CN1724529A - High purity cnidicin and preparation method thereof and be the pharmaceutical composition of activeconstituents with this compound - Google Patents
High purity cnidicin and preparation method thereof and be the pharmaceutical composition of activeconstituents with this compound Download PDFInfo
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- CN1724529A CN1724529A CN 200510014590 CN200510014590A CN1724529A CN 1724529 A CN1724529 A CN 1724529A CN 200510014590 CN200510014590 CN 200510014590 CN 200510014590 A CN200510014590 A CN 200510014590A CN 1724529 A CN1724529 A CN 1724529A
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Abstract
The invention discloses high purity cnidicin spectroscopic analysis structural identification, preparation technology's method, stability study, studies on acute toxicity, contain the pharmaceutical composition of osthole and the new pharmaceutical use of this natural product.With the Chinese medicine Fructus Cnidii through extraction using alcohol, macroporous adsorbent resin decolouring enrichment, and processing step such as resin separation purification handles, and obtains purity at the osthole more than 98%.Studies show that of osthole pharmacologically active of the present invention, it has tangible anti-liver cancer, anti-spontaneous lymphoma, tumour radiation sensitivity and leukopenia is had pharmacologically actives such as leukogenic effect.Osthole is expected to develop that to become low toxicity efficient and have the treatment and prevention of tumour new drug and a tumor aid treatment medication of the natural traditional Chinese medicine composition of multiple pharmacological effect.
Description
Technical field:
The extraction and separation technology of high purity cnidium monnieri hand element of the present invention belongs to the technology of preparing of natural drug monomer component in the field of medicaments.The extraction separation that particularly relates to high purity cnidicin, and the application of osthole in medicines such as the anti-curing oncoma of preparation, tumour radiation sensitivity, leukocyte increasing.
Background technology:
The macroporous adsorbent resin method is a novel technique that grows up nineteen seventies, because it has fast, efficient, convenient, sensitive, good selective, in recent years in extraction separation, the quality control of Chinese medicine preparation and the widespread use of analysis aspect of middle pharmaceutically active ingredient.Macroporous adsorption resin technology can shorten the production cycle, and required equipment is simple, has removed many operations consuming time such as staticly settling, concentrate from; The refining back of medicine volume-diminished, cost reduces, and has created condition for Chinese medicine enters the world market simultaneously; Macroporous adsorption resin technology, operating procedure is easy, and regeneration easily, can repeatedly use, and solvent can recycle and reuse.
As everyone knows, the hysteresis of traditional Chinese medicine extraction and process for refining causes Chinese medicine thick (impurity is many), big (dose is big), black (color is dark), is to restrict the industrialization of Chinese medicine development and expand one of principal element of world market.Therefore explore the sharp separation osthole with macroporous adsorbent resin, be fit to industrialization production.The high purity cnidicin extraction and separation process that the present invention developed has solved this technical barrier, can obtain purity quickly and easily greater than 98% osthole, and this technology can realize industrialization, for the Application and Development of this natural medicinal ingredients has been set up new preparation technology's method.
Osthole (Osthol) has another name called Osthole, and its chemical name is 7-methoxyl group-8-isopentene group tonka bean camphor, is a kind of coumarin compound that at first extraction separation obtains from samphire.Its structural formula is:
Content is higher in its dry mature fruit Fructus Cnidii at the samphire cnidium monnieri (Fructus Cnidii), and average content is about 2%.Osthole is a main active ingredient contained in the Chinese medicine Fructus Cnidii, has pharmacologically active widely, as effects such as osteoporosis, antiviral, anti-mutation, anticoagulant, raise immunity, treatment hepatitis and kidney invigorating and YANG supporting, thereby be subjected to people's common concern.By domestic and international patent and literature search are found, at home and abroad there is no a kind of stable commercial processes method that obtains high purity cnidicin at present, cause osthole to use, influenced normal performance, stability and the poor reproducibility of its curative effect with the medicament forms of total coumarins.The osthole purity of using supercritical extraction technique to obtain generally is no more than 87% at present.Can't make osthole as the one-component medicine in clinical use.At present, still none country researchs and develops it as medicine, introduces to the market.
