CN101347474B - Effective component of Chinese angelica and preparation and use thereof - Google Patents
Effective component of Chinese angelica and preparation and use thereof Download PDFInfo
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- CN101347474B CN101347474B CN2007101232716A CN200710123271A CN101347474B CN 101347474 B CN101347474 B CN 101347474B CN 2007101232716 A CN2007101232716 A CN 2007101232716A CN 200710123271 A CN200710123271 A CN 200710123271A CN 101347474 B CN101347474 B CN 101347474B
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- cell
- radix angelicae
- angelicae sinensis
- active component
- mobile phase
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Images
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to an effective component of angelica and the preparation method and usage. For the effective component of angelica of the present invention, the preparation process includes the following steps: step 1: the mixture of ethyl acetate and ethanol is used as a solvent to extract angelica; step 2: dregs are extracted by ethanol so as to obtain the extract; step 3: the extract goes through column chromatography to obtain the eluent; step 4: the mobile phase of the eluent obtained from the preparation of liquid chromatogram gradient elution is water and acetonitrile, the eluent is collected for 41.8 to 46.9 min so as to obtain the effective component.
Description
Technical field
The present invention relates to a kind of Chinese medicine extract for the treatment of tumor disease, relate in particular to the active component that from Radix Angelicae Sinensis, extracts, preparation and preparation method thereof and purposes.
Background technology
Tumor is a kind of commonly encountered diseases, frequently-occurring disease, and wherein malignant tumor is the most serious class disease of present harm humans health.At present in the industry to the treatment of malignant tumor mainly still based on operation, radiotherapy, chemotherapy, but many chemical anticarcinogenic drugs often involve normal cell when acting on target cell, cause serious side reaction.The genetoxic of plant amedica is not obvious, and Chinese herbal medicine is having special advantages and wide application prospect aspect the anticancer mutation, and Chinese medicine also plays the effect that can not be ignored in to the auxiliary treatment of tumor.Paclitaxel promptly is the good anticancer active native compound that has that typically obtains from plant, now has been developed as antitumor drug.The most serious tumor of China's hazardness is pulmonary carcinoma, nasopharyngeal carcinoma, the esophageal carcinoma, gastric cancer, colorectal cancer, hepatocarcinoma, breast carcinoma, cervical cancer, leukemia and lymphoma etc. at present.Particularly the incidence rate of hepatocarcinoma increases in recent years to some extent.Significant, the etiology of these tumors, pathogenesis and control thereof are the emphasis of China's tumor research.Cancer therapy drug and the anticancer ancillary drug of seeking high-efficiency low-toxicity are the important contents of current tumor research.
China's medicinal organism resource is very abundant, and its biological active substances is research and finds new drug guide chemicals, the natural treasure-house of developing new drug.At present, China extracts active substance from natural product, be used to be developed to treatment tumor disease, safety is good, toxicity is low new drug also seldom, extracts active substance from natural product, be developed to new drug, have significant application value and wide development prospect with antitumor curative effect.
Radix Angelicae Sinensis another name Radix Angelicae Sinensis, cloud are returned, XIGUI, Radix Angelicae Sinensis, oily Radix Angelicae Sinensis, white Radix Angelicae Sinensis, driedly return, Min returns.Its property is sweet, hot, bitter, warm, returns liver, the heart, spleen, lung meridian, has tonifying blood and regulating menstruation, promoting blood circulation and stopping pain, promoting pus discharge and tissue regeneration strengthening, effects such as relieving cough and asthma.Radix Angelicae Sinensis is used for the treatment of multiple disease clinically to be treated, pain due to blood stasis, arthralgia pain numbness, apoplexy and hemiplegia, pyogenic skin infection and carbuncle-abscess and skin sufferings, cough with asthma, severe palpitation, insomnia forgetfulness, irregularly intermittent and regularly intermittent pulse, jaundice etc. after, Light-headedness pale as: blood deficiency shallow complexion, lip and nail, menoxenia, infrequent menstruation amenorrhea, dysmenorrhea, menorrhagia, metrostaxis and gastrointestinal hemorrhage, traumatic injury, swelling and pain due to blood stasis, headache, the operation.
