CN105148258A - Composition and application thereof, and preparation containing composition - Google Patents
Composition and application thereof, and preparation containing composition Download PDFInfo
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- CN105148258A CN105148258A CN201510685186.3A CN201510685186A CN105148258A CN 105148258 A CN105148258 A CN 105148258A CN 201510685186 A CN201510685186 A CN 201510685186A CN 105148258 A CN105148258 A CN 105148258A
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Abstract
The invention relates to the field of medicines, and in particular relates to a composition and an application thereof, and a preparation containing the composition. The composition comprises thymosin and begonia fimbristipula. Begonia fimbristipula polysaccharide is extracted and purified. An in vitro cell experiment and an animal experiment prove that the begonia fimbristipula polysaccharide has relatively high activity of stimulating immunization and inhibiting tumor cell growth. Furthermore, an animal experiment result proves that enhanced immunocompetence of the begonia fimbristipula polysaccharide and a thymosin extract has synergistic effects.
Description
Technical field
The present invention relates to field of medicaments, particularly a kind of compositions and application thereof, the preparation containing said composition.
Background technology
Polypeptide and polysaccharide are the common active substances with enhancing immunization, active polypeptide and glycocalix are developed to multiple pharmaceutical preparation, be widely used in the auxiliary treatment of autoimmune disease, infectious disease and inflammation mediated disease, achieve good therapeutic effect.
Thymosin is clinical conventional immunotherapy medicaments, be widely used in the treatment of viral hepatitis, also for the auxiliary treatment of the infectious disease such as autoimmune row disease (vitiligo, rheumatoid arthritis etc.) and sepsis, pulmonary tuberculosis, bronchitis.
The thymosin applied clinically, mainly extracts preparation from animal thymus tissue, then makes the preparation of different dosage form.The patent that at present disclosed thymosin extracts and preparation technique is relevant has 20 (Thymosin alpha 1s and Thymosin β4 Patents except), relates generally to the extractive technique of thymosin, and different dosage form Thymopeptide technology.Wherein many composition and effectivenesses compound formulation Patents 4 of thymosin, relate separately to the composite injection (CN03105324.6) of thymosin and glutathion composite injection (CN02123538.4), thymosin and Thymosin alpha 1, the composite powder injection (CN201410382672.3) of thymosin and poloxamer enteric coatel tablets and composite injection agent (CN200810166927.7) and thymosin and methionine.
Activities of Some Plants polysaccharide has stronger enhancing immunity, antitumor action, is developed to immunotherapy medicaments.The domestic vegetable polysaccharides entering clinical practice has lentinan, Ganoderma polysaccharide, pachyman, ginseng polysaccharide, ganoderma spore polysaccharide at present, is mainly used in the auxiliary treatment for the treatment of the bad caused various disease of immunologic function and malignant tumor.
Radix Semiaquilegiae (Begoniafimbristipula), is the perennial acaulescence herbaceous plant of Begoniaceae Begonia, is the endemic species of China, belongs to medicinal and edible plant, both can be used as medicine, and is again a kind of well nutritive health-care vegetable.Pharmacy test shows, Radix Semiaquilegiae nature and flavor are sweet, cool, has heat-clearing and toxic substances removing, blood sugar lowering, a medical value such as relieving cough and diminishing inflammation of invigorating blood circulation, antiviral, infection and anti-malignant cell growth.But Radix Semiaquilegiae Application and Development is pharmaceutically less, has not yet to see Patents, do not have no the relevant report of thymosin and Radix Semiaquilegiae polysaccharide conbined usage yet.
Summary of the invention
In view of this, the present invention utilizes thymosin and the synergistic function of Radix Semiaquilegiae polysaccharide in enhancing human body immunity, provides a kind of compositions and application thereof, the compound oral administration preparation containing said composition.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of compositions, comprise thymosin and Radix Semiaquilegiae.
In specific embodiments more of the present invention, described compositions, in mass fraction, comprises
Thymosin 5 ~ 15 parts
Radix Semiaquilegiae 20 ~ 55 parts.
In specific embodiments more of the present invention, Radix Semiaquilegiae described in described compositions is the former medicine of Radix Semiaquilegiae or Radix Semiaquilegiae polyoses extract.
In specific embodiments more of the present invention, the preparation method of thymosin described in described compositions mixes with pepsin, water for getting animal thymus, extracts, purification;
Described purification adopts the polydextran gel affinity chromatograph system with affinity ligand to carry out chromatography, collects the component that active stimulation index is greater than 10;
Described affinity ligand is the one in Quercetin aglucon, gallic acid aglucon and beta-schardinger dextrin-aglucon.
In specific embodiments more of the present invention, described in described compositions, the preparation method of thymosin comprises the steps:
Step 1: get thymus and pepsin, water mixed extraction, extracts and obtains thymosin extracting solution;
Step 2: get beta-schardinger dextrin-aglucon polydextran gel, Quercetin aglucon polydextran gel, gallic acid aglucon polydextran gel and water in g/mL according to mass volume ratio for 1:5 mixes, the swelling 24h of temperature, remove unnecessary water, again with water in g/mL according to mass volume ratio for 1:2 mixes, leave standstill, remove unnecessary water and floating gel particle, repeat 2 times, obtain the gel after process;
Step 3: get the gel after process (by glue stereometer after swelling) and mix with the volume ratio of water according to 3:1, through ultrasonic degas, dress hollow chromatographic column, with 5 times of column volume water balance chromatographic columns, balance liquid flow velocity is 3 times of elution flow rate; The blade diameter length ratio of post is 1.5 ~ 2.5:20;
Step 4: get the upper surface that described thymosin extracting solution adds described chromatographic column, applied sample amount is 10 ~ 20% of bed volume, and eluent flow rate is 15 ~ 30mL/min;
Step 5: on-line monitoring under 214nm UV-detector, the eluting peak that collection vigor is greater than 10%.
