CN107033001A - A kind of Chlorogenic acid compound and the compound stranguria-treating and calculus-removing tablet containing the compound - Google Patents
A kind of Chlorogenic acid compound and the compound stranguria-treating and calculus-removing tablet containing the compound Download PDFInfo
- Publication number
- CN107033001A CN107033001A CN201710310320.0A CN201710310320A CN107033001A CN 107033001 A CN107033001 A CN 107033001A CN 201710310320 A CN201710310320 A CN 201710310320A CN 107033001 A CN107033001 A CN 107033001A
- Authority
- CN
- China
- Prior art keywords
- chlorogenic acid
- compound
- calculus
- stranguria
- treating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
- C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
- A61K33/12—Magnesium silicate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/11—Pteridophyta or Filicophyta (ferns)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
- A61K36/355—Lonicera (honeysuckle)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
- A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
- A61K9/2806—Coating materials
- A61K9/282—Organic compounds, e.g. fats
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/31—Extraction of the material involving untreated material, e.g. fruit juice or sap obtained from fresh plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Inorganic Chemistry (AREA)
- Emergency Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Preparation (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
A kind of compound stranguria-treating and calculus-removing tablet the present invention relates to Chlorogenic acid compound and containing the compound.2 θ angles of characteristic peak include 4.6 ± 0.2 °, 16.2 ± 0.2 °, 17.7 ± 0.2 °, 18.4 ± 0.2 °, 19.7 ± 0.2 °, 20.5 ± 0.2 °, 21.6 ± 0.2 °, 22.8 ± 0.2 °, 25.6 ± 0.2 °, 26.1 ± 0.2 ° and 30.7 ± 0.2 ° in the X x ray diffration pattern xs that described Chlorogenic acid compound is obtained using Cu K alpha ray measurements.The invention further relates to compound stranguria-treating and calculus-removing tablet containing the compound and preparation method thereof, the Compound Tablet is due to containing above-mentioned Chlorogenic acid compound, and bioavilability, which has, to be obviously improved, and has excellent effect in terms of row's calculus, diuresis, analgesia, anti-inflammatory.
Description
Technical field
The present invention relates to the field of Chinese medicines, specifically, it is related to a kind of Chlorogenic acid compound and the compound containing the compound
Stranguria-treating and calculus-removing tablet.
Background technology
Stone in urinary system belongs to the traditional Chinese medical science " pain in the back ", " sand pouring ", " hematuria " category.Clinically often cause renal colic and hematuria, and
Can be because of Incarcerated stone Obstruction Associated uronephrosis, scabies secondary infection；Renal insufficiency can occur for the later stage, be the multiple disease of urinary system.
This disease is caused by damp and hot more, and sick position is in kidney and bladder, and disease is just more real, the long then simulataneous insufficiency and excessive of disease, therefore should strengthen differentiating, must sample
It is dialectical to be combined with the differentiation of disease with controlling, shoot the arrow at the target.Clearing heat and promoting diuresis, vital energy regualting and blood circulation-promoting, treating stranguria fossil are used real example more；Asthenic symptoms are then adopted
With methods such as temperature male wind-supplying kidney, strengthening the spleen and replenishing qi.For stone in urinary system the treatment traditional Chinese medical science more emphasize distinguish the damp and hot, deficiency of vital energy, stasis blocking, liver
The causes of disease such as strongly fragrant, spleen kidney deficiency, are reviewed because treating, its effect is favourable.
Stranguria-treating and calculus-removing tablet is that Desmodium styracifolium clearing heat and promoting diuresis in dampclearing prescription, side, Tonglin Paishi are monarch drug in a prescription, separately containing pyrrosia lingua, Hai Jin
Husky, caulis lonicerae and talcum powder, with reducing fever and causing diuresis, the effect of Tonglin Paishi.For the heat gonorrhea caused by damp invasion of lower energizer, urolithiasis, disease
See that frequent micturition, urgent urination, odynuria or urine have sandstone；Lithangiuria, pyelonephritis is shown in above-mentioned patient.
This side is one of common Chinese patent drug for the treatment of urethral calculus, and its formulation and preparation method are carried out in the prior art
Some are explored.
The Chinese patent of Application No. 200810030936.3 discloses a kind of compound urolithiasis open capsule and its preparation technology,
The thick paste obtained by Desmodium styracifolium, pyrrosia lingua, Lygodium japonicum, caulis lonicerae, Desmodium styracifolium, pyrrosia lingua, Lygodium japonicum, the weight proportion of caulis lonicerae
For 3:1:1:1, auxiliary material then adds talcum powder, starch, its thick paste:Talcum powder:The weight proportion of starch is：14~15:1:1.5~
2.This method is extracted using the decocting method of traditional Chinese medicine technique, and some shadows can be caused for the fractions of active ingredient in medicinal material
Ring.
The Chinese patent of Application No. 201010511343.6 discloses a kind of preparation of Chinese patent medicine oral liquid used for removing urinary stony calculi
Method, 1.563g/ml containing Desmodium styracifolium, containing 0.3% sodium benzoate and 0.1~1% Steviosin, containing 5~20% ethanol or is free of
Ethanol.The monarch drug in a prescription Desmodium styracifolium in the logical prescription of traditional urolithiasis is extracted in this patent as main component, is not met in Chinese medicine preparation
The principle of monarch.
The Chinese patent of Application No. 201310168883.2 discloses a kind of new method for preparing compound stranguria-treating and calculus-removing tablet, will
The method that Desmodium styracifolium in prescription is extracted using 50% alcohol heat reflux is extracted.New technique both saved production into
This, reduces the loss of active ingredient, more improves the curative effect of product.This is prepared in the method for compound stranguria-treating and calculus-removing tablet, is still used
Decocting method in traditional Chinese medicine pharmacy, although the ethanol for introducing various concentrations extracts the active component of medicine, but lacks to spy
Determine the extraction of species active material, may be by other without effective component introducing preparation.
To solve the above problems, special propose the present invention.
The content of the invention
It is a first object of the present invention to provide a kind of more excellent in row's stone, diuresis, anti-inflammatory, ease pain drug effect
Compound stranguria-treating and calculus-removing tablet containing Chlorogenic acid compound；It is a second object of the invention to provide a kind of Chlorogenic acid compound so that
Compound stranguria-treating and calculus-removing tablet containing the Chlorogenic acid compound has higher bioavilability.
To realize the purpose of the present invention, adopt the following technical scheme that：
A kind of Chlorogenic acid compound, the diffraction for the x-ray diffraction pattern that the compound is obtained by Cu-K alpha ray measurements
When angle is 2 θ, characteristic peak includes：4.6±0.2°、16.2±0.2°、17.7±0.2°、18.4±0.2°、19.7±0.2°、
20.5 ± 0.2 °, 21.6 ± 0.2 °, 22.8 ± 0.2 °, 25.6 ± 0.2 °, 26.1 ± 0.2 ° and 30.7 ± 0.2 °.
The diffraction maximum of the x-ray diffraction pattern of above-mentioned Chlorogenic acid compound includes：
The X-ray diffracting spectrum of above-mentioned Chlorogenic acid compound is as shown in Figure 1.
The present invention further technical scheme be：Above-mentioned Chlorogenic acid compound is to extract to prepare from pyrrosia lingua and caulis lonicerae
Obtain.