Summary of the invention:
In view of the foregoing, the inventor has carried out a large amount of experimental studies to the processing method of extraction separation osthole from the Chinese medicine Fructus Cnidii, determined the macroporous adsorbent resin method, being used for osthole extracts, separate, preparation technology's method of purifying, and finish the research of structure analysis conclusive evidence and accelerated stability test, obtain purity and be stabilized in osthole more than 98%, thereby finished the technology invention of osthole, with the pharmaceutical composition that contains osthole, and said composition is at the anti-curing oncoma of preparation, application in tumour radiation sensitivity and the leukocyte increasing medicine.
One object of the present invention is, discloses to adopt the macroporous resin adsorption elution method to obtain purity greater than 98% osthole monomer.
Another object of the present invention is, discloses osthole in the application that is used to prepare aspect antitumor, tumour radiation sensitivity, the leukocyte increasing medicine.
Further object of the present invention is, discloses and has contained the osthole for the treatment of significant quantity, reaches the pharmaceutical composition of forming with one or more pharmaceutically acceptable carriers.
The present invention discloses the preparation technology method of purity greater than 98% osthole in further detail.Technical scheme of the present invention is achieved by following content:
A kind of high purity cnidicin, it is the Chinese medicine Fructus Cnidii to be boiled through ethanol carry, macroporous adsorbent resin decolouring enrichment, and by resin separation purification obtains purity greater than 98% osthole in conjunction with recrystallization.Described macroporous adsorbent resin is NKA-9, NKA-12, D4020 or AB-8.
Specifically undertaken by following processing step:
(1) extract: take by weighing a certain amount of Fructus Cnidii medicinal material, boil with 60～95% ethanol and carry 3～4 times, each 1～2 hour, merge ethanol extract, concentrate and reclaim ethanol;
(2) separate: ethanol extraction through macroporous resin column, is flowed out water soluble component-I with before washing, discarding, flow point-II before 30～45% ethanol eccysis part; With 50～90% ethanol elution part-III, for containing the tonka bean camphor composition of osthole more than 60%;
(3) refining:, choose the NKA-9 macroporous adsorbent resin and carry out separation and purification, with hexanaphthene, chloroform: sherwood oil (40: 1～10: 1), ethyl acetate: sherwood oil (40: 1～10: 1) or normal hexane wash-out obtain the osthole crude product; Osthole obtains purity greater than 98% osthole with activated carbon decolorizing with methanol-water, alcohol-water, sherwood oil, normal hexane or hexanaphthene recrystallization.
(4) resin regeneration: regenerate behind the pigment of resin column with the absorption of wash-outs such as ethyl acetate, acetone, methyl alcohol or chloroform, reuse.
The present invention repeatedly repeats extraction, separation and process for refining, has used 4 solvent elution systems.Used 4 kinds of macroporous adsorbent resins of opposed polarity to separate decolouring absorption: NKA-12, NKA-9, D4020 or AB-8; Being used for the purified polymeric adsorbent is NKA-9, has optimized extraction and separation process.Product purity all remains on more than 98%, portioned product purity even can reach 100% (HPLC result).Utilize many batches of products of this explained hereafter, stable, favorable reproducibility.
Its stability is carried out accelerated tests investigate (high temperature, high humidity, strong illumination), found that it to high temperature, high humidity, stable, the nothing decomposition of strong illumination, purity and physical and chemical index are constant.Technical indicator reaches the requirement of a kind new medicine declaration, and technology is fit to suitability for industrialized production.
Osthole accelerated stability test lot number: 0309
Osthole through IR, UV, MS,
1H-NMR, ultimate analysis, fusing point, TLC and character analysis, consistent with the document contrast.
Osthole has following physicochemical property:
Identify that at microscopically product is colourless column crystallization; Under ultraviolet lamp, be blue-fluorescence.
