiflori tablet of the present invention of every 100g culture medium.Changed a subculture in per 4 days, every day the time recording death toll, until whole death.With average life and maximum life span (average lifes of every group of last dead 5 fruit bats) is index, estimates the effect of lengthening the life (the results are shown in Table 6) of institute's reagent thing.
The various medicines of table 6 are to the influence of life span of drosophila melanogaster
Group |
The fruit bat number |
Average life (my god) |
The rate of lengthening the life (%) |
Maximum life span (my god) |
Blank group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
232 217 218 231 213 210 211 227 223 231 |
70.4±20.7 87.4±27.6** 88.1±25.1** 88.86±20.7** 89.25±21.5** 90.01±21.6** 90.77±20.9** 91.63±20.8** 79.25±20.3* 80.47±21.5* |
- 24.1 25.2 26.2 26.8 27.9 28.9 30.2 12.6 14.3 |
112.6 133.6 139.4 142.3 144.6 147.5 148.9 150.1 120.7 122.3 |
Annotate: compare * P<0.05, * * P<0.01 with the blank group.
From the experimental data of table 6 as can be seen: add seven kinds of fruit bats that oral liquid is turned out of the present invention the culture medium, average life and maximum life span are all than blank group significant prolongation, and more comprehensive along with oral liquid prescription, it is more obvious that the effect of life-saving embodies.α-mannatide oral liquid fleece-flower root sheet also has to a certain degree prolongation to life-span of fruit bat, but effect significantly is lower than seven kinds of oral liquids of the present invention.
3.7 the D-galactose is caused the influence of the inferior aging model of mice
The Km mice, body weight 22-25g, male and female half and half are divided into 11 groups at random by body weight, and promptly blank group, model control group, seven kinds of oral liquids of the present invention form seven groups, α-mannatide oral liquid group, Radix Polygoni Multiflori tablet group.Model group and each administration group mice cervical region every day subcutaneous injection 5%D-galactose 0.5ml/, continuous 4 weeks, matched group subcutaneous injection equal-volume normal saline.Began gastric infusion (be respectively 0.5ml distilled water, 0.5ml distilled water, 0.5ml seven kinds of oral liquids of the present invention, 0.5ml α-mannatide oral liquid, 0.5g Radix Polygoni Multiflori tablet and make solution), every day 1 time, totally 4 weeks the same day at injection of d-galactose.Cage was looked on and was examined mice outward appearance, active situation every day.Measure the learning and memory situation with the diving tower method when experiment finishes, eye socket blood sampling is then measured serum lipid peroxide (LPO) content with the thiobarbituricacid method, measures blood superoxide dismutase (SOD) activity with pyrogallol method.Get animal liver, cerebral tissue and make 5 μ m specimens paraffin embedding slices routinely, use lipofuscin content in Schmor ' the s reaction and display tissue, om observation, the result with the sxemiquantitative scoring formula divide 5 grades of expression (0 minute, do not see black-and-blue lipofuscin granule; 1 minute, the accidental fine particle that is dispersed in; 2 minutes, the fine particle of easily seeing; 3 minutes, the extensive fine grained that distributes; 4 minutes, the extensive fine grained that distributes, the thick engrain of part) (the results are shown in Table 7, table 8).
The various medicines of table 7 cause the influence of mouse memory acquired disturbance to the D-galactose
Group |
Number of animals |
Electric shock number of times in the training 5min |
Test is got an electric shock incubation period (S) |
Errors number in the training 5min |
Blank group model control group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
12 12 12 12 12 12 12 12 12 12 12 |
2.1±2.1 5.3±2.8## 2.7±1.2** 2.6±1.8** 2.5±1.4** 2.4±1.5** 2.2±1.1** 2.1±1.3** 2.0±1.0** 4.0±1.5* 3.9±1.6* |
152.3±81.6 81.5±65.6## 180.4±69.3* 186.7±70.1* 200.8±157.5* 209.2±68.5** 211.7±78.6** 224.5±89.5** 230.3±96.4** 120.3±58.6 124.2±88.4 |
2.0±1.5 4.8±2.7## 2.0±1.7* 1.9±1.3** 1.9±0.8** 1.8±1.3** 1.7±1.1** 1.7±1.3** 1.7±0.8** 3.5±1.3 3.5±1.5 |
Annotate: compare ##P<0.01 with the blank group, compare * P<0.05, * * P<0.01 with model control group.
Experimental result is found: after 4 weeks of D-galactose modeling, mice is movable to be reduced, behavior is slow, and fur is fluffy, and measuring its acquired memory with the diving tower method has obvious obstacle, get an electric shock and shorten incubation period, the test errors number of times increases, blood SOD reduced activity, and Serum LPO content increases, the generation of aging metabolite lipofuscin significantly increases in the cerebral tissue, shows with the D-galactose and successfully makes the animal aging model.After using seven kinds of oral liquid treatments of the present invention, the acquired dysmnesia of finding mouse aging have clear improvement, the electric shock prolongation of latency, the test errors number of times reduces, blood SOD increased activity, Serum LPO content descends, and the generation of aging metabolite lipofuscin significantly reduces in the cerebral tissue, and along with oral liquid prescription comprehensively, above-mentioned therapeutic effect is more remarkable.α-mannatide oral liquid fleece-flower root sheet can improve aging metabolite lipofuscin in the Serum LPO content of SOD activity, rising of test errors number of times, the reduction of electric shock incubation period, the increase of acquired dysmnesia, the prolongation of mouse aging and the cerebral tissue that increases to a certain extent, but evident in efficacyly is lower than seven kinds of oral liquids of the present invention.
