- A study on enhanced production of 3-demethylated colchicine by a novel strain of Bacillus endophyticus isolated from rhizospheric soils of Gloriosa superba
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A native strain of Bacillus endophyticus, PC was found to be effective in demethylation of colchicine. The strain was isolated from rhizospheric soils of Gloriosa superba from Erode, Tamilnadu, and was grown in optimised conditions of carbon and nitrogen. To intensify the demethylation, the strain was enriched and grown for 15 generations starting from low (0.5 mg/ml) to very high (10 mg/ml) concentrations of colchicine. Owing to enrichment, B. endophyticus showed an MIC of 25 mg/ml, and notable activity of demethylation of colchicine. Maximum production was observed at idiophase level, wherein PC produced around 71.97% of 3-DMC. Supplementary studies with the strain B. endophyticus PC proved the adverse effects of colchicine on the metabolic and structural portrait of cells. There was major variance in the cell shape and size, including the clipping of thickness of plasma membrane and cell wall. The number of polyhydroxybutyrate (PHB) rich inclusion bodies were found to be enhanced, suggesting their portrayal in activation of P450-BM3 enzyme which initiates demethylation.
- Das, Reena,Vasantharaja,Udayakumar, Prithika,Vaidyanathan, Rama
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- Colchicine is formed by para-para phenol coupling from autumnaline
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In order to distinguish between ortho-para and para-para phenol coupling in colchicine biosynthesis, [[3'-O14CH3]autumnaline was converted to [14C]colchicine in seeds of Colchicum autumnale and the product was then selectively demethylated by a bacterium that exclusively demethylates C-3 of colchicine. The retention of the radioactive label in 3-demethylcolchicine indicates that biosynthesis proceeds through para-para coupling.
- Maier, Ulrich H.,Zenk, Meinhart H.
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- Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70l bioreactor level
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A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5l bioreactor with 3l working volume. In 5l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70l bioreactor and resulted into ～80% conversion of 20mM colchicine in 48h with a volumetric productivity of 6.62mgl-1h-1. Scale-up factors were measured as volumetric oxygen transfer coefficient (kLa) i.e., 56h-1 and impeller tip velocity (Vtip) i.e., 7.065ms-1, respectively. The kinetic parameters Km, kcat, and kcat/Km of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271±30μM, 8533±25min-1, and 31.49μMmin-1, respectively, when IPTG induced recombinant E. coli culture was used.
- Dubey, Kashyap Kumar,Haque, Shafiul,Jawed, Arshad,Singh, Bhanu P.,Behera
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- Novel Heterogeneous Fenton's-Like Catalysis for Degradation of Colchicine Coupled with Extraction of Its Biologically Active Metabolite
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Nowadays, drug pollution; a form of water pollution caused by some pharmaceuticals and their metabolites resulting from consumers, industry and hospitals was reported. Colchicine (CLN) is considered one of the pharmaceutical wastewater contaminants which are not eliminated completely in municipal sewage treatment plants and are discharged into receiving water. Due to the higher toxicity of CLN, a novel heterogeneous Fenton's-like catalysis was established for complete degradation of CLN. So, a highly sensitive and specific liquid chromatographic method with quadrupole mass spectrometry (LC/Q-MS) was developed and validated for estimation of CLN in its pure form and in the presence of its degradation product. Herein, GraceSmart RP C18 column was utilized for separation of the cited drug (Retention time tR = 5.578 min) using methanol: water (55: 45, v/v) at 1.0 mL min?1. Detection was performed by Agilent 6120 Quadrupole MS detector in a positive ionization mode. Thereafter and for the first time, degradation of CLN by heterogeneous Fenton's-like catalysis using modified polyacrylonitrile (PAN) as a catalyst with H2O2 in aqueous acidic medium was performed. This process was firstly optimized by HPLC/UV detection at 248 nm using the aforementioned chromatographic conditions. As a result, CLN degraded completely within 30 min. The only observed degradation product was the biologically active, potent and less toxic antitumor metabolite of CLN (3- demethyl CLN) which was collected, extracted, and analyzed by Fourier Transfer- Infrared Spectroscopy (FTIR) and 13Carbon- Nuclear Magnetic Resonance (13C NMR). Finally, this method is eco-friendly and complies with the requirements of the green chemistry. It is suitable for complete removal of CLN and/or its metabolite contaminants from wastewater samples and estimation of the target drug without any interference from its degradation products. However, further study is required to expand the method applicability to the pharmaceutical wastewater treatment as well the production of 3- demethyl CLN on a large scale.
- Hosny, Noha M.,Huddersman, Katherine,Atia, Noha N.,El-Gizawy, Samia M.
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- Biological Effects of Modified Colchicines. Improved Preparation of 2-Demethylcolchicine, 3-Demethylcolchicine, and (+)-Colchicine and Reassignment of the Position of the Double Bond in Dehydro-7-deacetamidocolchicines
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A variety of colchicine, demecolcine, and isocolchicine derivatives were examined for their potency in the lymphocytic leukemia P388 screen in mice, for their toxicity in mice, and for their binding to microtubule protein.A qualitatively direct correlation was found between in vivo potency and toxicity; potency appeared to be less well correlated with tubulin binding.The most potent compounds were N-acylated analogues of colchicine and demecolcine.Among the monophenols, only 3-demethylcolchicine showed an appreciable effect in vitro and in vivo and was less toxic than colchicine.Improved methods were found for the preparation of 3- and 2-demethylcolchicine, which involved the use of 85percent phosphoric acid and concentrated sulfuric acid, respectively.Decoupling experiments with 1H NMR proved that the double bond of dehydro-7-deacetamidocolchiceine and its derived tropolonic methyl ethers 24 and 25 was in the 5,6 position, rather than the 6,7 position formerly tentatively assigned.
- Roesner, Manfred,Capraro, Hans-Georg,Jacobson, Arthur E.,Atwell, Louise,Brossi, Arnold,et al.
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p. 257 - 261
(2007/10/02)
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