CN101120950A - Application of realgar microorganism extracting liquid in preparing anti-tumor medicine - Google Patents
Application of realgar microorganism extracting liquid in preparing anti-tumor medicine Download PDFInfo
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- CN101120950A CN101120950A CNA2007102008258A CN200710200825A CN101120950A CN 101120950 A CN101120950 A CN 101120950A CN A2007102008258 A CNA2007102008258 A CN A2007102008258A CN 200710200825 A CN200710200825 A CN 200710200825A CN 101120950 A CN101120950 A CN 101120950A
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Abstract
The present invention relates to the application of the realgar microorganism leaching solution in anti tumor medicine preparation. The realgar microorganism leaching solution is prepared according to the exposed microorganism treating method in patent 2006102000675. The realgar leaching solution in the present invention has a profound inhabiting effect in a plurality of cancer disease comprising cancer of the liver, gastric cancer, leukemia and lung cancer. In addition, the application quantity is in level of microgram. Furthermore, because the realgar microorganism leaching solution is a dissoluble medicine, therefore the medicine can be applied respectively or applied in combination with other drugs, or with pharmaceutical acceptable excipient. The medicine is easy to be produced as various kinds of preparations, which comprises guttate pills, aerosol, vinum, syrups, oral application liquid, injection and injectable powder.
Description
Technical field
The present invention relates to the application of realgar microorganism extracting liquid in the preparation antitumor drug.
Background technology
Main Red Arsenic Sulfide (the As that contains of Realgar (Realgar)
4S
4), there is the people to think that also it is realgar (As that its master contains thing
2S
2), have another name called gold stone, be common minerals as Chinese medicine.Since Shennong's Herbal, in the medical history in existing two over thousands of years of China.Realgar has effects such as detoxifcation parasite killing, removing dampness and eliminating phlegm, preventing the attack (or recurrence) of malaria, is mainly used in carbuncle furuncle, snakeworm mosquito wound, abdominal pain due to worm stagnation, infantile convulsion, malaria etc.
[1], modern medicine thinks that Realgar has antitumor action, has been successfully used to chronic clinically and treatment acute myeloblastic leukemia
[2,3,4], therefore more and more be subjected to the extensive concern of Chinese scholars, become a focus of mineral Chinese medicine research.
Mineral drug is the culture rarity of the Chinese nation, but because its water-insoluble, causes bioavailability low, problems such as clinical consumption is big, toxicity height, the development that has seriously restricted mineral Chinese medicine with crack the international market
[5]
Realgar is the important component of many Chinese patent medicines as a kind of important mineral Chinese medicine, and the export trade of China's Chinese patent medicine is obstructed repeatedly because of Hg and As equal size exceed standard.Therefore, find out a kind of effectively stripping mineral drug effective ingredient, reduce the mineral drug use amount, increase curative effect, to reduce toxic mineral drug concocting method significant.
Compare Realgar ultramicro powder and nanometer grade powder with traditional processing procedure
[6]Preparation on certain degree, increased the dissolution characteristic of medicine with using, thereby make the aspects such as absorption, distribution, bioavailability of Realgar all obtain raising in various degree, strengthened drug effect.But this class is handled and still can only be used as medicine with suspension, remains the GI irritation that causes oral, takes problems such as difficulty, and produce nanometer-size realgar needs main equipment and advanced technology in addition, and universal use is still had any problem
[7]
At present, a high-new biotechnology microbial metallurgy technology is a dark horse
[8-10]Under certain air, pH, temperature conditions, the Ore lysate that some special antibacterials (thiobacillus ferrooxidant, thiobacillus thiooxidans etc.) can be changed insoluble sulphide ore into solubility reclaims required element then
[11-13]The maximum characteristics of this technology are simple, efficient, low-cost, pollution-free, mild condition, thereby metallurgical industry is extensively praised highly and used in the world, and 30% of world's copper output from the microbial metallurgy technology at present
[14]Realgar also is a kind of sulphide ore, has by the dissolved possibility of leaching microbacteria.Chinese invention patent 2006102000675 discloses the microbial treatment method of mineral Chinese medicines such as Cinnabaris and Realgar
[15]The present invention relates to the application of Realgar leachate in the preparation antitumor drug of microbial treatments, this leachate can play therapeutical effect to the multiple cancer that comprises hepatocarcinoma, gastric cancer, leukemia, pulmonary carcinoma etc.Because dissolving and conversion through microorganism, plurality of active ingredients in the Realgar is effectively dissolved, therefore this realgar microorganism extracting liquid can (Gamma Magnitude) play the obvious suppression effect to above-mentioned multiple cancer in extremely low concentration range, and reducing greatly of use amount also obviously improved the toxicity problem of realgar microorganism extracting liquid in treatment of cancer.