In specific embodiments more of the present invention, the preparation method of thymosin described in described compositions also comprises concentrated, cryodesiccated step.
In specific embodiments more of the present invention, in thymosin described in described compositions, content of peptides is 418mg/g ~ 436mg/g.
In specific embodiments more of the present invention, the preparation method of Radix Semiaquilegiae polyoses extract described in described compositions mixes with water for getting described Radix Semiaquilegiae, and obtained Radix Semiaquilegiae water extract, obtains through separation and purification.
In specific embodiments more of the present invention, separation and purification described in the preparation method of Radix Semiaquilegiae polyoses extract described in described compositions, comprises following characteristics step:
Step 1: described Radix Semiaquilegiae water extract through concentrating under reduced pressure, 80% alcohol settling 2 ~ 3 times;
Step 2: the precipitate getting step 1 acquisition mixes with water, upper macroporous resin column, the pure water eluting of 5 ~ 8 times of volumes collects eluent;
Step 3: eluent concentrating under reduced pressure, 80% alcohol settling 1 ~ 2 time, precipitation uses acetone and absolute ethanol washing 2 times more successively, and at 1 ~ 5Pa, vacuum drying under the condition of 30 ~ 60 DEG C, to obtain final product.
In specific embodiments more of the present invention, content >=80% of polysaccharide in Radix Semiaquilegiae polyoses extract described in described compositions, moisture≤5%.
Present invention also offers described compositions and prepare the application in medicine, health product and/or the food improving immunity.
Present invention also offers a kind of pharmaceutical preparation, comprise described compositions and pharmaceutically acceptable adjuvant.
In specific embodiments more of the present invention, described in described pharmaceutical preparation, pharmaceutically acceptable adjuvant comprises mannose and/or starch.
In specific embodiments more of the present invention, in mass parts in described pharmaceutical preparation, comprising:
In specific embodiments more of the present invention, in described pharmaceutical preparation, effective dose of every described pharmaceutical preparation is 100mg, and every day, formulation rate was 3 ~ 6.
The invention provides a kind of compositions, comprise thymosin and Radix Semiaquilegiae.The present invention has carried out extraction purification to Radix Semiaquilegiae polysaccharide, is found by cell assay in vitro and zoopery, and Radix Semiaquilegiae polysaccharide has the activity of stronger immune stimulatory and inhibition tumor cell growth.And results of animal shows, there is synergistic function in the enhancing immunocompetence of Radix Semiaquilegiae polysaccharide and thymosin extract.
Detailed description of the invention
The invention discloses a kind of compositions and application thereof, the preparation containing said composition, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
The invention provides a kind of compound oral administration preparation for immunocompromised patients's enhancing immunity, be made up of: thymosin 5 ~ 15%, Radix Semiaquilegiae polyoses extract 20 ~ 55%, mannitol 10 ~ 40% following raw material medicine, surplus is starch.
A kind of compound oral administration preparation improving immunity provided by the invention, the dosage form of described compound oral administration preparation is enteric coated capsule or oral tablet.
In specific embodiments more of the present invention, a kind of compound oral administration preparation improving immunity is with thymosin dry powder and Radix Semiaquilegiae polysaccharide dry powder for main pharmacodynamics composition, with manna sugar and starch for adjuvant.
In specific embodiments more of the present invention, the preparation method of described thymosin dry powder is:
(1) micro-yellow powder, easy moisture absorption, has fishy smell, content of peptides >=400mg/g, moisture≤5%.
(2) extracted by animal thymus and obtain thymus peptide solution, obtain through separation and purification, concentrated, lyophilization.
(3) animal thymus described in (1), is characterized in that any one or a few combination of pig, cattle, sheep, donkey, horse thymus.
(4) separation and purification described in (1), is characterized in that adopting the polydextran gel affinity chromatograph system with affinity ligand to carry out chromatography, collects the component that active stimulation index is greater than 10.
(5) affinity ligand described in (4), is characterized in that the one in Quercetin aglucon, gallic acid aglucon and beta-schardinger dextrin-aglucon.
In specific embodiments more of the present invention, the employing enzyme assisted extraction legal system of thymosin extracting solution is standby, and concrete operation method is as follows:
(1) FF calf thymus 1043 grams, remove fat and fascia, after rinsing well, weigh to obtain 1058 grams, add deionized water 1050 milliliters, homogenate 20 minutes under room temperature, until become milky white liquid.
(2) add the hydrochloric acid of 1mol/L to above-mentioned milky white liquid, regulate PH to 1.8, then add the pepsin adding 0.1% by raw material weight, 200 revs/min of stirrings, 40 DEG C of enzymolysis and extraction 3 hours, 100 DEG C of degeneration afterwards 15 minutes, be cooled to room temperature, obtain enzymolysis solution.
(3) above-mentioned enzymolysis solution 300 order non-woven fabrics filter, and filtrate, with the ultrafiltration of molecular cut off 10,000 dalton ultrafilter membrane, obtains micro-yellow clear transparent solutions, is thymosin extracting solution.
