CN107987089A - A kind of extract, preparation method and medical usage rich in chromene lactone - Google Patents
A kind of extract, preparation method and medical usage rich in chromene lactone Download PDFInfo
- Publication number
- CN107987089A CN107987089A CN201711275329.9A CN201711275329A CN107987089A CN 107987089 A CN107987089 A CN 107987089A CN 201711275329 A CN201711275329 A CN 201711275329A CN 107987089 A CN107987089 A CN 107987089A
- Authority
- CN
- China
- Prior art keywords
- extract
- netvein
- toxicity
- preparation
- root element
- 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
- 239000000284 extract Substances 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- -1 chromene lactone Chemical class 0.000 title claims abstract description 24
- BBFQZRXNYIEMAW-UHFFFAOYSA-N aristolochic acid I Chemical compound C1=C([N+]([O-])=O)C2=C(C(O)=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-N 0.000 claims abstract description 53
- 239000003814 drug Substances 0.000 claims abstract description 36
- 239000011347 resin Substances 0.000 claims abstract description 15
- 229920005989 resin Polymers 0.000 claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 11
- 231100000417 nephrotoxicity Toxicity 0.000 claims abstract description 6
- 239000000706 filtrate Substances 0.000 claims description 32
- 239000012043 crude product Substances 0.000 claims description 12
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 241001113317 Farfugium Species 0.000 claims description 8
- 210000003462 vein Anatomy 0.000 claims description 8
- 210000003734 kidney Anatomy 0.000 claims description 7
- 239000013078 crystal Substances 0.000 claims description 6
- 238000002425 crystallisation Methods 0.000 claims description 6
- 230000008025 crystallization Effects 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 238000002604 ultrasonography Methods 0.000 claims description 6
- 241000046585 Aristolochia clematitis Species 0.000 claims description 4
- 239000000469 ethanolic extract Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 101001031591 Mus musculus Heart- and neural crest derivatives-expressed protein 2 Proteins 0.000 claims description 2
- 231100000419 toxicity Toxicity 0.000 claims description 2
- 230000001988 toxicity Effects 0.000 claims description 2
- 239000000178 monomer Substances 0.000 abstract description 9
- 238000010979 pH adjustment Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000010898 silica gel chromatography Methods 0.000 abstract description 3
- 241000700159 Rattus Species 0.000 description 21
- 239000000243 solution Substances 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 11
- 206010023421 Kidney fibrosis Diseases 0.000 description 9
- 230000006907 apoptotic process Effects 0.000 description 9
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 206010016654 Fibrosis Diseases 0.000 description 5
- 230000004761 fibrosis Effects 0.000 description 5
- 238000003304 gavage Methods 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 210000005084 renal tissue Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 201000002793 renal fibrosis Diseases 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 241001369615 Aristolochia fangchi Species 0.000 description 2
- 241000758794 Asarum Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 244000292697 Polygonum aviculare Species 0.000 description 2
- 235000006386 Polygonum aviculare Nutrition 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000008157 edible vegetable oil Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 240000008027 Akebia quinata Species 0.000 description 1
- 235000007756 Akebia quinata Nutrition 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- BBFQZRXNYIEMAW-UHFFFAOYSA-M Aristolochate I Natural products C1=C([N+]([O-])=O)C2=C(C([O-])=O)C=C3OCOC3=C2C2=C1C(OC)=CC=C2 BBFQZRXNYIEMAW-UHFFFAOYSA-M 0.000 description 1
- 208000004884 Balkan Nephropathy Diseases 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 240000004980 Rheum officinale Species 0.000 description 1
- 235000008081 Rheum officinale Nutrition 0.000 description 1
- 244000303379 Styrax officinalis Species 0.000 description 1
- 235000001361 Styrax officinalis Nutrition 0.000 description 1
- 208000007271 Substance Withdrawal Syndrome Diseases 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000019382 gum benzoic Nutrition 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000008629 longdanxiegan Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000011617 nephropathy animal model Methods 0.000 description 1
- 230000004987 nonapoptotic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 210000004926 tubular epithelial cell Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
-
- 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)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
Abstract
The invention discloses a kind of extract, preparation method and medical usage rich in chromene lactone.Extract provided by the invention is not less than 90% rich in netvein goldenray root element A, netvein goldenray root element B and the sum of netvein goldenray root element C, three's content, or is not less than 85% rich in netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C and the sum of netvein goldenray root element, four contents；The monomer component of the extract is clear and definite, and content is high, easily controllable quality, can reduce the renal toxicity of aristolochic acid, can be used for preparing the single medicinal material containing aristolochic acid or Chinese medical extract or the toxicity-reducing medicament of compound Chinese medicinal preparation；Without Reusability silica gel column chromatography, these are not easy the separation means in factory's large-scale operation to the preparation method of extract of the present invention, only needed a macroreticular resin, and the extract of effective monomer component total content more than 85% can be prepared with reference to pH adjustment controls, it is easy to industrialization, easy to operate, cost is low.
