Summary of the invention
The objective of the invention is to overcome defective and the deficiency that prior art exists, a kind of " Actinidin " with antitumor action and its production and application is provided.
Purpose of the present invention is achieved through the following technical solutions: Actinidin is the total effective parts of a kind of compound anthraquinone glycoside etc., use 75% dissolve with ethanol, making becomes 1-10mg/ml alcohol liquid, point sample is (110 ℃ activate 1 hour) on high-efficient silica gel G thin layer chromatography (tlc) plate, the configuration chloroform: methanol: water: formic acid=3: 2: 0.2: 0.04 ratio is done developing solvent, the exhibition that launches is apart from 17cm, and taking-up is dried, and fluorescence down or sulphuric acid vanillin liquid spraying colour developing.Speckle (Fig. 1) with the Rf value in 0.00 to 0.100,0.167 to 0.300,0.400 to 0.533,0.700 to 0.833 or 0.900 to 0.967 scope shows that with methanol anion scanning the typical ion peak spectrum of Actinidin is 245,289,357,577,865amu (Fig. 2).
Actinidin preparation method and application comprise the steps:
1) Radix Actinidiae Chinensis decocting, decocting liquid is got supernatant concentration, adds chitosan clarification after-filtration, gets clear paste liquid;
2) clear paste liquid is through macroporous adsorption resin chromatography, and water is eluted to colourless, reuse organic solvent chromatography, and a stream part collection concentrates, and gets concentrated solution;
3) concentrated solution is adsorbed with polyamide, water is eluted to colourless, reuse organic solvent eluting, and eluent concentrates, and drying gets Actinidin.
4) Actinidin is added suitable adjuvant and additive after, make medicaments such as thin membrane coated tablet, hard capsule, mixture, soft capsule, granule, injection, liposome.
Water in the step 1) is deionized water or distilled water, and the 6-12 that adds water and be medical material weight doubly measures, and decocts each 1-3 hour 1-3 time; Be concentrated into 1: 1 or relative density 1.06-1.10 of medical material amount; Used chitosan be chitin take off the acetyl product, consumption is the 0.05-5% of medical material; Filter with filter cloth, filter paper, filter membrane.
Step 2) macroporous adsorbent resin in is D type or HPD type series; Granularity 20-60 order or 0.3mm-1.2mm; The aperture
Consumption is 0.5-2 a times of medical material amount; Flow velocity 0.5-10ml/g/ minute; Eluting water is deionized water or distilled water, and chromatography adopts organic solvent: 15%-30% ethanol, acetone or 50-85% ethanol, acetone; Collect the eluent of 50-85% ethanol or acetone, being concentrated into relative density is 1.100-1.350.
The polyamide consumption that adsorbs in the step 3) is 0.5-50 a times of concentrated solution; Granularity 100-120 order; The eluting organic solvent is ethanol or the acetone of 50-85%, preferred 50-75%; The concentrated solution relative density is 1.15-1.35; Dry vacuum available oven dry, vacuum-0.06-0.1Mpa, temperature≤80 ℃; Or centrifugal spray drying, centrifugal degree α=20000-50000; Inlet temperature 120-220 ℃, medicinal liquid flow velocity 10-200ml/ branch; Or vacuum lyophilization, vacuum-0.06-0.1Mpa, temperature-40 ℃-25 ℃, time 5-30 hour.
Adjuvant and additive that Actinidin in step 3) and the step 4) adds comprise cellulose family, stearic acid lipid, sodium benzoate, sucrose, starch, dextrin, cyclodextrin, lactose, the pre-powder process of Opadry film coating, sweet lactonaphthol, glycerol, sodium sulfite, PVP, PEG200-5000, two palmityl phosphatidyl alkali, two palmityl PHOSPHATIDYL ETHANOLAMINE, cholesterol, lecithin.Make medicaments such as thin membrane coated tablet, hard capsule, soft capsule, granule, injection, liposome.