In specific embodiments more of the present invention, described Radix Semiaquilegiae polysaccharide dry powder:
(6) polyoses content >=80%, moisture≤5%.
(7) obtained through separation and purification by Radix Semiaquilegiae water extract.
(8) separation and purification described in (7), comprises following characteristics step:
I) Radix Semiaquilegiae water extract through concentrating under reduced pressure, 80% alcohol settling 2 ~ 3 times;
II) after precipitate is dissolved in distilled water, upper macroporous resin column (chromatographic column internal diameter 3.6cm, post bed height 100cm, flow velocity 3 ml/min), the purified water eluting of 5 ~ 8 times of volumes, collects eluent;
III) eluent concentrating under reduced pressure, 80% alcohol settling 1 ~ 2 time, precipitation uses acetone and absolute ethanol washing 2 times more successively, vacuum drying (1 ~ 5Pa, 30 ~ 50 DEG C) and get final product.
In specific embodiments more of the present invention, the preparation method of Radix Semiaquilegiae water extract is: 500 grams of Radix Semiaquilegiae pulverizers are pulverized, and crosses 40 mesh sieves.Add 10 liters of distilled water, boil extraction 1.5 hours, repeat extraction 2 times, merge extractive liquid, obtain 16.8 liters of Radix Semiaquilegiae water extracts.
The present invention for adjuvant, utilizes the characteristic of mannitol not easily moisture absorption, good fluidity with mannitol and starch, and the plastic property of starch.The two does not interact after mixing with thymosin dry powder and Radix Semiaquilegiae polyoses extract, good stability, and the homogeneity of the rear powder of mixing and good fluidity, hydroscopicity is low, has higher biological activity.
Preferably, be made up of the crude drug of following weight portion: thymus peptide 10 part, Radix Semiaquilegiae polyoses extract 40 parts, mannose 15 parts, starch 35 parts.
Thymosin-Radix Semiaquilegiae polysaccharide composite the preparation of invention, can be used for hepatitis, chemotherapy and postoperative auxiliary treatment.During treatment, oral, every day 3 times, each 1-2 grain (every content of dispersion 100mg), surrounding is a course for the treatment of.
Compositions provided by the invention and application thereof, all can be buied by market containing raw materials used in the preparation of said composition and reagent.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1
The employing enzyme assisted extraction legal system of thymosin extracting solution is standby, and concrete operation method is as follows:
(1) FF calf thymus 1043 grams, remove fat and fascia, after rinsing well, weigh to obtain 1058 grams, add deionized water 1050 milliliters, homogenate 20 minutes under room temperature, until become milky white liquid.
(2) add the hydrochloric acid of 1mol/L to above-mentioned milky white liquid, regulate PH to 1.8, then add the pepsin adding 0.1% by raw material weight, 200 revs/min of stirrings, 40 DEG C of enzymolysis and extraction 3 hours, 100 DEG C of degeneration afterwards 15 minutes, be cooled to room temperature, obtain enzymolysis solution.
(3) above-mentioned enzymolysis solution 300 order non-woven fabrics filter, and filtrate, with the ultrafiltration of molecular cut off 10,000 dalton ultrafilter membrane, obtains micro-yellow clear transparent solutions, is thymosin extracting solution.
Thymosin extracting solution adopts beta-schardinger dextrin-aglucon polydextran gel adsorption by hydrogen bond thin layer chromatography to carry out purification, and concrete operation method is as follows:
(4) Gel pretreatment: the ratio to beta-schardinger dextrin-aglucon polydextran gel volume ratio 1:5 by weight adds distilled water, stirs slightly, leave standstill immersion after 24 hours, incline unnecessary water.Add the distilled water of 2 times of volumes again, stir gently, leave standstill, incline unnecessary water and floating gel particle, repeats 2 times.
(5) post is filled: the gel filler after washing mixes with the volume ratio of distilled water by 3:1, after ultrasonic degas, slowly pours hollow chromatographic column into.After dress post completes, with 5 times of column volume pure water equilibrium chromatographic columns, balance liquid flow velocity is 3 times of elution flow rate.After having balanced, require that filler distribution is even, tight, bubble-free, the blade diameter length ratio of post is 2:20.
(6) loading and eluting: ultrafiltrate is carefully added on the upper surface of this chromatographic column, applied sample amount is 20% of bed volume, and eluent flow rate is 30 ml/min.
(7) collect: on-line monitoring under 214nm UV-detector, the eluting peak that collection vigor is greater than 10%.
(8) concentrated, dry: under above-mentioned eluent 37 DEG C of conditions after concentrating under reduced pressure, lyophilization, obtains thymosin dry powder.
(9) after testing, thymosin dry powder is pale yellow powder, taste raw meat, and content of peptides is 418mg/g, and the polypeptide response rate is 65.29%.The dry percentage of water loss (105 DEG C, 3 hours) 3.15% of dry powder, easy moisture absorption.
Embodiment 2
Adopt enzyme assisted extraction legal system for thymosin extracting solution, concrete operation method is as follows:
(1) FF calf thymus 580 grams, remove fat and fascia, after rinsing well, weigh to obtain 576 grams, add deionized water 1150 milliliters, homogenate 20 minutes under room temperature, until become milky white liquid.
