Summary of the invention
Purpose of the present invention is used gallic acid exactly and is prepared anti-tumor drug, and the medicine of making can obviously suppress the growth of hepatoma carcinoma cell and human cervical carcinoma cell, and effect significantly.Also can be made into the pharmaceutical composition that contains gallic acid, this pharmaceutical composition can be one or more active component natural or chemosynthesis, also can be the pharmaceutical composition that one or more Chinese crude drugs or its active component and gallic acid are formed.
When the present invention was used to prepare anti-tumor drug, its oral or non-oral administration all was safe.Under oral situation, it can be any conventionally form administration, as powder, granule, tablet, capsule, pill, drop pill, soft capsule, leafing agent, oral liquid, suspension, syrup, buccal tablets, spray or aerosol etc.; When the non-oral administration of this medicine, can adopt any conventionally form, as suppository, injection: intravenous injection, intramuscular injection, ointment, inhalant etc.
It is that its excipient with solid or liquid constitutes that the present invention prepares anti-tumor drug, solid used herein or liquid excipient are well known in the art, lift several object lessons below, the excipient of solid preparation has lactose, starch, paste essence, calcium carbonate, synthetic or puritan filler aluminum, magnesium chloride, magnesium stearate, sodium bicarbonate, dry yeast etc.; The excipient of liquid preparation has water, glycerol, propylene glycol, simple syrup, ethanol, ethylene glycol, Polyethylene Glycol, Sorbitol etc.; The excipient of ointment can use fatty oil, agnolin, vaseline, glycerol, Cera Flava, wood are cured, liquid paraffin, resin, advanced wax etc. are combined into water-repelling agent or hydrophilizing agent.
Dosage of the present invention can be according to the mode of taking, patient's age and body weight and be in a bad way degree and other similar factor and change, and oral dose is: 20-300mg/ people, take for three times every day; The injection consumption is 10-200mg/ people, once a day.
Its effect of drugs is to show to the inhibiting observation of the hepatoma carcinoma cell of isolated culture with to the influence of human cervical carcinoma cell growth in vitro by gallic acid.
Experimental example 1 gallic acid is to the inhibitory action of the hepatoma carcinoma cell of isolated culture
1, materials and methods
1.1 the gallic acid that is subjected to the reagent thing to take traditional extraction process to obtain.
1.2 tumor strain human liver cancer cell QGY-7403.
1.3 reagent and instrument RPMT1640 cultivate powder, the GIBCO U.S. produces;
3H-TdR, Chinese Academy of Sciences Institute for Atomic Research provides; CO
2Incubator; PIKA-KOGYO Japan produces; 96 well culture plates; PKI-1002CO
2Incubator (PIKA-KOGYO Japan); LKB-1214 type ReckBeta liquid scintillation counter (Sweden).
1.4 gallic acid treats that to the conventional recovery cell of the inhibiting observation of the hepatoma carcinoma cell of isolated culture cell growth state is good, adjusting hepatoma carcinoma cell density is 1 * 10
6Individual/ml, the flat culture plate in 96 holes, every hole adds cell suspension 180 μ l, adds each 20 μ l of variable concentrations gallic acid simultaneously, and each concentration is all established three multiple holes, and the every hole of matched group adds 20 μ l culture fluid, places CO
2Every hole adds after cultivating 2h in the incubator
3H-TdR20 μ l places CO again
2Cultivate 1.5h in the incubator, with bull cell harvestor catcher collecting cell, filter membrane is used liquid flashing counting determining after doing
3The H-TdR incorporation, the result represents with Cpm, calculates and suppresses percentage rate.
2, gallic acid is to the inhibitory action of hepatoma carcinoma cell
Experiment shows that various dose is subjected to reagent thing and tumour inhibiting rate amount effect relationship, the results are shown in Table 1
Group dosage of cells incorporation tumour inhibiting rate
(g·L
-1) (X±S) (%)
Matched group 0 11428.37 ± 1287.05 0
Gallic acid 0.11 11355.25 ± 643.35 0.64
0.23 10951.05±118.30 4.17
0.47 10521.15±626.95 7.94
*
0.94 8889.18±262.41 22.22
*
1.88 7952.33±399.19 30.42
*
3.75 6510.20±1025.38 43.03
*
7.50 2749.19±517.82 75.49
**
15.00 138.68±46.51 98.79
**
*P<0.05,
**P<0.01,
The result shows that gallic acid has stronger inhibitory action to hepatoma carcinoma cell.
