CN101550081B - Effective components of Cacumen Platycladi and preparation method thereof - Google Patents
Effective components of Cacumen Platycladi and preparation method thereof Download PDFInfo
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- CN101550081B CN101550081B CN200810052596.4A CN200810052596A CN101550081B CN 101550081 B CN101550081 B CN 101550081B CN 200810052596 A CN200810052596 A CN 200810052596A CN 101550081 B CN101550081 B CN 101550081B
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- ethanol
- active principle
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- moving phase
- ethyl acetate
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Abstract
The invention relates to effective components of Cacumen Platycladi as well as a preparation method and a purpose thereof. The preparation process comprises the following steps: 1. the mixer of ethyl acetate and ethanol is used as a solvent for extracting Cacumen Platycladi; 2. eluent is obtained from an extracting solution through chromatographic column chromatography; 3. with the moving phase ofwater and acetonitrile, the eluent obtained by the phase color chromatographic gradient elution of a preparation solution is collected for 36.0-40.0 minutes, and the effective components are obtained from the eluent after 44.0-48.0 minutes.
Description
Technical field:
The present invention relates to a kind of Chinese medical extract for the treatment of tumor disease, relate in particular to the active principle extracting from Leafy twigs of Oriental Arborvitae, preparation and preparation method thereof and purposes.
Background technology:
Tumour is a kind of common disease, frequently-occurring disease, and wherein malignant tumour is the class disease that current harm humans health is the most serious.In the industry the treatment of malignant tumour mainly still be take to operation, radiotherapy, chemotherapy as main at present, 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 herbal medicine is having unique advantage and wide application prospect aspect anticancer anti-mutation, and Chinese medicine also plays the effect can not be ignored in to the assisting therapy of tumour.Taxol is the natural compounds with high anti-cancer activity typically obtaining from plant, has now been developed as antitumor drug.The most serious tumour of China's hazardness is lung cancer, nasopharyngeal carcinoma, the esophageal carcinoma, cancer of the stomach, large bowel cancer, liver cancer, mammary cancer, cervical cancer, leukemia and lymphoma etc. at present.Particularly the incidence of liver cancer increases in recent years to some extent.Significant, the nosetiology of these tumours, pathogenesis and control thereof, be the emphasis of China's tumor research.Cancer therapy drug and the anticancer ancillary drug of finding high-efficiency low-toxicity are the important contents of current tumor research.
China's medicinal organism resource is very abundant, and its physiologically active substance 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, for being developed to treatment tumor disease, security is good, toxicity is low new drug also seldom, extracts active substance from natural product, be developed to the new drug with antitumor curative effect, there is significant application value and wide development prospect.
Leafy twigs of Oriental Arborvitae is per nnial herb.Its chemical composition containing has, and rhizome is containing volatile oil, and in oil, principal constituent is: fenchone (fenchone), camphor, Bornyl acetate, terpene alcohol; And containing juniperic acid (juniperic acid) Quercetin, poplar luteoxanthin (myricetin), kaempferol, hinokiflavone (hinokiflavone), wax etc.Its nature and flavor: nature and flavor are cold in nature, bitter and puckery flavor.
Its function cures mainly as cooling blood for hemostasis, growing and blacking hair.For haematemesis, bleeding from five sense organs or subcutaneous tissue, spit blood, have blood in stool, uterine bleeding is more than, blood-head alopecia, early whitening of beard and hair.
Summary of the invention:
The object of the present invention is to provide the active principle of Leafy twigs of Oriental Arborvitae.
Another object of the present invention is to provide the preparation method of above-mentioned Leafy twigs of Oriental Arborvitae active principle.
The present invention also provides the preparation that contains above-mentioned Leafy twigs of Oriental Arborvitae active principle and the purposes of this component.
Leafy twigs of Oriental Arborvitae active principle of the present invention, its preparation process comprises the following steps:
Step 1: with ethyl acetate and alcohol mixture, as solvent, Leafy twigs of Oriental Arborvitae is extracted,
Step 2: extracting solution obtains elutriant through column chromatography;
Step 3: the elutriant obtaining with preparative liquid chromatography gradient elution, moving phase is water and acetonitrile, the elutriant of collecting 36.0-40.0 minute or 44.0-48.0 minute obtains active principle 1 (or being called C09), active principle 2 (or being called C11).