Present invention also offers a kind of preparation method of Chlorogenic acid compound, comprise the following steps：
(1) pyrrosia lingua and caulis lonicerae are taken, crushes and 50~80 mesh that sieve, is soaked in degreasing in low polar solvent；
(2) with the degreasing thing in 70% ethanol heating and refluxing extraction step (1), and filter while hot, adjusted and filtered with watery hydrochloric acid
Liquid pH be 4~6 after be concentrated to give pyrrosia lingua and Caulis Lonicerae extract crude product；
(3) pyrrosia lingua for obtaining step (2) is dissolved with Caulis Lonicerae extract crude product with 85% ethanol, filtering with microporous membrane,
By the upper macroporous absorbent resin of gained filtrate, thin-layer chromatography is eluted to 2.0ml/min speed as eluant, eluent with 40% ethanol
Tracking and monitoring without chlorogenic acid composition, will be collected obtained eluent and detected with thin-layer chromatography into eluent, merging with it is green
Ortho acid standard items Rf value identical eluents, recycling design obtains chlorogenic acid crude product；Washed different from chlorogenic acid standard items Rf values
De- liquid is decompressed to 0.08MPa recycling designs after merging under the conditions of 65~70 DEG C, and continue to be concentrated into relative density for 1.32~
1.35 medicinal extract C；
(4) the chlorogenic acid crude product for obtaining step (3) is dissolved in the mixed solution of methanol/ethyl acetate, wherein methanol with
The volume ratio of ethyl acetate is 2~4:1, the mass volume ratio of chlorogenic acid and the mixed solution is 1g:5~8ml, is adjusted with acetic acid
PH is 3~5, adds filtration sterilization after charcoal absorption, and filtrate is heated to after 45~60 DEG C with 0.5 DEG C/min speed to cool
Apply ultrasonic field progress crystallization of the frequency for 45~60kHz to filtrate simultaneously to 0~5 DEG C, crystal is obtained after being dried under reduced pressure washing.
Chlorogenic acid is a kind of phenylpropanoids that plant is produced during aerobic respiration through shikimic acid pathway, is
Caffeoyl guinic acid, oxidation resistance is strong, also emerging with anti AIDS virus, antitumor cell, antibacterial, raising maincenter
Put forth energy, cholagogic, teratogenesis, antiallergy and the function such as activity of adjusting Cytochrome P450 ligase.
Researcher of the present invention is had found in above-mentioned stranguria-treating and calculus-removing tablet, in addition to the curative effect that monarch drug in a prescription Desmodium styracifolium plays, pyrrosia lingua
The chlorogenic acid composition contained in caulis lonicerae of overall curative effect with to(for) the medicine has crucial effect, simultaneously because conventional method
Fairly simple for the extracting method of active component in pyrrosia lingua and caulis lonicerae in preparation, extract is the mixture of Multiple components,
Upgrading can not be carried out for a certain active material, therefore be introduced into large aperture adsorption resin to the chlorogenic acid composition in medicinal material
Purifying crystal is carried out, and is surprised to find that to be used to new chlorogenic acid crystal formation be greatly improved bioavilability in stranguria-treating and calculus-removing tablet.
In the step of above-mentioned preparation method (1), the frequency of the ultrasonic field is 50kHz.
Select different supersonic frequencies so that the degree of cavitation effect is different, can shorten the induction period of crystallization；Selection is different
Frequency the secondary nucleation process of crystallization can also be impacted, so as to accelerate crystalline rate.
In the step of above-mentioned preparation method (4), the power of the ultrasonic field is 300~600kW.
When changing ultrasonic power, certain influence can be produced on the granule size and size distribution of crystal, for chlorogenic acid
Tablet is made due to be engaged with main ingredient medicinal extract and pharmaceutic adjuvant in crystal, therefore its granule size and distribution directly affects medicine
The final effect of thing, therefore adjustment ultrasonic power is to produce one of committed step in chlorogenic acid crystal.
Present invention also offers a kind of compound stranguria-treating and calculus-removing tablet, the composition of described compound stranguria-treating and calculus-removing tablet includes：Desmodium styracifolium
1500g, Lygodium japonicum 500g, pyrrosia lingua 500g, caulis lonicerae 500g, talcum powder 25g, carboxyrnethyl starch sodium 10g, microcrystalline cellulose 30g, firmly
Fatty acid magnesium 3g and appropriate starch.
Microcrystalline cellulose is conventional tablet excipients, there is good compressibility, and has bonding concurrently, helps stream, calving disaggregation,
The tablet being pressed into has larger hardness, calving disaggregation, therefore adds in auxiliary material and select microcrystalline cellulose.
It is water extract-alcohol precipitation in view of extracting medicinal material in side, stickiness is stronger, makees excipients, Ke Nengda merely with microcrystalline cellulose
Less than good disintegration, another conventional excellent disintegrant-sodium carboxymethyl starch is selected.
In order that tabletting is smoothed out, the frictional force between sticking and reduction particle and particle, tablet and nib wall is reduced,
Make tablet smooth and beautiful appearance, appropriate lubricant need to be added, magnesium stearate is wide variety of lubricant, according to our factory's tablet manufacturing
Conventional amount used, each prescription adds magnesium stearate 3g.
Consider the outward appearance, hardness, disintegration time limited of tablet, various ratio of adjuvant influence little to the hardness of tablet；It is micro-
Crystalline cellulose consumption is in 3g/100 pieces, and tablet appearance is best in quality；Add carboxyrnethyl starch sodium larger on disintegration influence, can shorten
Disintegration time, consumption is convenient in 1g/100 pieces；Magnesium stearate consumption is fixed as 0.3g/100 pieces, and smoothly, no sticking shows for tabletting
As tablet surface is bright and clean.
Above-mentioned compound stranguria-treating and calculus-removing tablet also includes pyrrosia lingua and Caulis Lonicerae extract containing foregoing Chlorogenic acid compound of the invention.
Present invention also offers a kind of preparation method of compound stranguria-treating and calculus-removing tablet, comprise the following steps：
(1) take Desmodium styracifolium plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and in 65~70 DEG C of bars
0.08MPa concentrations are decompressed under part makes relative density be 1.10~1.12, adds 5 times of 85% ethanol of amount, stirs evenly, stand, filter
Cross, collect filtrate 0.08MPa is decompressed under the conditions of 65~70 DEG C and reclaim ethanol, and continue to be concentrated into relative density for 1.32~
1.35 extractum A；
With reference to former technique, Desmodium styracifolium is less using 5 times of 85% ethanol of amount, water extract-alcohol precipitation, loss of effective components is added, together
When the rate of extract can significantly reduce, so as to reduce the taking dose of medicine.Former process for refining is advanced rationally, therefore this preparation is still by former work
Skill is refined.
In addition, extract solution need to be concentrated into adds ethanol in right amount, different concentrating degrees have a significant impact to alcohol precipitation effect,
The present invention is according to alcohol precipitation of the knowhow to 1.08,1.10,1.12,1.15 (equal 50 DEG C of surveys) four different relative density concentrates
Effect is investigated, and when relative density of medicine liquid is between 1.10~1.12 (50 DEG C), the retention rate of active ingredient is higher, and
Cream rate can be met prepare tablet the need for.
(2) take Lygodium japonicum plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and in 65~70 DEG C of conditions
Under be decompressed to 0.08MPa concentration make relative density be 1.10~1.12, add 5 times amount 70% ethanol, stir evenly, stand, filtration,
Collect filtrate 0.08MPa be decompressed under the conditions of 65~70 DEG C and reclaim ethanol, and continue to be concentrated into relative density for 1.32~
1.35 medicinal extract B；
With reference to former technique, Lygodium japonicum, pyrrosia lingua, caulis lonicerae add 5 times of 70% ethanol of amount and Aqueous extracts are refined, water extracting alcohol
Heavy, loss of effective components is less, while the rate of extract can be significantly reduced, so as to reduce the taking dose of medicine.Former process for refining choosing
With condition rationally, due to having carried out independent extraction to pyrrosia lingua and caulis lonicerae in the present invention, then the extraction of Lygodium japonicum is still with reference to original
Technique.
In addition, extract solution need to be concentrated into adds ethanol in right amount, different concentrating degrees have a significant impact to alcohol precipitation effect,
The present invention is according to alcohol precipitation of the knowhow to 1.08,1.10,1.12,1.15 (equal 50 DEG C of surveys) four different relative density concentrates
Effect is investigated, and when relative density of medicine liquid is between 1.10~1.12 (50 DEG C), the retention rate of active ingredient is higher, and
Cream rate can be met prepare tablet the need for.