(2) with the salt acid for adjusting pH to 1.8 of 1mol/L, add the pepsin of 0.2%, 200 revs/min of stirrings by raw material weight, 40 DEG C of enzymolysis and extraction 3 hours, 100 DEG C of degeneration afterwards 15 minutes, are cooled to room temperature, obtain enzymolysis solution.
(3) above-mentioned enzymolysis solution 300 order non-woven fabrics filter, and filtrate, with the ultrafiltration of molecular cut off 10,000 dalton ultrafilter membrane, obtains micro-yellow clear transparent solutions, is thymosin extracting solution.
Thymosin extracting solution adopts Quercetin aglucon polydextran gel adsorption by hydrogen bond thin layer chromatography to carry out purification, and concrete operation method is as follows:
(4) Gel pretreatment: add distilled water in the ratio (w/v) of 1:5 in Quercetin aglucon polydextran gel, stir gently, room temperature is after swelling 24 hours, and incline unnecessary water.Add the distilled water of 2 times of volumes again, stir gently, leave standstill, incline unnecessary water and floating gel particle, repeats 2 times.
(5) post is filled: the filler after washing mixes with the volume ratio of distilled water by 3:1, after ultrasonic degas, slowly pours hollow chromatographic column into.After dress post completes, with 5 times of column volume pure water equilibrium chromatographic columns, balance liquid flow velocity is 3 times of elution flow rate.After having balanced, require that filler distribution is even, tight, bubble-free, the blade diameter length ratio of post is 1.5:20.
(6) loading and eluting: ultrafiltrate is carefully added on the upper surface of this chromatographic column, applied sample amount is 15% of bed volume, and eluent flow rate is 20 ml/min.
(7) collect: on-line monitoring under 214nm UV-detector, the eluting peak that collection vigor is greater than 10%.
(8) concentrated, dry: under above-mentioned eluent 37 DEG C of conditions after concentrating under reduced pressure, lyophilization, obtains thymosin dry powder.
(9) after testing, thymosin dry powder is pale yellow powder, taste raw meat, and content of peptides is 436mg/g, and the polypeptide response rate is 82.132%.The dry percentage of water loss (105 DEG C, 3 hours) 3.28% of dry powder, easy moisture absorption.
Embodiment 3
Enzyme assisted extraction method prepares thymosin extracting solution, and concrete operation method is as follows:
(1) FF calf thymus 2000 grams, remove fat and fascia, after rinsing well, weigh to obtain 2038 grams, add deionized water 2000 milliliters, homogenate 20 minutes under room temperature, until become milky white liquid.
(2) adjust above-mentioned milky white liquid pH to 1.8 with 0.1 hydrochloric acid solution, add the pepsin of 0.1% by raw material weight, 200 revs/min of stirrings, 50 DEG C of enzymolysis and extraction 3 hours, 100 DEG C of degeneration afterwards 15 minutes, are cooled to room temperature, obtain enzymolysis solution.
(3) above-mentioned enzymolysis solution crosses 300 order non-woven fabrics filtrations, and filtrate, with the ultrafiltration of molecular cut off 10,000 dalton ultrafilter membrane, obtains micro-yellow clear transparent solutions, is thymosin extracting solution.
Thymosin extracting solution adopts gallic acid aglucon polydextran gel adsorption by hydrogen bond thin layer chromatography to carry out purification, and concrete operation method is as follows:
(4) Gel pretreatment: gallic acid aglucon polydextran gel mixes with the distilled water of 5 times of w/vs, room temperature is after swelling 24 hours, and incline unnecessary water.Add the distilled water of 2 times of volumes again, stir gently, leave standstill, incline unnecessary water and floating gel particle, repeats 2 times.
(5) post is filled: the filler after washing mixes with the volume ratio of distilled water by 3:1, after ultrasonic degas, slowly pours hollow chromatographic column into.After dress post completes, with 5 times of column volume pure water equilibrium chromatographic columns, balance liquid flow velocity is 3 times of elution flow rate.After having balanced, require that filler distribution is even, tight, bubble-free, the blade diameter length ratio of post is 2.5:20.
(6) loading and eluting: ultrafiltrate is carefully added on the upper surface of this chromatographic column, applied sample amount is 10% of bed volume, regulates eluent flow rate to be 15 ml/min.
(7) collect: on-line monitoring under 214nm UV-detector, the eluting peak that collection vigor is greater than 10%.
(8) concentrated, dry: under above-mentioned eluent 37 DEG C of conditions after concentrating under reduced pressure, lyophilization, obtains thymosin dry powder.
(9) after testing, thymosin dry powder is pale yellow powder, taste raw meat, and content of peptides is 428mg/g, and the polypeptide response rate is 87.57%.The dry percentage of water loss (105 DEG C, 3 hours) 3.09% of dry powder, easy moisture absorption.
Embodiment 4
1,500 grams of Radix Semiaquilegiae pulverizers are pulverized, and cross 40 mesh sieves.Add 10 liters of distilled water, boil extraction 1.5 hours, repeat extraction 2 times, merge extractive liquid, obtain 16.8 liters of Radix Semiaquilegiae extracting solution.
2, after measured, in extracting solution, polyoses content is 1.19 grams per liters, and extracting yield is 4.0%.
3, Radix Semiaquilegiae extracting solution, in 0.1MPa, concentrating under reduced pressure under 60 DEG C of conditions, obtains concentrated solution 1.6 liters.