Mp：82-84℃
TLC R
fValue: 0.68 (to sherwood oil: 3: 1 systems of ethyl acetate)
Spectroscopic analysis:
1. infrared spectra:
IR
KBr MaxCm
-1: 2965,2910 (CH
3, CH
2); 1720 (α, the unsaturated delta-lactones of β);
1604,1461 (phenyl ring), 1383 (together with methyl)
2. UV scanning:
Shimadzu UV-3000 ultraviolet-visible spectrophotometer (day island proper Tianjin company).Osthole is dissolved in methyl alcohol, is made into the solution of 2mg/50ml, carry out UV scanning, wavelength is 200nm～800nm.The scanning result sample the last one absorption peak occurs at 220nm, a little acromion occurs at 254nm, becomes strong absorption peak band between 310～330nm.
3. proton nmr spectra:
Methyl 1.64 (3H s); (1.82 3H s); Methoxyl group 3.89 (3H s); Methylene radical 3.50 (2H J=g.3d); Methyne 5.20 (1H J=t); Phenyl ring 6.80,7.26 (2H J=8.7dd); The two keys 6.20,7.58 (2H J=9.4dd) of ester ring
4. ultimate analysis:
Molecular formula: C
15H
16O
3, molecular weight: 244.29;
Calculated value C% 73.75%; H% 6.60%
Measured value C% 73.64%; H% 6.50%
5. mass spectroscopy: 244 (molecular ion peaks); 229 (remove CH
3The peak); 201 (go
The peak); 189 (go
The peak); 175 (go
The peak).
6. gas-chromatography---the product solvent residues is analyzed:
A. GC conditions: chromatographic column is a quartz capillary column, and stationary liquid is SE-54, pillar specification 0.53mm * 30m; Column temperature is at 50 ℃ of sample introduction 1ml; The Sample Room temperature is 200 ℃; Detector is FID; Carrier gas is N
2, flow velocity is 50ml/min.
B. operation: sample headspace sample introduction, 60 ℃ of temperature, 30min.
C. gas chromatograph results: methyl alcohol standard substance: sample size 0.4214g, retention time 1.522min, peak shape BB, peak area 242 μ Vs osthole samples: sample size 0.78mg, retention time 1.546min, peak shape BB, peak area 38 μ Vs methanol content (38/242) * (0.78mg/0.4214g)=0.29mg/g, be 0.029%, residual quantity is low, meets the pharmacy requirement.
Purity testing:
1. high performance liquid chromatography: moving phase is 80% methyl alcohol, and the detection wavelength is 322nm, and C18 filled column HPLC tests many batch samples purity all more than 98%, and purity can reach 100% sometimes.
Pharmaceutical composition:
Of the present invention is the pharmaceutical composition of activeconstituents with the osthole, contains the osthole for the treatment of significant quantity, and one or more pharmaceutically acceptable carrier.Wherein, described osthole treatment significant quantity is 5～300mg; Be preferably 15～200mg; Best 25～150mg.
Described composition comprises oral preparations, injection finish or external preparation, and wherein oral preparations is tablet, various capsule, slow releasing tablet or dripping pill; Liquid preparation is the oil for injection preparation; External preparation is creme, paste, finish or glycerin preparation.
Described pharmaceutically acceptable carrier, comprise thinner, weighting agent (as N.F,USP MANNITOL, lactose, polyoxyethylene glycol) conventional in the preparation, tackiness agent (starch, Microcrystalline Cellulose), disintegrating agent (as carboxymethyl cellulose, the low hydroxypropylcellulose that replaces), lubricant (as talcum powder, Magnesium Stearate), wetting agent (as propylene glycol, ethanol), stablizer (EDTA-2Na, Sulfothiorine, Sodium Pyrosulfite, S-WAT, thanomin, sodium bicarbonate, niacinamide) or the like.Above-mentioned auxiliary material can be a common dose, mixes with osthole with proportioning commonly used, and after the osthole consumption was determined, the proportioning between each pharmaceutical excipient can suitably be regulated as required.
Compressing tablet is made the use of tablet, and the auxiliary material that is used as excipient has MgSO
4, W-Gum, talcum powder, specification has: 50mg, 100mg, 150mg, 200mg.
Osthole finish: osthole is dissolved in injection tea oil, soya-bean oil etc. is made into the injection finish.