(3) pyrrosia lingua and caulis lonicerae are taken, crushes and 50~80 mesh that sieve, is soaked in degreasing in low polar solvent；
(4) with the degreasing thing in 70% ethanol heating and refluxing extraction step (3), and filter while hot, adjusted and filtered with watery hydrochloric acid
Liquid pH be 4~6 after be concentrated to give pyrrosia lingua and Caulis Lonicerae extract crude product；
(5) pyrrosia lingua for obtaining step (4) is dissolved with Caulis Lonicerae extract crude product with 85% ethanol, filtering with microporous membrane,
By the upper macroporous absorbent resin of gained filtrate, thin-layer chromatography is eluted to 2.0ml/min speed as eluant, eluent with 40% ethanol
Tracking and monitoring without chlorogenic acid composition, will be collected obtained eluent and detected with thin-layer chromatography into eluent, merging with it is green
Ortho acid standard items Rf value identical eluents, recycling design obtains chlorogenic acid crude product；Washed different from chlorogenic acid standard items Rf values
De- liquid is decompressed to 0.08MPa recycling designs after merging under the conditions of 65~70 DEG C, and continue to be concentrated into relative density for 1.32~
1.35 medicinal extract C；
(6) the chlorogenic acid crude product for obtaining step (5) is dissolved in the mixed solution of methanol/ethyl acetate, wherein methanol with
The volume ratio of ethyl acetate is 2~4:1, the mass volume ratio of chlorogenic acid and the mixed solution is 1g:5~8ml, is adjusted with acetic acid
PH is 3~5, adds filtration sterilization after charcoal absorption, and filtrate is heated to after 40~60 DEG C with 0.5 DEG C/min speed to cool
It is 45~60kHz to apply frequency to filtrate simultaneously to 0~5 DEG C, and power is 300~600kW ultrasonic field crystallization, is dried under reduced pressure and washes
Crystal is obtained after washing.
(7) extractum A of combining step (1), the medicinal extract B of step (2), the medicinal extract C in step (5) and step (6) are obtained
Chlorogenic acid crystal, add talcum powder, carboxyrnethyl starch sodium, microcrystalline cellulose and starch, mix, particle is made, dry, stiffened fat
Sour magnesium and starch, tabletted, film coating are produced.
With reference to the technique of former compound stranguria-treating and calculus-removing tablet, talcum powder and appropriate amount of auxiliary materials are added to thick paste, mixes, particle is made,
Dry.Medicinal powder and auxiliary material are directly added into medicinal extract re-dry after granulation by former technique, and the drying heated time of particle is short, can reduce
Destruction of the medicinal extract convection drying process to active ingredient, effectively raises the retention rate of active ingredient.
Is sugar coated tablet is made in former technique, and complex process, cost is high, requires high to plain piece, being changed to Film coated tablets can reduce into
This, simplifies technique, therefore be prepared into Film coated tablets.
Researcher of the present invention improves the preparation technology of medicinal material on the basis of traditional preparation methods, to Desmodium styracifolium,
The extraction conditions of Lygodium japonicum have carried out orthogonal test and have drawn optimal scheme, and separate pyrrosia lingua and honeysuckle using macroporous absorbent resin
The extract of rattan, is purified and is crystallized to chlorogenic acid composition, and gained is crystallized and carried with the pyrrosia lingua without chlorogenic acid with caulis lonicerae
Take thing to be incorporated in medicine composition, form the compound stranguria-treating and calculus-removing tablet containing Chlorogenic acid compound so that the bioavilability of the medicine
Have and be obviously improved, and have excellent effect in terms of row's calculus, diuresis, analgesia, anti-inflammatory.
Brief description of the drawings
Fig. 1 is the X-ray diffracting spectrum of Chlorogenic acid compound prepared by the embodiment of the present invention 3.
When Fig. 2 is that the medicine drawn is administered to rat oral gavage in compound stranguria-treating and calculus-removing tablet prepared by the embodiment of the present invention 3 and comparative example 1
Concentration curve.
Embodiment
Be below the embodiment of the present invention, described embodiment be in order to further describe the present invention, rather than
The limitation present invention.
The extracting factor of Desmodium styracifolium in stranguria-treating and calculus-removing tablet and Lygodium japonicum is screened first, can through consulting literatures
Know that main active is flavonoids in Desmodium styracifolium and Lygodium japonicum, it is more reasonable using aqueous extraction-alcohol precipitation technology, to extraction time
Specified in more detail is all made that with extraction time, only lacks times amount that adds water, therefore it is studied, with times amount that determines to add water, and is selected
General flavone to rutin meter is preferred for index progress technique：
A. Determination Method of Flavone Content
The preparation precision of reference substance solution, which is weighed, to be dried under reduced pressure at 120 DEG C to the control substance of Rutin 10mg of constant weight, puts 50ml
In measuring bottle, add water appropriate, ultrasound makes dissolving, and is diluted to scale, shakes up, produces.(containing anhydrous rutin 0.2mg in per 1ml).
The preparation precision of standard curve draws reference substance solution 1.0,2.0,3.0,4.0,5.0ml, and 10ml measuring bottles are put respectively
In, 5ml, plus 5% sodium nitrite solution 0.5ml are respectively added water to, is shaken up, is placed 6 minutes, plus 10% aluminum nitrate solution 0.5ml, shake
It is even, place 6 minutes, plus sodium hydroxide test solution 4ml, add water to scale, shake up.Using corresponding solution as blank.According to light splitting light
Degree method (one annex of Chinese Pharmacopoeia version in 2015), determines absorbance, by ordinate of trap, dense at 510nm wavelength
Spend for abscissa, drafting standard curve.As shown in table 1.
The general flavone standard curve transverse and longitudinal coordinate value of table 1
Using absorbance as ordinate, concentration is abscissa, draws standard curve, and its regression equation is：
Y=11.786x-0.0018r=0.9997
Determination method precision measures the extract solution equivalent to 2g Desmodium styracifolium and 2g Lygodium japonicum, puts in 100ml measuring bottles, adds water
Scale is diluted to, is shaken up, precision measures 1ml, is put in 10ml measuring bottles, the method under sighting target directrix curve preparation " is added water to certainly
5ml " is risen, and determines trap immediately in accordance with the law, and the quite amount of rutin, calculating, is produced from need testing solution is read on standard curve.
B. trial test
Desmodium styracifolium 100g and Lygodium japonicum 100g accurately are weighed, is put in 5000ml round-bottomed flasks, plus 10 times of amount decoctings boil two
Secondary, 2 hours every time, filtration, filtrate merged.
Trial test conclusion：10 times of amount water 1000ml- of dregs of a decoction liquid absorption obtain liquid 779ml=imbibitions 221ml
100g=2.21 times of 221ml ÷ medicinal materials
May be selected 8,10,12 times of amounts, three levels carry out the water decocting process of preferred Desmodium styracifolium and Lygodium japonicum.
C. the different times comparative tests for amount that add water
Desmodium styracifolium 100g, Lygodium japonicum 100g accurately are weighed, three parts is weighed, puts respectively in 5000ml round-bottomed flasks, respectively
Plus 8 times of amounts, 10 times of amounts, 12 times of amount decoctings are boiled twice, 2 hours every time, collecting decoction, filtration collected filtrate, by general flavone content
The content that the method under item determines general flavone is determined, 2 are the results are shown in Table.
The Desmodium styracifolium of table 2 and Lygodium japonicum medicinal material decocting process preferred result table
It can be seen from upper table, with times increase for amount that adds water, general flavone content can increase therewith.But Jia 20 (10,10)
When water extraction is measured in amount water and 24 (12,12) times again, the content of general flavone is more or less the same, therefore, considers, and selects plus 10 times of amounts
Decocting is boiled twice, 2 hours every time.