4, in concentrated solution, add 8.53 liter of 95% ethanol, make concentration of alcohol reach 80%, leave standstill after 24 hours, buchner funnel sucking filtration, obtain crude polysaccharides precipitation 1.
5, above-mentioned polysaccharide precipitation 1 1.2 liters of distilled water dissolve, then add 6.4 liter of 95% ethanol, make concentration of alcohol reach 80%, and leave standstill after 24 hours, buchner funnel sucking filtration, obtains polysaccharide precipitation 2.
6, above-mentioned polysaccharide precipitation 21 liter of distilled water dissolution precipitation, adds 5.33 liter of 95% ethanol, makes concentration of alcohol reach 80%, leaves standstill after 24 hours, buchner funnel sucking filtration, obtains crude polysaccharides precipitation 3.
7, above-mentioned polysaccharide precipitation 3 is with 500 milliliters of 60 DEG C of hot water dissolvings, after being cooled to room temperature.Make the flow velocity of crude polysaccharides solution 3 ml/min by D101 macroporous resin chromatographic column (chromatographic column internal diameter 3.6cm, post bed height 100cm) with peristaltic pump, with 2.5 liters of distilled water with 5 ml/min flow velocity eluting, collect eluent.
8, above-mentioned eluent, 0.1MPa, 60 DEG C are concentrated into 0.5 liter of concentrate eluant.
9, adding 2.67 liter of 95% ethanol to above-mentioned concentrate eluant, is 80% to concentration of alcohol, hold over night, sucking filtration, and precipitation is dissolved in 0.5 liter of distilled water again, and after adding 95% ethanol repetition above-mentioned steps, sucking filtration obtains polysaccharide precipitation 4.
10, above-mentioned polysaccharide precipitation 4 respectively washes 2 times with 100 milliliters of acetone, dehydrated alcohol successively, and 50 DEG C of vacuum dryings obtain white Radix Semiaquilegiae polyoses extract 19.15 grams.
11, above-mentioned Radix Semiaquilegiae polyoses extract after testing, polyoses content 88.71%, and dry percentage of water loss (105 DEG C, 3 hours) is 2.88%.In purge process, polysaccharide recovery is 84.97%.
Embodiment 5
1,500 grams of Radix Semiaquilegiae pulverizers are pulverized, and cross 80 mesh sieves.Add 8 liters of distilled water, boil extraction 2 hours, repeat extraction 2 times, merge extractive liquid, obtain 13.5 liters of Radix Semiaquilegiae extracting solution.
2, after measured, in extracting solution, polyoses content is 1.38 grams per liters, and extracting yield is 3.73%.
3, Radix Semiaquilegiae extracting solution, in 0.1MPa, concentrating under reduced pressure under 60 DEG C of conditions, obtains concentrated solution 1.2 liters.
4, in concentrated solution, add 6.4 liter of 95% ethanol, make concentration of alcohol reach 80%, leave standstill after 24 hours, buchner funnel sucking filtration, obtain crude polysaccharides precipitation 1.
5, above-mentioned polysaccharide precipitation 11 liter of distilled water dissolution precipitation, adds 5.33 liter of 95% ethanol, makes concentration of alcohol reach 80%, leaves standstill after 24 hours, buchner funnel sucking filtration, obtains crude polysaccharides precipitation 2.
6, above-mentioned polysaccharide precipitation 2 is with 500 milliliters of 60 DEG C of hot water dissolvings, after being cooled to room temperature.Make the flow velocity of crude polysaccharides solution 3 ml/min by D101 macroporous resin chromatographic column (chromatographic column internal diameter 3.6cm, post bed height 100cm) with peristaltic pump, with 4 liters of distilled water with 5 ml/min flow velocity eluting, collect eluent.
7, above-mentioned eluent, in 0.1MPa, is concentrated into 0.5 liter of concentrate eluant under 60 DEG C of conditions.
8, adding 2.67 liter of 95% ethanol to above-mentioned concentrate eluant, is 80% to concentration of alcohol, hold over night, and sucking filtration, obtains polysaccharide precipitation 3.
9, above-mentioned polysaccharide precipitation 3 respectively washes 2 times, 1 ~ 5Pa with 100 milliliters of acetone, dehydrated alcohol successively, and 30 DEG C of vacuum dryings obtain white Radix Semiaquilegiae polyoses extract 18.84 grams.
10, above-mentioned Radix Semiaquilegiae polyoses extract after testing, polyoses content 85.42%, and dry percentage of water loss (105 DEG C, 3 hours) is 2.63%.In purge process, polysaccharide recovery is 86.38%.
Embodiment 6
1,160 grams of Radix Semiaquilegiae cross 60 mesh sieves after pulverizing with pulverizer, add 3.2 liters of distilled water, soak after 6 hours, boil extraction 1.5 hours, repeat extraction 3 times, merge and obtain 8.4 liters of Radix Semiaquilegiae extracting solution.
2, after measured, in extracting solution, polyoses content is 0.82 grams per liter, and extracting yield is 4.31%.
3, Radix Semiaquilegiae extracting solution, in 0.1MPa, concentrating under reduced pressure under 60 DEG C of conditions, obtains concentrated solution 0.8 liter.
4, in concentrated solution, add 4.26 liter of 95% ethanol, make concentration of alcohol reach 80%, leave standstill after 24 hours, buchner funnel sucking filtration, obtain crude polysaccharides precipitation 1.
