CN101347497B - Effective component of vietnamese sophora root and preparation and use thereof - Google Patents
Effective component of vietnamese sophora root and preparation and use thereof Download PDFInfo
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- CN101347497B CN101347497B CN2007101232684A CN200710123268A CN101347497B CN 101347497 B CN101347497 B CN 101347497B CN 2007101232684 A CN2007101232684 A CN 2007101232684A CN 200710123268 A CN200710123268 A CN 200710123268A CN 101347497 B CN101347497 B CN 101347497B
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- ethyl acetate
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Abstract
The present invention relates to an effective component of root of subprostrate sophora and the preparation method and usage. For the effective component of root of subprostrate sophora 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 the root of subprostrate sophora; step 2: the extract goes through column chromatography to obtain the eluent; step 3: the mobile phase of the eluent obtained from the preparation of liquid chromatogram gradient elution is water and acetonitrile, the eluent is collected for 36.0 to 40.0min 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 Sophorae Tonkinensis, 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 Sophorae Tonkinensis is an antipyretic, another name: root of subprostrate sophora, Herba Astragali Melilotoidis, the big Radix Sophorae Tonkinensis in mountain, medicinal part: root, meridian distribution of property and flavor: cold; Bitter; Attach to the lung and stomach meridians, its function cures mainly and is heat-clearing and toxic substances removing, the detumescence sore-throat relieving.Be used for fire-toxin and pent up, laryngopharynx swelling and pain, gingivitis.
Summary of the invention:
The object of the present invention is to provide a kind of active component of Radix Sophorae Tonkinensis.
Another object of the present invention is to provide the preparation method of above-mentioned Radix Sophorae Tonkinensis active component.
The present invention also provides the preparation that contains above-mentioned Radix Sophorae Tonkinensis active component and the purposes of this component.
Radix Sophorae Tonkinensis active component of the present invention, its preparation process may further comprise the steps:
Step 1: Radix Sophorae Tonkinensis is extracted as solvent with ethyl acetate and alcohol mixture,
Step 2: extracting solution gets eluent through column chromatography;
Step 3: with the eluent that the preparative liquid chromatography gradient elution obtains, mobile phase is water and acetonitrile, collects 36.0-40.0 minute eluent and obtains active component (or being called C09).
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 Sophorae Tonkinensis medical material, is solvent with ethyl acetate: ethanol=1-5: 1-5, and reflux, extract, is separated extracting solution with medicinal residues, and the extracting solution that obtains after the separation is an extract 1,
Step 2 is specially: extract 1 is crossed normal phase silicagel column, and use petroleum ether: ethyl acetate=47-53 earlier: 1 as the mobile phase eluting, changes chloroform then: methanol 8-13: 1 as mobile phase, gets eluent,
Step 3 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 flow velocity is 9-11ml/min, and column temperature is a room temperature, collects 36.0-40.0 minute eluent and obtains active component.
The program of gradient elution described in the step 3 is as follows: table 1 gradient table
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 36.0-40.0 minute, and solution obtains active component behind concentrate drying.
The preferred Radix Sophorae Tonkinensis active component of the present invention preparation method, comprise the following steps: and to add ethyl acetate and ethanol (1: 0.8-1.2) after the Radix Sophorae Tonkinensis pulverizing medicinal materials, reflux 0.8-1.2 hour, extract 1-3 time, merging filtrate gets extracting solution, extracting solution is condensed into extractum, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate (47-53:, get eluent I 1) as mobile phase, abandon it, change chloroform and methanol (8-13: 1) as mobile phase, get eluent II, will get sample behind the eluent II concentrate drying then; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is a preparative column, and mobile phase is water and acetonitrile, and gradient elution, flow velocity are 9-11ml/min, and column temperature is a room temperature.