Description
Technical field
The invention belongs to field of medicaments, it is related to a kind of extract rich in chromene lactone, preparation method and pharmaceutical
On the way.
Background technology
Netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C, netvein goldenray root element are isolated from net vein Farfugium kaemferi root earliest
Chromene lactone compound, chemical structural formula are as shown in the table.
It has been found that netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C, netvein goldenray root element can reduce the kidney of aristolochic acid
Toxicity, can be used for preparing the single medicinal material containing aristolochic acid or Chinese medical extract or the toxicity-reducing medicament of compound Chinese medicinal preparation
(application for a patent for invention submitted referring to applicant, Application No. 2017112638286).
But it was found from above-mentioned chemical constitution, netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C are isomer, this
Similitude in kind structure adds the purifying difficulty of monomer, and the cost of monomer patent medicine is too high.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of extract of net vein Farfugium kaemferi root, the extraction
Rich in netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C, netvein goldenray root element in thing, and provide the preparation method of the extract, with for
For netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C, netvein goldenray root element monomer patent medicine, pharmacy cost is reduced.
The above-mentioned purpose of the present invention is achieved by following technical solution：
A kind of extract rich in chromene lactone, the chromene lactone include netvein goldenray root element A, netvein goldenray root element B
It is not less than 90% with the sum of netvein goldenray root element C, three's content.
Preferably, the chromene lactone further includes netvein goldenray root element, and the sum of four contents are not less than 85%.
The preparation method of said extracted thing, includes the following steps：
Step S1, the dry root of net vein Farfugium kaemferi is crushed, is extracted with alcohol solution, collects extracting solution, filtering, collects filter
Liquid；
Step S2, macroporous resin column on filtrate, macroreticular resin model D101, first elutes 8 with 40% ethanol water
Column volume, then eluted with 60% ethanol water, the 5-6 column volume eluent is collected, is concentrated to give chromene lactone crude product；
Step S3, alkaline open loop：The buck ultrasound that crude product is 7.8-8.2 with pH is suspended, filtering, collects filtrate；
Step S4, acid closed loop：Filtrate pH is adjusted to filter to obtain two filtrates after 6.4-6.6 immediately, by two filtrate stand at low temperature
Crystallization；
Step S5, crystal obtained by step S4 is washed with deionized, kept dry.
Preferably, step S1 preferably 75% ethanol extracts.
Preferably, 0.45 μm of membrane filtration of step S3.
The preparation method of said extracted thing, includes the following steps：
Step S1, the dry root of net vein Farfugium kaemferi is crushed, is extracted with alcohol solution, collects extracting solution, filtering, collects filter
Liquid；
Step S2, macroporous resin column on filtrate, macroreticular resin model D101, first elutes 8 with 40% ethanol water
Column volume, then eluted with 60% ethanol water, the 5-7 column volume eluent is collected, is concentrated to give chromene lactone crude product；
Step S3, alkaline open loop：The buck ultrasound that crude product is 7.8-8.2 with pH is suspended, filtering, collects filtrate；
Step S4, acid closed loop：Filtrate pH is adjusted to filter to obtain two filtrates after 6.4-6.6 immediately, by two filtrate stand at low temperature
Crystallization；
Step S5, crystal obtained by step S4 is washed with deionized, kept dry.
Preferably, step S1 preferably 75% ethanol extracts.
Preferably, 0.45 μm of membrane filtration of step S3.
Said extracted thing is used to prepare the purposes of aristolochic acid toxicity-reducing medicament, is used to prepare in the single containing aristolochic acid
The purposes of the toxicity-reducing medicament of medicine, is used to prepare the purposes of the toxicity-reducing medicament of the Chinese medical extract containing aristolochic acid, is used to prepare
The purposes of the toxicity-reducing medicament of compound Chinese medicinal preparation containing aristolochic acid；The attenuation refers to the renal toxicity for reducing aristolochic acid.
