Synergistic effects of melphalan and Pinus kesiya Royle ex Gordon (Simaosong) extracts on apoptosis induction in human cancer cells.

BACKGROUND: This study aims to determine the synergistic effects of the chemotherapeutic drug melphalan and the phytoconstituents extracted from Pinus kesiya Royle ex Gordon (Simaosong) in human cancer cells. METHODS: P. kesiya twigs extracted from 50 % ethanol-water were evaluated alone (6-500 µg/mL) and in combination with melphalan (0.75-15 µg/mL). The cytotoxic effects of single extract or extract and melphalan combination were examined by a neutral red assay to investigate their antiproliferative and apoptosis induction effects in the U937 and HepG2 cell lines. Nuclei morphological change and DNA fragmentation were examined by DNA nuclei staining with 4´6-diamidino-2-phenylindole (DAPI) and agarose gel electrophoresis, respectively. The chemical constituents of the P. kesiya extract were assessed using gas chromatography-mass spectrometry (GC-MS) analysis. The synergistic effects of different IC50 ratios of the P. kesiya extract and melphalan combination were analyzed in each cancer cell line. The dose reduction index (DRI) was calculated to determine the extent of concentration reduction in the combination treatment compared with the concentration of each single treatment. RESULTS: The IC50 ratios for melphalan to P. kesiya extract that caused 75 % antiproliferation could be reduced after combination. This response was greater in the U937 cells than in the HepG2 cells (all P < 0.001). Melphalan and P. kesiya extract had a similar effect on apoptosis induction both singly and in combination. P. kesiya extract synergized the antiproliferation and apoptosis induction effects of melphalan. CONCLUSIONS: Combining the P. kesiya extract with melphalan reduced toxicity while retaining the therapeutic efficacy of melphalan.

In vitro alpha glucosidase inhibition and free-radical scavenging activity of propolis from Thai stingless bees in mangosteen orchard

ABSTRACTThe chemical component and biological activity of propolis depend on flora area of bee collection and bee species. In the study, the propolis from three stingless bee species, Lepidotrigona ventralis Smith, Lepidotrigona terminata Smith, and Tetragonula pagdeni Schwarz, was collected in the same region of mangosteen garden from Thailand. Total phenolic content, alpha glucosidase inhibitory effect, and free-radical scavenging activity using FRAP, ABTS, DPPH assays were determined. The most potent activity of propolis extract was investigated for bioactive compounds and their quantity. The ethanol extract of T. pagdeni propolis had the highest total phenolic content 12.83 ± 0.72 g of gallic acid equivalents in 100 g of the extract, and the strongest alpha glucosidase inhibitory effect with the IC50 of 70.79 ± 6.44 µg/ml. The free-radical scavenging activity evaluated by FRAP, ABTS, DPPH assays showed the FRAP value of 279.70 ± 20.55 µmol FeSO4 equivalent/g extract and the IC50 of 59.52 ± 10.76 and 122.71 ± 11.76 µg/ml, respectively. Gamma- and alpha-mangostin from T. pagdeni propolis extract were isolated and determined for the biological activity. Gamma-mangostin exhibited the strongest activity for both alpha glucosidase inhibitory effect and free-radical scavenging activity. Using HPLC quantitative analysis method, the content of gamma- and alpha-mangostin in the extract was found to be 0.94 ± 0.01 and 2.77 ± 0.08% (w/w), respectively. These findings suggested that T. pagdeni propolis may be used as a more suitable raw material for nutraceutical and pharmaceutical products and these mangostin derivatives as markers.

FTIR microspectroscopy discriminates anticancer action on human leukemic cells by extracts of Pinus kesiya; Cratoxylum formosum ssp. pruniflorum and melphalan.

Apoptosis is the principal molecular goal of chemotherapeutics for effective anticancer action. We studied the effect of 50% ethanolic-water extracts of Pinus kesiya, Cratoxylum formosum ssp. pruniflorum and melphalan on cytotoxicity and apoptosis induction for human leukemic U937 cells, and explored the mode of action using FTIR microspectroscopy. The number of viable U937 cells in vitro was decreased in a concentration-dependent manner by all tested compounds, although potency differed between the U937 and Vero cells. Melphalan and the extract of C. formosum exhibited relatively lower IC(50) values (15.0 ± 1.0 and 82.7 ± 3.2 µg/mL respectively) and higher selectivity (selective index>3) than the extract of P. kesiya (299.0 ± 5.2 µg/mL; selective index<3) on the U937 cells. All three compounds significantly induced apoptosis through the late stage - seen by the indicative DNA ladder - with the most effective being melphalan, then the P. kesiya and C. formosum extracts. FTIR microspectroscopy revealed that all three compounds raised the intensity of the ß-pleated sheet - higher than that of the untreated U937 cells - corresponding to a shift in the α-helix band associated with an alteration in the secondary structure of the protein band, confirming induction of apoptosis via pro-apoptotic proteins. The differences in intensity of the FTIR bands associated with lipids, proteins and nucleic acids were responsible for discrimination of the anticancer mode of action of each of the three compounds. The FTIR data suggest that the two plant extracts possessed anticancer activity with a different mode of action than melphalan.