List of references:
(1) Pharmacopoeia of the People's Republic of China version in 2005 is one one.
(2)Li J.E.，Wu W.L.，Wang Z.Y.，Sun G.L.Apoptotic effect of As
2S
2 on K562cells and its mechanism.Acta Pharmacol.Sinica，2002，23：991-996.
(3)Lu D.P.，Wang Q.Current study of APL treatment in China.InternationalJournal of Hematology，2002，76：316-318.
(4) on the sunny side, Huang Shilin, Guo Aixia etc. compound realgar natural indigo tablet treatment acute promyelocytic leukemia efficacy analysis [J]. clinical hematology magazine, 2003,13 (1): 11.
(5)Ying D.，Huibi X.et al.Size effects of realgar particles on apoptosisin a human vein endothelial cell line：ECV-304.Pharmacological Research，2001，44：513-519.
(6) Zhan Xiuqin.Modernization of Chinese medicine technology of preparing-professor Guo Liwei who grows with each passing hour talks nanometer Chinese medicine and micron Chinese medicine.Beijing University of Chinese Medicine's journal (natural science edition) .2002,18 (4): 198201.
(7)Ying D.，Huibi X.et al.Size effects of realgar particles on apoptosisin a human vein endothelial cell line：ECV-304.Pharmacological Research，2001，44：513-519.
(8)Torma A E.The role of Thiobacillus ferrooxidans in hydrometallurgialprocesses.Adv Biochem Eng.1977，6：1-37
(9)Colmer A R，Hinckel.M E.The role of microorganism in acid mine drainage.A preliminary report，Science，1947，106：253-256.
(10)Yuan，X，et al.Microbes processing technology study for non-metallicminerals.China Non-Metallic Mining Industry Herald，2000，4.
(11)VaAsquez M，Espejo R T.Chemolithotrophic bacteria in copper oresleached at high sulfuric acid concentration.Appl Environ Microbiol，1997，63：332-336.
(12)Espejo R T，Romero J.Bacterial community in copper sulfide oresinoculated and leached with solution from commercial-scale copper leachingplant.Appl Environ Microbiol，1997，63：1344-1352.
(13)Fowler T A，Crundwell F K.Leaching of zinc sulfide by Thiobacillusferrooxidans experiments with a controlled redox potential indicate no directbacterial mechanism.Appl Environ Microbiol，1998，64：3570-3575.
(14)Karavaiko G I.Microbiological processes for the leaching of metalsfrom ores.State-of-the-art review.In：Torma A E，editor.Moscow：Centre ofInternational Projects，GKNT，1985.
(15) Li Hongyu, Zhang Jinghong, Zhi Dejuan.2006102000675, the heavy metal removal method in the mineral drug.
Summary of the invention:
Realgar has been successfully used to chronic and treatment acute myeloblastic leukemia clinically, demonstrates good antitumor action.But because the defective of existing processing technique, the Realgar goods exist use amount big, the content height of poisonous metal or arsenic, thereby abroad piped off, and can't enter the foreign market.In addition, because the water-insoluble of Realgar now uses Realgar many based on oral clinically, patient takes difficulty, and problems such as patient's GI irritation is big have also seriously been restricted the application of Realgar in oncotherapy.Disclosed technical finesse Realgar in the Chinese invention patent 2006102000675 is adopted in enforcement of the present invention, makes realgar microorganism extracting liquid.This realgar microorganism extracting liquid can (Gamma Magnitude) play the obvious treatment effect to the multiple cancer that comprises hepatocarcinoma, gastric cancer, leukemia, pulmonary carcinoma etc. in extremely low concentration range.Be converted into solvable state through the Realgar behind the microbial treatments by insoluble state, this greatly reduces the use amount and the toxicity of realgar microorganism extracting liquid, has improved the drug effect in antineoplaston simultaneously greatly, has reached the target of high-efficiency low-toxicity.
In addition, because realgar microorganism extracting liquid is a kind of medicine of solubility, therefore can be separately or with the other medicines compatibility and add pharmaceutically acceptable adjuvant, be easy to make various preparations, comprise suppository, pill, granule, membrane, microcapsule, drop pill, aerosol, medicated wine, syrup, oral liquid, injection or injection powder injection etc., this is that the researcher in this field is understandable.