The preparation concrete operations of various pharmaceutical preparations are as follows:
(1). take by weighing and contain 10～70% ostholes by the configuration total amount, the weighting agent of adding 30～90%, tackiness agent, disintegrating agent, lubricant, correctives etc., fully mix and make particle, in the compacting in 2～4 hours of 60～70 ℃ of dryings in flakes, make every to contain osthole 40～240mg.
(2). the wet granular made directly 60～70 ℃ of dryings 2～4 hours, is filled in the hungry area softgel shell then, makes every capsules contain osthole 30～300mg.
(3). the preparation of finish: be to get 1%～10% osthole and oil for injection, be mixed with finish as tea oil, the packing sterilization makes.
(4). by the configuration total amount, take by weighing 10%～60% osthole, the weighting agent of adding 40%～90%, oxidation inhibitor etc., heating and melting temperature (40～90 ℃) makes the abundant consolute of raw material and auxiliary material, drips to make ball, makes every dripping pill contain osthole 5～30mg.
Formulation rate of the present invention can be according to all multifactor adjustment such as route of administration, patient age, body weight, disease type and severity, and per daily dose is generally 5～300mg/kg; Be preferably 15～200mg/kg; The best is 25～150mg/kg.Can show suitable change according to clinical case.
The invention also discloses high purity cnidicin in the application that is used to prepare aspect anti-curing oncoma, tumour radiation sensitivity, the leukocyte increasing medicine.Wherein, described anti-tumor activity comprises: the application of preparation treatment liver cancer, lymphoma, cervical cancer medicine aspect.For example osthole is to liver cancer, (spontaneous) lymphoma, (U
14) cervical cancer, right
157The Cs gamma-radiation causes the leukogenic effect of leukopenia.
Following pharmacological evaluation has confirmed that osthole has above-mentioned pharmacological action.
Pharmacodynamic study comprises:
(1) external anticancer experiment
(2) vivo antitumor experiment
(3) synergistic function of ray is tested
(4) leukocyte increasing experiment
(5) acute toxicity test
One, external tumor-inhibiting action:
Experimental technique: the MTT method of inspection is adopted in experiment.
(1) inoculating cell: the cell in vegetative period of taking the logarithm, after 0.02% EDTA digestion, add the RPMI-1640 solution that contains 10～15% foetal calf serums, cell dilution is become single cell suspension, counting is also adjusted cell count and is inoculated in 96 orifice plates.Postvaccinal cell put in 37 ℃ of CO2gas incubator cultivated 24 hours.
(2) dosing: set up sample A, B different concns such as separately 1.55～200ug/ml, every concentration is established 4 multiple holes, and multiple hole more than 8 is established in contrast.Test sample dilutes with RPMI-1640, uses the DMSO hydrotropy.Will put the interior continuation cultivation of 37 ℃ of CO2gas incubator 72 hours by cell after the dosing.
(3) add MTT:MTT and dissolve with RPMI-1640, every hole adds 50ul.Put 37 ℃ again and continue to cultivate taking-up after 4 hours.
(4) survey the OD value: the supernatant liquor in reject 96 orifice plates, every hole adds DMSO 150ul.Measure the OD value at microplate reader 630nm place.
Evaluation index: inhibitory rate of cell growth.Inhibiting rate=(the average OD value of the average OD value/control group of 1-experimental group) * 100%.Calculate IC with the straight-line regression method
50Result: to the cytotoxic effect of hepatoma cell line Be1-7402, IC
50Be 123.92ug/ml; To human lung adenocarcinoma A549, measure its IC
50Be 67.83ug/ml.
Two, tumor-inhibiting action in the body:
Suppress the spontaneous lymphoma experiment
1 material
1.1 animal is selected: IRM-2 mouse inbred lines that I breed voluntarily, 30, body weight 22-25g, male and female dual-purpose.
1.2 knurl kind: from the cultivation of going down to posterity of IRM-2 mouse inbred lines spontaneous lymphoma.
1.3 medicine preparation: get content and be 99.6% osthole, porphyrize and to be mixed with concentration be 0.095mg/ml, 0.14mg/ml, the suspension of 0.21mg/ml, solvent is a distilled water, jolting is even during administration.
