CN102697901A - Fructus crataegi leaf and novel application of extract of fructus crataegi leaf - Google Patents
Fructus crataegi leaf and novel application of extract of fructus crataegi leaf Download PDFInfo
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Abstract
The invention provides a fructus crataegi leaf and an application of an extract of the fructus crataegi leaf to preparation of a medicament for preventing and treating cataract. The fructus crataegi leaf and the extract thereof are used for increasing the oxidation resistance of an organism by inhibiting aldose reductase, so that the fructus crataegi leaf and the extract thereof can be used for preparing a medicament for preventing and treating cataract to fulfill the aim of treating cataract.
Description
Technical field
The present invention relates to the new purposes of Folium Crataegi and extract thereof.
Background technology
Cataract is because lenticular opacity; Lose the double lens effect, make light can not see through crystalline lens and arrive retina, a kind of common ophthalmic diseases that causes the patient to lose one's sight; Also being the first blinding factor in the world, is because aging and initiations such as dysbolismus such as diabetes mostly.Cataractous pathogenesis is complicated, thinks that at present the polyhydric alcohol path is activated under the hyperglycemia situation, causes polyhydric alcohol to gather; Cause the crystal fibre edema; Protein synthesis minimizing, protein decomposition, cracking and cyto-architectural destruction, last lens cortex is muddy with nuclear, and cataract forms.Aldose reductase (AR) is the crucial rate-limiting enzyme of polyhydric alcohol metabolic pathway, when hyperglycemia physiological status or excessive tissue utilization sugar, is activated, and impels the interior excessive glucose of body to be converted into sorbitol.Sorbitol is accumulated the permeability that can change cell membrane in a large number in cell, cause the cell permeability edema, and then diabetic complication occurs, especially at the higher sensitive part of AR content, GBM, retina and peripheral nervous etc.It is unusual that a large amount of zooperies and clinical research show that aldose reductase inhibitor (ARI) can effectively improve the polyhydric alcohol metabolic pathway, treatment and prevention cataract.
Modern day cataract extracapsular cataract extraction or phacoemulsification associating back room implantation of artificial lens are the cataractous main means of current treatment.Postoperative posterior lens capsule muddiness is that the vision that the generation of after cataract can make postoperative improve descends once again, influences surgical effect and expensive.Therefore, inquire into medicament and prevent and treat cataract, seek the emphasis that the cataract new drug just becomes pharmacy worker research of preventing and treating of determined curative effect, cheapness.
Folium Crataegi is the leaf of rosaceous plant Fructus Pyri Pashiae Crataegus pinnatifida Bge.var.major N.E.Br. or Fructus Crataegi C.pinnatifida Bge..The property sour, sweet, flat, main go into Liver Channel." Chinese pharmacopoeia version in 2005 is carried it and has the effect of " blood circulation promoting and blood stasis dispelling is regulated the flow of vital energy and promoted blood circulation ".Therefore cataractous prevention and treatment are used it in the further pharmacological action of research Folium Crataegi, are present stage new research directions, have the important clinical meaning.
Summary of the invention
Technical problem to be solved by this invention is to provide the new purposes of Folium Crataegi.
Another technical problem to be solved by this invention is to provide the new purposes of Folium Crataegi extract.
For solving the problems of the technologies described above, technical scheme of the present invention is:
The invention provides the purposes of Folium Crataegi in suppressing aldose reductase activity.
The invention provides Folium Crataegi strengthening lenticular oxidation resistance, reduce the purposes in the lenticular oxidative damage.
The invention provides the purposes of Folium Crataegi in preparation aldose reductase inhibitor thing.
The invention provides Folium Crataegi and prevent and treat the purposes in the cataractous medicine in preparation.
The invention provides the purposes of Folium Crataegi extract in suppressing aldose reductase activity.
The invention provides Folium Crataegi extract strengthening lenticular oxidation resistance, reduce the purposes in the lenticular oxidative damage.
The invention provides the purposes of Folium Crataegi extract in preparation aldose reductase inhibitor thing.