Summary of the invention:
The object of the present invention is to provide the active component of Radix Angelicae Sinensis.
Another object of the present invention is to provide the preparation method of above-mentioned Radix Angelicae Sinensis active component.
The present invention also provides the preparation that contains above-mentioned Radix Angelicae Sinensis active component and the purposes of this component.
Radix Angelicae Sinensis active component of the present invention, its preparation process may further comprise the steps:
Step 1: Radix Angelicae Sinensis is extracted as solvent with ethyl acetate and alcohol mixture,
Step 2: the medicinal residues ethanol extraction, obtain extracting solution,
Step 3: extracting solution gets eluent through column chromatography.
Step 4: with the eluent that the preparative liquid chromatography gradient elution obtains, mobile phase is water and acetonitrile, collects 41.8-46.9 minute eluent and obtains active component (or being called B08).
Wherein ethyl acetate described in the step 1 and alcoholic acid mixture, both ratios are ethyl acetate: ethanol=1-5: 1-5, are preferably ethyl acetate: ethanol=1-2: 1-2 most preferably is ethyl acetate: ethanol=1: 1.
In the described step, step 1 is specially: getting the Radix Angelicae Sinensis medical material, is solvent with ethyl acetate: ethanol=1-5: 1-5, and reflux, extract, is separated extracting solution with medicinal residues,
Step 2 is specially: medicinal residues obtain extracting solution with the ethanol extraction of 50-90%,
Step 3 is specially: described is with sample on 5% dissolve with methanol through column chromatography, crosses the ODS-C18 post, at first, adopts 5% methanol as mobile phase, changes 60% methanol then as mobile phase, gets eluent,
Step 4 is specially: continue to separate the eluent that obtains with preparative liquid chromatography, mobile phase is water-A and acetonitrile-B, carries out gradient elution, and described gradient elution program is as follows:
Flow velocity is 10ml/min, and column temperature is a room temperature; Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 41.8-46.9 minute, and solution obtains active component behind concentrate drying.
The preferred Radix Angelicae Sinensis active component of the present invention preparation method, comprise the following steps: the Radix Angelicae Sinensis pulverizing, add 6-10 and doubly measure ethyl acetate: ethanol=1: 1, reflux 1-2 hour, extract 1-3 time, medicinal residues add 6-10 and doubly measure 50-90% ethanol, reflux, filtrate merge extracting solution, after extracting solution concentrated, with sample on 5% dissolve with methanol, mistake ODS-C18 post, at first, adopt 5% methanol as mobile phase, change 60% methanol then, get eluent as mobile phase, continue separation eluent with preparative liquid chromatography, separation condition is: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water A and acetonitrile B, the gradient elution program is as follows:
Flow velocity is 10ml/min, and column temperature is a room temperature; Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 41.8-46.9 minute, and solution obtains active component behind concentrate drying.
The most preferred Radix Angelicae Sinensis active component of the present invention preparation method comprises the following steps:
Radix Angelicae Sinensis is pulverized, add 8 times of amount ethyl acetate: ethanol=1: 1, reflux 1 hour, extract 2 times, medicinal residues add 8 times of amount 70% ethanol, and reflux 1 hour is extracted 2 times, filtrate merge extracting solution, after extracting solution concentrated, with sample on 5% dissolve with methanol, mistake ODS-C18 post, at first, adopt 5% methanol as mobile phase, change 60% methanol then, get eluent as mobile phase, continue separation eluent with preparative liquid chromatography, separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water A and acetonitrile B, the gradient elution program is as follows
Flow velocity is 10ml/min, and column temperature is a room temperature; Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 41.8-46.9 minute, and solution obtains active component behind concentrate drying.
The active component composition such as the following table of collecting in 41.8-46.9 of the present invention minute
Table 2 component list
The present invention also provides the pharmaceutical composition that is prepared into as active constituents of medicine with Chinese medicine active component of the present invention, and pharmaceutical composition of the present invention comprises active component, and said composition can also add the medicine acceptable carrier as required.
