Abstract: Pereskia bleo (Cactaceae) was investigated for its cytotoxic effect against selected human cell lines, namely the Human Nasopharyngeal Epidermoid Carcinoma Cell Line (KB), Human Cervical Carcinoma Cell Line (CasKi), Human Colon Carcinoma Cell Line (HCT 116), Hormone-Dependent Breast Carcinoma Cell Line (MCF7) and non-cancer Human Fibroblast Cell Line (MRC-5) by using an in vitro neutral red cytotoxicity assay. All the crude methanolic and its fractionated extracts (hexane, ethyl acetate and water) exert no damage to the MRC-5 normal cells. The crude methanol extract and the ethyl acetate fraction of Pereskia bleo possessed notably high cytotoxic effect against KB cells with IC50 values of 6.5 and 4.5 μg mL-1, respectively. Four compounds isolated from the ethyl acetate fraction of Pereskia bleo were identified as phytol, β-sitosterol, 2,4-di-tert-butylphenol and vitamin E. All four compounds have not been reported for this plant.
INTRODUCTION
Pereskia bleo, commonly known as Jarum Tujuh Bilah (in Malay) and Cak Sing Cam (in Chinese) by the locals, belongs to the family Cactaceae. Pereskia bleo is a spiny shrub with distinct orange-red flowers and has been used as natural remedy in cancer-related diseases, either eaten raw or taken as a concoction brewed from fresh plant. The leaves were also consumed as vegetables by some people. Pereskia bleo is believed to have anti-cancer, anti-tumor, anti-rheumatic, anti-ulcer and anti-inflammatory properties. They are also used as remedy for the relief of gastric pain and for revitalising the body (Goh, 2000).
There were only two phytochemical and biological studies reported for this plant. The earliest phytochemical study was by Doetsch et al. (1980) who reported the isolation of four alkaloids, namely 3,4-dimethoxy-β-phenethylamine, mescaline, 3-methoxytyramine and tyramine from Pereskia bleo. A more recent investigation by Tan et al. (2005) reported that the methanol extract of Pereskia bleo possesed cytotoxic effect against T-47D cells and cell death was found to be apoptotic in nature mainly via the activation of caspase-3 and c-myc pathways.
In view of the traditional use of Pereskia bleo in cancer-related diseases and the initial investigation by Tan et al. (2005) which suggested the potential use of Pereskia bleo in the treatment of breast cancer, it was thus necessary to further expand this area of research to other cancer cell lines. An investigation on the cytotoxic effects of the methanol and fractionated extracts of Pereskia bleo against selected human cancer cell lines, namely the Human Nasopharyngeal Epidermoid Carcinoma Cell Line (KB), Human Cervical Carcinoma Cell Line (CasKi), Human Colon Carcinoma Cell Line (HCT 116), Hormone-Dependent Breast Carcinoma Cell Line (MCF7) and non-cancer Human Fibroblast Cell Line (MRC-5) using an in vitro neutral red cytotoxicity assay was thus pursued and the findings is reported in this communication. In addition, identification of isolated compounds present in the active fraction was also performed.
MATERIALS AND METHODS
Plant Material
The fresh leaves of Pereskia bleo were collected from Petaling
Jaya, Selangor, Malaysia in July to September 2006. They were identified
by Professor Dr. Halijah Ibrahim of Institute of Biological Sciences,
Faculty of Science, University of Malaya, Malaysia and voucher specimens
were deposited at the herbarium of the Institute of Biological Sciences,
Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
Instrumentation
NMR spectra were recorded on a JEOUL 400 MHZ FT NMR spectrometer at
400 MHZ for 1H NMR and at 100.40 MHZ for 13C NMR.
Internal standards used in 1H NMR spectra was TMS (δ:
0.00) for CDCl3; in 13C NMR was CDCl3 (δ:
77.0).