The invention provides Folium Crataegi extract and prevent and treat the purposes in the cataractous medicine in preparation.
Preferably, Folium Crataegi extract described in the such use is the Folium Crataegi ethanol extraction.
Preferably, the ethanol extraction of Folium Crataegi described in the such use is a Folium Crataegi 50%-95% ethanol extraction.
The present invention also provides the pharmaceutical composition of realizing such use, comprises Folium Crataegi and/or the Folium Crataegi extract and the optional acceptable excipient of pharmacy of treating effective dose.
The acceptable excipient of above-mentioned pharmacy can be the excipient of any routine in the field of pharmaceutical preparations; The selection of particular excipient will be depended on administering mode or disease type and the state that is used to treat particular patient, and the suitable drug preparation of compositions method that is used for the specific administration pattern is fully in drug world technical staff's the ken.For example, can be used as the acceptable excipient of pharmacy and comprise the conventional diluent of pharmaceutical field, carrier, filler, binding agent, wetting agent, disintegrating agent etc.
Above-mentioned composition can be processed various ways such as drop, tablet, powder, granule, capsule, oral liquid, injectable emulsion, sterile powder for injection pin, and the medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
The invention has the beneficial effects as follows:
Folium Crataegi and extract thereof are through suppressing aldose reductase, and the enhancing body oxidation resistance is treated cataractous medicine thereby be expected to be used for preparation, and then reached for cataractous therapeutic purposes.
Description of drawings
Figure 1A is a Folium Crataegi HPLC collection of illustrative plates;
Figure 1B is a Folium Crataegi extract HPLC collection of illustrative plates.
The specific embodiment
In order to make those skilled in the art better understand technical scheme of the present invention, technical scheme according to the invention is done further to specify below in conjunction with the specific embodiment.
Embodiment 1
The preparation of Folium Crataegi extract
Take by weighing dry Folium Crataegi 1kg, add 70% ethanol 10L, boiling reflux extracted 2 hours, filtered; Add 70% ethanol 10L in the residue, boiling reflux extracted 2 hours, filtered; Twice filtrating merges, and it is dried that 40 ℃ of heating in water bath are evaporated to, and obtains Folium Crataegi 70% ethanol extraction 214g.
Embodiment 2
The preparation of Folium Crataegi extract
Take by weighing dry Folium Crataegi 1kg, add 50% ethanol 10L, boiling reflux extracted 2 hours, filtered; Add 50% ethanol 10L in the residue, boiling reflux extracted 2 hours, filtered; Twice filtrating merges, and it is dried that 40 ℃ of heating in water bath are evaporated to, and obtains Folium Crataegi 50% ethanol extraction 235g.
Embodiment 3
The preparation of Folium Crataegi extract
Take by weighing dry Folium Crataegi 1kg, add 90% ethanol 10L, boiling reflux extracted 2 hours, filtered; Add 90% ethanol 10L in the residue, boiling reflux extracted 2 hours, filtered; Twice filtrating merges, and it is dried that 40 ℃ of heating in water bath are evaporated to, and obtains Folium Crataegi 90% ethanol extraction 201g.
Embodiment 4
The preparation of Folium Crataegi extract
Folium Crataegi medical material 1kg measures 70% ethanol extractions 2 times with 10 times, each 2h, and the extracting solution concentrating under reduced pressure is flung to ethanol, filters.Filtrating is used the D101 macroporous resin adsorption, and the resin bed volume is 1:1 (w/v) with the ratio of crude drug amount; After the last appearance, with the water washing of 2 times of resin bed volumes, discard water lotion earlier, with 70% ethanol elution of 4 times of resin bed volumes, the eluent concentrating under reduced pressure, vacuum drying promptly gets Folium Crataegi extract 88g.