Said extracted thing is used to prepare the purposes of aristolochic acid toxicity-reducing medicament, is used to prepare in the single containing aristolochic acid
The purposes of the toxicity-reducing medicament of medicine, is used to prepare the purposes of the toxicity-reducing medicament of the Chinese medical extract containing aristolochic acid, is used to prepare
The purposes of the toxicity-reducing medicament of compound Chinese medicinal preparation containing aristolochic acid；Attenuation refers to the renal toxicity for reducing aristolochic acid.
Advantages of the present invention：
1st, extract provided by the invention is rich in netvein goldenray root element A, netvein goldenray root element B and the sum of netvein goldenray root element C, three's content no
It is not less than less than 90%, or rich in netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C and the sum of netvein goldenray root element, four contents
85%；The monomer component of the extract is clear and definite, and content is high, and easily controllable quality, can reduce the renal toxicity of aristolochic acid, can
For preparing the single medicinal material containing aristolochic acid or Chinese medical extract or the toxicity-reducing medicament of compound Chinese medicinal preparation；
2nd, without Reusability silica gel column chromatography, these are not easy to grasp on a large scale in factory the preparation method of extract of the present invention
The separation means of work, it is only necessary to cross a macroreticular resin, and effective monomer component total content can be prepared with reference to pH adjustment controls
More than 85% extract, is easy to industrialization, easy to operate, and cost is low.
Brief description of the drawings
Fig. 1 is that extract prepared by embodiment 1 and embodiment 2 causes aristolochic acid what people's renal cells damaged
Protective effect, including suppress HK-2 Apoptosis；
Fig. 2 is the protection that extract prepared by embodiment 1 and embodiment 2 causes aristolochic acid renal interstitial fibrosis rat
Effect, suppresses renal fibrosis fibrosis area and the expression of markers of fibrosis PROTEIN C OL- I.
Embodiment
The specific guarantor for introducing essentiality content of the present invention, but the present invention not being limited with this with reference to the accompanying drawings and examples
Protect scope.
Embodiment 1：The preparation of extract 1
Include the following steps：
Step S1, the dry root of net vein Farfugium kaemferi is crushed, and is extracted with 75% ethanol, collects extracting solution, filtering, collects filter
Liquid；
Step S2, macroporous resin column on filtrate, macroreticular resin model D101, first elutes 8 with 40% ethanol water
Column volume, then eluted with 60% ethanol water, the 5-6 column volume eluent is collected, is concentrated to give chromene lactone crude product；
Step S3, alkaline open loop：The sodium hydroxide solution for being 8.0 by chromene lactone crude product pH value obtained by step S2
Ultrasound is suspended, 0.45 μm of membrane filtration, collects filtrate；
Step S4, acid closed loop：Filtrate pH filters to obtain two filtrates immediately after being adjusted to 6.5, by two filtrate, 4 DEG C of standing crystallizations；
Step S5, crystal obtained by step S4 is washed with deionized, kept dry.
Chromene lactone in the extract of high effective liquid chromatography for measuring drying, detects netvein goldenray root element A, netvein goldenray root
The sum of plain B, netvein goldenray root element C, netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C content are 91.4%.
Embodiment 2：The preparation of extract 2
Include the following steps：
Step S1, the dry root of net vein Farfugium kaemferi is crushed, and is extracted with 75% ethanol, collects extracting solution, filtering, collects filter
Liquid；
Step S2, macroporous resin column on filtrate, macroreticular resin model D101, first elutes 8 with 40% ethanol water
Column volume, then eluted with 60% ethanol water, the 5-7 column volume eluent is collected, is concentrated to give chromene lactone crude product；
Step S3, alkaline open loop：The sodium hydroxide solution for being 8.0 by chromene lactone crude product pH value obtained by step S2
Ultrasound is suspended, 0.45 μm of membrane filtration, collects filtrate；
Step S4, acid closed loop：Filtrate pH filters to obtain two filtrates immediately after being adjusted to 6.5, by two filtrate, 4 DEG C of standing crystallizations；
Step S5, crystal obtained by step S4 is washed with deionized, kept dry.
Chromene lactone in the extract of high effective liquid chromatography for measuring drying, detects netvein goldenray root element A, netvein goldenray root
Plain B, netvein goldenray root element C, the sum of netvein goldenray root element, netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C, netvein goldenray root cellulose content are
86.5%.
