Effect of precursors feeding and media manipulation on production of novel anticancer pro-drug camptothecin from endophytic fungus

We have established methodology for the isolation and characterization of a novel endophytic fungus from the inner bark of medicinal plant Nothapodytes foetida, which produced camptothecin in Sabouraud broth (SB) under shake flask conditions. Camptothecin and its related compounds are at present obtained by extraction from intact plants, but fungal endopytes may be an alternative source of production. In present study we have observed the effect of different nutrient combinations and precursors (tryptophan, tryptamine, geraniol, citral, mevalonic acid and leucine) on the accumulation of camptothecin by endophytic fungus Entrophospora infrequens. The precursors were fed either alone or in combinations (tryptophan and geraniol, tryptophan and citral, tryptophan and mevalonic acid, tryptophan and leucine). The highest camptothecin content was observed in the range of 503 ± 25µg/100g dry cell mass in Sabouraud medium. Camptothecin content in the medium was increased by 2.5 folds by the presence of tryptophan and leucine whereas the production with trytophan was also significantly different from other treatments. Furthermore, the effect of fungal camptothecin on the morphology of human cancer cell lines was also studied. The treated cells showed reduction in size, condensation of nucleus and the protoplasmic extensions were reduced. All these characteristics are found in apoptotic cells.

Fluorimetric analysis of camptothecin in Chinese herbal medicine common Camptotheca fruit / 药学学报

Three-dimensional (3D) fluorescence spectra and thin layer fluorescence chromatogram of common Camptotheca fruit (CCF) crude drug, camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) have been studied, and a novel fluorimetric method for determination of CPT in CCF crude drug has been established. In 3D fluorescence spectra, CPT presented 3 fluorescence peaks with excitation wavelengths lambdaex of 215, 255 and 365 nm, separately, and all peaks with emission wavelength lambdaem of 430 nm. HCPT presented 4 fluorescence peaks with lambdaex of 220, 265, 325 and 375 nm, separately, and all peaks with lambdaem of 555 nm. The fluorescence of CPT is much stronger than that of HCPT. Comparison of 3D fluorescence spectra and analysis of thin layer fluorescence chromatogram revealed that the main fluorescent component of CCF is CPT. HCPT and other components basically do not interfere with the fluorescence of CPT. Under the condition of pH 3.0-6.7, CCF aqueous solution can produce strong and steady fluorescence. Using methanol as solvent, the extracting solution of CCF was prepared, and diluted properly with water, then measured fluorescence intensity at lambdaex/lambdaem = 365/430 nm to determine the content of CPT. A linear calibration curve covered the concentration range 0.002 35-0.235 microg x mL(-1). The regression equation was IF = 9 + 30,844 c, with correlation coefficient r = 0.999 (n=9). The method has been applied to the analysis of CPT in a CCF sample, with a result of 0.127% and a spiked recovery rate of 102%. The reliability of the method has been verified by a thin layer chromatography-fluorescence scanning method. Experimental results demonstrated that this method can be used for quality evaluation of CCF crude drug.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkalo id accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

Effect of nitrogen on camptothecin content in Camptotheca acuminata seedlings / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of nitrogen concentration on the camptothecin (CPT) content in Camptotheca acuminata seedlings:</p><p><b>METHOD</b>The seedlings of C. acuminata with 6 pair of leaves were subjected to five nitrogen concentrations treatments by sand culture in a greenhouse. The CPT content in the seedlings was determined by HPLC on the 20th, 35th, 50th, 65th and 80th day respectively.</p><p><b>RESULT</b>The CPT content in the young leaves of C. acuminata seedlings supplied with different nitrogen concentration was significantly higher than that in other organs (P < 0.01), and it showed a single peak curve with the time course, the highest CPT content was observed on the 50th day after treatment. The CPT content in the young leaves obviously declined with increasing nitrogen concentration, and it reached the highest (6.72%) when nitrogen concentration was 4 mmol x L(-1), equal to 1.1 times that of 16 mmol x L(-1) nitrogen.</p><p><b>CONCLUSION</b>The results demonstrate that proper deficient nitrogen stress can significantly enhance CPT accumulation in young leaves of C. acuminata seedlings.</p>