GCMS analysis was performed using a Agilent Technologies 6980 N gas chromatograph equipped with a 5979 Mass Selective Detector (70 eV direct inlet); a HP-5ms (5% phenyl methyl siloxane) capillary column (30.0 m x250 μm x0.25 μm) initially set at 60 °C for 10 min, then programmed to 230 °C at 3 °C min-1 and held for 1 min at 230 °C using helium as the carrier gas at a flow rate of 1 mL min-1.
The GC analysis was performed on a Shidmadzu GC 14A equipped with a FID detector using fused-silica capillary HP-5 ms column (30.0 m length x250 μm; 0.25 μm film thickness) with helium as carrier gas at a flow rate of 1 mL min-1. Column temperature was programmed initially at 60 °C for 10 min, then programmed to 230 °C at 3 °C min-1 and held for 1 min at 230 °C.
Thin Layer Chromatography
Thin Layer Chromatography (TLC) analyses were carried out using precoated
TLC plates 60 F254 (20.25 mm thickness) purchased from Merck
and were visualized in UV light (254 and/or 343 nm) and/or iodine vapour.
Preparation of Plant Extracts for Cytotoxic Investigation
The leaves of Pereskia bleo (4526.79 g) were washed, dried
and ground to a fine powder (752.92 g) using a blender. The dried, ground
leaves (752.92 g) were then soaked in methanol (1.5 L) for 3 days at room
temperature. The solvent-containing extract was then decantered and filtered.
The extraction of the ground leaves were further repeated (2x) with methanol
(1.5 L each time). The filtrate from each extraction was combined and
the excess solvent was evaporated under reduced pressure using a rotary
evaporator to give a dark green crude methanol extract (79.81 g, 10.60%).
The methanol extract (33.58 g) was further extracted with hexane to give
a hexane-soluble fraction (2.19 g, 6.52%) and a hexane insoluble residue.
The hexane-insoluble residue was further partitioned between ethyl acetate-water
(1:1, 100 mL :100 mL) to give an ethyl acetate-soluble fraction (0.99
g, 2.95%). The water layer was freeze-dried to give a brown coloured water
fraction (18.14 g, 54.02%). The crude methanol and fractionated extracts
were dissolved in Dimethylsulfoxide (DMSO) with the exception of the water
fraction, to form stock solutions 20 mg mL-1 before testing.
The final concentration of DMSO in test wells was not in excess of 1 v/v%.
In vitro Cytotoxic Assays
Human Cell Lines
Human cancer cell lines used were the nasopharyngeal epidermoid carcinoma
KB, the cervical carcinoma CasKi, colon carcinoma HCT 116 and hormone-dependent
breast carcinoma MCF7. The normal fibroblast cell line MRC-5 was also
used. The cells were purchased from the American Tissue Culture Collection
(ATCC, USA). KB cells were maintained in Medium 199 (Sigma), CasKi cells
and MCF7 cells in RPMI 1640 medium (Sigma), HCT 116 in McCOY`S 5A Medium
(Sigma) and MRC-5 cells in EMEM (Eagle Minimum Essential Medium) (Sigma),
supplemented with 10% foetal bovine serum (FBS, PAA Lab, Austria), 100
μg mL-1 penicillin or streptomycin (PAA Lab, Austria)
and 50 μg mL-1 of fungizone (PAA Lab, Austria). The media
were filter sterilized using a 0.22 μm filter membrane (Schleicher
and Schuell). The cells were cultured in a 5% CO2 incubator
(Shel Lab water-jacketed) kept at 37 °C in a humidified atmosphere.
The culture was further subcultured every 2 or 3 days and routinely checked
under an inverted microscope (IMT-2 Olympus, Japan) for any contamination.
Neutral Red Cytotoxicity Assay
The neutral red cytotoxicity assay was used as described previously
by Borenfreund and Puerner (1984). The IC50 is the concentration
of extract that causes 50% inhibition or cell death and was obtained by
plotting the percentage of inhibition versus concentrations of plant samples.
The extract that gave an IC50 value of 20 μg mL-1
or less was considered active (Swanson and Pezzuto, 1990).