Embodiment 5
The mensuration of hyperin in Folium Crataegi and the Folium Crataegi extract
1 instrument and reagent
Agilent 1100 liquid chromatographic systems (VWD detector); Methanol, acetonitrile, oxolane are chromatographically pure; Hyperin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 111521-200303); The Folium Crataegi medical material picks up from mountain range, green hill, Ji County, Tianjin, and is accredited as the leaf of rosaceous plant Fructus Crataegi through the Chinese medicine academy Guo Junhua assistant researcher of Tianjin University Of Traditional Chinese Medicine.D101 macroporous resin (Tianjin sea light chemical industry company limited, article type only).
2 methods and result
2.1 the preparation of Folium Crataegi extract
The Folium Crataegi medical material is measured 70% ethanol extractions 2 times with 10 times, each 2h, and the extracting solution concentrating under reduced pressure is flung to ethanol, filters.Filtrating is used the D101 macroporous resin adsorption, and the resin bed volume is 1:1 (w/v) with the ratio of crude drug amount; After the last appearance, with the water washing of 2 times of resin bed volumes, discard water lotion earlier, with 70% ethanol elution of 4 times of resin bed volumes, the eluent concentrating under reduced pressure, vacuum drying promptly gets Folium Crataegi extract.
2.2 the preparation of need testing solution
2.2.1 hyperin assay need testing solution in the Folium Crataegi
Get Folium Crataegi medicinal powder 1.0g, the accurate title, decide, and adds 70% ethanol 100ml reflux, extract, 2 hours, supplies weightlessness, and 0.45 μ m filtering with microporous membrane promptly gets.
2.2.2 hyperin assay need testing solution in the Folium Crataegi extract
Get Folium Crataegi extract 20mg, the accurate title, decide, and puts in the 25ml measuring bottle, adds the about 20ml of methanol, and supersound process (power 250W, frequency 40kHz) 5 minutes is diluted to scale with methanol, shakes up, and 0.45 μ m filtering with microporous membrane promptly gets.
2.3HPLC condition
Dalian Yi Lite chromatographic column (Hypersil ODS, 4.6mm * 250mm, 5 μ m), mobile phase is acetonitrile: methanol: oxolane: 0.5% acetic acid=1:1:19.4:78.6 (v/v), flow velocity: 1mLmin-1; Detect wavelength 363nm.
2.4 methodological study
The preparation of the standard curve of hyperin: accurate to claim decide the hyperin reference substance an amount of, is made into the reference substance solution of series concentration with methanol, and by 2.3 chromatographic conditions mensuration, the regression equation of hyperin is Y
(A)=17997X
(C)+ 35.17, correlation coefficient r=0.9995, the range of linearity is 8-320 μ g.mL
-1
Hyperin LDL (LOD) and minimum quantitative limit (LOQ) are respectively 1 μ g.mL
-1With 3 μ g.mL
-1Day to day precision and withinday precision are all less than 4%.Average recovery is 96-104% (RSD% < 2%).
2.5 assay result
Through measuring, hyperin content is respectively 0.16% and 2.35% in Folium Crataegi and the Folium Crataegi extract.
Chromatogram is seen Figure 1A and Figure 1B.
The protein glycosylation dead end product is formed inhibition
1. related solution preparation
The 10mM beta-mercaptoethanol contains 135mM phosphate buffer (pH7.0) compound method: accurately take by weighing potassium dihydrogen phosphate 18.36g, sodium hydrogen phosphate 19.17g; Add respectively in the 1L distilled water; Two solution mix regulates pH value to 7.0; Press 74.18mg/100ml and add beta-mercaptoethanol, the vibration dissolving.
180mM phosphate buffer (pH7.0) compound method: accurately take by weighing potassium dihydrogen phosphate 24.48g, sodium hydrogen phosphate 25.56g, add respectively in the 1L distilled water, two solution mix regulates pH value to 7.0.
1.0M lithium sulfate solution compound method: accurately take by weighing lithium sulfate 1.1g and be dissolved in vibration dissolving in the 10ml distilled water.
10mM DL-glyceraldehyde solution compound method: accurately take by weighing 0.9gDL-glyceraldehyde and be dissolved in vibration dissolving in the 1L distilled water.
The 10mM imidazoles contains 6M sodium hydroxide solution compound method: accurately take by weighing the 240g dissolution of sodium hydroxide in the 1L distilled water, take by weighing 6.8g imidazoles vibration dissolving again.