Compositions of the present invention is the pharmaceutical dosage forms of unit dose, and described unit dosage form is meant the unit of preparation, as every of tablet, and capsular every capsules, every bottle of oral liquid, every bag of granule etc.
Compositions of the present invention active component wherein, its shared percentage by weight in preparation can be 0.1-99.9%, all the other are the medicine acceptable carrier.
Compositions of the present invention obtains by above-mentioned active component and medicine acceptable carrier are mixed with.
Compositions of the present invention, its pharmaceutical dosage forms can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, peroral dosage form preferably, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.
Compositions of the present invention, the preparation of its oral administration can contain excipient commonly used, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet in case of necessity.
The filler that is suitable for comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, for example sodium starch glycollate.Suitable lubricant comprises, for example magnesium stearate.The acceptable wetting agent of appropriate drug comprises sodium lauryl sulphate.
Can fill by mixing, the method that tabletting etc. are commonly used prepares solid oral composition.Mix repeatedly active substance is distributed in those compositionss of a large amount of filleies of whole use.
The form of oral liquid for example can be aqueous or oily suspensions, solution, Emulsion, syrup or elixir, perhaps can be a kind of available water before use or other suitable composite dry products of carrier.This liquid preparation can contain conventional additive, such as suspending agent, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible fat, emulsifying agent, for example lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), for example almond oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerol; Antiseptic, for example para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if desired, can contain conventional flavouring agent or coloring agent.
For injection, the liquid unit dosage forms of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this chemical compound can be suspended or dissolving.The preparation of solution is normally by being dissolved in active substance in a kind of carrier filter-sterilized before it is packed into a kind of suitable bottle or ampoule, sealing then.For example a kind of local anesthetic of adjuvant, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, can be after the bottle of packing into that this compositions is freezing, and under vacuum, water is removed.
Compositions of the present invention, when being prepared into medicament, optionally add suitable medicine acceptable carrier, described medicine acceptable carrier choosing is white: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, the EDTA disodium, EDTA calcium sodium, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, soil temperature 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, the phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Compositions of the present invention is determined usage and dosage according to patient's situation in use, but obeys every day three times, each 1-20 agent, as: 1-20 bag or grain or sheet.
The present invention also provides the application at anti-tumor aspect of Chinese medicine active component of the present invention and pharmaceutical composition.
Below be the data of pharmacological evaluation:
Medicine efficacy screening
Pharmacological model: HL-60 tumor cell
Cell culture and kind plateCell culture: use RPMI 1640 (Gibco) [adding the 2g/L sodium bicarbonate] 90%, hyclone (Ilex purpurea Hassk.[I.chinensis Sims) 10% Mixed culture HL 60 cells, density need be lower than 106/mL.Calculating needs cell total amount NT=ρ cellmL-1 * VT (ρ=2 * 104/mL), wherein VT=0.1mL * hole count+cell groove surplus.Suspension cell on the piping and druming culture bottle wall adds in the centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L Placenta Hominiss, viable count sum NL on the count plate, then the cell number N in the centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in the centrifugal preceding centrifuge tube.It is centrifugal that (900rad/min 10min), inhales and removes supernatant, adds culture fluid V1mL, and piping and druming makes the cell mixing, draws V2mL and joins in the cell groove, makes V2=NT/N * V1.The culture fluid that adds VT-V2mL in the cell groove again with volley of rifle fire piping and druming, mixing, is got this liquid, and every hole adds 150 μ L.Choose 4 holes behind the kind plate and add 200 μ L culture fluid as blank, the residue hole adds 200 μ L PBS, to reduce the evaporation of culture fluid.