The most preferred Radix Sophorae Tonkinensis active component of the present invention preparation method comprises the following steps: and will add ethyl acetate and ethanol (1: 1) after the Radix Sophorae Tonkinensis pulverizing medicinal materials that reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution; Extracting solution is condensed into extractum, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether and ethyl acetate (50: 1) as mobile phase, get eluent I, abandon it, change chloroform and methanol (10: 1) then as mobile phase, get eluent II, will get sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is preparative column (ZorbaxSB-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 36.0-40.0 minute, and solution obtains active component behind concentrate drying.
The active component composition such as the following table of collecting in 36.0-40.0 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 is selected from: 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 antitumor pharmacology experiment:
To the screening active ingredients of 36.0-40.0 minute section component, on cellular level, detect the growth inhibited effect of active component to kinds of tumor cells.
Screening active ingredients
On cellular level, detect the growth inhibited effect of active component to kinds of tumor cells.
Cell strain: HL-60 tumor cell
Sample preparation: according to the weight of the medicine of institute's weighing, add the DMSO dissolving of respective volume, 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 ℃.
Cell 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.
Experimental technique:
1 kind of plate:
(1) calculates and to need cell total amount NT=ρ cellmL-1 * VT (ρ=2 * 104/mL), wherein VT=0.1mL * hole count+cell groove surplus.
(2) 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 (3) 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.
(4) add the culture fluid of VT-V2mL again in the cell groove, with volley of rifle fire piping and druming, mixing, get this liquid, every hole adds 150 μ L.
(5) 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.
A) dosing
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).
B) 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 suppression ratio of 4 suppression 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 Sophorae Tonkinensis C09 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
| C09 | Negative | Blank | Positive | |
| Average cell survival number | 0.18 | 0.50 | 0.10 | 0.19 |
| Suppression ratio (%) | 80.75 | 0.00 | 100.00 | 78.06 |
| RSD(％) | 6.52 | 3.07 | 6.64 | 5.92 |
Cell strain: K562 tumor cell
Sample preparation: according to the weight of the medicine of institute's weighing, add the DMSO dissolving of respective volume, 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 ℃.
Cell 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.
Experimental technique:
1 kind of plate:
(1) calculates and to need cell total amount NT=ρ cellmL-1 * VT (ρ=8 * 103/mL), wherein VT=0.1mL * hole count+cell groove surplus.
(2) 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 (3) 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.
(4) add the culture fluid of VT-V2mL again in the cell groove, with volley of rifle fire piping and druming, mixing, get this liquid, every hole adds 100 μ L, hatches 24h.
(5) choose 4 holes behind the kind plate and add culture fluid as blank, the residue hole adds 100 μ L PBS, to reduce the evaporation of culture fluid.
2 dosings:
(1) 96 orifice plate changes liquid, and every hole adds fresh medium 150 μ L.
(2) culture fluid in adding 220 μ L/ holes in 96 new orifice plates, to draw 0.88 μ L medicinal liquid and add mixing, and dilute 250 times, the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, 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).
3SRB 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 suppression ratio of 4 suppression ratio is calculated as follows:
Drug effect the results are shown in Table 4.According to K562 inhibition rate of tumor cell result, Radix Sophorae Tonkinensis C09 active component has the highly significant effect to suppressing the K562 tumor cell proliferation.
Table 4, K562 inhibition rate of tumor cell result
| C09 | Negative | Blank | Positive | |
| Average cell survival number | 0.211 | 0.733 | 0.122 | 0.131 |
| Suppression ratio (%) | 84.91 | 0 | 100 | 98.47 |
| RSD(％) | 9.309 | 4.906 | 8.108 | 3.942 |
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 Sophorae Tonkinensis C09 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 Sophorae Tonkinensis C09 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 Sophorae Tonkinensis C09 component | Negative | Blank | Positive | |
| Average cell survival number | 0.095 | 0.267 | 0.102 | 0.104 |
| Suppression ratio (%) | 104.55 | 0 | 100 | 98.79 |
| RSD(％) | 3.614 | 1.687 | 4.816 | 3.765 |
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 Sophorae Tonkinensis C09 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 Sophorae Tonkinensis C09 active component has the highly significant effect to suppressing the KB tumor cell proliferation.