Extraction and Isolation of Compounds 1-4
Dried, ground leaves Pereskia bleo (1050.56 g) were extracted
(3x) with methanol (1.5 L each time). The methanol-containing extract
obtained was initially treated with charcoal, then filtered over celite
and the filtrate was evaporated under reduced pressure to give a crude
methanolic extract (99.44 g). Treatment with charcoal was necessary to
remove the high content of chlorophyll present in the extract. The presence
of chlorophyll interfered with efforts at chromatographic separation.
The crude methanol extract was then further partitioned with ethyl acetate
and water using a separating funnel. The solvent in the ethyl acetate-soluble
layer was then removed under reduced pressure to give the ethyl acetate
fraction (18.34 g). The ethyl acetate fraction was subjected to flash
column chromatography over Merck kieselgel 60 (0.063-0.200 mm size); elution
starting with chloroform with stepwise increment of methanol. The chloroform
fraction (3.47 g) obtained was then purified over a column (3x49 cm) packed
with 180 g of silica gel 60 (0.063-0.200 mm) and the components were separated
by successive elution with hexane followed by hexane enriched with increasing
percentages of dichloromethane, monitoring with TLC.
Compound 1 was isolated from the fraction obtained upon elution with d ichloromethane-hexane (1.6:8.4). Further elution with dichloromethane-hexane (3.5: 6.5) yielded a fraction (39.6 mg) from which compound 2 was obtained. Preparative-TLC on the dichloromethane-hexane (1:9) fraction (192.7 mg) using ethyl acetate: hexane (1:9) as developing solvent yielded compound 3 (21.0 mg). Repeated prep-TLC on the dichloromethane-hexane (1.6: 8.4) fraction (64.2 mg) using chloroform as the developing solvent yielded 4 (10.6 mg). The extraction and fractionation procedures leading to the isolation of compounds is shown in Fig. 1.
| Fig. 1: | Fractionation and isolation of bioactive compounds of Pereskia belo |
RESULTS AND DISCUSSION
Cytotoxic Activity of the Crude Methanol and Fractionated Extracts
The results of preliminary cytotoxicity screening of the methanol
and fractionated extracts are summarized in Table 1.
The methanol extract of Pereskia bleo demonstrated some cytotoxic
effect when screened against KB, CaSki, HCT 116 and MCF7 cancer cell lines,
the highest inhibitory effect was shown by KB cells (IC50
6.5 μg mL-1). The water fraction was found to have no
effect on all the cancer cell lines (IC50 >100 μg mL-1
in all cases). The ethyl acetate fraction was the only fraction that possessed
high inhibitory effect against KB cells with an IC50 value
of 4.5 μg mL-1. It can thus be concluded that the ethyl
acetate fraction was selectively toxic against KB cells. Even though the
mechanism of action has not been investigated in the present study, DNA
fragmentation studies are now being planned. Chemical investigation was
also directed to the ethyl acetate fraction. The active ingredients may
lead to valuable compounds that may have the ability to kill cancer cells
but exert no damage to normal cells (IC50 >100.0 μg
mL-1 against normal cells, MRC-5). At this stage, no conclusion
can be drawn regarding damage to normal cells as the effect of over-use
of Pereskia bleo would need chronic toxicity studies involving
experiments on animals.
| Table 1: | In vitro cytotoxic activity (IC50 μg mL-1) of crude methanolic and fractionated extracts of Pereskia bleo against various cancer and non-cancer cell lines |
The ethyl acetate fraction also displayed a slightly lower cytotoxic activity against HCT 116 and MCF7 cells (IC50 22.0 and 28.0 μg mL-1, respectively).The hexane fraction was found to exert a moderate effect on KB and MCF cells (IC50 28.0 and 25.0 μg mL-1, respectively). The cytotoxic effect on MCF7 was not consistent to that reported by Tan et al. (2005) on the effect of methanol extract of Pereskia bleo against T-47D cells. It is not possible to make a direct comparison of different cell lines. It would be advantageous to repeat the investigation to see the effect of Pereskia bleo on T-47D cells. All the fractions demonstrated weak cytotoxic activity against CaSki cells compared to other cell lines.