Dosage regimenRadix Angelicae Sinensis B08 active component adds the DMSO dissolving of respective volume, and concentration is about 50mg/mL.The concussion dissolving, if there are a large amount of drops to glue wall, can be suitably centrifugal.Can store-20 ℃.The culture fluid that adds 220 μ L/ holes in 96 new orifice plates will be drawn 0.88 μ L medicinal liquid and add mixing, dilute 250 times, and the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and this moment, drug dilution was 1000 times, and promptly final concentration is 50ng/mL.Hatch 48h.Each concentration is established 4 parallel multiple holes, and every plate is established negative control group (add blank solution in the cell, blank solution is joined method---add the culture fluid of 200 μ L, will draw 0.88 μ LDMSO and add mixing), reaches positive controls (cisplatin final concentration 4 μ g/mL).SRB dyeing: after cell culture finishes, take out culture plate, every hole adds trichloroacetic acid (TCA) the 100 μ L fixed cells of 40% (mass/volume), and room temperature is placed 5min, places 1h in 4 ℃ of refrigerators.Deionized water wash 5 times of each hole of culture plate are to remove TCA.Behind air drying, every hole adds 0.4% SRB100 μ L (dissolving of 1% chromatographically pure acetic acid), place 20min under the room temperature, discard in each hole and wash 5 times with 1% acetic acid behind the liquid, remove unconjugated dyestuff, with 10mmol/L Tris150 μ L/ hole dissolving, 5min vibrates behind the air drying, use microplate reader (ELx 800) to measure, used wavelength is 490nm.
The calculating of suppression ratioSuppression ratio is calculated as follows:
Drug effect the results are shown in Table 3.According to HL-60 inhibition rate of tumor cell result, Radix Angelicae Sinensis B08 active component has the highly significant effect to suppressing the HL-60 tumor cell proliferation.
Table 3, HL-60 inhibition rate of tumor cell result
| Radix Angelicae Sinensis B08 component | Negative | Blank | Positive | |
| Average cell survival number | 0.164 | 0.835 | 0.057 | 0.209 |
| Suppression ratio (%) | 86.279 | 0.000 | 100.0 | 80.463 |
| RSD(％) | 3.744 | 1.926 | 1.432 | 3.767 |
Pharmacological model: K562 tumor cell
Cell culture and kind plateCell culture: use RPMI 1640 (Gibco) [adding the 2g/L sodium bicarbonate] 90%, calf serum (match is happy) 10%, non essential amino acid (Gibco) 1% Mixed culture K 562 cells.Calculating needs cell total amount NT=ρ cellmL-1 * VT (ρ=8 * 103/mL), wherein VT=0.1mL * hole count+cell groove surplus.Suspension cell on the piping and druming culture bottle wall adds in the centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L Placenta Hominiss, viable count sum NL on the count plate, then the cell number N in the centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in the centrifugal preceding centrifuge tube.It is centrifugal that (900rad/min 10min), inhales and removes supernatant, adds culture fluid V1mL, and piping and druming makes the cell mixing, draws V2mL and joins in the cell groove, makes V2=NT/N * V1.The culture fluid that adds VT-V2mL in the cell groove again with volley of rifle fire piping and druming, mixing, is got this liquid, and every hole adds 100 μ L, hatches 24h.Choose 4 holes adding culture fluid as blank after planting plate, the residue hole adds 100 μ L PBS, to reduce the evaporation of culture fluid.
Dosage regimenRadix Angelicae Sinensis B08 active component adds the DMSO dissolving of respective volume, and concentration is about 50mg/mL.The concussion dissolving, if there are a large amount of drops to glue wall, can be suitably centrifugal.Can store-20 ℃.96 orifice plates change liquid, and every hole adds fresh medium 150 μ L.The culture fluid that adds 220 μ L/ holes in 96 new orifice plates will be drawn 0.88 μ L medicinal liquid and add mixing, dilute 250 times, and the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and this moment, drug dilution was 1000 times, and promptly final concentration is 50 μ g/mL.Hatch 48h.Each concentration is established 4 (2) individual parallel multiple holes, and every plate is established negative control group (add blank solution in the cell, blank solution is joined method---add the culture fluid of 200 μ L, will draw 0.88 μ LDMSO adding mixing), positive controls (amycin final concentration 4 μ g/mL).SRB dyeing: after cell culture finishes, take out culture plate, every hole adds trichloroacetic acid (TCA) the 70 μ L fixed cells of 40% (mass/volume), places 1h in 4 ℃ of refrigerators.Deionized water wash 5 times of each hole of culture plate are to remove TCA.Behind air drying, every hole adds 0.4% SRB100 μ L (dissolving of 1% chromatographically pure acetic acid), place 20min under the room temperature, discard in each hole and wash 5 times with 1% acetic acid behind the liquid, remove unconjugated dyestuff, with 10mmol/L Tris150 μ L/ hole dissolving, 5min vibrates behind the air drying, use microplate reader (ELx 800) to measure, used wavelength is 490nm.
