Optimization of cultural conditions for the growth and maximum production of camptothecin, an anticancer agent by an endophytic fungus, Glomerrella cingulata from Ophiorrhiza  mungus L.

Camptothecin (CPT) derived compounds are widely used clinically for chemotherapy of human cancer. Several plants such as Camptotheca acuminata, Nothapodytes foetida and Ophiorrhiza spp. are natural sources of CPT, but these plants produce very law amounts of CPT and its derivatives. Therefore, investigation into alternative sources of CPT has become important in recent years. Several reports are available on “in vitro” system of CPT production using tissue cultures of CPT-producing plants. Alternatively, endophytic fungi isolated from CPT producing plants were reported as promising sources of CPT in the recent years. Enterospora enfrequens isolated from Nothapodytes nimmoniana and Fusarium solani isolated from Camptotheca acuminate have been identified as CPT producers under “in vitro” conditions. However, no reports on the stability and optimization of cultural conditions for the growth and maximum production of CPT production by these fungi are available. Ophiorrhiza mungus L (Sinhala vernicular name; Dath katiya, family Rubiaceae) grown in Sri Lanka is also known to produce this alkaloid. In the present study, an endophytic fungus was isolated from field grown O. mungus. Stem tissues transplanted in Potato Dextrose Agar (PDA) medium resulted in the isolation of total of 19 individuals that belonged to six distinct species based on their colony characters. All 19 fungi were sub cultured in 100 ml Potato Dextrose Broth (PDB) and grown at 27 ^o C. Both the broth and mycelia were analyzed by HPLC for presence of CPT. only one fungal species (strain W3I28) was found to produce CPT, which was detected in the mycelium but not in the broth and content was similar to that produce by the host. The CPT was detected one week, two weeks as well as three weeks old cultures. Optimization of cultural conditions for the growth and maximum production of camptothecin by Glomerrella cingulate was performed by changing the physical and chemical parameters like incubation period, shaking conditions, incubation temperature and changing the chemical composition of the basal media (percentage of carbon source of the media). The highest content of CPT (9.70 ± 0.02 x 10^-4 %) (Fresh weight basis) was produced when the sucrose percentage of Potato Sucrose Broth (PSB) medium was adjusted to 3% when compared to that (2.45 ± 0.05 x 10^-4 %) (Fresh weight basis) produced in the standard PSB at 27^oc incubation temperature after 18 days from incubation. Further, the production of CPT was also observed in seven successive cultures of the fungus with no attenuation, indicating the production of CPT by the fungus is stable. This is the first report of the isolation and optimization of cultural conditions of CPT- producing endophytic fungus from Ophiorrhiza mungus.