The mixture of ethyl acetate described in step 1 and ethanol wherein, 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 described step, step 1 is specially: get Leafy twigs of Oriental Arborvitae medicinal material, take ethyl acetate: ethanol=1-5: 1-5 as solvent, and refluxing extraction, extracting solution is separated with the dregs of a decoction, and the extracting solution obtaining after separation is extract 1,
Step 2 is specially: extract 1 is crossed to ODS-C18 post, first, adopt 30%EtOH (5BV; 1BV ≈ 250ml) as moving phase, then change 95%EtOH (5BV) as moving phase, obtain elutriant,
Step 3 is specially: with preparative liquid chromatography, continue the separated elutriant obtaining, moving phase is water-A and acetonitrile-B, carries out gradient elution, and flow velocity is 9-11ml/min, column temperature is room temperature, and the elutriant of collecting 36.0-40.0 minute, 44.0-48.0 minute obtains active principle.
The program of gradient elution described in step 3 is as follows:
Table 1 gradient table
Flow velocity is 10ml/min, and column temperature is room temperature; Sample 100% dissolve with ethanol, separated through preparative liquid chromatography, at time period 36.0-40.0 minute or 44.0-48.0 minute, collect solution, solution obtains active principle after concentrate drying.
The preferred Leafy twigs of Oriental Arborvitae active principle of the present invention preparation method, comprise the following steps: after Leafy twigs of Oriental Arborvitae pulverizing medicinal materials, to add ethyl acetate and ethanol (1: 0.8-1.2), reflux 0.8-1.2 hour, extract 1-3 time, merging filtrate obtains extracting solution, and extracting solution is condensed into medicinal extract, crosses ODS-C18 post, first, adopt 30%EtOH (5BV; 1BV ≈ 250ml) as moving phase, obtain elutriant I, then change 95%EtOH (5BV) as moving phase, obtain elutriant II, will after elutriant II concentrate drying, obtain sample; With preparative liquid chromatography, continue the separated sample obtaining; The separation condition of preparative chromatography: chromatographic column is preparative column, moving phase is water and acetonitrile, gradient elution, flow velocity is 9-11ml/min, column temperature is room temperature.
The most preferred Leafy twigs of Oriental Arborvitae active principle of the present invention preparation method, comprises the following steps: after Leafy twigs of Oriental Arborvitae pulverizing medicinal materials, to add ethyl acetate and ethanol (1: 1), and reflux 1 hour, extracts 2 times, and merging filtrate obtains extracting solution; Extracting solution is condensed into medicinal extract, uses dissolve with ethanol loading, cross ODS-C18 post, first, adopt 30%EtOH (5BV; 1BV ≈ 250ml) as moving phase, obtain elutriant I, then change 95%EtOH (5BV) as moving phase, obtain elutriant II, will after elutriant II concentrate drying, obtain sample; With preparative liquid chromatography, continue the separated sample obtaining; The separation condition of preparative chromatography: chromatographic column is preparative column ZorbaxSB-C18; 21.2mm * 250mm, moving phase is water A and acetonitrile B, gradient elution program is as follows:
Flow velocity is 10ml/min, and column temperature is room temperature; Sample 100% dissolve with ethanol, separated through preparative liquid chromatography, at time period 36.0-40.0 minute or 44.0-48.0 minute, collect solution, solution obtains active principle after concentrate drying.
The active principle composition that collect 36.0-40.0 of the present invention minute, 44.0-48.0 minute is as following table
Table 2 component list
Leafy twigs of Oriental Arborvitae C09 interpretation of mass spectra result
Leafy twigs of Oriental Arborvitae C11 interpretation of mass spectra result
Related structure is identified and is combined chemical dictionary CD-ROM > > (The Combined Chemical Dictionary onCD-ROM) and affiliated pertinent literature with reference to < <.
The present invention also provides the pharmaceutical composition being prepared into as active constituents of medicine with Chinese medicine active principle of the present invention, and pharmaceutical composition of the present invention, comprises active principle, and said composition can also add medicine acceptable carrier as required.
