The Effect of Phyllanthus taxodiifolius Beille Extracts and its Triterpenoids Studying on Cellular Energetic Stage of Cancer Cells

Problem statement: The Multidrug Resistance phenomenon (MDR) is the c ause of unsuccessful in cancer chemotherapy. The tradition pla t is the population used as the alternative medicine in cancer therapy. Due to, P. taxodiifolius is medicinal Thai plant which is used as diuretic drug which has never been explored as the anticance r activities. Approach: Air-dried powder of P. taxodiifolius leaves and twigs were serially extracted by hexane , ethyl acetate, acetone and methanol. These four extracts were tested the antiproliferati ve activity on four cancer cell lines. These result s lead to successively purify two triternoids that are glo chidone ( 1) and lupeol ( 2). Both pure compounds were tested the anticancer properties on same cance r cell lines and further investigate the cellular energetic state perturbation by measuring the mitoc hondrial membrane potential modification. Results: Four crude extracts were extracted and two triterp enoids (glochidone and lupeol) were purified and identified from hexane extract. Our an tiproliferative activity of both compounds respecti vely showed in the IC50 value of K562, K562/Adr, GLC4 and GLC4/Adr equal t o 2.2±0.6, 4.2±1.5. 3.1±1.0 and 3.2±0.9 μg mL for glochidone and 2.3 ±0.6, 4.5±1.7, 2.3±0.5 and 2.6 ±0.5 μg mL for lupeol. The R value, which represents the multidrug resistance ph enotype, is about 2 for P-glycloprotein overexpress ion in K562/Adr and 1 for MRP1 overexpression in GLC4/A dr. Conclusion: All crude extractions and two triterpenoids show the clear evidence of anticancer activity of both sensitive and resistance of erythroleukemic and Small cell lung cancer cell lin es. Both compounds are not recognized by ABCB1 and ABCC1 proteins. Our results also indicated that lupeol initiate cell death by mitochondria membrane potential modification specially the sensi tive cell line.


INTRODUCTION
Cancer is one of the major death causes of disease. After the treatment, the Multidrug Resistance phenomenon (MDR) is a major obstacle to successful treatment outcome of many human malignancies cause by the reducing of intracellular drug target accumulation. MDR phenomena often associated with the over expression of protein transports, cellular energetic state and also cellular oxidative stress. This obstacle was wildly studied on its mechanism to find out the effective anticancer drug. New molecules either from the synthesis or pure compounds of plant extraction were explored (Monkodkaew et al., 2009: Nantapap et al., 2010Tangjai et al., 2008;Haque et al., 2006). Recently, the tradition plant is the population used as the alternative medicine in cancer therapy.
The Phyllanthus genus belongs to the Euphobiaceae family. By the diversified species of Phyllanthus, these plants have been wildly studied in various activities such as anti-inflammatory, antidiabetic, anti-diarrheal, antinociceptive, hepatoprotective activity and anticancer (Chudapongse et al., 2010;Kumar et al., 2008;Naaz et al., 2007;Shanbhag et al., 2010). The phytochemical study showed that the Phyllanthus genus consist triterpenoids, resins, steroids, alkaloids and phenolic compounds (Khatoon et al., 2006). The research on P. urinaria presented the apoptotic cell death in cancer cell lines. Anyway, P. taxodiifolius Beille which is used as diuretics drug in Thai traditional herbal medicine has not been study yet. In this work, we aim to study the anticancer properties by focusing its activity on mitochondrial.