The calculating of suppression ratioSuppression ratio is calculated as follows:
Drug effect the results are shown in Table 4.According to K562 inhibition rate of tumor cell result, Radix Angelicae Sinensis B08 active component has the highly significant effect to suppressing the K562 tumor cell proliferation.
Table 4, K562 inhibition rate of tumor cell result
| Radix Angelicae Sinensis B08 component | Negative | Blank | Positive | |
| Average cell survival number | 0.127 | 0.525 | 0.054 | 0.089 |
| Suppression ratio (%) | 84.61 | 0.00 | 100.0 | 92.52 |
| RSD(％) | 5.184 | 8.355 | 5.295 | 2.947 |
Pharmacological model: MCF-7 tumor cell
Cell culture and kind plateCell culture: use DMEM high glycoform (Gibco) [adding the 3.7g/L sodium bicarbonate] 90%, calf serum (match is happy) 10%, non essential amino acid (Gibco) 1% Mixed culture MCF-7 cell.Calculating needs cell total amount NT=ρ cellmL-1 * VT (ρ=2 * 103/mL), wherein VT=0.1mL * hole count+cell groove surplus.Suspension cell on the piping and druming culture bottle wall adds in the centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L Placenta Hominiss, viable count sum NL on the count plate, then the cell number N in the centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in the centrifugal preceding centrifuge tube.It is centrifugal that (900rad/min 10min), inhales and removes supernatant, adds culture fluid V1mL, and piping and druming makes the cell mixing, draws V2mL and joins in the cell groove, makes V2=NT/N * V1.The culture fluid that adds VT-V2mL in the cell groove again with volley of rifle fire piping and druming, mixing, is got this liquid, and every hole adds 100 μ L, hatches 24h.Choose 4 holes adding culture fluid as blank after planting plate, the residue hole adds 100 μ L PBS, to reduce the evaporation of culture fluid.
Dosage regimenRadix Angelicae Sinensis B08 active component adds the DMSO dissolving of respective volume according to the weight of the medicine of institute's weighing, and concentration is about 50mg/mL.The concussion dissolving, if there are a large amount of drops to glue wall, can be suitably centrifugal.Can store-20 ℃.96 orifice plates change liquid, and every hole adds fresh medium 150 μ L.The culture fluid that adds 220 μ L/ holes in 96 new orifice plates will be drawn 0.88 μ L medicinal liquid and add mixing, dilute 250 times, and the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and this moment, drug dilution was 1000 times, and promptly final concentration is 50 μ g/mL.Hatch 48h.Each concentration is established 4 (2) individual parallel multiple holes, and every plate is established negative control group (add blank solution in the cell, blank solution is joined method---add the culture fluid of 200 μ L, will draw 0.88 μ LDMSO adding mixing), positive controls (amycin final concentration 4 μ g/mL).The MTT colorimetric method for determining: take out culture plate, every hole, place to go supernatant, adding culture fluid-MTT mixed solution (culture fluid: 100 μ L MTT solution=10: 1), hatch 4h.Culture fluid is abandoned in suction, and every hole adds the DMSO of 150 μ L, vibration 10min, and 550nm microplate reader (Elx800) is measured.
The calculating of suppression ratioSuppression ratio is calculated as follows:
Drug effect the results are shown in Table 5.According to MCF-7 inhibition rate of tumor cell result, Radix Angelicae Sinensis B08 active component has the highly significant effect to suppressing the MCF-7 tumor cell proliferation.