2 inoculation methods: select tumor growth vigorous and do not have a diabrosis, the IRM-2 mouse that health condition is good, dislocation is put to death, be fixed on the operation plate, use the tincture of iodine, the alcohol disinfecting animal skin is shelled tumour in super clean bench under the aseptic condition, the incision tumour becomes the fritter about diameter 2mm, in the inguinal region place's subcutaneous vaccination of animal left side.0.3ml/ of inoculation back beginning in the 2nd day oral administration gavage administration, amounting to dosage is 1.11mg/kg, 1.67mg/kg, 2.25mg/kg, successive administration 10 days.
3 therapeutic evaluatioies: 24 hours execution animals are weighed after the drug withdrawal, dissect and peel off the knurl piece, claim knurl heavy.
Tumor control rate: C-T/C * 100%
T is that the average knurl of administration group is heavy in the formula, and C is that the average knurl of control group is heavy
The Fructus Cnidii rope is to the restraining effect of IRM-2 mouse spontaneous lymphoma
The result shows that osthole is 74.2% to lymphadenomatous inhibiting rate, and is suitable with the positive control drug endoxan when 2.50mg/kg, and toxic side effects obviously reduces.
The knurl that presses down to rat liver cancer H22 is tested
1 material
1.1 animal is selected: provided Kunming kind small white mouse by Chinese Academy of Medical Sciences's zooscopy, 72, body weight 21-23g, male and female half and half.
1.2 knurl kind: mouse hepatodynia H22 draws from Tianjin institute of Pharmaceutical Industry pharmacological room, the cultivation of going down to posterity.
1.3 medicine preparation: get content and be 99.6% osthole, porphyrize and to be mixed with concentration be 0.07mg/ml, 0.14mg/ml, the suspension of 0.28mg/ml, solvent is a distilled water, jolting is even during administration.
2 inoculation methods: dissect preceding laboratory with ultraviolet lamp sterilization 3 hours, select 6-7 days the mouse of going down to posterity of ascites growth, dislocation is put to death, use the tincture of iodine, alcohol disinfecting animal skin of abdomen is drawn ascites in super clean bench under the aseptic condition, shake up cancer cells numeration 3.2 * 10 by dilution in 1: 2 with physiological saline
7Individual/ml, at animal right upper extremity armpit place's subcutaneous vaccination 0.2ml/ only, 0.3ml/ of inoculation back beginning in the 2nd day oral administration gavage administration, amounting to dosage is 0.84mg/kg, 1.67mg/kg, 3.30mg/kg, successive administration 10 days.
3 therapeutic evaluatioies: put to death animal in 24 hours after the drug withdrawal, weigh, dissect and peel off the knurl piece, claim knurl heavy; And take by weighing thymic weight, spleen weight and liver weight.
Osthole is to the restraining effect of rat liver cancer H22 solid tumor
Kunming mouse connects the comparison of body weight and organ index and weight after the administration of H22 liver cancer
* with cis-platinum positive control ratio, significant difference is arranged, P＜0.01
Conclusion: the medicine antitumor activity is obvious, obviously reduces than positive control drug cis-platinum toxicity.Synergistic function to ray:
Osthole is to 1 * 5Gy
137Cs gamma-radiation irradiation mouse cervical cancer U14 presses down the knurl synergism
1, material
1.1 animal is selected: provided Kunming kind small white mouse by Chinese Academy of Medical Sciences's zooscopy, male and female half and half.
1.2 knurl strain: mouse cervical cancer U14 draws from Tianjin institute of Pharmaceutical Industry pharmacological room, the cultivation of going down to posterity.
1.3 medicine preparation: get content and be 99.6% osthole, porphyrize and to be mixed with concentration be 0.07mg/ml, 0.14mg/ml, the suspension of 0.28mg/ml, solvent is a distilled water, jolting is even during administration.
2, method:
2.1 inoculation: select 6～7 days the mouse of going down to posterity of ascites growth, dislocation is put to death, and is fixed on the operation plate, uses the tincture of iodine, and the alcohol disinfecting animal skin is drawn ascites in super clean bench under the aseptic condition, shakes up by dilution in 1: 2 with physiological saline, and the cancer cells numeration is 3 * 10
7Individual, at animal left side armpit place's subcutaneous vaccination 0.2ml.Inoculation back beginning in the 2nd day oral administration gavage administration 0.3ml/ only, amount to dosage and be 0.84mg/kg,
1.67mg/kg, 3.30mg/kg, successive administration 10 days.