Embodiment 1
(1) take Desmodium styracifolium 1500g plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and at 65 DEG C
Under the conditions of be decompressed to 0.08MPa concentration make relative density be 1.10, add 5 times amount 85% ethanol, stir evenly, stand, filter, receive
Collection filtrate is decompressed to 0.08MPa under the conditions of 65 DEG C and reclaims ethanol, and continues to be concentrated into the extractum A that relative density is 1.32；
(2) take Lygodium japonicum 500g plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and in 65 DEG C of conditions
Under be decompressed to 0.08MPa concentration make relative density be 1.10, add 5 times amount 70% ethanol, stir evenly, stand, filtration, collect filter
Liquid is decompressed to 0.08MPa under the conditions of 65 DEG C and reclaims ethanol, and continues to be concentrated into the medicinal extract B that relative density is 1.32；
(3) pyrrosia lingua 500g and caulis lonicerae 500g are taken, crushes and 50 mesh that sieve, is soaked in degreasing in low polar solvent；
(4) with the degreasing thing in 70% ethanol heating and refluxing extraction step (3), and filter while hot, adjusted and filtered with watery hydrochloric acid
Liquid pH be 4 after be concentrated to give pyrrosia lingua and Caulis Lonicerae extract crude product；
(5) pyrrosia lingua for obtaining step (4) is dissolved with Caulis Lonicerae extract crude product with 85% ethanol, filtering with microporous membrane,
By the upper macroporous absorbent resin of gained filtrate, thin-layer chromatography is eluted to 2.0ml/min speed as eluant, eluent with 40% ethanol
Tracking and monitoring without chlorogenic acid composition, will be collected obtained eluent and detected with thin-layer chromatography into eluent, merging with it is green
Ortho acid standard items Rf value identical eluents, recycling design obtains chlorogenic acid crude product；Washed different from chlorogenic acid standard items Rf values
De- liquid is decompressed to 0.08MPa recycling designs after merging under the conditions of 65 DEG C, and continues to be concentrated into the medicinal extract that relative density is 1.32
C；
(6) the chlorogenic acid crude product for obtaining step (5) is dissolved in the mixed solution of methanol/ethyl acetate, wherein methanol with
The volume ratio of ethyl acetate is 2:1, the mass volume ratio of chlorogenic acid and the mixed solution is 1g:5ml, is 5 with second acid for adjusting pH,
Filtration sterilization after charcoal absorption is added, filtrate is heated to after 60 DEG C to be cooled to 5 DEG C simultaneously to filter with 0.5 DEG C/min speed
It is 45kHz that liquid, which applies frequency, and power is 300kW ultrasonic field crystallization, is dried under reduced pressure after washing and obtains crystal, and gained chlorogenic acid is brilliant
The x-ray diffraction pattern that body is obtained by Cu-K alpha ray measurements is substantially similar to Fig. 1.
(7) extractum A of combining step (1), the medicinal extract B of step (2), the medicinal extract C in step (5) and step (6) are obtained
Chlorogenic acid crystal, add talcum powder 25g, carboxyrnethyl starch sodium 10g, microcrystalline cellulose 30g and starch 120g, mix, be made
Grain, is dried, and stiffened fatty acid magnesium 3g simultaneously adds starch to 350g, is pressed into 1000, film coating is produced.
Embodiment 2
(1) take Desmodium styracifolium 1500g plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and at 70 DEG C
Under the conditions of be decompressed to 0.08MPa concentration make relative density be 1.12, add 5 times amount 85% ethanol, stir evenly, stand, filter, receive
Collection filtrate is decompressed to 0.08MPa under the conditions of 70 DEG C and reclaims ethanol, and continues to be concentrated into the extractum A that relative density is 1.35；
(2) take Lygodium japonicum 500g plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and in 70 DEG C of conditions
Under be decompressed to 0.08MPa concentration make relative density be 1.12, add 5 times amount 70% ethanol, stir evenly, stand, filtration, collect filter
Liquid is decompressed to 0.08MPa under the conditions of 70 DEG C and reclaims ethanol, and continues to be concentrated into the medicinal extract B that relative density is 1.35；
(3) pyrrosia lingua 500g and caulis lonicerae 500g are taken, crushes and 80 mesh that sieve, is soaked in degreasing in low polar solvent；
(4) with the degreasing thing in 70% ethanol heating and refluxing extraction step (3), and filter while hot, adjusted and filtered with watery hydrochloric acid
Liquid pH be 5.5 after be concentrated to give pyrrosia lingua and Caulis Lonicerae extract crude product；
(5) pyrrosia lingua for obtaining step (4) is dissolved with Caulis Lonicerae extract crude product with 85% ethanol, filtering with microporous membrane,
By the upper macroporous absorbent resin of gained filtrate, thin-layer chromatography is eluted to 2.0ml/min speed as eluant, eluent with 40% ethanol
Tracking and monitoring without chlorogenic acid composition, will be collected obtained eluent and detected with thin-layer chromatography into eluent, merging with it is green
Ortho acid standard items Rf value identical eluents, recycling design obtains chlorogenic acid crude product；Washed different from chlorogenic acid standard items Rf values
De- liquid is decompressed to 0.08MPa recycling designs after merging under the conditions of 70 DEG C, and continues to be concentrated into the medicinal extract that relative density is 1.35
C；
(6) the chlorogenic acid crude product for obtaining step (5) is dissolved in the mixed solution of methanol/ethyl acetate, wherein methanol with
The volume ratio of ethyl acetate is 4:1, the mass volume ratio of chlorogenic acid and the mixed solution is 1g:7ml, is 4 with second acid for adjusting pH,
Filtration sterilization after charcoal absorption is added, filtrate is heated to after 52 DEG C to be cooled to 2 DEG C simultaneously to filter with 0.5 DEG C/min speed
It is 60kHz that liquid, which applies frequency, and power is 500kW ultrasonic field crystallization, is dried under reduced pressure after washing and obtains crystal, and gained chlorogenic acid is brilliant
The x-ray diffraction pattern that body is obtained by Cu-K alpha ray measurements is substantially similar to Fig. 1.
(7) extractum A of combining step (1), the medicinal extract B of step (2), the medicinal extract C in step (5) and step (6) are obtained
Chlorogenic acid crystal, add talcum powder 25g, carboxyrnethyl starch sodium 10g, microcrystalline cellulose 30g and starch 120g, mix, be made
Grain, is dried, and stiffened fatty acid magnesium 3g simultaneously adds starch to 350g, is pressed into 1000, film coating is produced.
Embodiment 3
(1) take Desmodium styracifolium 1500g plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and at 68 DEG C
Under the conditions of be decompressed to 0.08MPa concentration make relative density be 1.11, add 5 times amount 85% ethanol, stir evenly, stand, filter, receive
Collection filtrate is decompressed to 0.08MPa under the conditions of 67 DEG C and reclaims ethanol, and continues to be concentrated into the extractum A that relative density is 1.34；
(2) take Lygodium japonicum 500g plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and in 66 DEG C of conditions
Under be decompressed to 0.08MPa concentration make relative density be 1.10, add 5 times amount 70% ethanol, stir evenly, stand, filtration, collect filter
Liquid is decompressed to 0.08MPa under the conditions of 67 DEG C and reclaims ethanol, and continues to be concentrated into the medicinal extract B that relative density is 1.33；
(3) pyrrosia lingua and caulis lonicerae are taken, crushes and 60 mesh that sieve, is soaked in degreasing in low polar solvent；
(4) with the degreasing thing in 70% ethanol heating and refluxing extraction step (3), and filter while hot, adjusted and filtered with watery hydrochloric acid
Liquid pH be 6 after be concentrated to give pyrrosia lingua and Caulis Lonicerae extract crude product；
(5) pyrrosia lingua for obtaining step (4) is dissolved with Caulis Lonicerae extract crude product with 85% ethanol, filtering with microporous membrane,
By the upper macroporous absorbent resin of gained filtrate, thin-layer chromatography is eluted to 2.0ml/min speed as eluant, eluent with 40% ethanol
Tracking and monitoring without chlorogenic acid composition, will be collected obtained eluent and detected with thin-layer chromatography into eluent, merging with it is green
Ortho acid standard items Rf value identical eluents, recycling design obtains chlorogenic acid crude product；Washed different from chlorogenic acid standard items Rf values
De- liquid is decompressed to 0.08MPa recycling designs after merging under the conditions of 69 DEG C, and continues to be concentrated into the medicinal extract that relative density is 1.34
C；
(6) the chlorogenic acid crude product for obtaining step (5) is dissolved in the mixed solution of methanol/ethyl acetate, wherein methanol with
The volume ratio of ethyl acetate is 3:1, the mass volume ratio of chlorogenic acid and the mixed solution is 1g:8ml, is 3 with second acid for adjusting pH,
Filtration sterilization after charcoal absorption is added, filtrate is heated to after 40 DEG C to be cooled to 0 DEG C simultaneously to filter with 0.5 DEG C/min speed
It is 50kHz that liquid, which applies frequency, and power is 600kW ultrasonic field crystallization, is dried under reduced pressure after washing and obtains crystal, and gained chlorogenic acid is brilliant
The x-ray diffraction pattern that body is obtained by Cu-K alpha ray measurements is as shown in Figure 1.