The various medicines of table 8 cause the influence of the inferior aging model SOD of mice, LPO, lipofuscin to the D-galactose
Group |
Number of animals |
Erythrocyte sod (U/ml) |
Serum LPO MDAnmol/L |
Lipofuscin score value in the brain |
Blank group model control group first kind of oral liquid group second oral liquid of the present invention group the third oral liquid group of the present invention the 4th kind of oral liquid group of the present invention the 5th kind of oral liquid group of the present invention the 6th kind of oral liquid group of the present invention the 7th kind of oral liquid group α of the present invention-mannosans peptides oral liquor group shou wu pian group of the present invention |
12 12 12 12 12 12 12 12 12 12 12 |
34.0±5.0 14.9±8.7## 20.7±6.2* 21.6±7.8* 23.5±9.4* 26.4±9.5** 26.9±9.1** 27.3±8.3** 28.0±5.9** 16.8±11.5 17.8±8.6 |
35.2±32.9 71.2±31.7## 47.5±20.3* 45.3±21.3* 37.6±17.5** 36.5±18.4** 34.3±17.2** 32.5±14.6** 30.3±16.4** 60.3±18.6 59.2±14.5 |
0.5±0.4 2.6±0.7## 1.5±0.7* 1.4±0.5* 1.3±0.8* 1.2±0.9* 1.1±0.6** 1.1±0.4** 1.0±0.8** 2.0±1.3 2.1±1.5 |
Annotate: compare ##P<0.01 with the blank group, compare * P<0.05, * * P<0.01 with model control group.
4 discuss
Pharmacodynamic study is found, seven kinds of oral liquid gastric infusions of the present invention can obviously increase mice carbon clearance speed and mouse immune organ childhood (thymus, spleen) weight, promote the mice hemolytic antibody to generate, obviously prolong fruit bat average life and maximum life span, the inferior aging model of D-galactose induced mice, acetylphenylhydrazine hyperamization virtual model and biochemical weary source are caused model of qi-asthenia remarkable therapeutical effect is arranged, show this product have significantly set upright tonify deficiency, the anti-ageing pharmacological action that lengthens one's life, and evident in efficacy be better than single with α-mannatide oral liquid fleece-flower root sheet.
The clinical efficacy checking of anti-senility oral liquid of the present invention: Lee, woman, 55 years old, certain government functionary, fatigue and weak, limbs inaccurate coordination are often felt in readme in this year, visual deterioration and presbyopia, hypomnesis, tinnitus is frequent, poor sleeping quality, the self-induction passive protective physical fitness descends, often catch a cold, the yellowish-brown senile plaque appears in face, and blood pressure and blood fat are higher.Go to hospital to carry out symptomatic treatment, poor effect.Take anti-senility oral liquid of the present invention after 10 days, extended to present 7-8 hour from original 4-5 hour the length of one's sleep in night, and sleep state obviously improves; Blood pressure drops to present 110/70mmHg from original 160/90mmHg, recovers normal substantially; Mental status is clearly better, and continues to take two months, and the various discomforts of health are all improved, trick is light, activity freely, eyes are bright, memory is improved, tinnitus disappears, do not catch a cold once from taking medicine, passive protective physical fitness strengthens, and blood pressure and blood fat recover normal level, facial senile plaque fades away gradually, and skin smooth is ruddy.As seen anti-senility oral liquid of the present invention descends to senile physical function definite improvement effect comprehensively.
The statistics of anti-senility oral liquid clinical efficacy of the present invention: collect 349 old peoples' data, wherein male 200 examples, women 149 examples, age 55-65 year, the old people of every adding takes anti-senility oral liquid of the present invention, every day twice, each 10ml.Medication is added up after two months: 246 patients that the insomnia is arranged, and be 4-5 hour the average length of one's sleep between medication eve, and returned to 7-8 hour the average length of one's sleep at night after the medication, and sleep quality obviously improves; 198 hyperpietics, the mean blood pressure value is 150/80mmHg before the medication, and the mean blood pressure value is 100/70mmHg after the medication, and blood pressure returns to normal level substantially; 5 cancer patients' average survival period surpasses three and half, obviously prolongs than the average survival period of cancer patient of generally not taking this medicine, and quality of life improves; After 2 old man's medications the self-induction health change not obvious, all the other old men make moderate progress in all its bearings, good as the mental status, nutriture is good, and physique is strong, the ability of resisting disease strengthens, skin surface senile plaque desalination, audition, vision and memory all improve, and self original some diseases also all improves or recovers, each system's physiological function of health recovers normal, and the statistics total effective rate reaches 99.4%.