Table 5, MCF-7 inhibition rate of tumor cell result
| Radix Angelicae Sinensis B08 component | Negative | Blank | Positive | |
| Average cell survival number | 0.060 | 0.563 | 0.058 | 0.060 |
| Suppression ratio (%) | 99.653 | 0 | 100 | 99.604 |
| RSD(％) | 4.998 | 4.050 | 5.407 | 4.082 |
Pharmacological model: KB tumor cell
Cell culture and kind plateCell culture: use DMEM high glycoform (Gibco) [adding the 3.7g/L sodium bicarbonate] 90%, calf serum (match is happy) 10%, non essential amino acid (Gibco) 1% Mixed culture KB cell.Calculating needs cell total amount NT=ρ cellmL-1 * VT (ρ=2 * 103/mL), wherein VT=0.1mL * hole count+cell groove surplus.Suspension cell on the piping and druming culture bottle wall adds in the centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L Placenta Hominiss, viable count sum NL on the count plate, then the cell number N in the centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in the centrifugal preceding centrifuge tube.It is centrifugal that (900rad/min 10min), inhales and removes supernatant, adds culture fluid V1mL, and piping and druming makes the cell mixing, draws V2mL and joins in the cell groove, makes V2=NT/N * V1.The culture fluid that adds VT-V2mL in the cell groove again with volley of rifle fire piping and druming, mixing, is got this liquid, and every hole adds 100 μ L, hatches 24h.Choose 4 holes adding culture fluid as blank after planting plate, the residue hole adds 100 μ L PBS, to reduce the evaporation of culture fluid.
Dosage regimenRadix Angelicae Sinensis B08 active component adds the DMSO dissolving of respective volume according to the weight of the medicine of institute's weighing, and concentration is about 50mg/mL.The concussion dissolving, if there are a large amount of drops to glue wall, can be suitably centrifugal.Can store-20 ℃.96 orifice plates change liquid, and every hole adds fresh medium 150 μ L.The culture fluid that adds 220 μ L/ holes in 96 new orifice plates will be drawn 0.88 μ L medicinal liquid and add mixing, dilute 250 times, and the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and this moment, drug dilution was 1000 times, and promptly final concentration is 50 μ g/mL.Hatch 48h.Each concentration is established 4 (2) individual parallel multiple holes, and every plate is established negative control group (add blank solution in the cell, blank solution is joined method---add the culture fluid of 200 μ L, will draw 0.88 μ LDMSO adding mixing), positive controls (amycin final concentration 4 μ g/mL).The MTT colorimetric method for determining: take out culture plate, every hole, place to go supernatant, adding culture fluid-MTT mixed solution (culture fluid: 100 μ L MTT solution=10: 1), hatch 4h.Culture fluid is abandoned in suction, and every hole adds the DMSO of 150 μ L, vibration 10min, and 550nm microplate reader (Elx800) is measured.
The calculating of suppression ratioSuppression ratio is calculated as follows:
Drug effect the results are shown in Table 6.According to KB inhibition rate of tumor cell result, Radix Angelicae Sinensis B08 active component has the highly significant effect to suppressing the KB tumor cell proliferation.
Table 6, KB inhibition rate of tumor cell result
| Radix Angelicae Sinensis B08 component | Negative | Blank | Positive | |
| Average cell survival number | 0.060 | 0.563 | 0.058 | 0.060 |
| Suppression ratio (%) | 99.65 | 0 | 100 | 99.60 |
Pharmacological model: Hep G2 tumor cell
Cell culture and kind plateCell culture: use DMEM high glycoform (Gibco) [adding the 3.7g/L sodium bicarbonate] 90%, hyclone (Ilex purpurea Hassk.[I.chinensis Sims) 10%, non essential amino acid (Gibco) 1% Mixed culture Hep G2 cell.Calculating needs cell total amount NT=ρ cellmL-1 * VT (ρ=2 * 103/mL), wherein VT=0.1mL * hole count+cell groove surplus.Suspension cell on the piping and druming culture bottle wall adds in the centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L Placenta Hominiss, viable count sum NL on the count plate, then the cell number N in the centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in the centrifugal preceding centrifuge tube.It is centrifugal that (900rad/min 10min), inhales and removes supernatant, adds culture fluid V1mL, and piping and druming makes the cell mixing, draws V2mL and joins in the cell groove, makes V2=NT/N * V1.The culture fluid that adds VT-V2mL in the cell groove again with volley of rifle fire piping and druming, mixing, is got this liquid, and every hole adds 100 μ L, hatches 24h.Choose 4 holes adding culture fluid as blank after planting plate, the residue hole adds 100 μ LPBS, to reduce the evaporation of culture fluid.
