Figure 10.
(A) Effect of crude C. majus latex, alkaloid-rich fraction, and protein fraction on nitric oxide (NO) secretion by RAW 264.7 cells. Consecutive numbers (1–6) on the x axis correspond to different protein concentrations in the crude latex (S1) and protein fraction (S2) applied on cells: 1 = 1.3 × 10−5 mg/mL, 2 = 6.4 × 10−5 mg/mL, 3 = 3.2 × 10−4 mg/mL, 4 = 1.6 × 10−3 mg/mL, 5 = 1.6 × 10−2 mg/mL, 6 = 8.0 × 10−2 mg/mL. In the case of alkaloid-rich fraction (S3), consecutive numbers (1–6) correspond to its different dilutions: 1 = 1.6 × 10−6, 2 = 8 × 10−5, 3 = 4 × 10−4, 4 = 2 × 10−3, 5 = 2 × 10−2, 6 = 10−1. They were obtained using series of dilutions from concentrated samples. Cells treated with the most concentrated samples of crude latex (S1) and the protein fraction (S2) released NO at a concentration of approximately 2.72 ng/μL and 0.67 ng/μL, respectively. These concentrations were highly cytotoxic and induced the death of most cells. No NO secretion was observed for cells treated with the samples at non-toxic concentrations. LPS-treated cells (100 ng/µL) (positive control) secreted NO at a level of 0.57 ng/μL. (B) Simultaneous analysis of the effect of crude C. majus latex (S1) and its fractions (S2, S3) on RAW 264.7 cell viability. Among the tested samples, the strongest cytotoxicity was shown for the crude latex and the alkaloid-rich fraction in the range 1.6 × 10−3–8.0 × 10−2 mg/mL (numbers 4–6). The protein fraction exhibited the lowest toxicity to RAW 264.7 cells in all analyzed dilutions compared to the rest of the samples. All tested samples in the range of 1.3 × 10−5–3.2 × 10−4 mg/mL (numbers 1–3) showed the lowest cytotoxicity.