5, above-mentioned polysaccharide precipitation 1 0.8 liter of distilled water dissolution precipitation, adds 4.26 liter of 95% ethanol, makes concentration of alcohol reach 80%, leaves standstill after 24 hours, buchner funnel sucking filtration, obtains crude polysaccharides precipitation 2.
5, above-mentioned polysaccharide precipitation 2 0.5 liter of distilled water dissolution precipitation, adds 2.67 liter of 95% ethanol, makes concentration of alcohol reach 80%, leaves standstill after 24 hours, buchner funnel sucking filtration, obtains crude polysaccharides precipitation 3.
6, above-mentioned polysaccharide precipitation 3 is with 400 milliliters of 60 DEG C of hot water dissolvings, after being cooled to room temperature.Make the flow velocity of crude polysaccharides solution 3 ml/min by D101 macroporous resin chromatographic column (chromatographic column internal diameter 3.6cm, post bed height 100cm) with peristaltic pump, with 2.4 liters of distilled water with 5 ml/min flow velocity eluting, collect eluent.
7, above-mentioned eluent, in 0.1MPa, is concentrated into 0.2 liter of concentrate eluant under 60 DEG C of conditions.
8, adding 1.07 liter of 95% ethanol to above-mentioned concentrate eluant, is 80% to concentration of alcohol, hold over night, and sucking filtration, obtains polysaccharide precipitation 4.
9, above-mentioned polysaccharide precipitation 4 respectively washes 2 times, 1 ~ 5Pa with 50 milliliters of acetone, dehydrated alcohol successively, and 42 DEG C of vacuum dryings obtain white Radix Semiaquilegiae polyoses extract 6.58 grams.
10, above-mentioned Radix Semiaquilegiae polyoses extract after testing, polyoses content 88.53%, and dry percentage of water loss (105 DEG C, 3 hours) is 2.76%.In purge process, polysaccharide recovery is 84.57%.
Embodiment 7
Thymosin-Radix Semiaquilegiae polyoses extract makes enteric coated capsule, and adopt 3# capsule to load, every capsules fills 200 milligrams.
In every capsules, thymosin dry powder and Radix Semiaquilegiae polyoses extract dry powder are filled by a certain percentage, and overall control, at 100 milligrams, has been investigated mannose and starch proportion to the impact of filling powder mobility (angle of repose), the results are shown in Table 1.
The assay method of angle of repose (α): funnel is fixed on the diagram paper of horizontal positioned, hopper outlet and diagram paper distance are H, pour filling powder into funnel, until hopper outlet contacts with conical tip, measure cone base diameter (2R) and the height of cone, calculate angle of repose:
α=arctg (H/R), wherein R is chassis radius.
Table 1 mannose and content of starch are on the impact of filling powder angle of repose
Note: pharmacy can accept powder body should be less than 40 ° angle of repose.
From preparation angle, angle of repose, requirement was less than 40 °, and mobility just meets the requirements.Known according to table 1, the mobility of prescription 3-7 is better, is suitable for capsule-filling.Mannose price is higher, considers from cost-saving angle, can select 4,5 two prescriptions.
Adopt the mannitol in prescription 4 and starch proportion, change the ratio of thymosin and Radix Semiaquilegiae polysaccharide, obtain prescription 6,7, filling powder still compound formulation requirement angle of repose.
Embodiment 8---zoopery
1, experimental program
Healthy male SPF level ICR mice 96, body weight 20 ± 2g, is divided into 8 groups by weight average, often organizes 12.Experimental group setting and dosage regimen are in table 2.Medicine, after 30 days, carries out Turnover of Mouse Peritoneal Macrophages and engulfs chicken red blood cell test.
Table 2 drug effect cooperative experiment dosage regimen
Note: blank group gives normal saline.
2, data analysis and synergistic function judge
Experimental data adopts SPSS19.0 to carry out one factor analysis of variance, and P < 0.05 represents significant difference.
After experiment terminates, Q inspection is carried out to each group of macrophages phagocytic capacity, T lymhocyte transformation rate and NK cytoactive, analyze the synergism of thymosin and Radix Semiaquilegiae polysaccharide.Decision method is as follows:
Q=E
polypeptide-polysaccharide/ (E
polypeptide+ E
polysaccharide-E
polypeptide× E
polysaccharide) ... E is pharmacodynamics index observation
0.5<Q<0.85, antagonism; Q < 0.5, obvious antagonism
0.85<Q<1.15 is added merely
1.15<Q<20, collaborative enhancing; Q > 20, significantly strengthens.
3, experimental result
Each group of peritoneal macrophage phagocytic activity is in table 2.The macrophages phagocytic capacity of polysaccharide high dose group and thymosin group is significantly higher than matched group (P < 0.05), has immune-enhancing activity both proving.The immune-enhancing activity of polysaccharide group raises with the increase of polysaccharide consumption, and high dose group is significantly higher than low dose group (P < 0.05).The macrophages phagocytic capacity of each polysaccharide-polypeptide use in conjunction group is all significantly higher than corresponding polysaccharide dosage group.
According to Q assay, the Q-value of dosage in polysaccharide-polypeptide group and polysaccharide high dose-polypeptide group is respectively 1.25 and 1.21, when showing thymosin extract and Radix Semiaquilegiae polysaccharide (40-80mg/kg) drug combination of 20mg/kg dosage, there is obvious immune-enhancing activity.