Thin membrane coated tablet:
Actinidin 30-80%
Dextrin 1-20%
Lactose 1-20%
The pre-powder process 0.5-5% of Opadry film coating
PVP ethanol liquid 0.5-40%
Hard capsule:
Actinidin 30-90%
Dextrin 1-20%
Lactose 1-20%
PVP ethanol liquid 0.5-40%
Granule:
Actinidin 30-90%
Dextrin 1-20%
Lactose 1-20%
PVP ethanol liquid 0.5-40%
Mixture:
Actinidin 30-90%
Sucrose 1-20%
PEG400 1-10%
Antiseptic 0.5-1
Soft capsule:
Actinidin 30-90%
PEG4000 1-20%
Lactose 1-20%
Gelatin 1-20%
Glycerol 1-5%
Injection:
Actinidin 1-30%
Sodium sulfite 0.1-2%
Propylene glycol 0.1-10%
Water for injection 65-98%
Liposome:
Actinidin 1-30%
PEG200-5000 1-20%
Two palmityl phosphatidyl alkali 1-30%
Cholesterol 1-5%
Water for injection 0-96%
Actinidin of the present invention adopts mass spectral analysis, has determined the ion characteristic collection of illustrative plates, for further separation and purification provides definite target.Actinidin can be used for digestive tract tumor such as anti-treatment of esophagus cancer, gastric cancer, colorectal cancer, cancer of pancreas, hepatocarcinoma.
The invention provides the method for extracting Actinidin from Radix Actinidiae Chinensis, use this decocting method with deionized water or distilled water, impurity is few, effective ingredient rate of transform height; Chitosan clarification medicinal liquid is easy, economical, efficient, and loss of effective components is few; And chitosan is nontoxic, can not cause any bad side reaction to extract; Biggest advantage can be adsorbed objectionable impurities such as removing heavy metal when handling medicinal liquid.
Because macroporous adsorbent resin good separating effect, cost is low, easily produce, the present invention prepares in the method for Actinidin mainly the calculating according to D, DA series, HPD series plastics adsorbance, determine that the macroporous adsorbent resin consumption is the 0.5-2 optimum amount doubly of medical material amount, fully guarantee to adsorb effective ingredient, reduce the waste of resin;
The polyamide process for purification is that commercially available polyamide is milled into 100-120 purpose fine grained, more reasonably uses the polyamide consumption, improves the purity of raw material Actinidin, makes production operation easy, process stabilizing.
Specific implementation method
Concrete preparation method of the present invention illustrated by following routine embodiment, but protection scope of the present invention is not limited to this.
Embodiment 1
Get Radix Actinidiae Chinensis 100kg, add the 1200L deionized water, soaked 30 minutes, heated and boiled keeps boiling 3 hours, the leaching medicinal liquid; Decoction for the second time adds the 1000L deionized water, decocts 2 hours; Decoction for the third time adds the 800L deionized water, decocts 1 hour; Merge three times decocting liquid, standing over night, the leaching supernatant, vacuum concentration is to relative density 1.08; Clarifier chitosan 200g (with 1% acetum 20L dissolving), add in the water extracting liquid, stirring was left standstill 12 hours, and the leaching supernatant is by the pillar of D101-I type macroporous adsorbent resin 200kg, flow velocity 1L/ minute, be eluted to deionized water colourless, reuse 15% ethanol 200L eluting, and with 75% ethanol 500L eluting, collect 75% ethanol elution, reclaim ethanol, be concentrated into relative density 1.25 (70 ℃), admix the 25kg polyamide, the polyamide column of packing into, behind water and 15% ethanol elution,, collect 75% eluent with 75% ethanol 50L eluting, reclaim ethanol, be concentrated into relative density 1.35, at vacuum-0.06-0.1Mpa, 60 ℃ of following vacuum dryings of temperature, pulverize, obtain the Actinidin of 3.1kg.
Embodiment 2
Get Radix Actinidiae Chinensis 100kg, add the 1000L deionized water, soaked 45 minutes, heated and boiled keeps boiling 3 hours, the leaching medicinal liquid; Decoction for the second time adds the 800L deionized water, decocts 2 hours; Decoction for the third time adds the 800L deionized water, decocts 1 hour; Merge three times decocting liquid, standing over night, the leaching supernatant, vacuum concentration is to relative density 1.06; Clarifier chitosan 500g (with 1% acetum 50L dissolving) adds in the water extracting liquid, stirs and leaves standstill 12 hours, the leaching supernatant, by the pillar of HPD300-type macroporous adsorbent resin 200kg, flow velocity 1.2L/ minute, be eluted to colourless with deionized water, reuse 15% acetone 200L eluting, and, collect 75% acetone eluent with 75% acetone 500L eluting, reclaim acetone, be concentrated into relative density 1.30 (70 ℃), admix the 30kg polyamide, the polyamide column of packing into is behind water and the 15% acetone eluting, with 75% acetone 50L eluting, collect 75% eluent, reclaim acetone, be concentrated into relative density 1.15, centrifugal spray drying, centrifugal degree-20000-50000; Inlet temperature 120-220 ℃, medicinal liquid flow velocity 10-200ml/ branch; Obtain the Actinidin of 2.8kg.