Composition of the present invention, is the pharmaceutical dosage forms of unitary dose, and described unit dosage form refers to the unit of preparation, as every of tablet, and every capsules of capsule, every bottle of oral liquid, every bag of granule etc.
Composition of the present invention active principle wherein, its shared weight percent in preparation can be 0.1-99.9%, all the other are medicine acceptable carrier.
Composition of the present invention, by being mixed with above-mentioned active principle and medicine acceptable carrier to obtain.
Composition of the present invention, its pharmaceutical dosage forms can be any pharmaceutically useful formulation, and these formulations comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, paste, sublimed preparation, suspensoid, pulvis, solution, injection, suppository, ointment, plaster, creme, sprays, drops, patch.Preparation of the present invention, oral dosage form preferably, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, paste etc.
Composition of the present invention, the preparation of its oral administration can contain conventional vehicle, such as tackiness agent, weighting agent, thinner, tablet agent, lubricant, disintegrating agent, tinting material, seasonings and wetting agent, can carry out dressing to tablet if desired.
Applicable weighting agent comprises Mierocrystalline cellulose, mannitol, lactose and other similar weighting agent.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivative, for example sodium starch glycollate.Suitable lubricant comprises, for example Magnesium Stearate.The acceptable wetting agent of suitable medicine comprises sodium lauryl sulphate.
Can fill by mixing, the method that compressing tablet etc. are conventional is prepared solid oral composition.Repeatedly mix in those compositions that can make active substance be distributed in a large amount of weighting agents of whole use.
The form of oral liquid can be for example water-based or oily suspensions, solution, emulsion, syrup or elixir, or can be a kind of used water before use or the composite drying products of other suitable carrier.This liquid preparation can contain conventional additive, such as suspension agent, for example sorbyl alcohol, syrup, methylcellulose gum, gelatin, Natvosol, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible-fat, emulsifying agent, for example Yelkin TTS, anhydro sorbitol monooleate or gum arabic; Non-aqueous carrier (they can comprise edible oil), for example Prunus amygdalus oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerine; Sanitas, for example para hydroxybenzene methyl esters or propylparaben or Sorbic Acid, and if need, can contain conventional flavouring agent or tinting material.
For injection, the liquid unit dosage of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolve.The preparation of solution is normally by active substance being dissolved in a kind of carrier, and filter-sterilized before being packed into a kind of suitable bottle or ampoule, then seals.Auxiliary material for example a kind of local anesthetic, sanitas and buffer reagent also can be dissolved in this carrier.In order to improve its stability, can be after packing bottle into, this composition is freezing, and under vacuum, water is removed.
Composition of the present invention, when being prepared into medicament, optionally add applicable medicine acceptable carrier, described medicine acceptable carrier is selected from: N.F,USP MANNITOL, sorbyl alcohol, Sodium Pyrosulfite, sodium bisulfite, Sulfothiorine, cysteine hydrochloride, Thiovanic acid, methionine(Met), vitamins C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphoric acid salt or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium-chlor, Repone K, Sodium.alpha.-hydroxypropionate, Xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, Mierocrystalline cellulose and derivative thereof, alginate, gelatin, polyvinylpyrrolidone, glycerine, POLYSORBATE 80, agar, calcium carbonate, Calcium hydrogen carbonate, tensio-active agent, polyoxyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid material, kaolin, talcum powder, calcium stearate, Magnesium Stearate etc.
Composition of the present invention is determined usage and dosage according to patient's situation in use, can take 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 principle of the present invention and pharmaceutical composition.Be below the data of pharmacological evaluation:
Screening active ingredients:
Cell strain: HL-60 tumour cell
Cell cultures and kind plate cell cultures: use RPMI 1640 (Gibco) [adding 2g/L sodium bicarbonate] 90%, foetal calf serum (folium ilicis chinensis) 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 residual content.Suspension cell on piping and druming culturing bottle wall, adds in centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L placentas, viable count sum NL on count plate, the cell count N in centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in centrifugal front centrifuge tube.Centrifugal (900rad/min, 10min), sucks supernatant liquor, adds nutrient solution V1mL, and piping and druming, mixes cell, draws V2mL and joins in cell groove, makes V2=NT/N * V1.In cell groove, add the nutrient solution of VT-V2mL again, with the volley of rifle fire, blow and beat, mix, get this liquid, every hole adds 150 μ L.After kind plate, choose 4 holes and add 200 μ L nutrient solutions as blank, residue hole adds 200 μ L PBS, to reduce the evaporation of nutrient solution.