Table 3 peritoneal macrophage phagocytic activity
| Experimental group | Number of animals | Macrophages phagocytic capacity (%) | Q-value |
| Blank group | 12 | 10.20±1.11 a | / |
| Polysaccharide low dose group | 12 | 12.34±1.30 a | / |
| Dosage group in polysaccharide | 12 | 19.83±1.95 ab | / |
| Polysaccharide high dose group | 12 | 26.25±2.10 b | / |
| Thymosin group | 12 | 20.71±2.32 ab | / |
| Polysaccharide low dosage-polypeptide group | 12 | 29.22±2.90 b | 0.96 |
| Dosage in polysaccharide-polypeptide group | 12 | 45.83±3.15 c | 1.25 |
| Polysaccharide high dose-polypeptide group | 12 | 50.26±3.93 c | 1.21 |
Note: different alphabetical subscript represents significant difference (P < 0.05).
Indicate: according to summary of the invention, formulation rate every day of thymosin Radix Semiaquilegiae compound formulation is 3 ~ 6, and every effective dose is 100mg, dosage of being namely grown up is 5-10mg/kg (adult's body weight is in 60kg).According to people such as Huang Jihan, (dose,equivalent in pharmacological testing between animal and between animals and human beings body converts.Chinese Clinical pharmacology and therapeutics, 2004; 9 (9): 1069-1072.) method reported, mice dose,equivalent is 12.33 times of adult, i.e. 61.65-123.3mg/kg.In this experiment, when thymosin and Radix Semiaquilegiae polysaccharide drug combination, create synergistic function in the dosage interval of 60-100mg/kg.Therefore, the dosage of zoopery employing is consistent with the present invention.
The enteric coated capsule that the thymosin-Radix Semiaquilegiae polysaccharide of prescription 1 ~ 5 provided by the invention is made carries out above-mentioned animal experiment, and result of the test is close with the above results, without significant difference (P > 0.05).Show that enteric coated capsule provided by the invention can significantly improve immunity.
Embodiment 9
Clinical trial 1---thymosin-Radix Semiaquilegiae compound formulation is to heavy Type B viral hepatitis observation of curative effect
1, object
Hepatitis B patient 41 example, diagnostic criteria is with reference to " hepatitis B diagnostic criteria " (WS299-2008).All patient's courses of disease all exceed half a year, and its A type, the third type, hepatitis E virus (HAV, HCV, HEV) mark are all negative.
2, method
All patients are divided into 2 groups: (1) matched group 19 example, wherein man 17 example, female 2 example, and at 42.3 ± 5.9 years old age, wherein 16 examples merge spontaneous bacterial peritonitis (SBP); (2) treatment group, 21 examples, wherein man 19 example, female 2 example, at 40.1 ± 6.5 years old age, wherein 18 examples merge SBP.Before treatment, two groups in sex, age, clinical symptoms and lab index etc. without significant difference.
SBP diagnostic criteria: possess following any 2, gets rid of tuberculous peritonitis and cancer ascites.(1) heating, stomachache, abdominal part diffusivity tenderness, tension of abdominal muscle; (2) ascites leukocyte count > 0.5 × 10
9/ L or apocyte > 0.25 × 10
9/ L; (3) ascites bacteria culture is positive.
Matched group adopts general composite treatment, comprises hepatoprotective, infection, maintenance water and electrolyte balance and Supporting Therapy of suiting the medicine to the illness; Treatment group adds on the basis of the above with thymosin-Radix Semiaquilegiae compound enteric coated capsule (every capsules fills 200mg, wherein thymosin dry powder 20mg, Radix Semiaquilegiae polysaccharide 80mg, starch 70mg, mannitol 30mg), continues 8 weeks.Consumption is every day 3 times, each 1.
Observe treatment Patients Before And After serum bilirubin (TBIL), Prothrombin activity (PTA), SBP changes, the change of hepatitis B virus (HBV) mark.
3, result and evaluation
Observation of curative effect the results are shown in Table 4.
Front and back TBIL, PTA and SBP incidence rate treated by table 4
After treatment, TBIL, PTA and SBP incidence rate for the treatment of group, significantly lower than matched group (P < 0.05), shows that thymosin-Radix Semiaquilegiae polysaccharide composite enteric coated capsule has good curative effect to heavy hepatitis B.
The enteric coated capsule that all the other thymosins-Radix Semiaquilegiae polysaccharide of prescription 1 ~ 5 provided by the invention is made carries out above-mentioned human trial, and result of the test is close with the above results, without significant difference (P > 0.05).Show that enteric coated capsule provided by the invention has good curative effect to heavy hepatitis B.
Clinical trial 2---thymosin-Radix Semiaquilegiae polypeptide complex capsule is to the therapeutic effect of cancer patients patient
1, object
After Radical Colectomy for Carcinoma of Colon first the course for the treatment of chemotherapy 47 routine patients, be DukesB2 phase and C phase, without serious Organ dysfunction and chemotherapy taboo person.
2, method
Patient is divided into 2 groups at random.(1) chemotherapy group 19 example, wherein man 12 example, female 7 example, age (61.3 ± 11.5 years old); (2) thymosin compound formulation group 30 example, wherein man 19 example, female 11 example, age (63.8 ± 13.2 years old).Two groups of Age and sexes are without significant difference.