Embodiment 3：Said extracted thing causes aristolochic acid the protective effect that people's renal cells damages
First, experiment material
People's kidney proximal tubular cell line (HK-2) is purchased from China typical culture collection center；
Aristolochic acid-I (AA- I) is purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute；
Hyclone (FCS), Gibico companies；DMEM：HamsF-12 culture mediums (Hyclone)；Annexin-V reagents
Box, Invitrogen companies；
Enzyme-linked immunosorbent assay instrument, Thermofisher companies of the U.S.；Flow cytometer, U.S. company BD.
2nd, experimental method
1st, cell culture and packet
Well-grown HK-2 cells are taken, are suspended in the DMEM containing 10%FCS and 1% penicillin+streptomysin：HamsF-12
In culture medium, with 6 × 107/ L cell suspension inoculations are inoculated with 100 μ L per hole, in 37 DEG C, 5%CO in 96 orifice plates2, humidity
After cultivating 24h cell attachments under 100% environment, nutrient solution is abandoned in suction, continues to be incubated training by the following relative medicine that is separately added into
Support 48h.4 groups are set up in experiment altogether, and each group sets 8 multiple holes：
(1) control group：Nutrient solution is not added with AA- I；
(2) aristolochic acid group：AA- I is added in nutrient solution (25mg/L, each pharmaceutical intervention group are same)；
(3) 1 group of extract：Extract 1 (10mg/L, is final concentration, similarly hereinafter) and AA- I are added in nutrient solution at the same time；
(4) 2 groups of extract：Extract 2 (10mg/L) and AA- I are added in nutrient solution at the same time.
2nd, mtt assay measure cell survival rate
Each group 4h before culture terminates, the 20 μ L of MTT solution of 5mg/L are added per hole, after culture, 100 μ are added per hole
L DMSO, using the OD values that each hole is measured at enzyme-linked immunosorbent assay instrument 480nm.
3rd, flow cytometry detection apoptosis rate
After culture, cell 1 × 10 is collected6A/mL, 1000rpm centrifuge 5min, abandon supernatant, and cell is resuspended with PBS,
Operated according to Annexin-VFITC cell apoptosis detection kits specification, detect the percentage of apoptotic cell and non-viable non-apoptotic cell.
4th, statistical method
Analyzed using SPSS19.0 software statistics, data represent that comparison among groups uses single factor test variance with mean value ± deviation
Analysis.
3rd, experimental result
1st, mtt assay measure cell survival rate
Compared with control group, after AA- I acts on 48h, aristolochic acid group HK-2 cytoactives are obvious suppressed (P ＜ 0.05)；
Compared with aristolochic acid group, 2 groups of 1 group of extract, extract HK-2 cytoactives significantly improve (P ＜ 0.05).Each group 480nm
OD values under the conditions of wavelength detecting are shown in Table 1.
OD values under the conditions of 1 each group 480nm wavelength detectings of table
2nd, flow cytometry detection apoptosis rate
Compared with control group, after AA- I acts on 48h, aristolochic acid group HK-2 Apoptosis ratios substantially increase (P ＜
0.05)；Compared with aristolochic acid group, 2 groups of 1 group of extract, extract HK-2 Apoptosis ratios substantially reduce (P ＜
0.05).Each group apoptosis value is shown in Table 2 and Fig. 1.
2 each group HK-2 Apoptosis ratios of table
The embodiment illustrates that Aristolochic Acid in Antagonizing Human Renal Tubular epithelial cell HK-2 has obvious cytotoxicity, can cause
HK-2 survival rates reduce, apoptosis rate rise；Extract 1 provided by the invention, extract 2 can be small with antagonism Aristolochic Acid in Antagonizing Human Renal
The cytotoxicity of pipe epithelial cell HK-2, has prospect of the exploitation into aristolochic acid toxicity-reducing medicament.
Embodiment 4：Said extracted thing causes aristolochic acid the protective effect of renal interstitial fibrosis rat
First, experiment material
SPF grades of male SD rats, weight 280-300g, is purchased from Jiangning county Qinglongshan animal reproduction field；
3,4-methylenedioxy-8-methoxy-10-nitro-1-phenanthrenecarboxylic acid is dissolved with edible oil, as gavage working solution.