Dosage regimen Leafy twigs of Oriental Arborvitae active principle adds the DMSO of respective volume to dissolve, and concentration is about 50mg/mL.Concussion is dissolved, if there are a large amount of drops to glue wall, and can be suitably centrifugal.Can store-20 ℃.In 96 new orifice plates, add the nutrient solution in 220 μ L/ holes, absorption 0.88 μ L liquid is added and mixed, dilute 250 times, the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and now drug dilution is 1000 times, and final concentration is 50ng/mL.Hatch 48h.Each concentration is established 4 parallel multiple holes, every plate is established negative control group and (in cell, is added blank solution, blank solution is joined method---add the nutrient solution of 200 μ L, absorption 0.88 μ LDMSO is added and mixed), and positive controls (cis-platinum final concentration 4 μ g/mL).SRB dyeing: after cell cultures finishes, take out culture plate, every hole adds trichoroacetic acid(TCA) (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, to remove TCA.After air drying, every hole adds 0.4% SRB100 μ L (1% chromatographically pure acetic acid dissolves), under room temperature, place 20min, discard in each hole and with 1% acetic acid, wash 5 times after liquid, remove unconjugated dyestuff, after air drying, with 10mmol/L Tris150 μ L/ hole, dissolve, vibration 5min, use microplate reader (ELx 800) to measure, wavelength used is 490nm.
The calculating inhibiting rate of inhibiting rate is calculated as follows:
Drug effect the results are shown in Table 3.
Table 3
| Leafy twigs of Oriental Arborvitae C09 component | Negative | Blank | Positive | |
| Average cell survival number | 0.12 | 0.37 | 0.06 | 0.07 |
| Inhibiting rate (%) | 82.26 | 0.00 | 100.00 | 96.69 |
| RSD(％) | 1.23 | 4.48 | 2.69 | 8.49 |
Cell strain: K562 tumour cell
Cell cultures and kind plate cell cultures: use RPMI 1640 (Gibco) [adding 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 residual content.Suspension cell on piping and druming culturing bottle wall, adds in centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L placentas, viable count sum NL on count plate, the cell count N in centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in centrifugal front centrifuge tube.Centrifugal (900rad/min, 10min), sucks supernatant liquor, adds nutrient solution V1mL, and piping and druming, mixes cell, draws V2mL and joins in cell groove, makes V2=NT/N * V1.In cell groove, add the nutrient solution of VT-V2mL again, with the volley of rifle fire, blow and beat, mix, get this liquid, every hole adds 100 μ L, hatches 24h.After kind plate, choose 4 holes and add nutrient solution as blank, residue hole adds 100 μ L PBS, to reduce the evaporation of nutrient solution.
Dosage regimen Leafy twigs of Oriental Arborvitae active principle adds the DMSO of respective volume to dissolve, and concentration is about 50mg/mL.Concussion is dissolved, if there are a large amount of drops to glue wall, and can be suitably centrifugal.Can store-20 ℃.96 orifice plates change liquid, and every hole adds fresh medium 150 μ L.In 96 new orifice plates, add the nutrient solution in 220 μ L/ holes, absorption 0.88 μ L liquid is added and mixed, dilute 250 times, the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and now drug dilution is 1000 times, and final concentration is 50 μ g/mL.Hatch 48h.Each concentration is established 4 (2) individual parallel multiple holes, every plate is established negative control group and (in cell, is added blank solution, blank solution is joined method---add the nutrient solution of 200 μ L, absorption 0.88 μ LDMSO is added and mixed), positive controls (Zorubicin final concentration 4 μ g/mL).SRB dyeing: after cell cultures finishes, take out culture plate, every hole adds trichoroacetic acid(TCA) (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, to remove TCA.After air drying, every hole adds 0.4% SRB100 μ L (1% chromatographically pure acetic acid dissolves), under room temperature, place 20min, discard in each hole and with 1% acetic acid, wash 5 times after liquid, remove unconjugated dyestuff, after air drying, with 10mmol/L Tris150 μ L/ hole, dissolve, vibration 5min, use microplate reader (ELx800) to measure, wavelength used is 490nm.