(7) extractum A of combining step (1), the medicinal extract B of step (2), the medicinal extract C in step (5) and step (6) are obtained
Chlorogenic acid crystal, add talcum powder 25g, carboxyrnethyl starch sodium 10g, microcrystalline cellulose 30g and starch 120g, mix, be made
Grain, is dried, and stiffened fatty acid magnesium 3g simultaneously adds starch to 350g, is pressed into 1000, film coating is produced.
Producing for stranguria-treating and calculus-removing tablet is carried out using traditional handicraft, its product is as a comparison：
Comparative example 1
(1) Desmodium styracifolium 1500g plus 10 times of amount decoctings are taken to boil secondary, 2 hours every time, filtration, merging filtrate.
(2) filtrate decompression concentration (0.08MPa, 65~70 DEG C) to relative density is 1.10~1.12 (50 DEG C), adds 5
85% ethanol of amount, is stirred evenly again, is stood, and filtrate is collected in filtration.
(3) decompression filtrate recycling ethanol, and continue to be concentrated into the thick paste that relative density is 1.32~1.35 (50 DEG C).
(4) each 500g plus 10 times of amount decocting of pyrrosia lingua, Lygodium japonicum, caulis lonicerae is taken to boil secondary, 2 hours every time, filtration merged filter
Liquid.
(5) filtrate decompression concentration (0.08MPa, 65~70 DEG C) to relative density is 1.10~1.12 (50 DEG C), adds 5
70% ethanol of amount, is stirred evenly again, is stood, and filtrate is collected in filtration.
(6) filtrate decompression (0.08MPa, 65~70 DEG C) reclaims ethanol, and continue to be concentrated into relative density for 1.32~
The thick paste of 1.35 (50 DEG C).
(7) merge above-mentioned thick paste, add talcum powder 25g, carboxyrnethyl starch sodium 10g, microcrystalline cellulose 30g, plus starch 120g,
Mix, particle is made, dry, stiffened fatty acid magnesium 3g, plus starch is to 350g, is pressed into 1000, film coating is produced.
Comparative example 2
Chlorogenic acid crystal prepared by the method recorded according to embodiment 1 in Patent No. CN 102746153B document.
The solubility test of test example 1
Solubility is determined using balancing method, under the conditions of 25 DEG C of temperature, by obtained chlorogenic acid chemical combination in above-described embodiment
Thing constant temperature in pure water is stirred, and is measured the concentration of upper solution Content of Chlorogenic Acid using HPLC after standing and is scaled solubility.
The solubility of Chlorogenic acid compound made from the embodiment of the present invention 1~3 and comparative example 2 are compared, gained examination
Test and the results are shown in Table 3.
Solubility of the Chlorogenic acid compound of table 3 in water
As shown in Table 3, with the chlorogenic acid crystal phase ratio of the prior art of comparative example 2, the Chlorogenic acid compound that the present invention is provided
With higher solubility, it is more beneficial for that solution state formulation is made；Embodiment 1~3 increases the side of ultrasonic field in Crystallization Process
The solubility that legal system obtains Chlorogenic acid compound is substantially better than in comparative example 2 only using the obtained chlorogenic acid of organic solvent recrystallization
Compound.It follows that changing the effect for serving key in preparation method of the present invention to chlorogenic acid crystal formation, it is improved in water
Solubility.
The bioavilability of test example 2 is tested
The dense of chlorogenic acid of the compound stranguria-treating and calculus-removing tablet that is made of different process after being administered to rat oral gavage is detected by HPLC
Degree, and according to《Chemicals Non-clinical Pharmacokinetics investigative technique guideline (【H】GPT5-1)》Blood sampling time is set
Point, its bioavilability is determined by the pharmacokinetic to the chlorogenic acid in compound stranguria-treating and calculus-removing tablet.
First, tested medicine
By the compound stranguria-treating and calculus-removing tablet containing chlorogenic acid crystal made from the embodiment of the present invention 3 and comparative example 1 according to traditional work
The compound stranguria-treating and calculus-removing tablet sample composition that skill is made.
2nd, zoopery：
Take SD rats male and female half and half totally 20, be divided into 1 group of 3 groups of embodiment and comparative example, every group of male and female half and half 10 only according to
《Chemicals Non-clinical Pharmacokinetics investigative technique guideline (【H】GPT5-1)》Carry out and test, 24 is small before gastric infusion
When fasting can't help water, gastric infusion takes venous blood after rat eye after the different time points in 4 hours, adds 3.8% lemon
Acid centrifuges 15min at 3,000 rpm, and separated plasma directly removes removing protein with acetonitrile, supernatant is taken as test liquid, through liquid phase
Chromatograph detects that acquired results are computed being shown in Table 4 after arranging.
The pharmacokinetic parameter of compound stranguria-treating and calculus-removing tablet Content of Chlorogenic Acid compound is administered in the rat oral gavage of table 4
| Pharmacokinetic parameter | 3 groups of embodiment | 1 group of comparative example |
| Cmax(mg/L) | 10.42±1.74 | 9.23±2.06 |
| Tmax(min) | 14.83±2.13 | 29.76±1.96 |
| AUC0-4h(mg·L-1·min-1) | 776.85±51.23 | 614.90±43.65 |
| T1/2(min) | 45.31±2.11 | 35.29±3.47 |
As shown in Table 4, the bioavilability for the compound stranguria-treating and calculus-removing tablet that 1 group of Oral Administration in Rats comparative example is traditionally made
Relatively low (AUC0-4h=614.90 ± 43.65mgL-1·min-1), 3 groups of the embodiment that the present invention is provided possesses preferably biological profit
Expenditure (AUC0-4h=776.85 ± 51.23mgL-1·min-1)；And the compound stranguria-treating and calculus-removing tablet contrast being made compared to traditional handicraft
1 group of example, the chlorogenic acid that 3 groups of embodiment can reach higher blood concentration (C within the shorter timemax=10.42 ±
1.74mg/L, Tmax=14.83 ± 2.13min), also possess longer half-life period (T1/2=45.31 ± 2.11min).Thus may be used
Know, the chlorogenic acid extracting compound from pyrrosia lingua and caulis lonicerae used in the present invention is incorporated to the technical scheme of compound stranguria-treating and calculus-removing tablet,
The method that decocting herbs chlorogenic acid extracting composition is relied in the traditional handicraft that compares, has been significantly increased in compound stranguria-treating and calculus-removing tablet
The bioavilability of chlorogenic acid composition.
The compound stranguria-treating and calculus-removing tablet containing Chlorogenic acid compound prepared by other embodiments of the present invention is also carried out above-mentioned
Experiment, the result that it is obtained is similar.
Influence of the compound stranguria-treating and calculus-removing tablet of test example 3 to rat kidney stone
First, tested medicine
By the compound stranguria-treating and calculus-removing tablet containing chlorogenic acid crystal made from the embodiment of the present invention 1~3 and comparative example 1 according to tradition
The compound stranguria-treating and calculus-removing tablet sample composition that technique is made.