0.3mM NADPH solution compound method: accurately take by weighing 2.5mg NADPH and add 10ml 180mM phosphate buffer (pH7.0) vibration dissolving; Take out 1ml solution; Add 27ml 180mM phosphate buffer (pH 7.0), be made into the NADPH solution of 0.01M, ice bath is preserved.
2. sample solution preparation
With DMSO dissolving embodiment 1 said Folium Crataegi 70% ethanol extraction, be made into the solution of 64mg/ml ,-20 ℃ of preservations.
Before the experiment sample DMSO solution is diluted to 400 μ g/ml, 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 0 μ g/ml with 180mM phosphate buffer (pH 7.0), each sample solution contained DMSO volume when final concentration is equal and less than 2.5%.
3. aldose reductase preparation
(1) aldose reductase method for distilling
Wistar rat dislocation is put to death, and wins eyeball immediately, adopts posterior approach to win crystalline lens (carrying out) on ice and puts into the 20ml10mM beta-mercaptoethanol and contain 135mM phosphate buffer (pH 7.0); Homogenate; 100000 * g, 4 ° of centrifugal 30min of C get supernatant ,-20 ℃ of preservations.
(2) aldose reductase screening technique
Experiment is carried out in teat glass, establishes three groups, 4 every group multiple holes; Be respectively control group A, sample sets B, blank control group C, each sample testing group, the enzymatic solution 100 μ l of 180mM phosphate buffer (pH7.0) 225 μ l, 1.0M lithium sulfate solution 50 μ l, 10mM DL-glyceraldehyde solution 50 μ l, extraction and sample solution 25 μ l mix; 30 ℃ of preheating 3min; Add 0.3mMNADPH solution 50 μ l, 30 ℃ of reaction 30min, the hydrochloric acid 150 μ l cessation reactions of adding 0.5M; The standard curve group; The enzymatic solution 1400 μ l of 180mM phosphate buffer (pH7.0) 2450 μ l, 1.0M lithium sulfate solution 700 μ l, 10mM DL-glyceraldehyde solution 700 μ l, dimethyl sulfoxide 350 μ l, extraction and the hydrochloric acid 2100 μ l of 0.5M mix the back and take out 600 μ l; Adding NADP solution respectively, to make its final concentration be 0 μ M, 1 μ M, 2 μ M, 4 μ M, 8 μ M, 16 μ M; Each testing tube adds the 10mM imidazoles respectively and contains the 6M sodium hydroxide solution; 60 ℃ of reaction 20min, room temperature is put cold back in excitation wavelength 360nm, and emission wavelength 460nm detects the fluorescent absorption degree.
4 suppress active computational methods
Calculate the suppression ratio of each concentration of active component according to following formula, calculate IC50 according to the suppression ratio of each concentration.
Suppression ratio (%)=(A-B)/(A-C) * 100
Wherein A is the matched group trap, and B is the sample sets trap, and C is the blank control group trap.
Experimental result
Embodiment 7
1. laboratory animal
60 of the Wistar rats that was born 12 days (oneself breeding); Feed by female Mus; Raising is in Tianjin University Of Traditional Chinese Medicine's Experimental Animal Center, and environmental facility meets " Ministry of Health of the People's Republic of China's laboratory animal environmental facility standard " secondary standard, and the quality certification number is the moving word of doctor 01-19981109 number.
2. medicine compound method
Preparation eye drop: accurately take by weighing embodiment 1 said a certain amount of Folium Crataegi 70% ethanol extraction, be dissolved in the solution that the dissolving of vibrating in the distilled water is made into 1mg/ml, 2mg/ml, get eye drop behind the sucking filtration.
Animal divides into groups
The Wistar neonatal rat that was born 12 days; Male and female are regardless of, parallel normal group, model group, Folium Crataegi 70% ethanol extraction high dose group (4mg/kg), Folium Crataegi 70% ethanol extraction low dose group (2mg/kg), positive controls (Bernetine Sodium eye drop, the trade name: catalin of being divided into; Wuhan Yuanda Pharmaceutical Group Co., Ltd.; Lot number 080925), 12 every group, breast feeding.
