Screening has been and continues to be the most useful productive method of detecting antitumor agents. "In vitro" assay is less expensive and require less test material and time. The antitumor assay for screening plants extracts since 1960, by NCI (National Cancer Institute) has been sligthly modified. It has been used hela cell culture (cell line derived from human carcinoma of uterus) instead of the original KB (cell line derived from human carcinoma of nasopharynx). Hela cells were cultivated on Eagle's basal medium plus 10% serum added of antibiotics: penicillin 100 units/ml, streptomycin 100 mcg/ml and amphotericin 5 mcg/ml. To a dilute stock cells of 10-20 mcg/ml (protein values according to method of Oyama and Eagle) in complete media was simultaneously added test material (range concentration 1-100 mcg/ml) test material were either synthetics or natural products.
Total volume was approximately 4 ml. Also a blank and control was done. After 72 hours was conducted a protein analysis of tests, control and at least three protein standard and media blank tubes. Results were expressed as the dose that inhibts growth to 50% of control growth (ED 50). The criteria of activity for synthetics was an ED 50≤12 mcg/ml on the first test and an ED 50≤4 mcg/ml on the second and confirmation tests; and for natural products were an ED 50≤60 mcg/ml on the first test and an ED 50≤40 mcg/ml on the second and confirmation tests.