The specific embodiment:
Embodiment one
The preparation of realgar microorganism extracting liquid
Handle Realgar according to disclosed mineral drug bacterial treatment method in the Chinese invention patent 2006102000675, embodiment is as follows: take by weighing the 0.5g Realgar and cross in the 250mL conical flask of 200 purpose powder after sterilization, add the 9K iron-free liquid culture medium 90mL after sterilizing, transfer pH to 1.75 with sulphuric acid, after treating that pH is stable, the inoculation volume fraction is that 20% thiobacillus ferrooxidant and final concentration are 1.0g/L Fe
2+After taking by weighing quality, shake bottle at 30 ℃, hunting speed during for 135r/min and leach a 30d.Adopt the sulphuric acid of 1: 1 (volume ratio) to regulate pH value in the experimentation, replenish institute's moisture evaporated with distilled water.Shake bottle and leach after 30 days and stop, decompress filter is collected supernatant, regulates about leachate pH to 7, promptly gets realgar microorganism extracting liquid after crossing 0.22 micron filter membrane degerming
Embodiment two
The variable concentrations realgar microorganism extracting liquid is to the growth inhibited effect of hepatoma cell strain HepG-2
Cell culture: use the RPMI1640 culture medium, add 10% Ox blood serum, penicillin 100U/ml, streptomycin 100U/ml, put 37 ℃, 5%CO
2Cultivate under the condition.Changed one time culture fluid in per 2～3 days.
MTT test: with cell with about 5 * 10
4The cell density of individual/ml is inoculated on 96 orifice plates, every hole 100 microlitres, overnight incubation.Press default Concentraton gradient respectively at 24 hours, 48 hours and 72 hours and add realgar microorganism extracting liquid, at least 3 repetitions of each gradient.Continue to cultivate after 24 hours, MTT 20 microlitres that every hole adds 5mg/ml continued to place 37 ℃ of incubations 4 hours, quick turnover panel method abandoning supernatant, and the DMSO that every hole adds 150 microlitres detects on microplate reader, according to recording OD value calculation of half inhibitory concentration IC
50, the result as shown in Table 1.
Different action times of table one and concentration realgar microorganism extracting liquid are to the growth inhibited effect of hepatoma cell strain HepG-2
| Experimental group | Time | IC 50 |
| Arsenic trioxide | 24h | 2.08±0.64μg/ml |
| Arsenic trioxide | 48h | 1.13±0.23μg/ml |
| Arsenic trioxide | 72h | 0.54±0.32μg/ml |
| Realgar microorganism extracting liquid | 24h | 6.08±0.92μg/ml |
| Realgar microorganism extracting liquid | 48h | 0.98±0.14μg/ml |
| Realgar microorganism extracting liquid | 72h | 0.43±0.06μg/ml |
Embodiment three
Realgar microorganism extracting liquid is to the growth inhibited effect of stomach cancer cell line MGC-803
Cell culture: use the RPMI1640 culture medium, add 10% Ox blood serum, penicillin 100U/ml, streptomycin 100U/ml, put 37 ℃, 5%CO
2Cultivate under the condition.Changed one time culture fluid in per 2～3 days.
MTT test: with cell with about 10
5The density of individual/ml is inoculated on 96 orifice plates, every hole 100 microlitres, overnight incubation.Press default Concentraton gradient respectively at 24 hours, 48 hours and 72 hours and add realgar microorganism extracting liquid, at least 3 repetitions of each gradient.Continue to cultivate after 24 hours, MTT 20 microlitres that every hole adds 5mg/ml continued to place 37 ℃ of incubations 4 hours, quick turnover panel method abandoning supernatant, and the DMSO that every hole adds 150 Cheng detects on microplate reader, according to recording OD value calculation of half inhibitory concentration IC
50, the result as shown in Table 2.
Different action times of table two and concentration realgar microorganism extracting liquid are to the growth inhibited effect of stomach cancer cell line MGC-803
| Experimental group | Time | IC 50 |
| Arsenic trioxide | 24h | 1.98±0.69μg/ml |
| Arsenic trioxide | 48h | 1.03±0.28μg/ml |
| Arsenic trioxide | 72h | 0.49±0.22μg/ml |
| Realgar microorganism extracting liquid | 24h | 3.08±0.32μg/ml |
| Realgar microorganism extracting liquid | 48h | 0.78±0.21μg/ml |
| Realgar microorganism extracting liquid | 72h | 0.42±0.09μg/ml |
Embodiment four
Realgar microorganism extracting liquid is to the growth inhibited effect of leukemia cell line K562
Cell culture: use the RPMI1640 culture medium, add 10% Ox blood serum, penicillin 100U/ml, streptomycin 100U/ml, put 37 ℃, 5%CO
2Cultivate under the condition.Changed one time culture fluid in per 1～2 day.
