Isolation and cytotoxicity of triterpenes from the roots of Phyllanthus pulcher Wall. ex Müll. Arg. (Euphorbiaceae).

The dried powdered roots of Phyllanthus pulcher Wall. ex Müll. Arg. (Euphorbiaceae), were sequentially extracted with dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH). The extracts were tested for cytotoxic activity against three human cancer cell lines: MCF-7 (breast), NCI-H460 (lung) and DU-145 (prostate). The DCM extract exhibited the strongest cytotoxic activity compared with EtOAc and MeOH extracts. Hence from the DCM extract, five pentacyclic triterpenes, 3α-acetoxyl-25-hydroxyolean-12-en-28-oic acid (1), glochidone (2), 12(13)-dehydro-3α-acetoxyolean-28-oic acid (3), lupanyl acetate (4) and glochidonol (5) were isolated and identified by spectroscopic analyses (1H NMR, 13C NMR, FT-IR, UV, DEPT, HMQC, HMBC and HREIMS). This is the first report on the isolation of 4 from a natural source, whereas 1 and 3 have already been isolated from the families Hamamelidaceae and Compositae (Asteraceae), respectively. However this is the first study reporting the presence of 1 and 3 in the Euphorbiaceae family. The isolated tritepenes 1-5 were tested against the three human tumour cell lines as stated above. Only compounds 1 and 5 exhibited cytotoxic activity, 5 being most potent with IC50 values ranging 7.5–13.4 µg/mL (17.1–30.5 µM).


INTRODUCTION
The genus Phyllanthus is widely distributed throughout the tropical and subtropical parts of the world and has been extensively used in Asian traditional medicine (Burkill, 1935).The wide range of its pharmacological activities was discussed in a number of reviews (Unander et al., 1990(Unander et al., , 1992;;Bagalkotkar et al., 2006).The extensive phytochemical investigations on several Phyllanthus species revealed various bioactive constituents such as alkaloids, lignans, terpenes, flavones and tannins (Matsunga et al., 1993;Foo, 1995;Bila et al., 1996;Houghton et al., 1996;Lee et al., 1996;Zhang et al., 2001Zhang et al., , 2002;;Chang et al., 2003).In our continued drug discovery programme from natural Afr.J. Pharm.Pharmacol.products, screening of a few selected Phyllanthus species led to the identification of Phyllanthus pulcher with strong cytotoxic activity against several cancer cell lines.In Malaysia, P. pulcher is traditionally used for the treatment of stomach-ache and ulceration (Burkill, 1935).Only recently the biological activities of this species have been reported which includes anti-HIV-1 reverse transcriptase and anti-angiogenesis (Eldeen et al., 2010;Ng et al., 2010).However, there has been no report on the phytochemical investigation of this species.Hence in this article we report for the first time the isolation and structural elucidation of the main bioactive compounds from this plant that are believed to be responsible for the cytotoxic effects on cancer cell lines.These compounds are lupane-type triterpenoids and they share structural similarity to other well known triterpenoids such as betulinic acid and lupeol which were reported to possess good anticancer activity profile in vitro.In addition since there have been no previous reports on the identification of bioactive compounds from the roots of the plant, we believe the present study provides opportunities for researchers interested in developing standardised phytomedicines from this plant.This is necessary as this plant has been used in various traditional medicine systems as mentioned above and development of standardised products would be advantageous for human use.

General experimental procedures
Melting points were determined with Fisher-Johns aluminium heating stage melting point apparatus equipped with adjustable magnifier model 12-144 and are uncorrected.Optical rotations were measured with a JASCO DIP-370 digital polarimeter.UV spectra were obtained with a CARY 50 Conc UV-Visible spectrophotometer (Varian).The IR spectra were recorded on a Perkin Elmer RXI FT-IR spectrophotometer.The EIMS spectra were recorded on a Thermo Finnigan-POLARISQ mass spectrometer, with ionization induced by electron impact at 70 ev.HREIMS were measured on Finnigan/MAT 95XL-T Nier-Johnson geometry mass spectrometer. 1H and 13 C NMR spectra were obtained on a Varian unity INOVA-500MHz NMR spectrometer.2D NMR ( 1 H-1 H COSY, HSQC, HMBC and DEPT) spectra were recorded using standard pulse sequences.Column chromatography separations were carried out using Merck silica gel (230-400 mesh).Analytical thin-layer chromatography (TLC) was performed on 20×20 cm Merck silica gel 60 F254 aluminum plates.Doxorubicin (purity: >99%) was obtained from Pharmacia (Perth, Australia).

Plant material
The plant of P. pulcher was collected from the cultivation experimental plot of the Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, Malaysia between June -August of 2004 and 2005.A voucher specimen (No.SK 306/02) has been deposited at the Herbarium of the Institute of Biosciences, Universiti Putra Malaysia, Malaysia.

Cytotoxic activity
Microculture tetrazolium (MTT) cytotoxic assay was utilised to determine the cancer cell viability after addition of extracts or compounds.This assay is based on the ability of the dehydrogenase enzymes in living cells to convert soluble yellow MTT (into insoluble purple formazan) (Mosmann, 1983).The assay was carried out in the Laboratory of Pharmacotherapeutics, Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia.
Cancer cells MCF-7 (breast), NCI-H460 (non-small cell lung) and DU-145 (prostate) were grown in RPMI-1640 culture medium (Gibco, Auckland, New Zealand) supplemented with 10% heatinactivated foetal calf serum (PAA Laboratories, Linz, Austria), penicillin (100 IU/mL) and streptomycin (100 µg/mL) (Gibco, Auckland, New Zealand) in a humidified atmosphere with 5% CO2 at 37°C.The cells were subcultured using trypsin-EDTA every 3-4 days.Exponentially growing cells were plated in 96-well flat bottom microculture plates (TPP, Trasadingen, Switzerland) with cell numbers plated adjusted so that cells are subconfluent at the end of the assay.The cells were allowed to attach for about 24 h before the addition of extracts or isolated compounds.A stock concentration of 100 mM or 100 mg/mL for each test agent was made up in DMSO.The working concentrations ranging from 1 to 1000 µM or 0.1 µg/mL -100.0 µg/mL were obtained by serial dilution of stock in culture medium and 20 µL of each of the concentration was added into the appropriate wells in four replicates to obtain final concentrations ranging from 0.1 to 100 µM.The control cells were treated with the highest concentration of DMSO (0.1%) as vehicle control.The cells were exposed continuously to test agents for 96 h.This was followed by addition of MTT (PhytoTechnology Laboratories, Kansas, USA) to a final concentration of 0.4 mg/mL per well and the cells were further incubated for 4 h to allow conversion of MTT.The resulting formazan crystals were dissolved with dimethysulfoxide (Fisher Scientific, Leicestershire, United Kingdom) and the absorbance of the purple formazan was read at 550 nm using a 96-well microplate reader (VERSAmax, Molecular Devices, California, United States of America).Dose-response curves were constructed to obtain the 50% inhibitory concentrations (IC50) according to method of Jada et al. (2008).