The calculating inhibiting rate of inhibiting rate is calculated as follows:
Drug effect the results are shown in Table 4.
Table 4
| Leafy twigs of Oriental Arborvitae C09 component | Negative | Blank | Positive | |
| Average cell survival number | 0.11 | 0.52 | 0.06 | 0.09 |
| Inhibiting rate (%) | 89.57 | 0.00 | 100.00 | 93.64 |
| RSD(％) | 13.09 | 8.36 | 6.98 | 2.95 |
| Leafy twigs of Oriental Arborvitae C11 component | Negative | Blank | Positive | |
| Average cell survival number | 0.10 | 0.52 | 0.06 | 0.09 |
| Inhibiting rate (%) | 92.28 | 0.00 | 100.00 | 93.64 |
| RSD(％) | 8.14 | 8.36 | 6.98 | 2.95 |
Cell strain: Hep G2 tumour cell
Cell cultures and kind plate cell cultures: use DMEM high glycoform (Gibco) [adding 3.7g/L sodium bicarbonate] 90%, foetal calf serum (folium ilicis chinensis) 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 residual content.Suspension cell on piping and druming culturing bottle wall, adds in centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L placentas, viable count sum NL on count plate, the cell count N in centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in centrifugal front centrifuge tube.Centrifugal (900rad/min, 10min), sucks supernatant liquor, adds nutrient solution V1mL, and piping and druming, mixes cell, draws V2mL and joins in cell groove, makes V2=NT/N * V1.In cell groove, add the nutrient solution of VT-V2mL again, with the volley of rifle fire, blow and beat, mix, get this liquid, every hole adds 100 μ L, hatches 24h.After kind plate, choose 4 holes and add nutrient solution as blank, residue hole adds 100 μ L PBS, to reduce the evaporation of nutrient solution.
Dosage regimen Leafy twigs of Oriental Arborvitae active principle, according to the weight of weighed medicine, according to the weight of weighed medicine, adds the DMSO of respective volume to dissolve, and concentration is about 50mg/mL.Concussion is dissolved, if there are a large amount of drops to glue wall, and can be suitably centrifugal.Can store-20 ℃.96 orifice plates change liquid, and every hole adds fresh medium 150 μ L.In 96 new orifice plates, add the nutrient solution in 220 μ L/ holes, absorption 0.88 μ L liquid is added and mixed, dilute 250 times, the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, and now drug dilution is 1000 times, and final concentration is 50 μ g/mL.Hatch 48h.Each concentration is established 4 (2) individual parallel multiple holes, every plate is established negative control group and (in cell, is added blank solution, blank solution is joined method---add the nutrient solution of 200 μ L, absorption 0.88 μ LDMSO is added and mixed), positive controls (Zorubicin final concentration 4 μ g/mL).MTT colorimetric method for determining: take out culture plate, every hole, place to go supernatant, add nutrient solution-MTT mixing solutions (nutrient solution: MTT solution=10: 1) 100 μ L, hatch 4h.Nutrient solution 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 inhibiting rate of inhibiting rate is calculated as follows:
Drug effect the results are shown in Table 5.
Table 5
| Leafy twigs of Oriental Arborvitae C09 component | Negative | Blank | Positive | |
| Average cell survival number | 0.33 | 0.57 | 0.16 | 0.19 |
| Inhibiting rate (%) | 59.76 | 0.00 | 100.00 | 91.71 |
| RSD(％) | 3.92 | 7.59 | 8.49 | 6.34 |
Cell strain: MCF-7 tumour cell
Make up a prescription: according to the weight of weighed medicine, add the DMSO of respective volume to dissolve, concentration is about 50mg/mL.Concussion is dissolved, if there are a large amount of drops to glue wall, and can be suitably centrifugal.Can store-20 ℃.
Cell cultures: use DMEM high glycoform (Gibco) [adding 3.7g/L sodium bicarbonate] 90%, calf serum (match is happy) 10%, non-essential amino acid (Gibco) 1% mixed culture MCF-7 cell.