Embodiment 3
Get Radix Actinidiae Chinensis 100kg, add the 1000L deionized water, soaked 45 minutes, heated and boiled keeps boiling 3 hours, the leaching medicinal liquid; Decoction for the second time adds the 800L deionized water, decocts 2 hours; Decoction for the third time adds the 800L deionized water, decocts 1 hour; Merge three times decocting liquid, standing over night, the leaching supernatant, vacuum concentration is to relative density 1.06; Clarifier chitosan 750g (with 1% acetum 75L dissolving), add in the water extracting liquid, stirring was left standstill 24 hours, and the leaching supernatant is by the pillar of DA201-type macroporous adsorbent resin 200kg, flow velocity 1.0L/ minute, be eluted to deionized water colourless, reuse 15% acetone 200L eluting, and with 75% acetone 500L eluting, collect 75% acetone eluent, reclaim acetone, be concentrated into relative density 1.30 (70 ℃), admix the 25kg polyamide, the polyamide column of packing into, behind water and the 15% acetone eluting,, collect 75% eluent with 75% acetone 50L eluting, reclaim acetone, be concentrated into relative density 1.15, vacuum lyophilization, vacuum-0.1Mpa, temperature-40 ℃ rises to 25 ℃, 25 hours time.Obtain the Actinidin of 3.8kg.
Embodiment 4
Actinidin, use 70% dissolve with ethanol, making becomes 1-10mg/ml alcohol liquid, point sample is (110 ℃ activate 1 hour) on high-efficient silica gel G thin layer chromatography (tlc) plate, the configuration chloroform: methanol: water: formic acid=3: 2: 0.2: 0.04 ratio is done developing solvent, the exhibition that launches is apart from 17cm, and taking-up is dried, and fluorescence down or sulphuric acid vanillin liquid spraying colour developing.Speckle with the Rf value in 0.00 to 0.100,0.167 to 0.300,0.400 to 0.533,0.700 to 0.833 or 0.900 to 0.967 scope.
Embodiment 5
Actinidin 400g
Dextrin 40g
Lactose 60g
The pre-powder process 10g of Opadry film coating
PVP ethanol liquid 0.5-40%
Make 1000
Embodiment 6
Actinidin 400g
Dextrin 40g
Lactose 60g
PVP ethanol liquid 0.5-40%
Make 1000 capsules
Embodiment 7
Actinidin 800g
Dextrin 1000g
Lactose 1200g
PVP ethanol liquid 0.5-40%
Make the 3g/1000 bag
Embodiment 8
Actinidin 100g
Sucrose 200g
PEG400 20g
Antiseptic 0.5-1%
Distilled water adds to full dose
Make 1000ml
Embodiment 9
Actinidin 400g
PEG4000 200g
Lactose 100g
Gelatin 100g
Glycerol 50g
Make 1000
Embodiment 10
Actinidin 10g
Sodium sulfite 2g
Propylene glycol 100ml
Water for injection adds to full dose
Make 1000ml
Embodiment 11
Actinidin 50g
PEG200-5000 50g
Two palmityl phosphatidyl alkali 100g
Cholesterol 5g
Make pro-liposome
Embodiment 12
With pro-liposome, add the injection water, cross the sterilization of 0.22 microporous filter membrane, fill N2, embedding gets lipidosome injection.
Embodiment 13
With pro-liposome, add the mannitol of injection water and 10%, cross the sterilization of 0.22 μ microporous filter membrane, packing, lyophilization gets injectable powder.
Embodiment 14
Movable normal behind the oral Actinidin extractum of mice, the end sees that animal has overt toxicity reaction and death condition, growth promoter normal (seeing below the body weight gain table).Huge inspection is dissected in off-test, and obvious pathological change is seen at the main organs end.
Experiment conclusion:
When the disposable oral Actinidin of mice is equivalent to Radix Actinidiae Chinensis 750g crude drug/kg, do not see that this medicine has any toxic reaction, it is safe taking in the dosage more than 370 times according to one day clinical consumption conversion Actinidin of people.
Embodiment 15
The extracorporeal anti-tumor function of Actinidin
1, is subjected to the reagent thing
Actinidin sample preparation: after the DMSO dissolving, add solution or uniform suspension that PBS is made into 100 μ g/ml, dilute with PBS then.