Table 6, KB inhibition rate of tumor cell result
| Radix Sophorae Tonkinensis C09 component | Negative | Blank | Positive | |
| Average cell survival number | 0.216 | 1.242 | 0.148 | 0.289 |
| Suppression ratio (%) | 93.83 | 0 | 100 | 87.14 |
| RSD(％) | 5.652 | 2.550 | 5.773 | 2.779 |
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 Sophorae Tonkinensis C09 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 Sophorae Tonkinensis C09 active component has the highly significant effect to suppressing the HepG2 tumor cell proliferation.
Table 7, Hep G2 inhibition rate of tumor cell result
| Radix Sophorae Tonkinensis C09 component | Negative | Blank | Positive | |
| Average cell survival number | 0.175 | 0.665 | 0.164 | 0.196 |
Beneficial effect of the present invention is:
1. use normal phase silicagel column in the extraction and separation process of the present invention, can remove impurity such as desaccharide, protein, aminoacid effectively, improved content of effective, adopted preparative hplc simultaneously, can obtain effective ingredient fast and accurately.
2. Radix Sophorae Tonkinensis 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 C09 active component first from the Radix Sophorae Tonkinensis 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 Sophorae Tonkinensis 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 Sophorae Tonkinensis active component
Get Radix Sophorae Tonkinensis medical material 250g, it is pulverized the back add ethyl acetate and ethanol (1: 1), reflux 1 hour is extracted 2 times, filtrate merge extracting solution.Extracting solution is condensed into extractum gets 9.8g, with extractum and silica gel mixed sample, separate with normal phase silicagel column, at first use petroleum ether and ethyl acetate (50: 1) as mobile phase, get eluent I, change chloroform and methanol (10: 1) then as mobile phase, get eluent II, get the 1.5g sample behind the concentrate drying.Continue to separate the sample that obtains with preparative liquid chromatography.The separation condition of preparative hplc: chromatographic column is Agient preparative column (Zorbax SB-C
1821.2mm * 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 36.0-40.0 minute, obtains active component component 0.03g behind the concentrate drying.
The analysis of embodiment 2 Radix Sophorae Tonkinensis active components
HPLC-ELSD coupling to the Radix Sophorae Tonkinensis active component of embodiment 1 is analyzed
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 Sophorae Tonkinensis active component preparations
Get Radix Sophorae Tonkinensis active component 0.5g and 10.5g Polyethylene Glycol-6000 mix homogeneously of embodiment 1, 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 Sophorae Tonkinensis active component preparations
Get Radix Sophorae Tonkinensis active component 0.5g, glucose 4.5g, sodium thiosulfate 0.9g and the distilled water 1ml of embodiment 1, behind the said components mix homogeneously, lyophilization, 500 of packing, promptly.
Embodiment 5 Radix Sophorae Tonkinensis 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 Radix Sophorae Tonkinensis active component 0.5g, mannitol 5.5g, calcium disodium edetate 0.9g and the distilled water 2ml of embodiment 1 again, behind the said components mixing, lyophilization, 300 of packing, promptly.
The preparation of embodiment 6 Radix Sophorae Tonkinensis active components
Operating procedure is with embodiment 1, and condition changes into: ethyl acetate and ethanol (1: 5), normal phase silicagel column separate, and at first use petroleum ether and ethyl acetate 47: 1 as mobile phase, eluent I, change chloroform and methanol (8: 1) then as mobile phase.
The preparation of embodiment 7 Radix Sophorae Tonkinensis active components
Operating procedure is with embodiment 1, and condition changes into: ethyl acetate and ethanol (5: 1), normal phase silicagel column separate, and at first use petroleum ether and ethyl acetate 53: 1 as mobile phase, eluent I, change chloroform and methanol (13: 1) then as mobile phase.