Cataract modelling and administration
Become cup-shaped in the neonatal rat eyelid of opening neonatal rat gently with tweezers on the 12nd day of being born; Normal group and model group splash into distilled water, and the administration group splashes into the Folium Crataegi 70% ethanol extraction eye drop of above-mentioned variable concentrations, and positive controls splashes into Bernetine Sodium eye drop 2ml/kg body weight; Keep eyes to open about 1min; Prevent that medicinal liquid from overflowing outside the eye, every 8h administration 1 time, continuous 7 days.In neonatal rat the 14th day subcutaneous injection sodium selenite 19mol/kg body weight of being born, perusal cataract degree behind the 96h, chloral hydrate anesthesia, is gathered crystalline lens ,-80 ℃ of preservations at abdominal aortic blood.
4 sample collections
Behind the abdominal aortic blood, win eyeball rapidly, adopt the posterior approach method to peel off crystalline lens, blot, weigh-80 ℃ of preservations with coarse filter paper.
Detection method
1 serum index detects
Blood 3500rmin
-1, 4 ℃ of centrifugal 10min isolate serum.With ultra oxygen compound dismutase test kit, malonaldehyde test kit, hydrogen peroxide enzyme reagent kit (be Nanjing and build up biological company limited production), on multi-functional plate reading machine, detect ultra oxygen compound dismutase (SOD), malonaldehyde (MDA), catalase (CAT) level.
2 crystalline lens biochemistry detection
Crystalline lens is weighed in the rearmounted homogenate pipe, adds ice 1.0ml 180mM phosphate buffer (pH7.0), homogenate, 3500rmin
-14 ℃ of centrifugal 10min; Get supernatant,, on multi-functional plate reading machine, detect SOD, MDA, GSH, CAT with ultra oxygen compound dismutase test kit, malonaldehyde test kit, glutathion test kit, hydrogen peroxide enzyme reagent kit (be Nanjing and build up biological company limited production); Fluorescence spectrophotometry detects aldose reductase activity.
4 experimental results
4.1 serum index testing result
The activity of neonatal rat SOD in serum, CAT and MDA content are seen table 1; But perception model group neonatal rat activity of SOD in serum reduces than significance than normal group, and Bernetine Sodium eye drop, Folium Crataegi 70% ethanol extraction high dose group and Folium Crataegi 70% ethanol extraction low dose group be the activity of the inductive cataract neonatal rat of ability significance raising sodium selenite SOD in serum all; The activity of Bernetine Sodium eye drop, Folium Crataegi 70% ethanol extraction high dose group neonatal rat SOD in serum is apparently higher than normal group, and the effect of Folium Crataegi 70% ethanol extraction high dose is superior to Bernetine Sodium eye drop group.Model group neonatal rat change of serum C AT activity descends than significance with normal group; Bernetine Sodium eye drop, Folium Crataegi 70% ethanol extraction high dose group be the activity of ability significance raising neonatal rat change of serum C AT all, and Folium Crataegi 70% ethanol extraction low dose group has the active trend of the neonatal rat change of serum C AT of raising.Each dose groups neonatal rat Content of MDA does not have significant difference.
The activity of table 1 neonatal rat SOD in serum, CAT and MDA content
With model control group relatively have significant difference * p 0.05, * * p < 0.01
4.2 crystalline lens biochemistry detection result
Neonatal rat crystalline lens SOD, CAT, AR activity and GSH content are seen table 2; Model group crystalline lens CAT activity reduces with the active significance of normal group crystalline lens CAT, and the equal ability of the high low dose treatment group of positive controls and Folium Crataegi 70% ethanol extraction significance rising crystalline lens CAT is active but still do not make it return to normal level.Model group crystalline lens SOD activity reduces than significance with normal group; Bernetine Sodium eye drop treatment group, the high low dose group of Folium Crataegi 70% ethanol extraction all can improve crystalline lens SOD activity by significance, and all the other each groups all can make it return to normal level except that Folium Crataegi low dose treatment group.Model group crystalline lens GSH content descends than significance with normal group crystalline lens GSH content, and the high low dose treatment group of positive controls and Folium Crataegi 70% ethanol extraction all can significance rising crystalline lens GSH content and made it reach normal level basically.