2nd, experimental method
1st, aristolochic acid kidney fibrosis rat model and experiment packet are established
Aristolochic acid kidney fibrosis modeling method (bibliography：The foundation of Chronic Aristolochic Acid Nephropathy animal model and ox
Sulfonic acid acts on its early protection, the special collection of Chinese combination of Chinese tradiational and Western medicine magazine in June, 2003 fundamental research of volume 23, and Beijing is big
Study medicine department of pathology of the department of the Chinese Academy of Sciences)：Rat is first pressed to dosage the continuous gavage 5d, drug withdrawal 9d of 20mg/kg/d 3,4-methylenedioxy-8-methoxy-10-nitro-1-phenanthrenecarboxylic acids；Hereafter agent
Amount reduces to 15mg/kg/d, every other week gastric infusion, until the 11st week.
SPF grades of male SD rat adaptability are fed after a week, 4 groups, every group 10 are randomly divided into according to weight：
(1) control group：Gavage gives isometric solvent (edible oil)；
(2) aristolochic acid group：Modeling according to the method described above；
(3) 1 group of extract：Modeling as stated above, and from first week the next day gavage 25mg/kg/d extracts 1；
(4) 2 groups of extract：Modeling as stated above, and from first week the next day gavage 25mg/kg/d extracts 2.
2nd, renal fibrosis pathologic finding
After culture, put to death rat, take out rat kidney, PBS buffer elutes totally on ice, conventional dehydration, embed,
After section and Masson dyeing, light microscope (× 100) gathers renal interstitial image (every rat random acquisition of cortical section
15 visuals field), carry out automatic measurement point with multi-functional true color pathological image analysis system (BJ University of Aeronautics ＆ Astronautics manufactures)
Analysis, the kidney region fibrosis relative area=renal interstitial Lv Ran areas area/visual field gross area × 100%.
3rd, the detection that renal fibrosis marker protein COL- I is expressed
After culture, rat is put to death, rat kidney is taken out, is positioned on ice, prevents protein degradation.Clip about 35mg
Tissue, adds the lysate that 350 μ L concentration are 100mg/mL, is fully ground with grinding rod, stands 20min on ice, opposite at 4 DEG C
Centrifugal force 5.67 × 104× g centrifuges 15min.Protein supernatant is drawn, discards precipitation.Relative centrifugal force 5.67 × 10 at 4 DEG C4
× g centrifuges 15min, draws protein supernatant again, discards precipitation.Using GAPDH albumen as internal reference, using immunoblotting
(westernblot) relative expression levels of each group renal tissues of rats COL- I are measured.
4th, statistical method
Analyzed using SPSS19.0 software statistics, data represent that comparison among groups uses single factor test variance with mean value ± deviation
Analysis.
3rd, experimental result
1st, each group renal interstitial fibrosis rat relative area
Compared with control group, aristolochic acid group renal interstitial fibrosis rat relative area significantly raises (P ＜ 0.05)；With horse
Pocket bell acid group compares, and 2 groups of 1 group of extract, extract renal interstitial fibrosis rat relative areas substantially reduce (P ＜ 0.05).
Each group renal interstitial fibrosis rat relative area is shown in Table 3 and Fig. 2.
3 renal interstitial fibrosis rat relative area (%) of table
2nd, I expressions of each group renal tissues of rats markers of fibrosis PROTEIN C OL-
Compared with control group, I protein expression levels of COL- significantly raise (P ＜ in aristolochic acid group renal tissues of rats
0.05)；Compared with aristolochic acid group, I protein expression levels of COL- are obvious in 2 groups of 1 group of extract, extract renal tissues of rats
Reduce (P ＜ 0.05).The relative expression levels of each group rat COL- I are as shown in Figure 2.
The embodiment illustrates that aristolochic acid can cause renal tissues of rats fibrosis；Extract 1 provided by the invention, extraction
Thing 2 can have prospect of the exploitation into aristolochic acid toxicity-reducing medicament with the internal renal toxicity of antagonism aristolochic acid.
Embodiment 5：Pharmaceutical composition is made with single medicinal material
A kind of pharmaceutical composition, is the combination of single medicinal material and 1 or 2 extract of embodiment；The single medicinal material can be closed
The Chinese medicine containing aristolochic acid such as akebi, birthwort, aristolochia fangchi, dutchmanspipe root, Ciliatenerve Knotweed Root, berba aristolochiae mollissimae, herba aristolochiae, Cortex Magnoliae Officinalis, asarum.