2nd, zoopery
The SD rats male and female half and half totally 60 for taking body weight to be 180~200g, are divided into blank group, model group, embodiment 1,2,3
1 group of group and comparative example, every group of male and female half and half 10, remaining each group continuous gavage administration 30 days in addition to model group, 1 time a day,
During this in addition to blank group, feed of the daily feeding of other groups containing 2.5% ethylene glycol and 2.5% ammonium chloride controls drink simultaneously
Water, and intramuscular injection vitamine D3 causes kidney stone model weekly.Experiment puts to death animal in full 30 days, takes out double kidney observations outer
Shape, and longitudinally split observation and whether there is obvious crystalline deposit thing, free calculus and calcified plaque and judge calculus degree, after by kidney organ
It is placed in after being homogenized in 4ml 1mol/L hydrochloric acid and soaks 24 hours, determines kidney calcium, kidney oxalic acid content, kidney urea nitrogen and kidney creatinine and contain
Amount, as a result as shown in table 5.
Influence of the compound stranguria-treating and calculus-removing tablet of table 5 to Rat renal calcium, kidney oxalic acid, kidney urea nitrogen and kidney creatinine
| Group | Kidney calcium (umol/g) | Kidney oxalic acid (nmol/g) | Kidney urea nitrogen (umol/g) | Kidney creatinine (umol/g) |
| Blank group | 19.2±2.6** | 59.6±6.5** | 30.4±5.2** | 0.6±0.1** |
| Model group | 24.7±6.1 | 101.3±28.6 | 42.4±10.8 | 1.6±0.4 |
| 1 group of embodiment | 20.7±4.2* | 65.5±12.3** | 31.4±8.1** | 0.5±0.4** |
| 2 groups of embodiment | 21.0±5.1* | 64.9±11.7** | 32.4±6.9** | 0.4±0.3** |
| 3 groups of embodiment | 19.3±4.9** | 61.4±10.8** | 30.9±7.3** | 0.5±0.2** |
| 1 group of comparative example | 23.3±6.1* | 73.6±13.7** | 36.3±10.2** | 0.9±0.3** |
Note：Compared with model group, * represents P<0.05, * * represents P<0.01
As shown in Table 5, by embodiment 1, the rat of 2,3 groups and 1 group of gastric infusion of comparative example, kidney calcium has with kidney oxalic acid
Decline, illustrate that decline of the compound stranguria-treating and calculus-removing tablet to calculus main component serves effect；And the decline of kidney urea nitrogen and kidney creatinine
Illustrate that Renal Function in Rats has recovered upon administration.Further analysis finds that embodiment 1, kidney oxalic acid, the kidney of 2,3 groups of rats are urinated
Plain nitrogen and kidney creatinine declines by a big margin the difference (P with highly significant<, and effect is better than using traditional handicraft system 0.01)
Into 1 group of comparative example；Embodiment 1, the kidney calcium fall of 2 groups of rats have significant difference (P<0.05), embodiment is 3 groups big
The kidney calcium fall of mouse has the difference (P of highly significant<, and effect is better than the contrast being made using traditional handicraft 0.01)
1 group of example.To sum up, the effect treated using the compound stranguria-treating and calculus-removing tablet containing Chlorogenic acid compound to kidney stone modeling rat will
Better than the compound stranguria-treating and calculus-removing tablet prepared using traditional handicraft, it can thus be appreciated that Chlorogenic acid compound is in enhancing compound stranguria-treating and calculus-removing tablet row's stone
Effect in serve key effect.
Influence of the compound stranguria-treating and calculus-removing tablet of test example 4 to rat urine volume
First, tested medicine
By the compound stranguria-treating and calculus-removing tablet containing chlorogenic acid crystal made from the embodiment of the present invention 1~3 and comparative example 1 according to tradition
The compound stranguria-treating and calculus-removing tablet sample composition that technique is made.
2nd, zoopery
The SD rats male 30 that body weight is 180~200g is taken, is divided into blank group, embodiment 1,2,3 groups and comparative example 1
Group, every group 6.Fasting be can't help after water 15h, and every group of gavage gives 5% glucose saline of Water l oad after 30 minutes in addition to blank group
Gastric infusion, oppresses the remaining urine of rat lower abdomen emptying after administration, urine is collected in 5h, after the time arrives more than compressing rat lower abdomen emptying
Urine measurement urine volume, as a result as shown in table 6.
Influence of the compound stranguria-treating and calculus-removing tablet of table 6 to rat urine volume
| Group | Urine volume (ml/5h) |
| Blank group | 1.81±0.63 |
| 1 group of embodiment | 4.41±1.23** |
| 2 groups of embodiment | 4.22±1.37** |
| 3 groups of embodiment | 4.37±1.15** |
| 1 group of comparative example | 3.28±1.09** |
Note：Compared with blank group, * represents P<0.05, * * represents P<0.01
As shown in Table 6, by embodiment 1, the rat of 2,3 groups and 1 group of gastric infusion of comparative example, contrast is without the sky being administered
White group, average urine volume has rising in its five hours, illustrates the effect of compound stranguria-treating and calculus-removing tablet diuresis.Further analysis finds,
Embodiment 1, the urine volume of 2,3 groups of rats have the difference (P of highly significant compared to blank group<, and effect is better than and used 0.01)
1 group of the comparative example that traditional handicraft is made.To sum up, the profit carried out using the compound stranguria-treating and calculus-removing tablet containing Chlorogenic acid compound to rat
Urine effect is better than the compound stranguria-treating and calculus-removing tablet prepared using traditional handicraft, it can thus be appreciated that Chlorogenic acid compound is in enhancing compound urolithiasis
The effect of key is served in the effect of logical piece diuresis.
Influence of the compound stranguria-treating and calculus-removing tablet of test example 5 to rat paw edema
First, tested medicine
By the compound stranguria-treating and calculus-removing tablet containing chlorogenic acid crystal made from the embodiment of the present invention 1~3 and comparative example 1 according to tradition
The compound stranguria-treating and calculus-removing tablet sample composition that technique is made.
2nd, zoopery
50 male and female half and half of SD rats for taking body weight to be 180~200g, are divided into blank group, embodiment 1,2,3 groups and contrast
1 group of example, every group of 10 male and female half and half.The gastric infusion in addition to blank group, 1 time a day, continuous 4d are right per mouse after last dose 1h
The egg clear solution 0.1ml of vola pedis skin injection 10% afterwards, respectively at 1h, 2h, 3h before inflammation and after inflammation, measures vola pedis girth during 4h
And result is recorded, calculate swelling rate as shown in table 7.
Influence of the compound stranguria-treating and calculus-removing tablet of table 7 to rat paw edema
Note：Compared with blank group, * represents P<0.05, * * represents P<0.01
As shown in Table 7, by embodiment 1, the rat of 2,3 groups and 1 group of gastric infusion of comparative example, contrast is without the sky being administered
White group, swelling rate increasing degree declines to a great extent its right vola pedis in 4 hours after inflammation, illustrates compound stranguria-treating and calculus-removing tablet anti-inflammatory
Effect.Further analysis finds, embodiment 1, the foot swelling rate of 2,3 groups of rats after inflammation before 1h and 2h when, compared to blank group
With significant difference (P<, and effect is better than 1 group of comparative example being made using traditional handicraft 0.05)；Embodiment 1,2,3 groups
The foot swelling rate of rat after inflammation before 1h and 2h when, compared to the difference (P that blank group has highly significant<, and effect 0.01)
It is better than 1 group of comparative example being made using traditional handicraft.To sum up, using the compound stranguria-treating and calculus-removing tablet containing Chlorogenic acid compound to big
The antiphlogistic effects that mouse is carried out are better than the compound stranguria-treating and calculus-removing tablet prepared using traditional handicraft, it can thus be appreciated that Chlorogenic acid compound is increasing
The effect of key is served in the effect of strong compound stranguria-treating and calculus-removing tablet anti-inflammatory.