Dosage regimenRadix Angelicae Sinensis B08 active component is according to the weight of the medicine of institute's weighing, according to the weight of the medicine of institute's weighing, adds the DMSO dissolving of respective volume, and concentration is about 50mg/mL.The concussion dissolving, if there are a large amount of drops to glue wall, can be suitably centrifugal.Can store-20 ℃.96 orifice plates change liquid, and every hole adds fresh medium 150 μ L.The culture fluid that adds 220 μ L/ holes in 96 new orifice plates will be drawn 0.88 μ L medicinal liquid and add mixing, dilute 250 times, and the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and this moment, drug dilution was 1000 times, and promptly final concentration is 50 μ g/mL.Hatch 48h.Each concentration is established 4 (2) individual parallel multiple holes, and every plate is established negative control group (add blank solution in the cell, blank solution is joined method---add the culture fluid of 200 μ L, will draw 0.88 μ LDMSO adding mixing), positive controls (amycin final concentration 4 μ g/mL).The MTT colorimetric method for determining: take out culture plate, every hole, place to go supernatant, adding culture fluid-MTT mixed solution (culture fluid: 100 μ L MTT solution=10: 1), hatch 4h.Culture fluid is abandoned in suction, and every hole adds the DMSO of 150 μ L, vibration 10min, and 550nm microplate reader (Elx800) is measured.
The calculating of suppression ratioSuppression ratio is calculated as follows:
Drug effect the results are shown in Table 7.According to Hep G2 inhibition rate of tumor cell result, Radix Angelicae Sinensis B08 active component has the highly significant effect to suppressing Hep G2 tumor cell proliferation.
Table 7, Hep G2 inhibition rate of tumor cell result
| Radix Angelicae Sinensis B08 component | Negative | Blank | Positive | |
| Average cell survival number | 0.127 | 0.613 | 0.068 | 0.185 |
| Suppression ratio (%) | 89.17 | 0 | 100 | 87.09 |
| RSD(％) | 2.802 | 2.755 | 6.898 | 3.237 |
Radix Angelicae Sinensis active component of the present invention and preparation thereof can be used in preparation treatment, prophylaxis of tumours medicine.
Beneficial effect of the present invention is:
1. use the reverse phase silica gel post in the extraction and separation process of the present invention, can remove the impurity that easily formation is extremely adsorbed on the preparative hplc post such as chlorophyll effectively, improved content of effective, can obtain effective ingredient fast and accurately.
2. Radix Angelicae Sinensis active component chemical constituent provided by the invention is simply clear and definite, is easier to illustrate its mechanism of action on pharmacological research, is easier to the quality control of medicine aborning.
Method provided by the invention obtains containing the B08 active component first from the Radix Angelicae Sinensis medical material, and first it is carried out medicine efficacy screening on various tumor cell strains, because composition is definite, content is clear and definite, preparation technology is convenient, and is active good, the suitable antitumor new Chinese medicine that is developed to.
Description of drawings
Fig. 1 is the HPLC analysis chart of Radix Angelicae Sinensis active component of the present invention.
The specific embodiment
Further describe flesh and blood of the present invention below in conjunction with embodiments of the invention, this embodiment only is used to the present invention is described and the present invention is not limited.
The preparation of embodiment 1 Radix Angelicae Sinensis active component
Radix Angelicae Sinensis is pulverized, add 8 times of amount ethyl acetate: ethanol=1: 1, reflux 1 hour, extract 2 times, medicinal residues add 8 times of amount 70% ethanol, and reflux 1 hour is extracted 2 times, filtrate merge extracting solution, after extracting solution concentrated, with sample on 5% dissolve with methanol, mistake ODS-C18 post, at first, adopt 1250ml 5% methanol as mobile phase, change 1250ml 60% methanol then, get eluent as mobile phase, continue separation eluent with preparative liquid chromatography, separation condition: chromatographic column is Agilent preparative column (Zorbax SB-C18; 21.2mm * 250mm), mobile phase is water A and acetonitrile B, the gradient elution program is as follows
Flow velocity is 10ml/min, and column temperature is a room temperature; Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 41.8-46.9 minute, and solution obtains active component behind concentrate drying.