Embodiment 6：Pharmaceutical composition is made with Chinese medical extract
A kind of pharmaceutical composition, is the combination of Chinese medical extract and 1 or 2 extract of embodiment；The Chinese medicine can be Guan Mu
The Chinese medicine containing aristolochic acid such as logical, birthwort, aristolochia fangchi, dutchmanspipe root, Ciliatenerve Knotweed Root, berba aristolochiae mollissimae, herba aristolochiae, Cortex Magnoliae Officinalis, asarum.
The pharmaceutical composition can be used for pharmacy or the products such as slim tea be made into.
Embodiment 7：Pharmaceutical composition is made with Chinese medicine compound prescription
A kind of pharmaceutical composition, is Chinese medicine compound prescription and the combination of 1 or 2 extract of embodiment；The compound can be that rheum officinale is clear
The Chinese medicine containing aristolochic acid such as stomach ball, children-welfare tablet, Fenqing Wulin pill, Longdan Xiegan wan, shixiang fansheng pill, storax pill for treating coronary heart disease
Compound.
To sum up, extract provided by the invention is rich in netvein goldenray root element A, netvein goldenray root element B and netvein goldenray root element C, San Zhehan
The sum of amount is not less than 90%, or rich in netvein goldenray root element A, netvein goldenray root element B, netvein goldenray root element C and the sum of netvein goldenray root element, four contents no
Less than 85%；The monomer component of the extract is clear and definite, and content is high, easily controllable quality, can reduce the kidney poison of aristolochic acid
Property, it can be used for preparing the single medicinal material containing aristolochic acid or Chinese medical extract or the toxicity-reducing medicament of compound Chinese medicinal preparation；This
Without Reusability silica gel column chromatography, these are not easy the separation means in factory's large-scale operation to the preparation method of invention extract,
A macroreticular resin was only needed, and the extraction of effective monomer component total content more than 85% can be prepared with reference to pH adjustment controls
Thing, is easy to industrialization, easy to operate, and cost is low.
The effect of above-described embodiment is the essentiality content for specifically introducing the present invention, but those skilled in the art should know
Protection scope of the present invention, should not be confined to the specific embodiment by road.
Claims (10)
- A kind of 1. extract rich in chromene lactone, it is characterised in that：The chromene lactone includes extract 1, carries The sum of thing 2 and netvein goldenray root element C, three's content is taken to be not less than 90%.
- 2. extract according to claim 1, it is characterised in that：The chromene lactone further include netvein goldenray root element, four The sum of person's content is not less than 85%.
- 3. the preparation method of extract described in claim 1, it is characterised in that include the following steps：Step S1, the dry root of net vein Farfugium kaemferi is crushed, is extracted with alcohol solution, collects extracting solution, filtering, collects filtrate；Step S2, macroporous resin column on filtrate, macroreticular resin model D101, first elutes 8 cylinders with 40% ethanol water Product, then eluted with 60% ethanol water, the 5-6 column volume eluent is collected, is concentrated to give chromene lactone crude product；Step S3, alkaline open loop：The buck ultrasound that crude product is 7.8-8.2 with pH is suspended, filtering, collects filtrate；Step S4, acid closed loop：Filtrate pH is adjusted to filter to obtain two filtrates after 6.4-6.6 immediately, by two filtrate stand at low temperature crystallizations；Step S5, crystal obtained by step S4 is washed with deionized, kept dry.
- 4. preparation method according to claim 3, it is characterised in that：Step S1 preferably 75% ethanol extracts.
- 5. preparation method according to claim 3, it is characterised in that：0.45 μm of membrane filtration of step S3.
- 6. the preparation method of extract described in claim 2, it is characterised in that include the following steps：Step S1, the dry root of net vein Farfugium kaemferi is crushed, is extracted with alcohol solution, collects extracting solution, filtering, collects filtrate；Step S2, macroporous resin column on filtrate, macroreticular resin model D101, first elutes 8 cylinders with 40% ethanol water Product, then eluted with 60% ethanol water, the 5-7 column volume eluent is collected, is concentrated to give chromene lactone crude product；Step S3, alkaline open loop：The buck ultrasound that crude product is 7.8-8.2 with pH is suspended, filtering, collects filtrate；Step S4, acid closed loop：Filtrate pH is adjusted to filter to obtain two filtrates after 6.4-6.6 immediately, by two filtrate stand at low temperature crystallizations；Step S5, crystal obtained by step S4 is washed with deionized, kept dry.
