CN103483351B - The extracting method of a kind of bilobalide and application - Google Patents
The extracting method of a kind of bilobalide and application Download PDFInfo
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- CN103483351B CN103483351B CN201310471503.2A CN201310471503A CN103483351B CN 103483351 B CN103483351 B CN 103483351B CN 201310471503 A CN201310471503 A CN 201310471503A CN 103483351 B CN103483351 B CN 103483351B
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
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- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The present invention provides the extracting method of a kind of bilobalide, supercritical extraction and microwave extracting is adopted to be prepared from, content is improved a lot, present invention also offers and provide bilobalide to suppress the application in rat glioma C 6 cells hyperproliferation agent and preparation to increase the application in bone density medicine in preparation.
Description
Technical field
The present invention relates to technical field of extraction of Chinese traditional medicine, be specifically related to extracting method and the application of a kind of bilobalide.
Background technology
Ginkgolid belongs to terpenoid, is made up of sesquiterpene lactones and diterpenoid-lactone, is the important active component of Semen Ginkgo Ye Zhongyi class.Bilobalide (hilobalide;BB) belong to sesquiterpene lactones, be the unique sesquiterpene lactones compound found from Folium Ginkgo at present.Ginkalide A (ginkgolideA；GA), ginkalide B (ginkgolideB；GB), ginkalide C (ginkgolideC；GC), Semen Ginkgo lactone M (ginkgolideM；GM), Semen Ginkgo lactone J (ginkgolideJ；GJ) for diterpene-kind compound, its difference be in that containing hydroxy number different with the position that hydroxyl connects, extraction and the purification process of bilobalide have: solvent extraction, post extraction method, solvent extraction-post extraction method, super critical extraction and chromatograph or column chromatography purification method etc..
In prior art, not yet there is bilobalide to adopt the use in conjunction report of supercritical and microwave technology in extracting preparation, and adopt said method, technique is coarse, backward, and impurity is many, causes that patient's consumption is excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
Summary of the invention
Goal of the invention: in order to solve the problems referred to above, it is an object of the invention to provide the extracting method of a kind of bilobalide.
Further object is that and provide bilobalide to suppress the application in rat glioma C 6 cells hyperproliferation agent and preparation to increase the application in bone density medicine in preparation.
Technical scheme: it is an object of the invention to by following scheme realization:
The extracting method of a kind of bilobalide, takes Folium Ginkgo, joins CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO2Flow 1-3ml/g crude drug min, extraction time 150-180min, obtain supercritical extract, add 70% ethanol, put into and microwave extracting apparatus carries out microwave extracting, extract power 400-600W, extract 2 times, each 4-8 minute, 2 times that amount is medical material weight of 70% ethanol every time added, obtain extract, concentration, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain bilobalide extract.
The extracting method of above-mentioned bilobalide, described CO2It is 5% that supercritical extraction entrainer accounts for the percent by volume of total extractant.
The extracting method of above-mentioned bilobalide, described CO2The extracting pressure 20MPa of supercritical extraction, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 160min.
The extracting method of above-mentioned bilobalide, described microwave extracting power 500W, extract 6 minutes every time.
Above-mentioned bilobalide suppresses the application in rat glioma C 6 cells hyperproliferation agent in preparation, and the extraction step of bilobalide is: takes Folium Ginkgo, joins CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO2Flow 1-3ml/g crude drug min, extraction time 150-180min, obtain supercritical extract, add 70% ethanol, put into and microwave extracting apparatus carries out microwave extracting, extract power 400-600W, extract 2 times, each 4-8 minute, 2 times that amount is medical material weight of 70% ethanol every time added, obtain extract, concentration, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain bilobalide extract.
Above-mentioned bilobalide suppresses the application in rat glioma C 6 cells hyperproliferation agent, CO described in the extracting method of bilobalide in preparation2It is 5% that supercritical extraction entrainer accounts for the percent by volume of total extractant, described CO2The extracting pressure 20MPa of supercritical extraction, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 160min, described microwave extracting power 500W, extract 6 minutes every time.
Above-mentioned bilobalide increases the application in bone density medicine in preparation, and the extraction step of bilobalide is: takes Folium Ginkgo, joins CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO2Flow 1-3ml/g crude drug min, extraction time 150-180min, obtain supercritical extract, add 70% ethanol, put into and microwave extracting apparatus carries out microwave extracting, extract power 400-600W, extract 2 times, each 4-8 minute, 2 times that amount is medical material weight of 70% ethanol every time added, obtain extract, concentration, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain bilobalide extract.
Above-mentioned bilobalide increases the application in bone density medicine, CO described in the extracting method of bilobalide in preparation2It is 5% that supercritical extraction entrainer accounts for the percent by volume of total extractant, described CO2The extracting pressure 20MPa of supercritical extraction, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 160min, described microwave extracting power 500W, extract 6 minutes every time.