2.2 irradiation: with 1Gy
137The Cs gamma-radiation began irradiation on the 3rd day after inoculation, shone altogether 5 days for 1 time every day.
2.3 therapeutic evaluation: put to death animal in 24 hours after the drug withdrawal, weigh, dissect and peel off the knurl piece, claim knurl heavy.Get thymus gland, spleen is weighed, and observes thymus index and spleen index; Get a side femur, 5mM calcium chloride solution flushing marrow is measured dna content.
Osthole is right
137Cs gamma-radiation irradiation cervical cancer U14 presses down the knurl synergism
Function of increasing leukocyte:
1. experiment material:
1.1 animal: 40 of ICR mouse, body weight 22～24g, male and female half and half are purchased in Beijing dimension tonneau China laboratory animal company limited.
1.2 experimental technique:
1.2.1 medicine preparation: get content and be 99.6% osthole, porphyrize and to be mixed with concentration be 0.07mg/ml, the suspension of 0.14mg/ml, solvent is a distilled water, jolting is even during administration.
1.2.2 grouping and administration: animal is divided into 4 groups at random, 10/group, is respectively according to group, ethinylestradiol positive controls and osthole (99.8%) 1.67mg/kg and 0.84mg/kg dosage group.Continuous 3 administrations in the 3rd, 2,1 day of irradiation back, successive administration 5 days again after the irradiation.Oral administration gavage, 0.3ml/, control group gives distilled water.
1.2.3 the condition of causing injury:
137The disposable full-body exposure of Cs-gamma-rays, dose rate be 89.1404 human relations/minute, dosage is 4Gy.
1.2.4 observation index:
(1) white blood cell count(WBC): the 9th day eye socket in irradiation back got blood, the white corpuscle diluted, and electron microscope is the counting leukocyte count down.
4Gy Cs
137Gamma-radiation full-body exposure ICR mouse anti is put test-results
Annotate: * P＜0.01; Femur has the nuclear numeration for * 10
9/ L.
(2) dna content: irradiation back was taken off vertebra with the mouse neck on the 9th day and is put to death, and got a side femur, 5mM calcium chloride solution flushing marrow.The perchloric acid reaction solution that adds 0.2N is considered liquid and is measured absorbancy with ultraviolet spectrophotometer.
(3) get thymus gland, spleen, observe thymus index and spleen index.
Conclusion: osthole is to 4Gy Cs
137Gamma-rays full-body exposure ICR mouse anti is put test-results and is shown, osthole lowly has significant leukogenic effect to the white corpuscle that ray causes, than control group rising 32.5% (P＜0.01).
Acute toxicity test:
6 every group of Kunming mouses, male and female half and half, respectively (soup is thickness very) all do not have death when oral administration gavage administration 800mg/kg, 1600mg/kg, 2000mg/kg, and diet, spirit are all normal.Effectively to press down cancer dosage be 1.00～2.50mg/kg and it is best, and medicine is very safe.
High purity cnidicin of the present invention and extracting method thereof are compared with prior art has following advantage:
(1) utilizes Fructus Cnidii extraction separation osthole, have the content height, the characteristics that extract yield is high.Yield is between 1～2%, and drug resource is abundant, cheap and easy to get.
(2) it is simple to adopt macroporous adsorbent resin method extraction separation osthole to have technology, separates and can accomplish scale production fast, and process stabilizing can repeat; Extracts active ingredients is complete, avoids the waste of drug resource; Resin can be regenerated, and solvent can recycle and reuse and the characteristics that reduce cost.
(3) to have effective constituent definite for osthole of the present invention, purity height, good stability, the characteristics of efficacy stability.
(4) because effective constituent is definite, the purity height, impurity is few, and low solvent residue, medicine have the characteristics of system stable and controllable for quality.
Description of drawings:
Fig. 1 is the high performance liquid phase collection of illustrative plates of 0309 batch of osthole, and content is 99.8%.