- 7. preparation method according to claim 6, it is characterised in that：Step S1 preferably 75% ethanol extracts.
- 8. preparation method according to claim 6, it is characterised in that：0.45 μm of membrane filtration of step S3.
- 9. the extract described in claim 1 is used to prepare the purposes of aristolochic acid toxicity-reducing medicament, it is used to prepare containing birthwort The purposes of the toxicity-reducing medicament of the single medicinal material of acid, is used to prepare the use of the toxicity-reducing medicament of the Chinese medical extract containing aristolochic acid On the way, it is used to prepare the purposes of the toxicity-reducing medicament of the compound Chinese medicinal preparation containing aristolochic acid；The attenuation refers to reduction aristolochic acid Renal toxicity.
- 10. the extract described in claim 2 is used to prepare the purposes of aristolochic acid toxicity-reducing medicament, it is used to prepare containing birthwort The purposes of the toxicity-reducing medicament of the single medicinal material of acid, is used to prepare the use of the toxicity-reducing medicament of the Chinese medical extract containing aristolochic acid On the way, it is used to prepare the purposes of the toxicity-reducing medicament of the compound Chinese medicinal preparation containing aristolochic acid；Attenuation refers to the kidney for reducing aristolochic acid Toxicity.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711275329.9A CN107987089B (en) | 2017-12-06 | 2017-12-06 | A kind of extract, preparation method and medical usage rich in chromene lactone |
| PCT/CN2018/084057 WO2019109576A1 (en) | 2017-12-05 | 2018-04-23 | Use of benzopyran compound |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711275329.9A CN107987089B (en) | 2017-12-06 | 2017-12-06 | A kind of extract, preparation method and medical usage rich in chromene lactone |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107987089A true CN107987089A (en) | 2018-05-04 |
| CN107987089B CN107987089B (en) | 2018-09-04 |
Family
ID=62036401
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711275329.9A Active CN107987089B (en) | 2017-12-05 | 2017-12-06 | A kind of extract, preparation method and medical usage rich in chromene lactone |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107987089B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109700843A (en) * | 2019-02-01 | 2019-05-03 | 云南省药物研究所 | A kind of net vein Farfugium kaemferi extract and its application in prevention and treatment stomach trouble |
| WO2019109576A1 (en) * | 2017-12-05 | 2019-06-13 | 赵腾骅 | Use of benzopyran compound |
| CN111072619A (en) * | 2019-12-11 | 2020-04-28 | 云南中烟工业有限责任公司 | Compound with drug-resistant bacterium resisting effect and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107890466A (en) * | 2017-12-05 | 2018-04-10 | 赵腾骅 | A kind of chromene lactone is used for the purposes that for aristolochic acid and the Chinese medicine containing aristolochic acid is attenuated |
-
2017
- 2017-12-06 CN CN201711275329.9A patent/CN107987089B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107890466A (en) * | 2017-12-05 | 2018-04-10 | 赵腾骅 | A kind of chromene lactone is used for the purposes that for aristolochic acid and the Chinese medicine containing aristolochic acid is attenuated |
Non-Patent Citations (3)
| Title |
|---|
| 赵树年，等: "岩天麻化学成分的研究(Ⅰ)", 《中草药》 * |
| 陈于澍，等: "岩天麻化学成分的研究（Ⅱ）", 《中草药》 * |
| 陈于澍，等: "岩天麻化学成分的研究（Ⅱ）", 《云南大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019109576A1 (en) * | 2017-12-05 | 2019-06-13 | 赵腾骅 | Use of benzopyran compound |
| CN109700843A (en) * | 2019-02-01 | 2019-05-03 | 云南省药物研究所 | A kind of net vein Farfugium kaemferi extract and its application in prevention and treatment stomach trouble |
| CN111072619A (en) * | 2019-12-11 | 2020-04-28 | 云南中烟工业有限责任公司 | Compound with drug-resistant bacterium resisting effect and preparation method and application thereof |
| CN111072619B (en) * | 2019-12-11 | 2022-11-15 | 云南中烟工业有限责任公司 | Compound with drug-resistant bacterium resisting effect and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107987089B (en) | 2018-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10086032B2 (en) | Method for preparing a Camellia nitidissima Chi lipid-lowering and hypoglycemic agent | |
| CN107987089B (en) | A kind of extract, preparation method and medical usage rich in chromene lactone | |
| CN101791336B (en) | Extract from long pepper and preparation method and application thereof | |
| JP5622222B2 (en) | Hypolipidemic composition and use thereof | |
| CN102370635B (en) | Application and preparation method of novel nardosinone | |
| CN107890466B (en) | A kind of chromene lactone is used for as aristolochic acid and the purposes of the attenuation of the Chinese medicine containing aristolochic acid | |
| CN105198943B (en) | A kind of entitled tea hill how glycosides A acylated flavonoids glucosides and its preparation method and application | |
| CN102526219B (en) | Extract for Relinqing Granule, as well as preparation method and application of same | |
| CN107951880B (en) | A kind of benzopyrone is used for as aristolochic acid and the purposes of the attenuation of the Chinese medicine containing aristolochic acid | |
| CN102526218B (en) | Method for preparing Polygonum capilalum extract and Polygonum capilalum extract | |
| CN104224952A (en) | Preparation method for total anthraquinones of rheum officinale with stable and uniform proportions of all components | |
| CN105230836B (en) | A kind of health tea that rhinitis is had auxiliary therapeutic action and preparation method and detection method | |
| CN107334793A (en) | The purposes of wintersweet platymiscium helicobacter pylori resistant | |
| CN100464760C (en) | Use of cocklebur fruit total glucoside extract in preparing anti inflammation reaction product | |
| CN1994352A (en) | Use of total glucosides extract of cocklebur fruit in preparation of product for resisting inflammatory reaction | |
| CN103760271B (en) | The detection method of brain heart clearing capsule | |
| CN107028965A (en) | Application and product of the yuenkanin or derivatives thereof in fat-reducing medicament is prepared | |
| CN106109862A (en) | A kind of Rhizoma Paridis extract treating viral influenza | |
| CN105213364B (en) | The application of khellactone | |
| CN102188464A (en) | Coumarin and phenylchromone extract from angelica polymorpha maxim and preparation method and use thereof | |
| CN101757009B (en) | Application of notoginseng saponins Rg 1 in preparing medicine for treating pulmonary interstitial fibrosis | |
| CN100574754C (en) | A kind of preparation method of total coumarins extract from peucedanum praeruptorm | |
| CN108014152A (en) | A kind of pharmaceutical composition of diastole uterine smooth muscle and preparation method thereof | |
| CN1927250B (en) | Pharmaceutical composition, its preparation method and application | |
| CN107260773B (en) | The purposes of wintersweet platymiscium anti-herpesvirus |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180802 Address after: 210012 21 flower god Avenue, Yuhuatai District, Nanjing, Jiangsu Applicant after: NANJING DARRA PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Address before: 210009 China Medicine University, 639 Jiangning dragon road, Jiangning, Nanjing. Applicant before: Zhao Tenghua |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Extract enriched in benzopyran lactone as well as preparation method and medical application of extract Effective date of registration: 20190805 Granted publication date: 20180904 Pledgee: Bank of Jiangsu Limited by Share Ltd. Taishan Nanjing road subbranch Pledgor: NANJING DARRA PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Registration number: Y2019320000006 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20200615 Granted publication date: 20180904 Pledgee: Bank of Jiangsu Limited by Share Ltd. Taishan Nanjing road subbranch Pledgor: NANJING DARRA PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Registration number: Y2019320000006 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Extract enriched in benzopyran lactone as well as preparation method and medical application of extract Effective date of registration: 20200616 Granted publication date: 20180904 Pledgee: Bank of Jiangsu Limited by Share Ltd. Taishan Nanjing road subbranch Pledgor: NANJING DARRA PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Registration number: Y2020980003127 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210617 Granted publication date: 20180904 Pledgee: Bank of Jiangsu Limited by Share Ltd. Taishan Nanjing road subbranch Pledgor: NANJING DARRA PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Registration number: Y2020980003127 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231017 Address after: Rooms 1503 and 1504, 15th floor, Building F, Life Science Park, No. 22 Tiansheng Road, Jiangbei New District, Nanjing, Jiangsu Province, 210000 Patentee after: Nanjing Dao'er Medical Research Institute Co.,Ltd. Address before: 210012 21 flower god Avenue, Yuhuatai District, Nanjing, Jiangsu Patentee before: NANJING DARRA PHARMACEUTICAL TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right |