Analgesic activity of the compound stranguria-treating and calculus-removing tablet of test example 6 to rat
First, tested medicine
By the compound stranguria-treating and calculus-removing tablet containing chlorogenic acid crystal made from the embodiment of the present invention 1~3 and comparative example 1 according to tradition
The compound stranguria-treating and calculus-removing tablet sample composition that technique is made.
2nd, zoopery
Kunming mouse male 30 is taken, is divided into blank group, embodiment 1,2,3 groups and 1 group of comparative example, every group 6.Except sky
The white outer gastric infusion of group, 1 time a day, continuous 5d, after last dose 1h, every group of 0.6% acetum of intraperitoneal injection 0.2ml/,
Mouse writhing number of times in 10min is recorded, each group analgesia percentage is calculated, the results are shown in Table shown in 8.
The influence that the compound stranguria-treating and calculus-removing tablet of table 8 is reacted mouse writhing
| Group | Writhing number of times (secondary/10min) | Ease pain percentage (%) |
| Blank group | 25.3±6.2 | - |
| 1 group of embodiment | 12.5±3.9** | 37.2% |
| 2 groups of embodiment | 13.1±4.1** | 35.6% |
| 3 groups of embodiment | 12.7±4.8** | 36.9% |
| 1 group of comparative example | 16.2±5.2** | 26.4% |
Note：Compared with blank group, * represents P<0.05, * * represents P<0.01
As shown in Table 8, by embodiment 1, the rat of 2,3 groups and 1 group of gastric infusion of comparative example, contrast is without the sky being administered
Writhing number of times declines to a great extent in white group, its 10min, illustrates that compound stranguria-treating and calculus-removing tablet analgesic is acted on.Further analysis finds,
The writhing number of times of embodiment 1,2,3 groups of rats in the 10min after injection acetic acid, compared to the difference that blank group has highly significant
(P<, and its percentage (37.2%, 35.6%, 36.9%) that eases pain is much larger than the comparative example 1 that is made using traditional handicraft 0.01)
Group (26.4%).To sum up, the analgesic effect carried out using the compound stranguria-treating and calculus-removing tablet containing Chlorogenic acid compound to rat is better than
The compound stranguria-treating and calculus-removing tablet prepared using traditional handicraft, it can thus be appreciated that Chlorogenic acid compound is in enhancing compound stranguria-treating and calculus-removing tablet analgesic effect
The effect of key is served in fruit.
The data and result of summary test example, it is known that the compound stranguria-treating and calculus-removing tablet of the invention containing Chlorogenic acid compound
The compound stranguria-treating and calculus-removing tablet prepared compared to traditional handicraft, can preferably improve its bioavilability, and row's calculus, diuresis,
There is excellent effect in terms of analgesia, anti-inflammatory.Improvement of the compound stranguria-treating and calculus-removing tablet Content of Chlorogenic Acid compound crystal form of the present invention to medicine
Serve the effect of key.
The present invention is described in detail above, its object is to allow those skilled in the art to understand this
The content of invention is simultaneously carried out, and it is not intended to limit the scope of the present invention, all Spirit Essence institutes according to the present invention
The equivalent change or modification done, should all cover within the scope of the present invention.
Claims (10)
1. a kind of Chlorogenic acid compound, it is characterised in that described Chlorogenic acid compound passes through the X- that Cu-K alpha ray measurements are obtained
When the angle of diffraction of x ray diffration pattern x is 2 θ, characteristic peak includes：4.6±0.2°、16.2±0.2°、17.7±0.2°、18.4±
0.2 °, 19.7 ± 0.2 °, 20.5 ± 0.2 °, 21.6 ± 0.2 °, 22.8 ± 0.2 °, 25.6 ± 0.2 °, 26.1 ± 0.2 ° and 30.7
±0.2°。
2. Chlorogenic acid compound according to claim 1, it is characterised in that the X-ray diffraction of the Chlorogenic acid compound
The diffraction maximum of figure includes：
3. Chlorogenic acid compound according to claim 2, it is characterised in that the X-ray diffraction of the Chlorogenic acid compound
Collection of illustrative plates is as shown in Figure 1.
4. Chlorogenic acid compound according to claims 1 to 3, it is characterised in that described Chlorogenic acid compound is from stone
Extract what is prepared in Wei and caulis lonicerae.
5. the preparation method of the Chlorogenic acid compound described in a kind of claim 4, it is characterised in that comprise the following steps：
(1) pyrrosia lingua and caulis lonicerae are taken, crushes and 50~80 mesh that sieve, is soaked in degreasing in low polar solvent；
(2) with the degreasing thing in 70% ethanol heating and refluxing extraction step (1), and filter while hot, filtrate pH is adjusted with watery hydrochloric acid
To be concentrated to give pyrrosia lingua and Caulis Lonicerae extract crude product after 4~6；
(3) pyrrosia lingua for obtaining step (2) is dissolved with Caulis Lonicerae extract crude product with 85% ethanol, filtering with microporous membrane, by institute
The upper macroporous absorbent resin of filtrate is obtained, being eluted to thin-layer chromatography as eluant, eluent with 2.0ml/min speed with 40% ethanol tracks
Monitoring is into eluent without chlorogenic acid composition, and the eluent that collection is obtained is detected with thin-layer chromatography, is merged and chlorogenic acid
Standard items Rf value identical eluents, recycling design obtains chlorogenic acid crude product；By the eluent different from chlorogenic acid standard items Rf values
0.08MPa recycling designs are decompressed to after merging under the conditions of 65~70 DEG C, and it is 1.32~1.35 to continue to be concentrated into relative density
Medicinal extract C；
(4) the chlorogenic acid crude product for obtaining step (3) is dissolved in the mixed solution of methanol/ethyl acetate, wherein methanol and acetic acid
The volume ratio of ethyl ester is 2~4:1, the mass volume ratio of chlorogenic acid and the mixed solution is 1g:5~8ml, be with second acid for adjusting pH
3~5, filtration sterilization after charcoal absorption is added, filtrate is heated to after 45~60 DEG C to be cooled to 0 with 0.5 DEG C/min speed
~5 DEG C apply frequency to filtrate simultaneously and carry out crystallization for 45~60kHz ultrasonic field, and crystal is obtained after being dried under reduced pressure washing.
6. preparation method according to claim 5, it is characterised in that in step (1), the frequency of the ultrasonic field is
50kHz。
7. preparation method according to claim 5, it is characterised in that in step (4), the power of the ultrasonic field is 300
~600kW.
8. a kind of compound stranguria-treating and calculus-removing tablet, it is characterised in that the composition of described compound stranguria-treating and calculus-removing tablet includes：Desmodium styracifolium 1500g,
Lygodium japonicum 500g, pyrrosia lingua 500g, caulis lonicerae 500g, talcum powder 25g, carboxyrnethyl starch sodium 10g, microcrystalline cellulose 30g, magnesium stearate
3g and appropriate starch.
9. compound stranguria-treating and calculus-removing tablet according to claim 8, it is characterised in that described compound stranguria-treating and calculus-removing tablet, which includes containing, has the right
Profit require the pyrrosia lingua of Chlorogenic acid compound made from preparation method described in Chlorogenic acid compound or claim 5 described in 1 or 2 with
Caulis Lonicerae extract.