Structural Determination of Isolated Compounds 1-4
Compound 1 was identified as phytol, 2 as β-sitosterol, 3 as
2,4-di-tert-butylphenol and 4 as Vitamin E. All four compounds have not
been reported for this plant. Further studies are in progress on the effect
of the isolated compounds on KB cells and to explore its mode of action.
Compound 1 (Phytol). Yellow viscous oil; EI-MS m/z (%): 278 (2, [M+-H2O]), 137 (5, [C10H18]+), 123 (40, [M-CH2]+), 109 (15, [M-CH2]+), 95 (36, [M-CH2]+), 81 (38, [M-CH2]+), 71 (100, (CH3)2CHCH2CH2+), 57 (62, (CH3)2CHCH2+); 1H-NMR (CDCl3, 399.65 MHZ)δ: 0.82 (12H, H-16, H-17, H-18, H-19, methyls), 0.95-1.61 (19H, m, methyls and methyline protons), 1.68 (3H, s, H-2), 2.00 (2H, t, J = 7.6 Hz, H-4), 4.15 (2H, d, J = 7.1 Hz, H-1), 5.42 (1H, t, J = 7.1 Hz, H-2); 13C-NMR (CDCl3, 100.40 MHZ)δ: 59.4 (C-1), 123.1 (C-2), 140.3 (C-3), 39.9 (C-4), 25.1 (C-5), 36.6 (C-6), 32.8 (C-7), 37.3 (C-8), 24.5 (C-9), 37.4 (C-10), 32.7 (C-11), 37.3 (C-12), 24.8 (C-13), 39.4 (C-14), 28.0 (C-15), 22.6 (C-16), 22.7 (C-17), 19.7 (C-18), 19.7 (C-19), 16.2 (C-20).
Compound 1, present as the major component in this plant, was identified by comparison of its spectral data as written above with that reported by Ming et al. (2004). Phytol (1) showed significant anti-tumor activity against P388 mouse lymphocytic leukemia cells (Phutdhawong et al., 2004), molt 4B lymphoid leukemia cells (Hibasami et al., 2002), HT-29 human colon cancer cells, MG-63 osteosarcoma cells and AZ-521 gastric cancer cells (Lee et al., 1999). The cytotoxic activity of phytol was due to an iduction of apoptosis (Komiya et al., 1999). Based on these reported studies, it can be concluded that phytol (1) maybe responsible for the remarkable cytotoxic effect of the ethyl acetate fraction against the KB cancer cell lines.
Compound 2 (β-sitosterol). White coloured needles; EI-MS m/z (%): 414 (100, M+), 396 (57, [M-H2O]+), 381 (43, [M-H2O-CH3]+); 1H-NMR (CDCl3, 399.65 MHZ)δ: 0.69 (3H, s, H-18), 0.82 (3H, s, H-27), 0.83 (3H, s, H-26), 0.85 (3H, s, H-29), 0.94 (3H, br, H-19), 1.02 (3H, s, H-21), 0.97-2.28 (m, CH2, CH), 3.54 (H-3), 5.36 (br, H-6); 13C-NMR (CDCl3, 100.40 MHZ)δ: 37.2 (C-1), 31.7 (C-2), 71.8 (C-3), 42.3 (C-4), 140.7 (C-5), 121.7 (C-6), 31.9 (C-7), 32.2 (C-8), 50.1 (C-9), 36.5 (C-10), 21.1 (C-11), 39.8 (C-12), 42.3 (C-13), 56.8 (C-14), 24.3 (C-15), 28.2 (C-16), 56.0 (C-17), 11.8 (C-18), 19.4 (C-19), 36.1 (C-20), 18.8 (C-21), 33.9 (C-22), 26.0 (C-23), 45.8 (C-24), 29.1 (C-25), 19.8 (C-26), 19.2 (C-27), 23.1 (C-28), 12.0 (C-29). These data were in fair agreement to that reported by Nes et al. (1992). In agreement with literature data (Block et al. (2004), Chaturvedula et al. (2002), Awad et al. (2000), Moghadasian (2000) and Jackson et al. (2000), β-sitosterol (2) was not known to be cytotoxic to several cell lines.