Plant extractions:
The P. taxodiifolius Beille leaves and twigs were collected from Amnatcharoen, Thailand and identified by voucher specimen (BKF no. 127614). It has been deposited at the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand before using. These leaves and twings (5 kg) were air-dried and grinded to be fine powder before marinate sequentially from hexane (16 L×3 days ×6 times), ethyl acetate (17 L×3 days ×6 times), acetone (16 L×3 days ×7 times) and methanol (16 L×3 days ×7 times) for extraction. All four extractions were evaporated to collect crude extraction and tested the antiproliferation on cancer cell lines. This bioactive information guided us to further purify and identify pure compounds from hexane extraction and then investigate the bioactivity on same cancer cell lines. From the bioassay-guided fractionation, the hexane fraction was separated by chromatographic technique. The hexane extract (40.2 g) was separated by column chromatography over silica gel 300 g. Gradient elution was conducted initially with n-hexane, gradually enriched with ethyl acetate, followed by increasing amount of methanol in ethyl acetate and finally with methanol. Fractions (500 mL each) were collected and combined on the basis of TLC behavior. Fraction 2 and 3 which eluted by 10% ethyl acetate-methanol, was obtained as two white needles which identified as glochidone (1) and lupeol (2), respectively.
Cell and cell culture: The bioactivity of compounds were performed in four cancer cell lines (the erythroleukemia sensitive (K562) and its adriamycin resistance cell line (K562/Adr) and the small cell lung cancer sensitive (GLC4) and its adriamycin resistance cell line (GLC4/Adr)). Cells were cultivated in RPMI-1640 medium complement with 10% fetal bovine serum albumin and 1% antibiotic at 37°C, 5% CO 2 and 95% humidity incubator for 3days before experiment. The K562/Adr and GLC4/Adr are characterized as multidrug resistance phenotype by the over expression of plasma membrane proteins that is P-glycoprotein (ABCB1) and MRP1 (ABCC1), respectively.
Cytotoxic assay: The toxicity of compounds on cell growth was tested by incubate each cell line (5000 cells) with various concentration of each compound from 0 up to 250 µg mL −1 in RPMI-1640 containing 10% fetal bovine serum albumin and 1% antibiotic then cultured in 37°C for 72 h. Cell viability was determined by using MTT-colorimetric assay. In this assay MTT will be reduced to be formazan crystal (OD560) by the activity of mitochondria. At 72 h incubation time, 2.5 mg mL −1 of MTT was added in to each sample and incubated at 37°C for 4 h. After that the excessive MTT was removed and cells were washed twice with phosphate buffer solution pH 7.0. Then dimethyl sulfoxide solution was added to dissolve the formazan crystal. The cytotoxic activity (Puapairoj et al., 2005) of compound was presented as the IC 50 value, which represent the concentration of compound in which the cellular proliferation was inhibited to 50%. The percentage of cell-growth inhibition (IC%) was obtained from the following equation: The Resistance factor (R value) represent the multidrug resistance phenotype of such compound can be calculated as the IC 50 value of resistance cell divide by IC 50 value of its corresponding sensitive cell line.
Determination of the mitochondria membrane potential (∆ ∆ ∆ ∆Ψ Ψ Ψ Ψ m ): The mitochondria membrane potential which reflects to cellular energetic state of each cell line was determined by using the kinetic uptake of rhodamine B. The rhodamine B is a fluorescence probe that can be uptake into cell and accumulated in mitochondria by the defect of it potential. The fluorescence intensity at 582 nm (excited at 553 nm) of rhodamine B in mitochondria can be determined due to the local quencher by formazan crystals. The initial rate of rhodamine B fluorescence extinction could be determined and calculated to predict the ∆Ψ m (mVolt) value of intact cell as descript by Reungpatthanaphong et al. (2003). The effect of 2 pure compounds on mitochondrial membrane potential modification was studies on four cancer cell lines. The 2×10 6 cells of each cell were incubated with pure compound at the IC 50 concentration in 2 mL of Luckhoff Na + pH 7.3 at 37°C for 30 min before mitochondria membrane potential determination.

DISCUSSION
Phyllanthus taxodiifolius is a Thai plant used as diuretics drug in Thai traditional herbal medicine. The cytotoxic on cancer cell line not only show the anticancer activity of all extractions and pure compounds but also the anti multidrug resistance. The certain part of extractions and purification, we obtained triterpenoids compound glochidone and lupeol. Lupeol is a triterpenoids existent in foliage, flowering and fruit trees. Its bioactive was reported as an anti-leukemia and anti-inflammatory (Saleem, 2009). Our result showed that lupeol can disturb the cellular energetic state by the mitochondria membrane potential modification of two sensitive cancer cell lines which involve in the initiation of apoptotic cell death. These results are supported by several studied on triterpenoids also lupeol which are reported as the apoptotic potential by caspase activation and also effect via mitochondria (Chaturvedi et al., 2008;Li et al., 2005;Nigam et al., 2009;Reyes-Zurita et al., 2009).

CONCLUSION
All crude extractions and two triterpenoids (glochidone and lupeol) purified from hexane fraction of P. taxodiifolius leaves and twigs show the clear evidence of anticancer activity of both sensitive and resistance of erythroleukemic and small cell lung cancer cell lines. Our results also indicated that lupeol initiated cell death by mitochondria membrane potential modification.

ACKNOWLEDGEMENT
The researchers very grateful to Center for Innovation in Chemistry and the Thailand Research Fund and the Commission on Higher Education (Grant No. MRG5080217) and Lampang Rajabhat University for financial support. Moreover, Center of Excellent for Molecular Imaging (CEMI), Department of Radiologic Technology, Faculty of Associated Medical Sciences, Chiang Mai University for convenient use on flow cytometer, COULTER EPICS CL-MCLTM (Coultronics France SA) and Spectrofluorometer (PerkinElmer).