Experimental technique:
1 kind of plate:
(1) calculate and to need cell total amount NT=ρ cellmL-1 * VT (ρ=2 * 103/mL), wherein VT=0.1mL * hole count+cell groove residual content.
(2) suspension cell on piping and druming culturing bottle wall, adds in centrifuge tube.Get 10 μ L, add the blue dilution of 10 μ L placentas, viable count sum NL on count plate, the cell count N in centrifuge tube is: NL/4 * 104 * 2 * V, wherein V is the liquor capacity in centrifugal front centrifuge tube.
(3) centrifugal (900rad/min, 10min), sucks supernatant liquor, adds nutrient solution V1mL, and piping and druming, mixes cell, draws V2mL and joins in cell groove, makes V2=NT/N * V1.
(4) in cell groove, add the nutrient solution of VT-V2mL again, with the volley of rifle fire, blow and beat, mix, get this liquid, every hole adds 100 μ L, hatches 24h.
(5) after kind plate, choose 4 holes and add nutrient solution as blank, residue hole adds 100 μ L PBS, to reduce the evaporation of nutrient solution.
2 dosings:
(1) 96 orifice plate changes liquid, and every hole adds fresh medium 150 μ L.
(2) in 96 new orifice plates, add the nutrient solution in 220 μ L/ holes, absorption 0.88 μ L liquid is added and mixed, dilute 250 times, the medicine that above-mentioned dilution is good has every hole, hole of cell to add 50 μ L to kind, now drug dilution is 1000 times, and final concentration is 50 μ g/mL.Hatch 48h.Each concentration is established 4 (2) individual parallel multiple holes, every plate is established negative control group and (in cell, is added blank solution, blank solution is joined method---add the nutrient solution of 200 μ L, absorption 0.88 μ LDMSO is added and mixed), positive controls (Zorubicin final concentration 4 μ g/mL).
3 MTT colorimetric method for determining:
(1) take out culture plate, every hole, place to go supernatant, add nutrient solution-MTT mixing solutions (nutrient solution: MTT solution=10: 1) 100 μ L, hatch 4h.
(2) inhale and abandon nutrient solution, every hole adds the DMSO of 150 μ L, vibration 10min, and 550nm microplate reader (Elx800) is measured.
The calculating inhibiting rate of 4 inhibiting rates is calculated as follows:
Drug effect the results are shown in Table 6.
Table 6
| , Leafy twigs of Oriental Arborvitae C09 component | Negative | Blank | Positive | |
| Average cell survival number | 0.09 | 0.15 | 0.06 | 0.09 |
| Inhibiting rate (%) | 67.31 | 0.00 | 100.00 | 70.33 |
| RSD(％) | 2.47 | 1.47 | 4.51 | 5.86 |
| , Leafy twigs of Oriental Arborvitae C11 component | Negative | Blank | Positive | |
| Average cell survival number | 0.09 | 0.15 | 0.06 | 0.09 |
| Inhibiting rate (%) | 71.43 | 0.00 | 100.00 | 70.33 |
| RSD(％) | 3.42 | 1.47 | 4.51 | 5.86 |
Beneficial effect of the present invention is:
1. in extraction and separation process of the present invention, use reverse phase silica gel post, can effectively remove the impurity that easily formation is extremely adsorbed on preparative chromatography post such as chlorophyll, improved the content of effective constituent, can obtain fast and accurately effective constituent.
2. Leafy twigs of Oriental Arborvitae active principle chemical composition provided by the invention is simply clear and definite, is easier to illustrate its mechanism of action on pharmacological research, is easier to aborning the quality control of medicine.
Method provided by the invention obtains containing C09, C11 active principle first from Leafy twigs of Oriental Arborvitae medicinal material, and it is carried out in various tumor cell strains to medicine efficacy screening first, because composition is definite, content is clear and definite, preparation technology is convenient, active good, the suitable anti-tumor Chinese medicine new drug that is developed to.
Accompanying drawing explanation
Fig. 1 is the HPLC analysis chart of Leafy twigs of Oriental Arborvitae active principle 1 of the present invention.
Fig. 2 is the HPLC analysis chart of Leafy twigs of Oriental Arborvitae active principle 2 of the present invention.