In prior art, bilobalide adopts the method for alcohol extraction, technique is coarse, backward, and impurity is many, causes that patient's consumption is excessive, be inconvenient to take, bilobalide yield prepared by the present invention increases, and content is high, and purity reaches 97-98%, prior art does not find that it suppresses the application in rat glioma C 6 cells hyperproliferation agent and preparation to increase the application in bone density medicine in preparation, it was shown that this product is hopeful to make such medicine.
Detailed description of the invention
Form by the following examples, the foregoing of the present invention is described in further detail again, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below, and all technology realized based on foregoing of the present invention belong to the scope of the present invention.
Embodiment 1
Take Folium Ginkgo 1000g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 4% that entrainer accounts for the percent by volume of total extractant, extracting pressure 15MPa, temperature 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 150min, obtain supercritical extract, add 70% ethanol, put into and microwave extracting apparatus carries out microwave extracting, extract power 400W, extract 2 times, each 4 minutes, 70% amount of alcohol every time added was 2000g, obtains extract, concentration, it is added on D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain bilobalide extract, be 97.8% through the content of high effective liquid chromatography for measuring bilobalide a, b, c.
Embodiment 2
Take Folium Ginkgo 1000g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 6% that entrainer accounts for the percent by volume of total extractant, extracting pressure 30MPa, temperature 50 C, CO2Flow 3ml/g crude drug min, extraction time 180min, obtain supercritical extract, add 70% ethanol, put into and microwave extracting apparatus carries out microwave extracting, extract power 600W, extract 2 times, each 8 minutes, 70% amount of alcohol every time added was 2000g, obtains extract, concentration, it is added on D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain bilobalide extract, be 98.1% through the content of high effective liquid chromatography for measuring bilobalide a, b, c.
Embodiment 3
Take Folium Ginkgo 1000g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 20MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 160min, obtain supercritical extract, add 70% ethanol, put into and microwave extracting apparatus carries out microwave extracting, extract power 500W, extract 2 times, each 6 minutes, 70% amount of alcohol every time added was 2000g, obtains extract, concentration, it is added on D101 macroporous adsorptive resins, 50% ethanol elution, collect 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain bilobalide extract, be 98.3% through the content of high effective liquid chromatography for measuring bilobalide a, b, c.
In above-described embodiment, Folium Ginkgo is the dried leaves of Ginkgoaceae Semen Ginkgo (maidenhair tree, Gong Sunshu) GinkgobilobaL..
Embodiment 4: bilobalide suppresses the experimentation data of rat glioma C 6 cells propagation
1 experiment material
1.1 experiment cell strains
Rat glioma (C6), Nanjing Zhengliang Pharmaceutical Technology Co., Ltd.'s laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: bilobalide of the present invention: prepare by embodiment 3 method.
Medicinal liquid liquid storage: weigh 100mg bilobalide, is dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.
1.3 experiment reagents
DMEM(GIBCO company Cat.No.12100-061Lot.No.758137)；Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419)；NaHCO3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration)；Trypsin(AMRESCO company lot number: 2010/04)；EDTA(AMRESCO company lot number: 2009/10)；PenicillinGSodiumSalt(AMRESCO company lot number: 2010242)；StreptomycinSulfate(AMRESCO company lot number: 2010382)；Dehydrated alcohol (Nanjing Chemistry Reagent Co., Ltd.'s lot number: 080310182)；MTT (Biosharp lot number: 0793);PBS(laboratory autogamy)；1.4 experiment equipments
Lycra inverted microscope (Germany Leica model: DM1L)；Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190)；CO2 incubator (FORMA model: 3111)；Super-clean bench (safe and sound company of Su Jing group manufactures model: SW-CJ-ZFD)；Pure water instrument (Spring company of U.S. model: S/N020579)；Accurate pipettor (Gilson Inc of France model: P2)；Electronic balance (Sai Duolisi company limited of Germany model: BT323S)；Full-automatic high-pressure autoclave (SANYO company of Japan model: MLS-3020)；Table electrothermal air dry oven (Shanghai precision experimental facilities company model: DHG9123A)；Refrigerator (Siemens Company's model: KG18V21TI)；Liquid nitrogen container (CBS model: 2001)；Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model: KA-1000)；0.2 μm of filter (MILLIPORE model: SLGP033RB)；10cm culture dish (NEST company), 96 well culture plates (NEST company)；Cell counting count board；Centrifuge tube, pipet, Tips are some.
2 experimental techniques
1) C6 cell DMEM+10%FBS in 37 DEG C, 5%CO2 carry out cellar culture (10cm culture dish), when Growth of Cells to logarithmic (log) phase, collect cell, discard culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, it is added thereto in 5ml complete medium and reaction, is proceeded in centrifuge tube after piping and druming cell, 1000rpm is centrifuged 5min, adjusts 3 × 104/ml of concentration of cell suspension.