Chemotherapy group single file chemotherapy.Chemotherapy regimen is: cisplatin 60mg/m2 and mitomycin 10mg/m2 intravenous injection, first day; 5-fluorouracil 1000mg/m2 intravenous drip, 1 times/day.Thymosin compound formulation group carries out the chemotherapy of same approach, and (every capsules fills 200mg, wherein thymosin dry powder 20mg to Oral thymopeptidin compound enteric coated capsule simultaneously, Radix Semiaquilegiae polysaccharide 80mg, starch 70mg, mannitol 30mg), 2 tablets/time, every day 3 times, successive administration 4 weeks.
3, result and evaluation
Analyze first 1 day of Chemotherapy in Patients, chemotherapy starts latter 14th day, the cellular immune function of the 28th day and humoral immune function.The results are shown in Table 5, table 6.
Immunological Markers for Cell-Mediated Immunity change before and after table 5 liang group treatment
As shown in Table 5, after chemotherapy the 14th day and 28 days, chemotherapy group peripheral blood CD3, CD4, CD8 and CD4/CD8 and NK cells ratio are lower than before chemotherapy, and chemotherapy 28 days, the CD3 of thymosin group, CD4, CD8 and CD4/CD8 and NK cells ratio are all higher than before chemotherapy.The CD3 of thymosin group, CD4 and CD4/CD8 and NK cells ratio is significantly higher than chemotherapy group (P < 0.05).
Immunological indices change before and after table 6 liang group treatment
The result of table 6 shows, chemotherapy the 28th day, thymosin group serum IgG is significantly higher than chemotherapy group.
These results illustrate, thymosin-Radix Semiaquilegiae polysaccharide composite preparation can strengthen the immunity after colorectal cancer patients chemotherapy.
The enteric coated capsule that all the other thymosins-Radix Semiaquilegiae polysaccharide of prescription 1 ~ 5 provided by the invention is made carries out above-mentioned human trial, and result of the test is close with the above results, without significant difference (P > 0.05).Show enteric coated capsule provided by the invention can strengthen colorectal cancer patients chemotherapy after immunity.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a compositions, is characterized in that, comprises thymosin and Radix Semiaquilegiae.
2. compositions according to claim 1, is characterized in that, in mass fraction, comprises
Thymosin 5 ~ 15 parts
Radix Semiaquilegiae 20 ~ 55 parts;
Described Radix Semiaquilegiae is the former medicine of Radix Semiaquilegiae or Radix Semiaquilegiae polyoses extract.
3. compositions according to claim 1 and 2, is characterized in that, the preparation method of described thymosin mixes with pepsin, water for getting animal thymus, extracts, purification, concentrated, lyophilization and obtaining;
Described purification adopts the polydextran gel affinity chromatograph system with affinity ligand to carry out chromatography, collects the component that active stimulation index is greater than 10;
Described affinity ligand is the one in Quercetin aglucon, gallic acid aglucon and beta-schardinger dextrin-aglucon.
4. the compositions according to any one of claims 1 to 3, is characterized in that, in described thymosin, content of peptides is 418mg/g ~ 436mg/g.
5. the compositions according to any one of Claims 1-4, is characterized in that, the preparation method of described Radix Semiaquilegiae polyoses extract mixes with water for getting described Radix Semiaquilegiae, and obtained Radix Semiaquilegiae water extract, obtains through separation and purification.
6. compositions according to claim 4, is characterized in that, described separation and purification, comprises following characteristics step:
Step 1: obtain Radix Semiaquilegiae water extract; Through concentrating under reduced pressure, 80% alcohol settling 2 ~ 3 times;
Step 2: the precipitate getting step 1 acquisition mixes with water, upper macroporous resin column, the pure water eluting of 5 ~ 8 times of volumes collects eluent;
Step 3: eluent concentrating under reduced pressure, 80% alcohol settling 1 ~ 2 time, precipitation uses acetone and absolute ethanol washing 2 times more successively, and at 1 ~ 5Pa, vacuum drying under the condition of 30 ~ 60 DEG C, to obtain final product.
7. the compositions according to any one of claim 1 to 6, is characterized in that, in described Radix Semiaquilegiae polyoses extract, and content >=80% of polysaccharide, moisture≤5%.
8. the compositions according to any one of claim 1 to 7 is preparing the application in the medicine of raising immunity, health product and/or food.
9. a pharmaceutical preparation, is characterized in that, comprises the compositions as described in any one of claim 1 to 8 and pharmaceutically acceptable adjuvant;
Described pharmaceutically acceptable adjuvant comprises mannose and/or starch.
10. pharmaceutical preparation according to claim 9, is characterized in that, in mass parts, comprising:
Effective dose of every described pharmaceutical preparation is 100mg, and every day, formulation rate was 3 ~ 6.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108042665A (en) * | 2018-01-23 | 2018-05-18 | 德兴市百草园生态健康养生有限公司 | A kind of kidney rehabilitation traditional Chinese medicine powder and its preparation method and application |
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| CN109568345A (en) * | 2019-01-04 | 2019-04-05 | 湖南海济药业有限公司 | A kind of strengthen immunity, oral solution of anti-aging and preparation method thereof |
| CN110339347A (en) * | 2019-08-01 | 2019-10-18 | 山东晟奇生物科技有限公司 | One kind being used for immunoregulatory compound preparation |
| CN111685311A (en) * | 2020-07-02 | 2020-09-22 | 中国计量大学 | Processing method of gynura bicolor and lentinus edodes lotus root starch and product thereof |
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