2, cell strain
Human breast carcinoma (Bcap-37), the former leukemia of the chronic marrow of people (K562), human breast carcinoma (MCF-7), human colon carcinoma (HT-29), murine melanoma (K111), become human lung fibroblast (WI38), Chinese hamster pneumonocyte (CHL), l cell (3T3), people's hepatocarcinoma (SMMC7721), human leukemia cell (W256), the above-mentioned cell strain of Humanmachine tumour (SKMEL28) frozen and go down to posterity by this laboratory.
3, culture medium
(1) RPMI (1640) cell culture medium (GIBCO): contain 10% deactivation newborn calf serum (Saida biological Pharmaceutical Co., Ltd., Shanghai city); The L-glutaminase (the import packing, SANGON); Sodium Pyruvate; 1 * 10
5U.L
-1Penicillin, 100mg.L
-1Streptomycin; Aseptic filtration, 4 ℃ of preservations.
(2) 0.25% trypsin solutions (Trypsin): available from Invitrogen company ,-20 ℃ of preservations.
(3) phosphate buffer (PBS): NaCl8g, KCl0.2g, Na
2HPO
41.15g, KH
2PO
40.2g, be dissolved in the 1L distilled water, 121 ℃ of autoclave sterilization 20min, 4 ℃ of preservations.
(4) MTT (AMRESCO) solution: be made into 5mg/ml solution with PBS.
(5) lysate: every 100ml deionization distilled water contains SDS10g, isobutanol 5ml, concentrated sulphuric acid 0.12ml.
4, experimental technique
Cytotoxicity to above-mentioned tumor cell line records by mtt assay.Concrete steps are as follows:
(1) sample preparation: sample is made gradient dilution with PBS, obtain the dilute sample that concentration is respectively 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml, 0.01 μ g/ml, 0.001 μ g/ml.
(2) will dilute good sample and add in flat 96 orifice plates, every hole 10 μ l make two parallel testings at every.The corresponding work of DMSO added in the entering plate, behind the gradient dilution in contrast.
(3) get the cell that is in exponential phase, cell is suspended in the RPMI-640 culture medium that contains 10% calf serum after trypsinization and washing, through the blue dyeing of Placenta Hominis exclusive method meter viable count, and regulates cell suspending liquid density to 2 * 10
5Cell/ml.
(4) in flat 96 orifice plates, every hole adds 90 microlitre cells, overnight incubation in 37 ℃, 5%CO2 cell culture incubator.
(5) flat 96 orifice plates that will add cell insulation 48 hours in 37 ℃, 5%CO2 cell culture incubator.
(6) add 20 μ l 5mg/mlMTT solution in every hole, continue in incubator, to be incubated 3~4 hours.
(7) every hole adds 100 μ l DMSO, continues incubated overnight in incubator, next day, surveys the optical density (can adopt the DG3022 enzyme-linked immunosorbent assay instrument) in each hole with the A570 wavelength.Each grade test solution hole average and negative control value relatively get the percent inhibition (establishing certain density standard antineoplastic agent simultaneously as positive control) of each grade concentration.Computing formula is:
IC%=(C-T)/Cx100%
T is the negative matched group OD of a test solution group OD value C value
According to each IC% that detects each grade of liquid concentration, try to achieve IC then with regression curve
50Value.
5, experimental result
The results are shown in following table 1, Actinidin has the good antitumor effect to the former leukemia of the chronic marrow of people (K562), human colon carcinoma (HT-29), people's hepatocarcinoma (SMMC7721), Humanmachine tumour (SKMEL28), murine melanoma (K111), human breast carcinoma (MCF-7), murine melanoma (K111), human colon carcinoma (HT-29) cell strain.
Table .1 sample water is carried, ethanol extract is to the in-vitro multiplication inhibitory action of each cell strain:
Embodiment 16
The zoopery that Actinidin suppresses tumor sees Table 2
Method:
1, laboratory animal: Kunming mouse, ♀, 19-21g, by Shanghai Si Laike animal responsibility company limited.The quality certification number: SCXK (Shanghai) 2003-003
2, tumor source source: S180 tumor source has this laboratory to go down to posterity to protect kind
3, experimental technique:
Eugonic mice S180 ascites cells is diluted by 1: 5 with normal saline, mice one side oxter inoculation 0.2ml diluent, random packet, every group of 10 mices.Different extract medication groups, blank group are inoculated administration in the 2nd day, and continuous 7 days, put to death mice on the 9th day, take off the tumor piece and weigh, calculate tumour inhibiting rate.