Experimental example 2, gallic acid are to the influence of human cervical carcinoma Hela cell's growth in vitro
1, materials and methods
1.1 the gallic acid that is subjected to the reagent thing to take traditional extraction process to obtain.Quantitatively take by weighing before the experiment, ultraviolet radiation sterilization.The dissolving of 10% calf serum PRMI-1640 culture fluid ,-20 refrigerators are preserved standby.
1.2 tumor strain human cervical carcinoma Hela cell strain.
1.3 reagent and instrument: RPMI-1640 culture medium (U.S. Sigma company), pancreatin (U.S. Sigma company), newborn calf serum, tetramethyl azo azoles indigo plant (U.S. Sigma company), 96 well culture plates (Denmark), fluorouracil/Fluorouracil/5-fluoro-2,4 (
1H,
3H)-hybar X (Tianjin 030512).Enzyme-linked immunosorbent assay instrument (Japan), CO
2Incubator (U.S.), inverted microscope (Japanese olympua), optical microscope (Japanese olympua), super-clean bench (Shanghai), centrifuge (U.S. Sokuall RC5B type)
1.4 cell culture: tumor cell Hela cell strain is available from Jilin Province's treatment and prevention of tumour institute, and 10% newborn calf serum, RPMI-1640 basic culture solution are put 37 ℃, 5% CO
2Cultivate in the incubator, saturated humidity went down to posterity once in 3-4 days.
2, tetrazolium salts (MTT) colorimetry detects the influence of gallic acid to human cervical carcinoma Hela cell's growth
Get human cervical carcinoma Hela cell's exponential phase of growth, after the trypsinization,, make 2 * 10 to contain the RPMI-1640 culture fluid diluting cells of 10% calf serum
4/ ml cell suspension adds in 96 well culture plates, and every hole 100 μ l put 37 ℃, 5%CO
2, cultivate 24~28 hours in the saturated humidity incubator after, the gallic acid culture fluid that adds variable concentrations is the dosage group, adds the positive matched group of 5 fluorouracil culture fluid that contains 25ug/mL, and the culture fluid that adds equivalent is made the blank group, each concentration is established 3 holes, puts 37 ℃, 5%CO
2Cultivated 30 hours in the incubator, in finishing preceding 4 hours of cultivation, every hole adds MTT20 μ l, then, continues to cultivate 4 hours, stops cultivation, the centrifugal supernatant of abandoning, and every hole adds dimethyl sulfoxide 150 μ l, micro-oscillator concussion 5min, microplate reader λ automatically
490nmMeasure light absorption value, calculate the variable concentrations gallic acid, see Table 2 the Hela inhibitory rate of cell growth.
The light absorption value OD that tetrazolium salts (MTT) colorimetry records represents that with x ± s data are checked with F check and q and carried out statistical disposition, dosage and effect relation linear regression analysis and correlation analysis, and the logarithm probit method is asked IC
50
3, result
Experiment shows, variable concentrations be subjected to the reagent thing to the Hela cell have stronger toxicity (
*P<0.01 or
*P<0.05) and tangible dose-effect relationship arranged.Behind the variable concentrations gallic acid effect 30h, cell growth is suppressed, and exists significantly to rely on effect between concentration and suppression ratio, and both be proportionate (r=0.9860, P<0.01) are shown in the correlation regression analysis; Regression equation is y=0.2132x-0.0092, and calculating can act on the half effective inhibition concentration IC of 30h
50Be 10.90ug/mL.Statistical analysis shows that each processed group has significant difference (P<0.05) to the Hela cyto-inhibition between following each processed group of 100ug/mL dosage group.
Table 2 variable concentrations gallic acid is bred influence to the human cervical carcinoma Hela cell
Group dosage (ug/mL) OD
490nmValue (the tumour inhibiting rate (%) of x ± s)
Negative control 0 0.392 ± 0.0982 0
Positive control 25 0.165 ± 0.0273 57.91%
Gallic acid 500 0.024 ± 0.0041 93.88%
*
250 0.025±0.0049 93.62%
**
100 0.035±0.0041 91.07%
**
50 0.048±0.0196 87.76%
**
25 0.106±0.0114 72.96%
**
12.5 0.188±0.0300 52.04%
**
6.25 0.257±0.0713 34.44%
*
3.125 0.299±0.0361 23.72%
*
With compare
*P<0.05,
*P<0.01; Relatively (respectively organize) P<0.05 between the medication group below the 100ug/mL