The analysis of embodiment 2 Radix Angelicae Sinensis active components
Chromatographic conditionChromatographic column Agilent Zorbax SB-C18 post (4.6mm * 150mm, 5 μ m); Adopt gradient elution, mobile phase A is 0.2% glacial acetic acid aqueous solution mutually, and Mobile phase B is mutually for containing the acetonitrile solution of 0.2% glacial acetic acid; The gradient elution program is as follows: in the time of 0 minute, mobile phase A is that 90% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 10% 0.2% glacial acetic acid; In the time of 10 minutes, mobile phase A is that 50% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 50% 0.2% glacial acetic acid; In the time of 30 minutes, mobile phase A is that 5% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile solution of 95% 0.2% glacial acetic acid; In the time of 35 minutes, mobile phase A is that 5% 0.2% glacial acetic acid aqueous solution, Mobile phase B are the acetonitrile of 95% 0.2% glacial acetic acid.Solution flow rate 0.5mLmin
-1It is long to detect the wavelength all-wave; 30 ℃ of column temperatures; ELSD condition: 105 ℃ of drift tube temperatures; Nitrogen flow rate 2.0L/min
The preparation of need testing solutionTake by weighing active component of the present invention, in volumetric flask, be diluted to scale, shake up, promptly with dissolve with methanol solution.
Assay methodThe accurate need testing solution of drawing injects chromatograph of liquid, measures.
Embodiment 3 Radix Angelicae Sinensis active component preparations
Get the Radix Angelicae Sinensis active component of embodiment 1,0.5g and 10.5g Polyethylene Glycol-6000 mix homogeneously, heating and melting moves in the drop pill drip irrigation behind the change material, and medicine liquid droplet is to 6-8 ℃ of liquid paraffin, and oil removing makes 400 of drop pill.
Embodiment 4 Radix Angelicae Sinensis active component preparations
Get the Radix Angelicae Sinensis active component of embodiment 1,0.5g, glucose 4.5g, sodium thiosulfate 0.9g and distilled water 1ml, behind the said components mix homogeneously, lyophilization, 500 of packing, promptly.
Embodiment 5 Radix Angelicae Sinensis active component preparations
Get Lignum Dalbergiae Odoriferae oil 1.5g, join in the saturated hydroxypropyl of 13ml, stirring and dissolving filters, the filter leaf cold drying, Lignum Dalbergiae Odoriferae oil and the clathrate powder of hydroxypropyl.Except that above-mentioned Lignum Dalbergiae Odoriferae oil closes the clathrate powder of hydroxypropyl, get the Radix Angelicae Sinensis active component of embodiment 1 again, 0.5g, mannitol 5.5g, calcium disodium edetate 0.9g and distilled water 2ml, behind the said components mixing, lyophilization, 300 of packing, promptly.
Claims (1)
1. Radix Angelicae Sinensis active component with antitumor action, its preparation process may further comprise the steps:
Radix Angelicae Sinensis is pulverized, add 8 times of amount ethyl acetate: ethanol=1: 1, reflux 1 hour, extract 2 times, medicinal residues add 8 times of amount 70% ethanol, and reflux 1 hour is extracted 2 times, filtrate merge extracting solution, after extracting solution concentrated, with sample on 5% dissolve with methanol, mistake ODS-C18 post, at first, adopt 5% methanol as mobile phase, change 60% methanol then, get eluent as mobile phase, continue separation eluent with preparative liquid chromatography, separation condition: chromatographic column is Agilent preparative column Zorbax SB-C18; 21.2mm * 250mm, mobile phase is water A and acetonitrile B, and the gradient elution program is as follows
Flow velocity is 10ml/min, and column temperature is a room temperature; Sample 100% dissolve with ethanol separates through preparative liquid chromatography, collects solution at time period 41.8-46.9 minute, and solution obtains active component behind concentrate drying.
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