Fig. 2 is the high performance liquid phase collection of illustrative plates of 0403 batch of osthole, and content is 99.6%.
Fig. 3 is the process flow sheet of osthole extraction separation.
Embodiment:
In order to explain enforcement of the present invention more fully, provide following preparation embodiment.These embodiments only are to explain rather than limit the scope of the invention.Preparation can adopt any a collection of compound activity composition among the present invention.The present invention is described further below by embodiment.
Embodiment 1:
(1) 100g Fructus Cnidii medicinal material adds 80% alcohol reflux 3 times of 500～800ml, refluxes 1 hour at every turn.Merge ethanol liquid rotary evaporation, reclaim ethanol in 65 ℃, the muddy liquid 220ml of residual water goes up macroporous adsorbent resin and lives, effective column length 43cm, column diameter 6cm (NKA-12), last sample and with 1500ml water elution (water liquid I discards).Then with 40% ethanol 1500ml wash-out, obtain brown eluate, the HPLC demonstration does not contain osthole (obtaining II).Obtain the tonka bean camphor composition (obtaining III) of osthole based on 75% ethanol elution.Regenerate behind the pigment of resin column with the absorption of acetone wash-out.The partially recycled solvent of III obtains the light yellow solid material after 60 ℃ of oven dried, HPLC detects osthole content more than 60%, separate with macroporous resin column, choose NKA-9 macroporous resin 120g, effective column length 60cm, diameter 4.5cm is with the normal hexane wash-out, obtain osthole, treat that TLC monitoring osthole goes out to the greatest extent.Change with the acetone wash-out, remove other compositions of absorption, resin is regenerated.Reclaim solvent, get osthole 1.7 grams, yield is 1.7%.Through activated carbon decolorizing, alcohol-water recrystallization, HPLC detect osthole content at 99.8% (lot number 0309 is seen accompanying drawing 1).
(2) 200g Fructus Cnidii medicinal material adds 85% alcohol reflux 3 times of 1000～1500ml, refluxes 2 hours at every turn.Merge ethanol liquid rotary evaporation, 65 ℃ are reclaimed ethanol, and the muddy liquid 300ml of residual water goes up macroporous adsorbent resin and lives, effective column length 60cm, column diameter 7.5cm (AB-8), last sample and with 1500ml water elution (water liquid I discards).Then with 35% ethanol 2500ml wash-out, obtain brown eluate, the HPLC demonstration does not contain osthole (obtaining II).Obtain the tonka bean camphor composition (III) of osthole based on 70% ethanol elution.Regenerate behind the pigment of resin column with eluent ethyl acetate absorption.Behind the partially recycled solvent of III, with organic solvent ethyl acetate extraction enrichment osthole, HPLC detects osthole content more than 65%, separate with macroporous resin, choose NKA-9 macroporous resin 200g, effective column length 75cm, diameter 6.5cm, with hexanaphthene, obtain osthole, treat that TLC monitoring osthole goes out to the greatest extent.Change with the acetone wash-out, remove other compositions of absorption, resin regeneration.Reclaim solvent, get osthole 3.9 grams, yield is 1.95%.Through activated carbon decolorizing, methanol-water recrystallization, HPLC detect osthole content at 99.6% (lot number 0403 is seen accompanying drawing 2).
Embodiment 2:
Get osthole 50.0g, medical starch 50.0g, 50% ethanol is an amount of, granulates, whole grain, oven dry, dress 2# capsule, every contains osthole 0.1g.
Embodiment 3:
Get osthole 50.0g, medical starch 30.0g, dextrin 30.0g, 50% ethanol is an amount of, above-mentioned raw materials is fully mixed make particle, 60～70 ℃ of dryings 2～4 hours, makes 1000, and every contains osthole 0.05g.
Embodiment 4:
Get osthole 40g, polyoxyethylene glycol-4000 20g, polyoxyethylene glycol-6000 60g, Sodium Pyrosulfite is an amount of, and heating and melting temperature (40～90 ℃) makes raw material and auxiliary material fully miscible, drip and make 20000 dripping pills, make every dripping pill contain osthole 1.0～3.0mg.