Compound 3 (2,4-ditert-butylphenol). Yellow coloured crystalline powder; EI-MS m/z (%): 206 (15, M+), 191 (100, [M-CH3]+); 1H-NMR (CDCl3, 399.65 MHZ)δ: 1.24 (9H, s, three methyls), 1.36 (9H, s, three methyls), 6.53 (d, J = 8.39 Hz, H-6), 7.02 (dd, J = 8.39, 2.40 Hz, H-5), 7.24 (d, J = 2.40 Hz, H-3); 13C-NMR (CDCl3, 100.40 MHZ)δ: 151.7 (C-1), 135.2 (C-2), 123.5 (C-3), 143.0 (C-4), 124.0 (C-5), 115.9 (C-6), 30.0 (C-7), 34.7 (C-8), 31.6 (C-9, 10, 11), 31.8 (C-12,13,14). The spectral data was in agreement with that reported by Kamitori et al. (1984). Compound 3 is structurally related to the well known antioxidant Butylated Hydroxyanisole (BHA). It is highly probable that compound 3 has antioxidant properties too. Phenolic antioxidants were reported to exert anticarcinogenic activity, presumably through the induction of phase II detoxifying enzymes such as glutathione S-transferases and quinone reductase, which provides prevention of tumour initiation (Yoshioka et al., 1995; Yu et al., 1997). They can also decrease oxidative stress-induced carcinogenesis by a direct scavenging of Reactive Oxygen Species (ROS) (Tanaka et al., 1997) and by inhibiting cell proliferation secondary to the inhibition of protein phosphorylation (Schreck et al., 1992).
Compound 4 (Vitamin E). Yellow viscous oil; EI-MS m/z (%): 430 (34, [M+]), 205 (8, C13H17O2), 165 (100, C10H13O2). Compound 4 was identified by comparison of its mass spectral data with that reported by Pereira et al. (2002).
Vitamin E (4) is a dietary anti-oxidant capable of eliminating free radical damage, inducing apoptosis and decreasing oncogene expression (Bermudez et al., 2007). Previous studies indicated that Vitamin E decrease the toxicity of chemotherapy without reducing its effectiveness (Lamson and Brignall, 1999; Drisko et al., 2003). Bermudez et al. (2007) suggested that Vitamin E might be an important protective agent against ovarian cancer cell growth as well as potentially effective therapeutic adjuvant. Structures of compounds 1-4 are shown in Fig. 2.
| Fig. 2: | Chemical compounds isolated from the ethyl acetate fraction of Pereskia bleo |
CONCLUSIONS
Pereskia bleo, which was selected for the present study based on its traditional medicinal use, showed remarkable inhibitory activity against KB cells. The ethyl acetate fraction showed very good cytotoxic activity against the nasopharyngeal epidermoid carcinoma KB cells but however, a study on the mechanism of signal transduction such as cell cycle and cell death would provide a more convincing evidence. An investigation into this phenomena is now underway. In addition this fraction also showed no cytotoxicity against the normal cell line; if this also occurs in vivo, the use of this plant by locals for cancer treatment would have scientific support. Thus, Pereskia bleo could be further developed for use in the treatment of the nasopharyngeal epidermoid carcinoma. Studies on the effect of the isolated components (present in the ethyl acetate fraction) on KB cells are now underway. The resulting information will certainly contribute to a better understanding of the anti-carcinogenic activity of the natural constituents in Pereskia bleo.
ACKNOWLEDGMENT
This study was supported by a research fund from the University of Malaya (Vote F F0155/2005D) and the Ministry of Science, Technology and Innovation (MOSTI) (e-sciencefund 1202032026). We are also grateful to Prof A. Hamid A Hadi for use of his laboratory space.