10. the preparation method of compound stranguria-treating and calculus-removing tablet described in a kind of claim 8 or 9, it is characterised in that comprise the following steps：
(1) take Desmodium styracifolium plus 10 times to measure decoctings and boil secondary, 2 hours every time, filtration, merging filtrate and under the conditions of 65~70 DEG C
Being decompressed to 0.08MPa concentrations makes relative density be 1.10~1.12, adds 5 times of 85% ethanol of amount, stirs evenly, stand, filter, receive
Collection filtrate is decompressed to 0.08MPa under the conditions of 65~70 DEG C and reclaims ethanol, and it is 1.32~1.35 to continue to be concentrated into relative density
Extractum A；
(2) Lygodium japonicum plus 10 times of amount decoctings are taken to boil secondary, 2 hours every time, filtration, merging filtrate simultaneously subtracted under the conditions of 65~70 DEG C
Being depressed into 0.08MPa concentrations makes relative density be 1.10~1.12, adds 5 times of 70% ethanol of amount, stirs evenly, stand, filter, collect
Filtrate is decompressed to 0.08MPa under the conditions of 65~70 DEG C and reclaims ethanol, and it is 1.32~1.35 to continue to be concentrated into relative density
Medicinal extract B；
(3) pyrrosia lingua and caulis lonicerae are taken, crushes and 50~80 mesh that sieve, is soaked in degreasing in low polar solvent；
(4) with the degreasing thing in 70% ethanol heating and refluxing extraction step (3), and filter while hot, filtrate pH is adjusted with watery hydrochloric acid
To be concentrated to give pyrrosia lingua and Caulis Lonicerae extract crude product after 4~6；
(5) pyrrosia lingua for obtaining step (4) is dissolved with Caulis Lonicerae extract crude product with 85% ethanol, filtering with microporous membrane, by institute
The upper macroporous absorbent resin of filtrate is obtained, being eluted to thin-layer chromatography as eluant, eluent with 2.0ml/min speed with 40% ethanol tracks
Monitoring is into eluent without chlorogenic acid composition, and the eluent that collection is obtained is detected with thin-layer chromatography, is merged and chlorogenic acid
Standard items Rf value identical eluents, recycling design obtains chlorogenic acid crude product；By the eluent different from chlorogenic acid standard items Rf values
0.08MPa recycling designs are decompressed to after merging under the conditions of 65~70 DEG C, and it is 1.32~1.35 to continue to be concentrated into relative density
Medicinal extract C；
(6) the chlorogenic acid crude product for obtaining step (5) is dissolved in the mixed solution of methanol/ethyl acetate, wherein methanol and acetic acid
The volume ratio of ethyl ester is 2~4:1, the mass volume ratio of chlorogenic acid and the mixed solution is 1g:5~8ml, be with second acid for adjusting pH
3~5, filtration sterilization after charcoal absorption is added, filtrate is heated to after 40~60 DEG C to be cooled to 0 with 0.5 DEG C/min speed
~5 DEG C apply frequency to filtrate simultaneously and carry out crystallization for 45~60kHz ultrasonic field, and crystal is obtained after being dried under reduced pressure washing；
(7) what the extractum A of combining step (1), the medicinal extract B of step (2), the medicinal extract C in step (5) and step (6) were obtained is green
Ortho acid crystal, adds talcum powder, carboxyrnethyl starch sodium, microcrystalline cellulose and starch, mixes, particle is made, and dries, stiffened fatty acid magnesium
And starch, tabletted, film coating, produce.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710310320.0A CN107033001B (en) | 2017-05-05 | 2017-05-05 | Chlorogenic acid compound and compound stranguria-treating and calculus-removing tablet containing same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710310320.0A CN107033001B (en) | 2017-05-05 | 2017-05-05 | Chlorogenic acid compound and compound stranguria-treating and calculus-removing tablet containing same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107033001A true CN107033001A (en) | 2017-08-11 |
| CN107033001B CN107033001B (en) | 2020-07-14 |
Family
ID=59538305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710310320.0A Active CN107033001B (en) | 2017-05-05 | 2017-05-05 | Chlorogenic acid compound and compound stranguria-treating and calculus-removing tablet containing same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107033001B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709256A (en) * | 2018-12-19 | 2019-05-03 | 金花企业(集团)股份有限公司西安金花制药厂 | Scutelloside and chlorogenic acid thin layer identify detection method in a kind of Compound Jinyinhua Granules |
| CN111089912A (en) * | 2019-05-21 | 2020-05-01 | 江苏万略医药科技有限公司 | Method for researching drug metabolism based on molecular and cell level |
| WO2020143746A1 (en) * | 2019-01-11 | 2020-07-16 | 四川九章生物科技有限公司 | Use of chlorogenic acid in preparing medicine or pharmaceutical composition for preventing or treating pain |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101249135A (en) * | 2008-03-28 | 2008-08-27 | 湖南长沙宝鉴生物工程有限公司 | Compound urolithic stranguria unlock capsules and preparation technique thereof |
| CN102755366A (en) * | 2012-07-19 | 2012-10-31 | 浙江维康药业有限公司 | Honeysuckle soft capsule containing chlorogenic acid compound |
-
2017
- 2017-05-05 CN CN201710310320.0A patent/CN107033001B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101249135A (en) * | 2008-03-28 | 2008-08-27 | 湖南长沙宝鉴生物工程有限公司 | Compound urolithic stranguria unlock capsules and preparation technique thereof |
| CN102755366A (en) * | 2012-07-19 | 2012-10-31 | 浙江维康药业有限公司 | Honeysuckle soft capsule containing chlorogenic acid compound |
Non-Patent Citations (1)
| Title |
|---|
| 李望等: "栽培植物的绿原酸研究进展", 《作物研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709256A (en) * | 2018-12-19 | 2019-05-03 | 金花企业(集团)股份有限公司西安金花制药厂 | Scutelloside and chlorogenic acid thin layer identify detection method in a kind of Compound Jinyinhua Granules |
| WO2020143746A1 (en) * | 2019-01-11 | 2020-07-16 | 四川九章生物科技有限公司 | Use of chlorogenic acid in preparing medicine or pharmaceutical composition for preventing or treating pain |
| CN111089912A (en) * | 2019-05-21 | 2020-05-01 | 江苏万略医药科技有限公司 | Method for researching drug metabolism based on molecular and cell level |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107033001B (en) | 2020-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107033001A (en) | A kind of Chlorogenic acid compound and the compound stranguria-treating and calculus-removing tablet containing the compound | |
| CN102068507A (en) | Preparation method of Chinese medicinal capsules for tonifying lung and activating blood circulation | |
| CN102319357A (en) | Lipid-lowering dispersible tablets and preparation technology thereof | |
| CN1813860A (en) | Naoxinqing Chinese medicine dispersible tablet and its preparing process | |
| CN1772085A (en) | Extract for treating functional indigestion and its medicine composition | |
| CN1931233B (en) | Medicine composition of red sage and epimedium for treating cardiac and cerebral vascular diseases | |
| CN102652819B (en) | Fuyankang dispersible tablet and preparation method thereof | |
| CN101843668B (en) | Chinese medicinal granules for treating femoral head necrosis and preparation method thereof | |
| CN104771595B (en) | A kind of Chinese medicine composition of strengthen immunity and preparation method thereof | |
| CN101797365B (en) | Preparation technology of Kaixiong Shunqi Jiaonang | |
| CN1931337A (en) | Bock greenbrier rhizome micro pill and its prepn process | |
| CN100482267C (en) | Preparation for diabetes and its making method | |
| CN111228419A (en) | Mailuoshutong micro pill preparation and its preparing method | |
| CN1311852C (en) | Yuquan extractum and its medicine composition and use | |
| CN100434092C (en) | Prescription containing sweet clover component and its formulation | |
| CN108159377A (en) | A kind of Chinese medicine preparation for anti-trioxypurine | |
| CN102631422B (en) | Drug composition for treating acute gastroenteritis/chronic gastroenteritis | |
| CN105288173A (en) | Traditional Chinese medicine pellet preparation for regulating menstruation and removing freckles and preparation method thereof | |
| CN103263581A (en) | Shuangshen capsule for reducing blood sugar | |
| CN112717078B (en) | Traditional Chinese medicine composition, preparation thereof, preparation method and application | |
| CN111603501B (en) | Traditional Chinese medicine composition for primary liver cancer and application thereof | |
| CN107823502A (en) | A kind of tablet form treats high fat of blood Chinese medicine | |
| CN107625894A (en) | A kind of preparation method of compound ginseng stilbene dispersible tablet | |
| CN100589800C (en) | Chinese medicine preparation of total coumarins extract from peu cedanum praeruptorum dunn, and its preparing method and use | |
| CN100512827C (en) | Chinese medicine tablet of total coumarins extract from peucedanum praeruptorm and its preparing method and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |