Cytotoxic Effects of Chlorophyllides in Ethanol Crude Extracts from Plant Leaves

Abstract

Chlorophyllide (chlide) is a natural catabolic product of chlorophyll (Chl), produced through the activity of chlorophyllase (chlase). The growth inhibitory and antioxidant effects of chlide from different plant leaf extracts have not been reported. The aim of this study is to demonstrate that chlide in crude extracts from leaves has the potential to exert cytotoxic effects on cancer cell lines. The potential inhibitory and antioxidant effects of chlide in crude extracts from 10 plant leaves on breast cancer cells (MCF7 and MDA-MB-231), hepatocellular carcinoma cells (Hep G2), colorectal adenocarcinoma cells (Caco2), and glioblastoma cells (U-118 MG) were studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and DPPH (1,1-diphenyl-2-picrylhydrazyl) assays. The results of the MTT assay showed that chlide in crude extracts from sweet potato were the most effective against all cancer cell lines tested. U-118 MG cells were the most sensitive, while Caco2 cells were the most resistant to the tested crude extracts. The cytotoxic effects of chlide and Chl in crude extracts from sweet potato and of commercial chlorophyllin (Cu-chlin), in descending order, were as follows: chlide > Chl > Cu-chlin. Notably, the IC₅₀ of sweet potato in U-118 MG cells was 45.65 μg/mL while those of Chl and Cu-chlin exceeded 200 μg/mL. In the DPPH assay, low concentrations (100 μg/mL) of chlide and Cu-chlin from crude extracts of sweet potato presented very similar radical scavenging activity to vitamin B2. The concentration of chlide was negatively correlated with DPPH activity. The current study was the first to demonstrate that chlide in crude extracts from leaves have potential cytotoxicity in cancer cell lines. Synergism between chlide and other compounds from leaf crude extracts may contribute to its cytotoxicity.

Figure 1

HPLC analysis profiles of Chl and chlase-treated ethanol crude extracts from different plant species. The products were separated by HPLC and detected at 667 nm from 0 to 80 min. Chl and chlide were detected within 30 min and 10 min.

Figure 2

Cytotoxicity of chlase-treated ethanol crude extracts on NIH/3T3, MCF7, MDA-MB-231, Hep G2, Caco2, and U-118MG cells determined by MTT assays. The cells (5 × 10⁴/well) were stimulated with different doses of chlide (50, 80, 100, 150, and 200 μg/mL). The bars represent the percentage growth inhibition of cells. All measurements made in 96-well plates were carried out using five technical replicates. When compared with the control, the results were statistically significant (∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001).

Figure 3

Correlation Analyses. IC50 values were evaluated by linear regression analysis. Correlation coefficients between cytotoxic activity and Chl/chlide contents were calculated by Pearson's correlation coefficient and showed here. High correlation: 0.7-0.99 (correlation coefficients); moderate correlation: 0.4-0.69; modest correlation: 0.1-0.39; weak correlation: 0.01-0.09.

Figure 4

IC ₅₀ of Chl and chlase-treated ethanol crude extracts from sweet potato in cancer cell lines. IC₅₀ values were calculated based on the results of the MTT assay and are shown in % of Cu-chlin. The IC₅₀ values of different cell lines are shown at the bottom.

Figure 5

DPPH assay. DPPH radical scavenging activity was determined for Chl and chlide in sweet potato leaf ethanol extracts and Cu-chlin. The IC₅₀ value of Chl, chlide, and Cu-chlin are shown at the bottom.