2) entering in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, and culture plate puts into (37 DEG C, 5%CO2) cellar culture in cell culture incubator.
3) according to cell growth status, general long to 50%-70%, add to obtain raw cook solution, continue to cultivate 24h.
4) add 20 μ lMTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, makes crystal fully dissolve.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) arranging background (being not added with cell, only add culture fluid), control wells (cell, the medicine dissolution medium of same concentrations, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes simultaneously.
7) suppression ratio of cell is represented by result with medicine:
Cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value)/control wells OD value × 100%.Experiment repeats 3 times.
3 statistical dispositions
Adopting the correlation analysis in MicrosoftExcel2003 software and Studentt inspection, data represent with mean ± S.D..
4 experimental results
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to C6 cell inhibitory effect variant (P < 0.05), dosage this difference when 10mg/ml has significance (P < 0.01), has pole significant difference (P < 0.001) when dosage reaches 15-20mg/ml.
Table 1 bilobalide is to C6 cell inhibitory effect influence research
Note: compare with matched group, * P < 0.05；* P < 0.01
5 experiment conclusion
Bilobalide can suppress C6 cell proliferation, reduces the Growth of Cells number of C6 cell, and this effect is dose dependent.
Embodiment 5: the pharmacodynamic study on the impact of the bone density of laboratory animal
1 sample preparation
Bilobalide of the present invention: prepare by embodiment 3 method.Precious strong SHENGU JIAONANG, Shandong can elixir industry Group Co., Ltd, lot number: 20120711.Tretinoin sheet, Shandong Liang Fu pharmaceutical Co. Ltd, lot number: 130120
2 animals
Wistar rat, male, body weight 180-240g, Shanghai Slac Experimental Animal Co., Ltd. provides.
The foundation of 3 experiment packets and model rat with osteoporosis
Take normal Wistar rats 30 and be only randomly divided into 5 groups, take wherein 6 for NS group；Take and wherein 6 be only used as model group, press tretinoin 70mg/kg gavage two weeks merely；Take and wherein 6 be only used as precious strong SHENGU JIAONANG matched group；Remaining 12 rats are as high low dose group of the present invention.Gavage corresponding medicine while gavaging tretinoin as ZHENMUSHENGU JIAONANG matched group and high and low dose group of the present invention by the dosage of 70mg/kg, withdraw tretinoin after gavaging two weeks and continue two weeks mensuration indices.Rat sacrificed by decapitation, takes its femur by organizing arrangement, uses X-line mammary gland video camera production mating plate, on the greater trochanter of femur, femoral head, neck of femur, intertrochanteric line and under intertrochanteric line with black and white densimeter pinpoints mensuration and record bone density value in 5 positions.
The present invention impact on rat bone density
Note: compare with model group, * P < 0.05；* P < 0.01
4 experimental results
The present invention compares with model group, has significant difference, illustrates that the present invention can improve bone density, treatment fracture, and effect is better than precious strong SHENGU JIAONANG, can be used for preparation and increases the application in bone density medicine.
Claims (2)
1. bilobalide extract increases the application in bone density medicine in preparation, it is characterised in that the extraction step of bilobalide extract is: takes Folium Ginkgo, joins CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO2Flow 1-3m1/g crude drug min, extraction time 150-180min, obtain supercritical extract, add 70% ethanol, put into and microwave extracting apparatus carries out microwave extracting, extract power 400-600W, extract 2 times, each 4-8 minute, 2 times that amount is medical material weight of 70% ethanol every time added, obtain extract, concentration, be added on D101 macroporous adsorptive resins, 50% ethanol elution, collects 5 times amount column volume eluents, decompression recycling ethanol, concentrate and dry, obtain bilobalide extract.
2. bilobalide extract increases the application in bone density medicine in preparation according to claim 1, it is characterised in that CO described in the extracting method of bilobalide extract2It is 5% that supercritical extraction entrainer accounts for the percent by volume of total extractant, described CO2The extracting pressure 20MPa of supercritical extraction, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 160min, described microwave extracting power 500W, extract 6 minutes every time.
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Non-Patent Citations (3)
| Title |
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| 潘炘等.超临界CO_2萃取银杏叶中银杏内酯工艺的研究.《食品工业科技》.2010,第31卷(第12期),第250-252页. * |
| 舒血宁注射液预防老年骨折患者术后谵妄的疗效;吴坚等;《上海医学》;20101231;第33卷(第5期);第472-473页 * |
| 银杏叶中萜内酯类化合物的研究进展;刘彬果等;《药学实践杂志》;20111125;第29卷(第6期);第421-425页 * |
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