MTT test: with cell with about 10
5The density of individual/ml is inoculated on 96 orifice plates, every hole 100 microlitres.Press default Concentraton gradient respectively at 24 hours, 48 hours and 72 hours and add realgar microorganism extracting liquid, at least 3 repetitions of each gradient.Continue to cultivate after 24 hours, MTT 20 microlitres that every hole adds 5mg/ml continued to place 37 ℃ of incubations 4 hours, added three liquid, 50 microlitres, and 37 ℃ of overnight incubation detect in microplate reader, according to recording OD value calculation of half inhibitory concentration IC
50, the result as shown in Table 4.
Different action times of table three and concentration realgar microorganism extracting liquid are to the growth inhibited effect of leukemia cell line K562
| Experimental group | Time | IC 50 |
| Arsenic trioxide | 24h | 1.85±0.84μg/ml |
| Arsenic trioxide | 48h | 1.27±0.84μg/ml |
| Arsenic trioxide | 72h | 0.72±0.84μg/ml |
| Realgar microorganism extracting liquid | 24h | 1.69±0.08μg/ml |
| Realgar microorganism extracting liquid | 48h | 1.04±0.25μg/ml |
| Realgar microorganism extracting liquid | 72h | 0.42±0.09μg/ml |
Embodiment five
Realgar microorganism extracting liquid is to the growth inhibited effect of leukemia drug-resistant cell strain K562/ADM
Cell culture: use the RPMI1640 culture medium, add 10% Ox blood serum, penicillin 100U/ml, streptomycin 100U/ml, put 37 ℃, 5%CO
2Cultivate under the condition.Changed one time culture fluid in per 1～2 day.
MTT test: with cell with about 10
5The density of individual/ml is inoculated on 96 orifice plates, every hole 100 microlitres.Press default Concentraton gradient respectively at 24 hours, 48 hours and 72 hours and add realgar microorganism extracting liquid, at least 3 repetitions of each gradient.Continue to cultivate after 24 hours, MTT 20 microlitres that every hole adds 5mg/ml continued to place 37 ℃ of incubations 4 hours, added three liquid, 50 microlitres, and 37 ℃ of overnight incubation detect in microplate reader, according to recording OD value calculation of half inhibitory concentration IC
50, the result as shown in Table 4.
Different action times of table four and concentration realgar microorganism extracting liquid are to the growth inhibited effect of leukemia drug-resistant cell strain K562/ADM
| Experimental group | Time | IC 50 |
| Arsenic trioxide | 24h | 2.56±0.36μg/ml |
| Arsenic trioxide | 48h | 1.91±0.93μg/ml |
| Arsenic trioxide | 72h | 0.92±0.04μg/ml |
| Realgar microorganism extracting liquid | 24h | 2.06±0.54μg/ml |
| Realgar microorganism extracting liquid | 48h | 1.34±0.25μg/ml |
| Realgar microorganism extracting liquid | 72h | 0.56±0.14μg/ml |
Embodiment six
Realgar microorganism extracting liquid is to the growth inhibited effect of lung cancer cell line Lewis
Cell culture: use the RPMI1640 culture medium, add 10% Ox blood serum, penicillin 100U/ml, streptomycin 100U/ml, put 37 ℃, 5%CO
2Cultivate under the condition.Changed one time culture fluid in per 1～2 day.
MTT test: with cell with about 10
5The density of individual/ml is inoculated on 96 orifice plates, every hole 100 microlitres.Press default Concentraton gradient respectively at 24 hours, 48 hours and 72 hours and add realgar microorganism extracting liquid, at least 3 repetitions of each gradient.Continue to cultivate after 24 hours, MTT 20 microlitres that every hole adds 5mg/ml continued to place 37 ℃ of incubations 4 hours, added three liquid, 50 microlitres, and 37 ℃ of overnight incubation detect in microplate reader, according to recording OD value calculation of half inhibitory concentration IC
50, the result as shown in Table 5.