Embodiment
Below in conjunction with embodiments of the invention, further describe flesh and blood of the present invention, this embodiment does not only limit the present invention for the present invention is described.
Embodiment 1 Leafy twigs of Oriental Arborvitae active principle preparation
Leafy twigs of Oriental Arborvitae active principle preparation method of the present invention, comprises the following steps: after Leafy twigs of Oriental Arborvitae pulverizing medicinal materials, to add ethyl acetate and ethanol (1: 1), and reflux 1 hour, extracts 2 times, and merging filtrate obtains extracting solution; Extracting solution is condensed into medicinal extract, uses dissolve with ethanol loading, cross ODS-C18 post, first, adopt 30%EtOH (5BV; 1BV ≈ 250ml) as moving phase, obtain elutriant I, then change 95%EtOH (5BV) as moving phase, obtain elutriant II, will after elutriant II concentrate drying, obtain sample; With preparative liquid chromatography, continue the separated sample obtaining; The separation condition of preparative chromatography: chromatographic column is preparative column ZorbaxSB-C18; 21.2mm * 250mm, moving phase is water A and acetonitrile B, gradient elution program is as follows:
Flow velocity is 10ml/min, and column temperature is room temperature; Sample 100% dissolve with ethanol, separated through preparative liquid chromatography, at time period 36.0-40.0 minute or 44.0-48.0 minute, collect solution, solution obtains active principle 1,2 after concentrate drying.
The analysis of embodiment 2 Leafy twigs of Oriental Arborvitae active principles
HPLC-ELSD combination analysis to the Leafy twigs of Oriental Arborvitae active principle of embodiment 1
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 the acetonitrile solution containing 0.2% Glacial acetic acid mutually; Gradient elution program is as follows: in the time of 0 minute, and the acetonitrile solution of 0.2% Glacial acetic acid that 0.2% glacial acetic acid aqueous solution that mobile phase A is 90%, Mobile phase B are 10%; In the time of 10 minutes, the acetonitrile solution of 0.2% Glacial acetic acid that 0.2% glacial acetic acid aqueous solution that mobile phase A is 50%, Mobile phase B are 50%; In the time of 30 minutes, the acetonitrile solution of 0.2% Glacial acetic acid that 0.2% glacial acetic acid aqueous solution that mobile phase A is 5%, Mobile phase B are 95%; In the time of 35 minutes, the acetonitrile of 0.2% Glacial acetic acid that 0.2% glacial acetic acid aqueous solution that mobile phase A is 5%, Mobile phase B are 95%.Solution flow rate 0.5mLmin
-1; Detect wavelength all-wave long; 30 ℃ of column temperatures; ELSD condition: 105 ℃ of drift tube temperatures; Nitrogen flow rate 2.0L/min
the preparation of need testing solutiontake active principle of the present invention, with dissolve with methanol solution, in volumetric flask, be diluted to scale, shake up, obtain.
measuring methodthe accurate need testing solution of drawing, injection liquid chromatography, measures.
Embodiment 3 Leafy twigs of Oriental Arborvitae active principle preparations
Get the Leafy twigs of Oriental Arborvitae active principle 1 or 2 of embodiment 1,0.5g mixes with 10.5g PEG-4000, and heating and melting moves to after material in dripping pill drip irrigation, and liquid drops in 6-8 ℃ of whiteruss, and oil removing makes 400 of dripping pills.
Embodiment 4 Leafy twigs of Oriental Arborvitae active principle preparations
Get the Leafy twigs of Oriental Arborvitae active principle 1 or 2 of embodiment 1,0.5g, glucose 4.5g, Sulfothiorine 0.9g and distilled water 1ml, after said components mixes, lyophilize, 500 of packing, obtain.
Embodiment 5 Leafy twigs of Oriental Arborvitae active principle preparations
Get Lignum Dalbergiae Odoriferae oil 1.5g, join in the hydroxypropylβ-cyclodextrin that 13ml is saturated, stirring and dissolving, filters, filter leaf cryodrying, Lignum Dalbergiae Odoriferae oil and the inclusion compound powder of hydroxypropylβ-cyclodextrin.Except above-mentioned Lignum Dalbergiae Odoriferae oil closes the inclusion compound powder of hydroxypropylβ-cyclodextrin, then get the Leafy twigs of Oriental Arborvitae active principle 1 or 2 of embodiment 1,0.5g, N.F,USP MANNITOL 5.5g, Calcium Disodium Edetate 0.9g and distilled water 2ml, after said components mixes, lyophilize, 300 of packing, obtain.