Effects of nitrogen forms on camptothecin content and its metabolism-related enzymes activities in Camptotheca acuminata seedlings / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of nitrogen forms on the camptothecin (CPT) content, tryptophan synthase (TSB) and tryptophan decarboxylase (TDC) activities in Camptotheca acuminata seedlings.</p><p><b>METHOD</b>The seedlings of C. acuminata with 6 pairs of leaves were subjected to 5 different NH4(+) -N/NO3(-) -N ratio (0 : 100, 75 : 25, 50 : 50, 25 : 75, 100 : 0) treatments by sand culture in a greenhouse. The CPT content, TSB activity in the young leaves and TDC in the stem barks of the seedlings were determined by HPLC on the 15th, 30th, 45th, 60th and 75th day, respectively.</p><p><b>RESULT</b>The obvious relationship between CPT content and nitrogen forms was observed. When NH4(+) - N /NO3(-) -N ratio was 25 : 75, CPT accumulation in young leaves displayed the best advantages (the highest value is 5.69 per thousand) and increased in the early 30 days of treatment and then declined. There was no obvious relationship between TSB activity in the young leaves and nitrogen forms. TDC activity in the stem bark was the highest when NH4(+) -N /NO3(-) -N ratio was 25 : 75, and the change of TDC activity paralleled to CPT content in the young leaves.</p><p><b>CONCLUSION</b>A short-term treatment that NH4(+) -N /NO3(-) -N ratio was 25:75 may gain high CPT content in the young leaves through enhancing the TDC activity in the stem bark of C. acuminata seedlings.</p>

Isolation and purification of 10-hydroxycamptothecin and vincoside-lactam from Camptotheca acuminata seed by polyamide / 中国中药杂志

To investigate the technological parameters of the isolation and purification of 10-hydroxycamptothecin and vincoside-lactam from Camptotheca acuminata seed by polyamide. The static arid dynamic adsorption characteristics of 10-hydroxycamptothecin and vincoside-lactam on polyamide were studied, and the contents were determined by HPLC. The optimum parameters for adsorption were as follows: the contents of 10-hydroxycamptothecin and vincoside-lactam in the extracts were 0.189 g x L(-1) and 0.334 g x L(-1), respectively, pH 6, flow rate was 1.0 mL x min(-1), processing volume was 3 BV; for desorption: ethanol-water (60:40), flow rate was 1.0 mL x min(-1), 5 BV as an eluent. After treated with polyamide, the contents of 10-hydroxycamptothecin and vincoside-lactam were 17.52% and 32.87%, respectively, the recovery yields were 66.05% and 75.86%, respectively. Results showed that polyamide revealed a good ability to separate 10-hydroxycamptothecin and vincoside-lactam. Therefore, we concluded that results in this study may provide scientific references for the large-scale production of 10-hydroxycamptothecin and vincoside-lactam extracted from C. acuminata seed.

Callus induction and cultivation of Camptotheca acuminata / 中国中药杂志

<p><b>OBJECTIVE</b>To study the callus induction from leaf, stem segments and stem segments with axillary's bud and the subculture conditions of callus in Camptotheca acuminata.</p><p><b>METHOD</b>The explants were inoculated into the media of MS with different concentrations of 6-BA, NAA and 2, 4-D by orthogonal experiment.</p><p><b>RESULT AND CONCLUSION</b>The optimal medium for callus induction was MS with 6-BA 2 mg x L(-1), NAA 2 mg x L(-1) and 2,4-D 2 mg x L(-1). It had better effects in leaf than in other explants. The induction ratio in leaf reached above 80%. The callus subcultured on the medium of MS with 6-BA 1 mg x L(-1) and 2, 4-D 1 mg x L(-1) grew vigorous and more quickly than that in other media. It was loose, friable in consistency and suitable for cell culture.</p>

Effects of cu2+ on biosynthesis of camptothecin in cell cultures of Camptotheca acuminata / 生物工程学报

Camptothecin is a strong anti-tumor compound isolated from Camptotheca acuminata. One of the most important way for the production of Camptothecin is by cell cultures of Camptotheca acuminata. The effect of Cu2+ on camptothecin accumulation in Camptotheca acuminata cell line was described in this paper. The results showed that the optimum CuCl2 concentration in B5 medium was 0.008 mg/mL, which increased camptothecin production for 30 times compare to the control while has no inhibitive effects on cell growth, at the same time, the peroxidase activity was increased and the anthocyanidin accumulation was inhibited. The promotive effects of Cu2+ on camptothecin accumulation in light was higher than that in dark.