The preparation of embodiment 8 Radix Sophorae Tonkinensis active components
Operating procedure is with embodiment 1, and condition changes into: ethyl acetate and ethanol (1: 2), normal phase silicagel column separate, and at first use petroleum ether and ethyl acetate 47: 1 as mobile phase, eluent I, change chloroform and methanol (8: 1) then as mobile phase.
The preparation of embodiment 9 Radix Sophorae Tonkinensis active components
Operating procedure is with embodiment 1, and condition changes into: ethyl acetate and ethanol (2: 1), normal phase silicagel column separate, and at first use petroleum ether and ethyl acetate 53: 1 as mobile phase, eluent I, change chloroform and methanol (13: 1) then as mobile phase.
Claims (6)
1. a Radix Sophorae Tonkinensis active component is characterized in that, its preparation process may further comprise the steps:
Step 1 is: getting the Radix Sophorae Tonkinensis medical material, is solvent with ethyl acetate: ethanol=1-5: 1-5, and reflux, extract, is separated extracting solution with medicinal residues, and the extracting solution that obtains after the separation is an extract 1,
Step 2 is: extract 1 is crossed normal phase silicagel column, and use petroleum ether earlier: ethyl acetate=47～53: 1 as the mobile phase eluting, changes chloroform then: methanol 8～13: 1 as mobile phase, gets eluent,
Step 3 is: continue to separate the eluent that obtains with preparative liquid chromatography, mobile phase is water-A and acetonitrile-B, carries out gradient elution, 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 36.0-40.0 minute, and solution obtains active component behind concentrate drying.
2. the active component of claim 1 is characterized in that, ethyl acetate described in the step 1 and alcoholic acid mixture, and both ratios are ethyl acetate: ethanol=1-2: 1-2.
3. the active component of claim 1 is characterized in that, ethyl acetate described in the step 1 and alcoholic acid mixture, and both ratios are ethyl acetate: ethanol=1: 1.
4. the pharmaceutical composition that contains any one active component of claim 1-3.
5. the preparation method of the active component of claim 1 is characterized in that, its preparation process may further comprise the steps:
Step 1 is: getting the Radix Sophorae Tonkinensis medical material, is solvent with ethyl acetate: ethanol=1-5: 1-5, and reflux, extract, is separated extracting solution with medicinal residues, and the extracting solution that obtains after the separation is an extract 1,
Step 2 is: extract 1 is crossed normal phase silicagel column, and use petroleum ether earlier: ethyl acetate=47～53: 1 as the mobile phase eluting, changes chloroform then: methanol 8～13: 1 as mobile phase, gets eluent,
Step 3 is: continue to separate the eluent that obtains with preparative liquid chromatography, 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 36.0-40.0 minute, and solution obtains active component behind concentrate drying.
6. the preparation method of the active component of claim 5 is characterized in that, step is as follows:
To add ethyl acetate after the Radix Sophorae Tonkinensis pulverizing medicinal materials: ethanol=1: 1, reflux 1 hour is extracted 2 times, and merging filtrate gets extracting solution; Extracting solution is condensed into extractum, and itself and silica gel mixed sample, with normal phase silicagel column it is separated, at first use petroleum ether: ethyl acetate=50: 1 is as mobile phase, eluent I, abandon it, change chloroform then: methanol=10: 1 is as mobile phase, get eluent II, will get sample behind the eluent II concentrate drying; Continue to separate the sample that obtains with preparative liquid chromatography; The separation condition of preparative hplc: chromatographic column is 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 36.0-40.0 minute, and solution obtains active component behind concentrate drying.
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| CN103690595B (en) * | 2013-01-07 | 2016-04-27 | 中国医学科学院药用植物研究所 | A kind of ethyl acetate extract of Radix Sophorae Tonkinensis and preparation, detection method and application |
| CN108864026A (en) * | 2018-09-20 | 2018-11-23 | 广西壮族自治区药用植物园 | Genistein composition and its application |
| CN109106749A (en) * | 2018-09-20 | 2019-01-01 | 广西壮族自治区药用植物园 | The preparation method of sophorae subprostratae radix extract and the application of sophorae subprostratae radix extract |
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