Embodiment 5:
Get osthole 25g under the aseptic condition, join among the injection tea oil 5000ml, stir about dissolving in 30 minutes, embedding, sterilization promptly get and inject finish.
After the preferred embodiment that describes in detail, being familiar with this technology personage can be well understood to, can carry out various variations and modification not breaking away under above-mentioned claim and the spirit, all foundations technical spirit of the present invention all belongs to the scope of technical solution of the present invention to any simple modification, equivalent variations and modification that above embodiment did.And the embodiment that the present invention also is not subject in the specification sheets to be given an actual example.
Claims (9)
1, a kind of high purity cnidicin is characterized in that, the Chinese medicine Fructus Cnidii is through 60～95% extraction using alcohols, and macroporous adsorbent resin decolouring, enrichment, and macroporous adsorbent resin separation and purification obtain purity greater than 98% osthole.
2, the preparation method of the described osthole of claim 1 is characterized in that being undertaken by following processing step:
(1) extract: take by weighing a certain amount of Fructus Cnidii medicinal material, boil with 60～95% ethanol and carry 3～4 times, each 1～2 hour, merge ethanol extract, concentrate and reclaim ethanol;
(2) separate: ethanol extraction by adsorption resin column, is discarded preceding flow point with water elution, obtain brown effluent, obtain osthole content at the tonka bean camphor more than 60% with 50～90% ethanol elutions again with 30～45% ethanol elutions;
(3) refining: choose NKA-9 macroporous adsorbent resin dress post, with hexanaphthene, chloroform: sherwood oil 40: 1～10: 1, ethyl acetate: sherwood oil 40: 1～10: 1 or normal hexane wash-out carry out separation and purification, obtain the osthole effluent; With activated carbon decolorizing,, obtain purity greater than 98% osthole with methanol-water, alcohol-water, sherwood oil, normal hexane or hexanaphthene recrystallization;
(4) resin regeneration: adsorption resin column is regenerated after with ethyl acetate, acetone, methyl alcohol or chloroform wash-out, reuses.
3, preparation method as claimed in claim 2, wherein, separating the used polymeric adsorbent of decolouring is NKA-9, NKA, AB-8 or D4020; Refining used polymeric adsorbent is NKA-9.
4, with the osthole be the pharmaceutical composition of activeconstituents, said composition contains the osthole for the treatment of effective dose, and one or more pharmaceutically acceptable carrier.
5, pharmaceutical composition as claimed in claim 4, wherein, said composition can be the form of oral preparations, injection or external preparation.
6, pharmaceutical composition as claimed in claim 5, wherein, oral preparations is tablet, various capsule, slow releasing tablet or pill; Injection is the liquid oils preparation; External preparation is creme, paste or finish.
7, the described high purity cnidicin of claim 1 is in the application that is used to prepare aspect anti-curing oncoma, tumour radiation sensitivity, the leukocyte increasing medicine.
8, the described high purity cnidicin of claim 1 is being used for preparing the application that prevents and treats liver-cancer medicine.
9, the described high purity cnidicin of claim 1 is being used for preparing the application that prevents and treats lymphoid tumor medicament.
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| CN102641314A (en) * | 2011-02-22 | 2012-08-22 | 广州大光制药有限公司 | Medicine composition for preventing and/or treating gynecological diseases |
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| CN108976193A (en) * | 2018-09-19 | 2018-12-11 | 南方医科大学 | A kind of Osthole extracting method |
| CN108976193B (en) * | 2018-09-19 | 2023-04-11 | 南方医科大学 | Method for extracting osthole |
| CN114890974A (en) * | 2022-06-09 | 2022-08-12 | 湖北医药学院 | Method for extracting osthole by response surface design method |
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Assignee: T.J.Biotechnologies (Tianjin) Co., Ltd. Assignor: Radiology Research Institute ,Chinese Academy of Medical Sciences Contract fulfillment period: 2009.1.6 to 2017.1.6 contract change Contract record no.: 2009120000210 Denomination of invention: High purity cnidicin and its preparation method and medicinal composition using said compound as active component Granted publication date: 20080723 License type: Exclusive license Record date: 2009.9.14 |
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