RESULTS AND DISCUSSION
The investigation of phytochemicals of the roots of this species with cytotoxic activity led to the isolation of five triterpenes 1-5 (Figure 1).Compound 4 is reported for the first time from a natural Bagalkotkar et al. 185 source while 1 and 3 are isolated for the first time from Euphorbiaceae family.Compounds 1, 2 and 5 were identified by comparison of their spectral data with the literature values, which supported the structures as 3αacetoxyl-25-hydroxyolean-12-en-28-oic acid (Chun et al., 2002) and, glochidone and glochidonol (Puapairoj et al., 2005), respectively.Although 12(13)-dehydro-3α-acetoxyolean-28-oic acid (3) was previously reported (Bohlmann et al., 1979), here we are representing the complete data for the first time.
The 1 H NMR spectrum also showed a signal at δ 3.38 (1H, dd, J ax/ax = 11.2,J ax/eq 4.1 Hz) due to coupling with two methylene protons at CH 2 -2, was attributed to H-3, and it was confirmed by its HMBC correlations with δ C 23.8 (C-2), 37.5 (C-4), 54.9 (C-5), 29.2 (C-23), 16.6 (C-24) and 171.8 (C=O) (Figure 2).The chemical shift of this proton showed that the stereochemistry was assignable to either axial (α) or equatorial (β) position.The values of the chemical shift couplings (diaxial (11.2 Hz) and axial/equatorial (4.1 Hz) interactions suggested that H-3 was in an axial (α) position, hence the acetate was in an equatorial (β) position.On the basis of above spectroscopic analyses, 4 was confirmed as lupanyl acetate, which was formerly reported as a synthetic product from lupenyl acetate (Heilbron et al., 1938).However, this is the first report of its occurrence as a natural product.DCM extract of the roots of P. pulcher was most potent and its activity was more selective towards MCF-7 cells when compared with DU-145 and H460 (non-small cell lung) cell lines (Table 1).The cytotoxic activity of the isolated compounds 1-5 was evaluated in vitro against the same three human tumour cell lines as above.Compound 1 exhibited cytotoxic activity against MCF-7 and DU-145 cell lines with IC 50 values of 26.4 and 39.9 µg/mL, respectively, but it failed to exhibit activity against H460 cells (Table 2).Compound 5 showed cytotoxic activity against MCF-7, DU-145 and H460 cell lines with Afr.J. Pharm.Pharmacol.the IC 50 values of 7.5, 13.4 and 9.5 µg/mL, respectively (Table 2).Among all the isolated compounds only compound 5 exhibited cytotoxic activity against H460cells (Table 2).Compounds 2, 3 and 4 failed to exhibit cytotoxic effect against all the tested cell lines (Table 2).
Compound 5 had stronger activity against MCF-7 cells by nearly 3-fold compared with 1.
Testing the compounds against the three cancer cell lines provided insights into the selectivity profile.This is vital as selectivity could be used to predict the extent of toxicity to normal tissues.Compound 5, which was identified as lupane-type triterpenoid, was the most potent agent against the panel of three cell lines tested and rewardingly the compound exhibited selectivity ` Bagalkotkar et al. 187   towards MCF-7 cells (IC 50 17.1 µM).Search of the literature revealed that lupane-type triterpenoids such as lupeol possess in vitro antitumour, antiangiogenic and apoptosis inducing properties in cancer cells (Moriarty et 1998;You et al., 2003;Aratanechemuge et al., 2004) which is in accordance with our findings.Interestingly, betulinic acid, another lupane-type triterpenoid with good in vitro anticancer activity was reported to be less cytotoxic to normal cells such as normal human astrocytes and melanocytes (Cichewicz and Kouzi, 2004) Basing on the above evidences, we propose that compound 5 would be less toxic against normal tissues.Afr.J. Pharm.Pharmacol.However, the most appropriate strategy to assess the toxicity of a compound to normal tissues of the whole body is through evaluation in animal models.Therefore, in vivo toxicity study would be advantageous.
By comparing the IC 50 values (in µg/mL) of DCM extract with that of compounds 1 and 5, it would seem obvious that the cytotoxicity exhibited by DCM extract could mainly be due to the presence of 5 as the major compound in the extract.However, there may exist minor bioactive compounds such 1 in the DCM extract which could have also contributed to the cytotoxicity of the extract.

Conclusion
The DCM extract of roots of P. pulcher displayed a strong cytotoxic activity against MCF-7, DU-145 and H460 cell lines, with IC 50 values of 9.8, 13.4 and 26.0 µg/mL, respectively.This prompted us to perform chemical investigation to identify the bioactive compounds responsible for the cytotoxicity of the extract which led to the isolation of five triterpenoids.We found that lupanetype triterpenoids accumulated in the roots of this species with 1 and 5 being the cytotoxic compounds.Upon a closer examination to relate the bioactivity with that of the compounds' chemical structures revealed both compounds possessed a free hydroxyl moiety.Such a feature is also apparent among the biologically active lupanetype triterpenoids such as betulinic acid and lupeol.Hence, we strongly believe this could be an important pharmacophore to be considered for cytotoxicity in this class of compounds.Comparison of the structures of the compounds 2 and 5 bear close resemblance to each other.The only notable difference is the additional hydroxyl group in 5, which might have been derived biosynthetically from the hydroxylation of 2. In this investigation, 5 has been identified as the most promising compound due to its selective cytotoxicity against breast cancer cells.

Figure 1 .
Figure 1.Chemical structures of compounds 1-5 from the roots of Phyllanthus pulcher.