4. experimental result: table 2
Group |
Sex |
Quantity |
Dosage (crude drug g/kg) |
Tumour inhibiting rate (%) |
1L |
♀ |
10 |
4 |
16.36 |
1H |
♀ |
10 |
12 |
34.71 |
2L |
♀ |
10 |
8 |
16.48 |
2H |
♀ |
10 |
24 |
30.87 |
3L |
♀ |
10 |
20 |
34.71 |
3H |
♀ |
10 |
50 |
44.61 |
4L |
♀ |
10 |
20 |
52.80 |
4H |
♀ |
10 |
50 |
60.98 |
Annotate: (1) water extract 10g crude drug/ml; (2) ethanol extraction 10g crude drug/g; (3) macroporous resin eluate 15g crude drug/g; (4) Actinidin.
Embodiment 17
1. test objective
The test Actinidin is to the preliminary curative effect (table 3) of rat liver cancer (HAC) solid tumor
2. be subjected to the reagent thing
Actinidin; Compound method:, now with the current with normal saline dilution (0.1g/ml).
3. animal
Source and kind: Kunming mouse is provided by the court's Animal House
The quality certification number: Shanghai is moving closes the card word No. 107
Body weight: 19-21 gram
Sex: female
Each treated animal number: 10
4. transplanted tumor
Rat liver cancer (HAC cell) is that pharmacological room of Shanghai Institute of Pharmaceutical Industry goes down to posterity and keeps
5. Shi Yan key step and method
Get well-grown rat liver cancer (HAC cell), after normal saline dilution in 1: 4, every mice axil subcutaneous vaccination 0.2ml, random packet is established normal saline matched group and cyclophosphamide positive controls (100mg/kg, ip * 3 (1,3,5)), lot number 980401, sample sets (6.25,12.5,25ml/kg).Work giving different pharmaceutical next day, 0.5ml/ Mus, continuous irrigation stomach 7 days.Inoculate back 10 days and take off neck execution animal, dissect and get the tumor piece, relatively the size of each dosage group tumor weight.The result judges according to following formula:
6. result
Table 3 Actinidin is to the tumor-inhibiting action of rat liver cancer (HAC)
Sample |
Dosage (ml/kg) |
Dosage regimen |
Number of animals all the time |
Heavy (g) x ± SD of tumor |
Tumour inhibiting rate % |
Blank (normal saline) |
25 |
po×7 |
1010 |
3.18±0.77 |
|
Actinidin |
6.25 12.5 25 |
po×7 po×7 po×7 |
1010 1010 1010 |
2.13±0.33 1.93±0.73 1.50±0.32 |
33.02
** 39.31
** 52.83
** |
CTX |
100mg/kg |
ip×3 |
1010 |
0.17±0.07 |
94.66
** |
Compare with the blank group:
*P<0.01.
From last table 3 as seen, Actinidin has certain inhibitory action to rat liver cancer (HAC) when 25ml/kg dosage, and tumour inhibiting rate is 52.83%, has compared significant difference (P<0.01=with the blank group.
Attached: test report
One. test apparatus:
The ABI 3200Q Trap level Four bar mass spectrograph (u.s.a. applied biosystem company) of connecting
Two. test procedure:
1. sample configuration: take by weighing sample 1.0mg, be dissolved in 1mL methanol, be made into 1mg/mL solution; Being diluted to concentration with methanol is 5ug/mL solution.
2. get methanol and do positive and negative ion scanning, sweep limits: 100~1000amu respectively;
3. get above-mentioned 5ug/mL sample solution and do positive and negative ion scanning, sweep limits: 100~1000amu respectively.
Three. test results and analysis:
1. result of the test is seen accompanying drawing 2-5.
2. interpretation of result
Compare with methanol cation scintigram, the scanning of sample cation does not see that strong characteristic ion peak is arranged.
With methanol anion scintigram relatively, in the scanning of sample anion occurs, occur 289, the strong quasi-molecular ions of 577amu, have characteristic.Simultaneously, because therefore 289=(577+1)/2 infers that 289 and 577 structures have certain dependency.In addition 245,357,865amu also has the characteristic ion peak
The typical ion peak spectrum of Actinidin is 245,289,357,577,865amu.
For further separation and purification provides definite target to Actinidin.
3. fill up the shortage of pharmaceutical preparation of the effective site of Radix Actinidiae Chinensis, develop the novel form of single medicinal material, reach the purpose of declaring the new drug dosage form.