Claims (6)
1. a Leafy twigs of Oriental Arborvitae active principle, is characterized in that, its preparation process comprises the following steps:
Step 1 is: gets Leafy twigs of Oriental Arborvitae medicinal material, take ethyl acetate: ethanol=1-5:1-5 as solvent, and refluxing extraction, extracting solution is separated with the dregs of a decoction, and the extracting solution obtaining after separation is extract 1,
Step 2 is: extract 1 is crossed to ODS-C18 post, first, adopt the 30%EtOH of 5BV as moving phase, 1BV=250ml; Then change the 95%EtOH of 5BV as moving phase, obtain elutriant,
Step 3 is: with preparative liquid chromatography, continue the separated elutriant obtaining, chromatographic column is preparative column ZorbaxSB-C18; 21.2mm * 250mm, moving phase is water-A and acetonitrile-B, carries out gradient elution,
Described gradient elution program is as follows:
Flow velocity is 10ml/min, and column temperature is room temperature; Sample 100% dissolve with ethanol, separated through preparative liquid chromatography, at time period 36.0-40.0 minute or 44.0-48.0 minute, collect solution, solution obtains active principle after concentrate drying respectively.
2. the active principle of claim 1, is characterized in that, the mixture of ethyl acetate described in step 1 and ethanol, and both ratios are ethyl acetate: ethanol=1-2:1-2.
3. the active principle of claim 1, is characterized in that, the mixture of ethyl acetate described in step 1 and ethanol, and both ratios are ethyl acetate: ethanol=1:1.
4. the pharmaceutical composition that contains any one active principle of claim 1-3.
5. a preparation method for Leafy twigs of Oriental Arborvitae active principle, is characterized in that, its preparation process comprises the following steps:
Step 1 is: gets Leafy twigs of Oriental Arborvitae medicinal material, take ethyl acetate: ethanol=1-5:1-5 as solvent, and refluxing extraction, extracting solution is separated with the dregs of a decoction, and the extracting solution obtaining after separation is extract 1,
Step 2 is: extract 1 is crossed to ODS-C18 post, first, adopt the 30%EtOH of 5BV as moving phase, 1BV=250ml; Then change the 95%EtOH of 5BV as moving phase, obtain elutriant,
Step 3 is: with preparative liquid chromatography, continue the separated elutriant obtaining, chromatographic column is preparative column ZorbaxSB-C18; 21.2mm * 250mm, moving phase is water-A and acetonitrile-B, carries out gradient elution,
Described gradient elution program is as follows:
Flow velocity is 10ml/min, and column temperature is room temperature; Sample 100% dissolve with ethanol, separated through preparative liquid chromatography, at time period 36.0-40.0 minute or 44.0-48.0 minute, collect solution, solution obtains active principle after concentrate drying respectively.
6. the preparation method of claim 5, is characterized in that, step is as follows:
To after Leafy twigs of Oriental Arborvitae pulverizing medicinal materials, add ethyl acetate: ethanol=1:1, reflux 1 hour, extracts 2 times, and merging filtrate obtains extracting solution; Extracting solution is condensed into medicinal extract, crosses ODS-C18 post, first, adopt the 30%EtOH of 5BV as moving phase, 1BV=250ml; Then change the 95%EtOH of 5BV as moving phase, obtain elutriant II, will after elutriant II concentrate drying, obtain sample; With preparative liquid chromatography, continue the separated sample obtaining; The separation condition of preparative chromatography: chromatographic column is preparative column Zorbax SB-C18; 21.2mm * 250mm, moving phase is water A and acetonitrile B, gradient elution program is as follows:
Flow velocity is 10ml/min, and column temperature is room temperature; Sample 100% dissolve with ethanol, separated through preparative liquid chromatography, at time period 36.0-40.0 minute or 44.0-48.0 minute, collect solution, solution obtains active principle after concentrate drying respectively.
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