Synergistic anticancer effect of the extracts from Polyalthia evecta caused apoptosis in human hepatoma (HepG2) cells.

OBJECTIVE: To evaluate the anticancer activity of the extract fraction of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep and the synergistic anticancer effect of the extracts from P. evecta by using the ATR/FT-IR spectroscopy. METHODS: The 50% ethanol-water crude leaf extract of P. evecta (EW-L) was prepared and was further fractionated to isolate various fractions. The anticancer activity was investigated from cytotoxicity against HepG2 using a neutral red assay and apoptosis induction by evaluation of nuclei morphological changes after DAPI staining. Synergistic anticancer effects of the extracts from P. evecta were performed using the ATR/FT-IR spectroscopy. RESULTS: The result showed that the EW-L showed higher cytotoxicity and apoptosis induction in HepG2 cells than its fractionated extracts. The hexane extract exhibited higher cytotoxicity and apoptosis induction than the water extracts, but less than the EW-L. The combined water and hexane extracts apparently increased cytotoxicity and apoptosis induction. The %apoptotic cells induced by the extract mixture were increased about 2-fold compared to the single hexane extract. CONCLUSIONS: The polar extract fraction is necessary for the anticancer activity of the non-polar extract fraction. The ATR/FT-IR spectra illustrates the physical interaction among the constituents in the extract mixture and reveals the presence of polyphenolic constituents in the EW-L, which might play a role for the synergistic anticancer effect.

Anticancer effect of the extracts from Polyalthia evecta against human hepatoma cell line (HepG2).

OBJECTIVE: To investigate the anticancer activity of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep against human hepatoma cell line (HepG2). METHODS: The anticancer activity was based on (a) the cytotoxicity against human hepatoma cells (HepG2) assessed using a neutral red assay and (b) apoptosis induction determined by evaluation of nuclei morphological changes after DAPI staining. Preliminary phytochemical analysis of the crude extract was assessed by HPLC analysis. RESULTS: The 50% ethanol-water crude leaf extract of P. evecta (EW-L) showed greater potential anticancer activity with high cytotoxicity [IC50 = (62.8 ± 7.3)µg/mL] and higher selectivity in HepG2 cells than normal Vero cells [selective index (SI) = 7.9]. The SI of EW-L was higher than the positive control, melphalan (SI = 1.6) and the apoptotic cells (46.4 ± 2.6) % induced by EW-L was higher than the melphalan (41.6 ± 2.1)% (P<0.05). The HPLC chromatogram of the EW-L revealed the presence of various kinds of polyphenolics and flavonoids in it. CONCLUSIONS: P. evecta is a potential plant with anticancer activity. The isolation of pure compounds and determination of the bioactivity of individual compounds will be further performed.

Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2) cell line.

BACKGROUND: Six plants from Thailand were evaluated for their cytotoxicity and apoptosis induction in human hepatocarcinoma (HepG2) as compared to normal African green monkey kidney epithelial cell lines. METHODS: Ethanol-water crude extracts of the six plants were tested with neutral red assay for their cytotoxicity after 24 hours of exposure to the cells. Apoptotic induction was tested in the HepG2 cells with diamidino-2-phenylindole staining. DNA fragmentation, indicative of apoptosis, was analyzed with agarose gel electrophoresis. Alkylation, indicative of DNA damage, was also evaluated in vitro by 4-(4'-nitrobenzyl) pyridine assay. RESULTS: The extract of Pinus kesiya showed the highest selectivity (selectivity index = 9.6) and potent cytotoxicity in the HepG2 cell line, with an IC50 value of 52.0 ± 5.8 µg/ml (mean ± standard deviation). Extract of Catimbium speciosum exerted cytotoxicity with an IC50 value of 55.7 ± 8.1 µg/ml. Crude extracts from Glochidion daltonii, Cladogynos orientalis, Acorus tatarinowii and Amomum villosum exhibited cytotoxicity with IC50 values ranging 100-500 µg/ml. All crude extracts showed different alkylating abilities in vitro. Extracts of P. kesiya, C. speciosum and C. orientalis caused nuclei morphological changes and DNA laddering. CONCLUSION: The extracts of C. speciosum, C. orientalis and P. kesiya induced apoptosis. Among the three plants, P. kesiya possessed the most robust anticancer activity, with specific selectivity against HepG2 cells.