Study of Potential Anticancer Compound Leaves Polyalthia glauca (Hassk.) Boerl .: Isolation and Identification of Compounds Cytotoxic Mechanism in Cell Cycle and Apoptosis of Cervical Cancer HeLa Cells
ABSTRACT: Polyalthia (Annonaceae) is considered to be a medicinal importance because of the presence of clerodane diterpenoids and alkaloids in various parts of the plant. They have many bioactivities such as cytotoxicity on many variate of cancer cell line, antiinflamation, analgesic, anti-HIV, antibacteria, and antioksidan. Compound as an anticancer activity in P. glauca has not been reported. Therefore the objective of this research were to isolate and identify the structure of cytotoxic compound, to study the cytotoxic activity of it on the HeLa cell line, to study the effect of it on cell cycle and apoptotic induction, and to investigate the influence it on p53, pRb, Bcl-2, caspase-9 and caspase-3 expression on HeLa cell line in vitro of P. glauca leaves The bioactive compound isolation was done by bioassay guided fractionation. Monitoring compound was done by TLC and cytotoxic effect by MTT assay. The dried powder leaves of P. glauca was extracted by maserated using chloroform, methanol and water.The active extract was fractionated by vacuum liquid column chromatography using 16 eluents that increased in polarity (n-hexane: ethyl acetate, v/v). The fractionation produced 16 fractions , and the fractions that had similar pattern on KLT were combined and obtaining 6 combined fractions. MTT assay results showed that the most active fraction (FIII) was the one that was eluted by n-heksan : ethyl acetat (6:4;v/v) This fraction was further separation by preparative thin layer chromatography and obtained 3 compounds. Compounds was tested for purity by TLC, further active compound structures were elucidated by UV, IR, NMR (1D and 2D) and Mass Spectroscopy. Active compounds were evaluated for their cytotoxic activities on HeLa cell lines by MTT assay method. Effect of active compounds on HeLa cell cycle were analyzed by flow cytometry. Apoptosis induction on HeLa cell line by the active compounds were analyzed by observation of DNA fragmentation. Expression of p53, pRb, Bcl-2, caspase-9 and caspase-3 were detected by immunocytochemistry technique, then analyzed semiquantitatively using the Allred scoring. The results showed that cytotoxic compound in P. glauca leaves belonging to the group of secolignan, the active compound 1 as Glaucinitinitin A was 4 - [(3,4-Dimethoxy-phenyl) - (3,4,5-trimethoxy-phenyl) -methyl] -3-methylene-dihydro-furan-2 -one, the active compound 2 as Glaucinitinitin B was 4 - [(3,4-Dimethoxy-phenyl) - (3,4,5-trimethoxy-phenyl) -methyl] -3-methylene-dihydro-furan-2-one, and the active compound 3 as Glaucinitinitin C was 4- [Bis- (3,4,5-trimethoxy-phenyl) -methyl] -3-methylene-dihydro-furan-one, and cytotoxicity assay of those three compounds on HeLa cells showed IC50 was 12,11�±0,29 �¼g/mL, 6,62�±0,2 �¼g/mL, 9,02�±0,2�¼g/mL respectively. Results on the molecular assay showed that the Glaucinitin A was able to inhibit the cell cycle at G0-G1 phase amounted to 70.65%, while the active Glaucinitin B and Glaucinitin C affect at the G2-M phase amounted to 19,03 and 19,1 %. The active compounds also proved to be able to induce apoptotic on HeLa cells and capable of inducing the expression of p53, pRb, caspase-9, caspase-3, and could suppress the expression of Bcl-2 HeLa cells. From the research can be concluded that three new cytotoxic compound of P. glauca leaves have been isolated which potential to modulate cell cycle and capable to induce apoptosis in cervical cancer cells HeLa.
ABSTRACT: Polyalthia (Annonaceae) is considered to be a medicinal importance because of the presence of clerodane diterpenoids and alkaloids in various parts of the plant. They have many bioactivities such as cytotoxicity on many variate of cancer cell line, antiinflamation, analgesic, anti-HIV, antibacteria, and antioksidan. Compound as an anticancer activity in P. glauca has not been reported. Therefore the objective of this research were to isolate and identify the structure of cytotoxic compound, to study the cytotoxic activity of it on the HeLa cell line, to study the effect of it on cell cycle and apoptotic induction, and to investigate the influence it on p53, pRb, Bcl-2, caspase-9 and caspase-3 expression on HeLa cell line in vitro of P. glauca leaves The bioactive compound isolation was done by bioassay guided fractionation. Monitoring compound was done by TLC and cytotoxic effect by MTT assay. The dried powder leaves of P. glauca was extracted by maserated using chloroform, methanol and water.The active extract was fractionated by vacuum liquid column chromatography using 16 eluents that increased in polarity (n-hexane: ethyl acetate, v/v). The fractionation produced 16 fractions , and the fractions that had similar pattern on KLT were combined and obtaining 6 combined fractions. MTT assay results showed that the most active fraction (FIII) was the one that was eluted by n-heksan : ethyl acetat (6:4;v/v) This fraction was further separation by preparative thin layer chromatography and obtained 3 compounds. Compounds was tested for purity by TLC, further active compound structures were elucidated by UV, IR, NMR (1D and 2D) and Mass Spectroscopy. Active compounds were evaluated for their cytotoxic activities on HeLa cell lines by MTT assay method. Effect of active compounds on HeLa cell cycle were analyzed by flow cytometry. Apoptosis induction on HeLa cell line by the active compounds were analyzed by observation of DNA fragmentation. Expression of p53, pRb, Bcl-2, caspase-9 and caspase-3 were detected by immunocytochemistry technique, then analyzed semiquantitatively using the Allred scoring. The results showed that cytotoxic compound in P. glauca leaves belonging to the group of secolignan, the active compound 1 as Glaucinitinitin A was 4 - [(3,4-Dimethoxy-phenyl) - (3,4,5-trimethoxy-phenyl) -methyl] -3-methylene-dihydro-furan-2 -one, the active compound 2 as Glaucinitinitin B was 4 - [(3,4-Dimethoxy-phenyl) - (3,4,5-trimethoxy-phenyl) -methyl] -3-methylene-dihydro-furan-2-one, and the active compound 3 as Glaucinitinitin C was 4- [Bis- (3,4,5-trimethoxy-phenyl) -methyl] -3-methylene-dihydro-furan-one, and cytotoxicity assay of those three compounds on HeLa cells showed IC50 was 12,11�±0,29 �¼g/mL, 6,62�±0,2 �¼g/mL, 9,02�±0,2�¼g/mL respectively. Results on the molecular assay showed that the Glaucinitin A was able to inhibit the cell cycle at G0-G1 phase amounted to 70.65%, while the active Glaucinitin B and Glaucinitin C affect at the G2-M phase amounted to 19,03 and 19,1 %. The active compounds also proved to be able to induce apoptotic on HeLa cells and capable of inducing the expression of p53, pRb, caspase-9, caspase-3, and could suppress the expression of Bcl-2 HeLa cells. From the research can be concluded that three new cytotoxic compound of P. glauca leaves have been isolated which potential to modulate cell cycle and capable to induce apoptosis in cervical cancer cells HeLa.