Table 2 neonatal rat crystalline lens SOD is active, active, the GSH content of CAT
With model control group relatively have significant difference * p 0.05, * * p < 0.01
Conclusion:
1, Folium Crataegi 70% ethanol extraction can reduce the inductive cataract neonatal rat of selenium SOD in serum, CAT activity by significance, and the whole oxidation resistance of enhancing body can reduce the cataract incidence rate, points out this medicine can be used for cataract therapy and prevention.
2, Folium Crataegi 70% ethanol extraction can strengthen the inductive cataract neonatal rat of selenium crystalline lens SOD, CAT activity and GSH content by significance, strengthens lenticular oxidation resistance, reduces lenticular oxidative damage, prevents and treats cataract.
Method for preparing: according to the above ratio that Folium Crataegi 70% ethanol extraction, lactose and corn starch is mixed, cross 200 mesh sieves, the even moistening of water after sieve, adds magnesium stearate to the mixture drying after the moistening, then the mixture tabletting is promptly got.
Embodiment 9
Capsule: embodiment 1 said Folium Crataegi 70% ethanol extraction 20mg
Galactose 188mg
Magnesium stearate 2mg
Method for preparing: according to the above ratio with Folium Crataegi 70% ethanol extraction and galactose, magnesium stearate uniform mixing, cross 200 mesh sieves,, promptly get the mixture that the obtains capsule of packing into No. 1.
Oral liquid: embodiment 1 said Folium Crataegi 70% ethanol extraction 2g
Formula ratio Folium Crataegi 70% ethanol extraction added in 400 ml distilled waters dissolve, filter, add an amount of simple syrup, sodium benzoate, adding distil water to 1000 milliliter again, cold preservation filters, and is packaged in 10 milliliters of ampoules, and 100 ℃ of sterilizations 30 minutes promptly get.
Embodiment 11
Granule: embodiment 1 said Folium Crataegi 70% ethanol extraction 1000mg
Formula ratio Folium Crataegi 70% ethanol extraction is added in the 600g sucrose, and mix homogeneously is crossed 200 mesh sieves, and 80% ethanol soft material is granulated drying, 6g/ bag.
Above-mentioned detailed description of the new purposes of this Folium Crataegi being carried out with reference to the specific embodiment; Be illustrative rather than determinate; Can enumerate out several embodiment according to institute's limited range; Therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (3)
1. Folium Crataegi prevents and treats the purposes in the cataractous medicine as unique active component in preparation.
2. the Folium Crataegi ethanol extraction prevents and treats the purposes in the cataractous medicine as unique active component in preparation.
3. purposes according to claim 2 is characterized in that said Folium Crataegi ethanol extraction is a Folium Crataegi 50%-95% ethanol extraction.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060127412A1 (en) * | 2004-06-07 | 2006-06-15 | Kao Corporation | Aromatase activator |
| WO2006088385A2 (en) * | 2005-02-18 | 2006-08-24 | S.C. Biotehnos S.A. | Bioactive complex of triterpene acids, its production process and medicinal products with therapeutical uses |
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- 2010-06-23 CN CN2012102033142A patent/CN102697901A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060127412A1 (en) * | 2004-06-07 | 2006-06-15 | Kao Corporation | Aromatase activator |
| WO2006088385A2 (en) * | 2005-02-18 | 2006-08-24 | S.C. Biotehnos S.A. | Bioactive complex of triterpene acids, its production process and medicinal products with therapeutical uses |
Non-Patent Citations (1)
| Title |
|---|
| 贾敏如: "《国际传统药和天然药物》", 31 October 2006, article "欧山楂", pages: 145-146 * |
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Application publication date: 20121003 |