Different action times of table five and concentration realgar microorganism extracting liquid are to the growth inhibited effect of lung cancer cell line Lewis
| Experimental group | Time | IC 50 |
| Arsenic trioxide | 24h | 2.38±0.66μg/ml |
| Arsenic trioxide | 48h | 1.53±0.13μg/ml |
| Arsenic trioxide | 72h | 0.74±0.42μg/ml |
| Realgar microorganism extracting liquid | 24h | 1.98±0.36μg/ml |
| Realgar microorganism extracting liquid | 48h | 1.12±0.54μg/ml |
| Realgar microorganism extracting liquid | 72h | 0.54±0.08μg/ml |
Claims (3)
1. a realgar microorganism extracting liquid is characterized in that this realgar microorganism extracting liquid in the multiple cancer of preparation treatment, comprises in hepatocarcinoma, gastric cancer, leukemia, the lung-cancer medicament and using.
2. a kind of realgar microorganism extracting liquid according to claim 1 is characterized in that this leachate is the leachate of microbial treatments gained, prepares according to Chinese invention patent application 2006102000675 disclosed methods.
3. according to the described a kind of realgar microorganism extracting liquid of claim 1～2, it is characterized in that this realgar microorganism extracting liquid can be separately or with the other medicines compatibility and add pharmaceutically that the acceptable adjuvant is prepared into suppository, tablet, pill, granule, membrane, microcapsule, drop pill, aerosol, medicated wine, syrup, oral liquid, injection or injection powder injection.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101554384A (en) * | 2009-05-15 | 2009-10-14 | 李红玉 | Method for preparing realgar injection and use thereof |
| CN102119958A (en) * | 2010-01-11 | 2011-07-13 | 李红玉 | Medicinal composition containing realgar |
| CN101537014B (en) * | 2009-02-27 | 2015-01-07 | 兰州大学 | Anti-tumor arsenic combined drug |
| CN107982315A (en) * | 2016-10-26 | 2018-05-04 | 兰州大学 | A kind of Chinese medicine composition for treating leukaemia and preparation method thereof |
| CN108236613A (en) * | 2016-12-27 | 2018-07-03 | 兰州大学 | Realgar microorganism extracting liquid is preparing the application in inhibiting angiogenesis drug |
| CN109528765A (en) * | 2018-11-12 | 2019-03-29 | 兰州大学 | Native copper leachate is in cultured osteoblast-like cells in vitro proliferation and the purposes in mature mineralising treatment |
| CN109718248A (en) * | 2017-10-30 | 2019-05-07 | 兰州大学 | A kind of application of realgar microorganism extracting liquid in the drug of preparation degradation cancer protein |
| CN111012799A (en) * | 2018-10-09 | 2020-04-17 | 兰州大学 | Application of realgar microbial leaching as PML-RAR α protein inhibitor |
| CN114053303A (en) * | 2021-12-02 | 2022-02-18 | 兰州大学 | Application of realgar microbial leaching solution in preparation of drug for reducing phosphorylation level of protein kinase B |
-
2007
- 2007-06-15 CN CNA2007102008258A patent/CN101120950A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101537014B (en) * | 2009-02-27 | 2015-01-07 | 兰州大学 | Anti-tumor arsenic combined drug |
| CN101554384A (en) * | 2009-05-15 | 2009-10-14 | 李红玉 | Method for preparing realgar injection and use thereof |
| CN102119958A (en) * | 2010-01-11 | 2011-07-13 | 李红玉 | Medicinal composition containing realgar |
| CN102119958B (en) * | 2010-01-11 | 2014-10-29 | 李红玉 | Medicinal composition containing realgar |
| CN107982315A (en) * | 2016-10-26 | 2018-05-04 | 兰州大学 | A kind of Chinese medicine composition for treating leukaemia and preparation method thereof |
| CN108236613A (en) * | 2016-12-27 | 2018-07-03 | 兰州大学 | Realgar microorganism extracting liquid is preparing the application in inhibiting angiogenesis drug |
| CN109718248A (en) * | 2017-10-30 | 2019-05-07 | 兰州大学 | A kind of application of realgar microorganism extracting liquid in the drug of preparation degradation cancer protein |
| CN111012799A (en) * | 2018-10-09 | 2020-04-17 | 兰州大学 | Application of realgar microbial leaching as PML-RAR α protein inhibitor |
| CN109528765A (en) * | 2018-11-12 | 2019-03-29 | 兰州大学 | Native copper leachate is in cultured osteoblast-like cells in vitro proliferation and the purposes in mature mineralising treatment |
| CN114053303A (en) * | 2021-12-02 | 2022-02-18 | 兰州大学 | Application of realgar microbial leaching solution in preparation of drug for reducing phosphorylation level of protein kinase B |
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